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J Cancer 2021; 12(12):3548-3557. doi:10.7150/jca.50292
Lenvatinib induces anticancer activity in gallbladder cancer by targeting AKT
1. Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052 P.R. China.
2. Key Lab of Digestive Organ Transplantation of Henan Province, Open and Key Laboratory of Hepatobiliary and Pancreatic Surgery and Digestive Organ Transplantation at Henan Universities, Zhengzhou Key Laboratory of Hepatobiliary and Pancreatic Disease and Organ Transplantation, Zhengzhou, Henan 450052.
3. Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.
4. Department of Histology and Embryology, Medical College of Zhengzhou University, Zhengzhou 450052, P.R. China.
*These authors contributed equally to this work.
Ye J, Qi L, Liang J, Zong K, Liu W, Li R, Feng R, Zhai W. Lenvatinib induces anticancer activity in gallbladder cancer by targeting AKT. J Cancer 2021; 12(12):3548-3557. doi:10.7150/jca.50292. Available from https://www.jcancer.org/v12p3548.htm
Gallbladder cancer (GBC) is characterized by poor prognosis, early metastasis, and high recurrence rates, which seriously threaten human health. The effect of lenvatinib, a widely used drug in anti-hepatocellular carcinoma in China, on GBC progress, as well as its underlying molecular mechanism, remains unclear. Therefore, the present study investigated the effect of lenvatinib on human GBC GBC-SD and NOZ cells and its underlying mechanisms. A series of experiments, including cell proliferation clone formation, wound healing, and cell migration and invasion assays, as well as flow cytometry, were performed to investigate the anticancer effect of lenvatinib on GBC. Western blotting was used to detect alterations in protein expression, of CKD2, CKD4, cyclin D1, caspase-9, matrix metalloproteinase (MMP)-2, cell migration-inducing protein (CEMIP) and phospho-AKT (p-AKT). In addition, the chemosensitivity of lenvatinib-treated GBC cells to gemcitabine (GEM) and whether the activation of phosphoinositide 3 kinase (PI3K)/AKT contributed to the chemoresistance, were determined. Finally, the anticancer effect of lenvatinib in vivo was detected using a xenograft mouse model. These data showed that treatment with lenvatinib inhibited cell proliferation, colony formation ability, migration, induced apoptosis, regulated cell cycle and resulted in decreased resistance to GEM. Treatment with lenvatinib decreased the expression of MMP-2, CEMIP, CDK2, CDK4 and cyclin D1, and increased the expression of cleaved caspase-9, which was mediated by the inactivation of the PI3K/AKT pathway in vitro. In addition, lenvatinib inhibited autophagy in GBC-SD and NOZ cells. Besides, Lenvatinib suppresses GBC cell growth in vivo by targeting p-AKT. In combination, the present data indicated that lenvatinib plays a potential anticancer role in GBC by downregulating the expression of p-AKT.
Keywords: lenvatinib, gallbladder cancer, proliferation, apoptosis, migration, AKT
Gallbladder cancer (GBC), one of the most common malignant tumors of the digestive system and has an overall 5-year survival rate of <5% . Currently, surgical resection is the most effective treatment for GBC, but its recurrence and mortality rates remain high . Other treatment options for advanced GBC include chemotherapy and radiotherapy. However, the therapeutic efficacy of those are limited and the prognosis of advanced GBC patients still very poor. It is therefore necessary to develop new treatments for advanced GBC patients.
In the past decade, sorafenib, multi-kinase kinase inhibitor [against vascular endothelial growth factor receptor 1 (VEGFR1)-3, fms-like tyrosine kinase 3, RET and RAF kinases, platelet-derived growth factor (PDGF) and KIT], has crucially contributed to the systemic therapy of patients with advanced or metastatic hepatocellular carcinoma (HCC) or clear cell carcinoma of the kidney [3, 4]. However, the overall response rate of sorafenib in the treatment of advanced HCC is not high (only 2.3%), the improvement of patients' main symptoms is not obvious, the side effects are obvious and there is a lack of effective molecular markers for prediction, which, combined with its high price, limit its use in patients with advanced HCC . As a result, several clinical trials on molecularly-targeted drugs, such as sunitinib  and brivanib , emerged but failed. Recently, lenvatinib has become another Food and Drug Administration-approved molecularly-targeted drug for the first-line treatment of advanced or metastatic hepatocellular carcinoma. In the REFLECT clinical trial, 954 HCC patients were randomly divided into the lenvatinib and sorafenib groups. As shown in that study, lenvatinib has been shown to obtain satisfactory results in increasing the OS, progression-free survival (PFS) and TTP, as compared with sorafenib in unresectable HCC patients . In addition, the subgroup analysis of the REFLECT study demonstrated that the efficacy of lenvatinib in patients with advanced HCC in China was more significant than in that in the global population, which suggested that lenvatinib can serve as another alternative first-line targeted drug for advanced HCC.
Lenvatinib, another new oral, molecularly-targeted anti-cancer drug, possesses an anti-cancer effect in HCC by inhibiting multi-targeted tyrosine kinase inhibitors against VEGFR1-3, fibroblast growth factor receptor (FGFR)1-4, PDGFRα, RET and KIT . Previous studies have that lenvatinib suppressed HCC and anaplastic thyroid cancer (ATC) cell growth in vitro and in vivo [9, 10]. In addition, treatment with lenvatinib and I-131 significantly inhibited cell growth and induced apoptosis by activating endoplasmic reticulum-associated apoptosis mechanisms . Heptanomide-lenvatinib combination treatment inhibited drug resistance by inhibiting the FGFR signaling pathway in ATC cancer stem cells . Although the anti-cancer effect of lenvatinib against HCC and ATC have been reported, both the effect and the underlying molecular mechanism of lenvatinib are yet to be investigated in GBC cells.
The phosphoinositide 3 kinase (PI3K)/AKT pathway is one of the most important signaling pathways that plays a key role in cell biological behavior, including cell growth, apoptosis, invasion, differentiation and metabolism . Increasing evidence has shown that the PI3K/AKT pathway is upregulated and involved in GBC [14, 15]. It is also known to play an important role in imparting chemoresistance, and its activation has been shown to increase the chemoresistance of cancer cells [16, 17]. A previous study reported that the expression of PI3K/AKT was increased in sorafenib-resistant liver cancer cells, and that the inactivation of AKT can sensitize cells to sorafenib-mediated apoptosis . However, the role of lenvatinib in the activation of PI3K/AKT remains unclear.
In the present study, in order to further investigate the role of lenvatinib in cell biological behavior, as well as the PI3K/AKT pathway in GBC cells, in vitro and in vivo assays were performed. The data from this study demonstrated that lenvatinib inhibited cell growth, colony formation, migration and invasion, and induced cell apoptosis and regulated cell cycle arrest progression. Besides, we first reported that treatment with lenvatinib significantly suppressed autophagy. In addition, treatment with lenvatinib enhanced the sensitivity of GBC to gemcitabine (GEM) by downregulating the activation of the PI3K/AKT pathway. The present findings suggested that lenvatinib treatment may have a therapeutic potential in patients with GBC.
Materials and methods
The human GBC GBC-SD and NOZ cell lines were obtained from Procell Life Science & Technology Co., Ltd. The GBC-SD cells were cultured in Rosewell Park Memorial Institute (RPMI)-1640 medium (cat. no. 31800; Beijing Science & Technology Co., Ltd.) and NOZ were cultured in DMEM/F12 (cat. no. SH30023.01; HyClone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products) at 37 °C in a humidified atmosphere with 5% CO2.
Cell viability assay
Lenvatinib was purchased from Selleck Chemicals (cat. no. S5240) and dissolved in dimethyl sulfoxide. Cell viability was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay. Human GBC-SD and NOZ cells were seeded onto 96-well plates at a density of 5×103 cells/well. After 24 h, the medium was removed and replaced with medium containing 0, 10, 20, 40 or 80 µM lenvatinib for different times (0, 24, 48 and 72 h). Following treatment, the cells were incubated with 10 μl CCK-8 solution in a 5% CO2 incubator at 37 °C for 1 h. The OD values were then obtained by measuring the absorbance at a wavelength of 450 nm using a micro-plate reader (Thermo Fisher Scientific, Inc.).
Colony formation assay
GBC-SD and NOZ cells were seeded onto 6-well plates at a density of 5×102 cells/well and culture medium containing lenvatinib was replaced every 3 days and incubated for 2 weeks until clones were visible. Next, cells were washed with PBS and fixed with methanol for 20 min, following staining with 0.1% crystal violet (Institute of Biotechnology) for 30 min at room temperature. Colonies containing >50 cells were counted under an inverted microscope. (IX71; Olympus Corporation).
For cell apoptosis analysis, GBC-SD and NOZ cells were detected using an Annexin V/fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences), according to the manufacturer's instructions. Human GBC-SD and NOZ cells were harvested following treatment with 25 and 50 µM lenvatinib for 48 h. The cell suspension was transferred to a culture tube and 5 µl propidium iodide (PI) fluorescent dye was added. Cells were then incubated at room temperature for 15 min in the dark, and 400 µl binding buffer was then added. Flow cytometry results were analyzed using a FACS Calibur system (BD Biosciences). For cell cycle arrest analysis, human GBC-SD and NOZ cells were collected following treatment with lenvatinib, washed with cold PBS and fixed in cold 75% ethanol at 4 °C overnight. Subsequently, GBC-SD and NOZ cells were washed with cold PBS, incubated with RNase and stained with PI in the dark for 15 min. The proportion of cells at the G0/G1, S and G2/M phases was determined using the FACS Calibur system, according to the manufacturer's instructions. Data were analyzed by BD FACS Diva 8.0.1 software.
Wound healing assay
Human GBC‑SD and NOZ cells were cultured in 6‑well plates overnight at 37 °C. Once the GBC‑SD and NOZ cells were spread over the plates, a vertical long wound was scratched into the cells using a 200-μl pipette tip. Following this, the scratched cells were washed twice with PBS and images of the cells were captured using an inverted microscope (IX71; Olympus Corporation). The cells were treated with 25 μM lenvatinib and cultured in medium for 48 h at 37 °C, following which a further image was obtained using an inverted microscope. The wound closure ratio was calculated as follows: (0 h width‑48 h wound width)/0 h wound width.
Migration and invasion assay
For the migration assay, GBC‑SD and NOZ cells were suspended in 100 μl serum‑free RPMI-1640 medium and placed in the upper chamber of a Transwell chamber coated with (for the invasion assay) or without (for the migration assay) Matrigel (BD Biosciences). In addition, 600 µl medium containing 10%FBS was added in the lower chamber of a Transwell. Subsequently, 25 or 50 μM lenvatinib was applied to the chamber and incubated for 24 h at 37 °C. Invasive cells on the lower surface of the membrane were washed with PBS and fixed in methanol for 20 min. The invasive cells were stained with 0.1% crystal violet for 20 min at room temperature. The cellular migration or invasion through the membrane was visualized using an inverted microscope (IX71; Olympus Corporation).
Cells were collected and total protein was extracted from GBC-SD and NOZ cells on ice using RIPA lysis buffer (Institute of Biotechnology) with a protease inhibitor cocktail (104 mM AEBSF, 80 μM Aprotinin, 5 mM Bestatin, 1.5 mM E-64, 2 mM Leupeptin and 1.5 mM Pepstatin A; MedChemExpress) for 20 min. The protein concentration was determined using a BCA-kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millpore,cat.no IPVH00010). The membranes were immunoblotted with the following primary antibodies: Matrix metalloproteinase (MMP)-2 (dilution, 1:800; cat. no., 10737-2-AP), cell migration-inducing and hyaluronan-binding protein (dilution, 1:600; cat. no., 21129-1-AP), CDK2 (dilution, 1:2,000; cat. no., 10122-1-AP), CDK4 (dilution, 1:1,000; cat. no., 11026-1-AP), Cyclin D1 (dilution, 1:5,000; cat. no., 60186-1-Ig), Caspase-9 (dilution, 1:600; cat. no., 66169-1-Ig) and β-actin (dilution, 1:5,000; cat. no., 60008-1-Ig) (all were purchased from ProteinTech, China), and p-AKT (dilution, 1:1,000; cat. no., 4060; Cell Signaling Technology, Inc.) at 4 °C overnight. Following incubation with a horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution, 1:5,000; cat. no., IH-0011) or anti-mouse IgG antibodies (dilution, 1:5,000; cat. no., IH-0031; both from DingGuo BioTech Co., Ltd.) for 1 h at room temperature, the proteins were visualized using an enhanced chemiluminescence western blotting detection kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. β-Actin or GAPDH served as the internal control.
Tumor xenograft mouse models
Eighteen female immunodeficient BALB/c nude mice (age, 4-6 weeks; weight, 16±2 g) were purchased from Vital River Laboratory Animal Technology Company Limited. All mice were raised under pathogen‑free conditions and maintained in a controlled environment (temperature, 25±2 °C; relative humidity, 70±5%; 12-h light/dark cycle) and fed standard laboratory food and water. The tumor xenografts were generated by a subcutaneous injection of GBC-SD cells (5×107/ml × 125 μl) into the right flank of female nude mice. The mice were randomly divided into three groups of 6 mice: The control and the lenvatinib groups, administered with 15 or 30 mg/kg (intraperitoneal injection, daily) [9, 19]. Tumor size and weight were measured using calipers and electronic balance every 2 or 3 days. The tumor volume was determined according to the following formula: Tumor volume (mm3) = 0.5× (length × width2). The maximum tumor diameter in the present study was 15.74 mm, which was allowable by ethical guidelines. After 22 days, the animals were anesthetized with 1% pentobarbital (50 mg/kg) and sacrificed by breaking their neck. The tumor tissues were removed and measured. Xenograft tumors were collected and fixed in 10% formalin for 24 h at room temperature, following which they were paraffin-embedded and cut into 4-μm sections for immunohistochemical analysis.
Lenvatinib inhibited proliferation and colony formation in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with different concentrations of lenvatinib for 24, 48 and 72 h. (A and B) Cell viability was examined by Cell Counting Kit-8 assay. (C) Colony formation. *P<0.05 vs. 0 µM. NC, negative control.(Click on the image to enlarge.)
The slides were incubated in heated antigen retrieval solution (10 mmol/l citrate buffer, pH 6.0), and subsequently treated with 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidases. Following washing with PBS, the slides were incubated with a diluted primary antibody p-AKT (dilution, 1:100; cat. no., 4060; Cell Signaling Technology, Inc.) at 4 °C overnight and then with the secondary antibody (1:500 dilution; cat. no., PV-9000; ZSGB-BIO) for 20 min at room temperature. The reaction was developed using a 3,3'‑diaminobenzidine kit (1:50 dilution in buffer; cat. no., ZLI-9017; ZSGB-BIO). Finally, hematoxylin was applied to counterstain the nuclei prior to dehydration and mounting. The stained slides were observed under a light microscope (CX31; Olympus Corporation; original magnification, ×200).
All data are presented as the mean ± standard deviation using SPSS 17.0 software (SPSS Inc.) from at least three independent experiments. Statistical differences between groups were determined by Student's t-test followed by Shapiro-Wilk W test, or one-way ANOVA followed by Bonferroni test. P<0.05 was considered to indicate a statistically significant difference.
Lenvatinib suppresses the viability and colony formation of GBC-SD and NOZ cells
To determine the cytotoxicity of lenvatinib, GBC-SD and NOZ cells were exposed to different concentrations of lenvatinib (0-80 µM) for different time intervals (0, 24, 48 and 72 h). A CCK-8 assay was then carried out to detect the effects of lenvatinib on GBC-SD and NOZ cells. Data from Fig. 1A and 1B showed that cellular growth was significantly suppressed by lenvatinib in a dose- and time-dependent manner. The IC50 values of lenvatinib in GBC-SD and NOZ cells at 24, 48 and 72 h were 155.275, 69.659 and 33.403 µM; 8618.871, 28.642 and 10.035 µM, respectively, and the concentrations of 25 and 50 µM for 48 h were chosen for the following study. The effect of lenvatinib on the colony formation ability of GBC-SD and NOZ cells was also observed. As shown in Fig. 1C, treatment with lenvatinib exhibited a significant decrease in the colony number, when compared with the negative control (NC) group. These data demonstrated that treatment with lenvatinib effectively inhibited cell viability and colony formation in GBC-SD and NOZ cells.
Lenvatinib induced apoptosis and regulated cell cycle arrest progression in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. (A) Quantification of apoptotic rate. (B) Distribution of cell cycle. (C and D) Protein expression of caspase-9, CytoC, Bax, CDK2, CDK4 and cyclin D1 were detected by western blotting. β-actin or GAPDH was used as a loading control. *P<0.05 vs. NC. Len, Lenvatinib; FITC, fluorescein isothiocyanate; CytoC, cytochrome c; Bax, Bcl-2-associated X protein; NC, negative control.(Click on the image to enlarge.)
Lenvatinib induced cell apoptosis and inhibited cell cycle arrest at the G0/G1 phase in GBC-SD and NOZ cells
To detect the effect of lenvatinib on cell apoptosis, GBC-SD and NOZ cells were stained with Annexin V-FITC/PI and quantified by flow cytometry. As shown in Fig. 2A, the GBC-SD and NOZ cell apoptotic rate of the NC and 25 and 50 µM lenvatinib treatment groups treated for 48 h was 5.9±1.1, 19.8±5.8, and 20.3±4.1%; 3.3±0.7, 17.3±1.3, and 21.0±0.4%, respectively. These data suggested that treatment with lenvatinib significantly promoted cell apoptosis in GBC-SD and NOZ cells. Furthermore, as shown in Fig. 2B, treatment with 50 µM lenvatinib also inhibited cell cycle progression by inducing G0/G1 phase arrest in GBC-SD cells. While treatment with 25 or 50 µM lenvatinib also suppressed cell cycle progression by inducing S phase arrest in NOZ cells. In addition, the expression of caspase-9, cytochrome c (CytoC), Bcl-2-associated X protein (Bax) and relative cell cycle proteins CDK2, CDK4 and cyclin D1 were investigated by blotting to explore the mechanism of lenvatinib-induced apoptosis and inhibited cell cycle. As shown in Fig. 2C and 2D, following the culture of 25 and 50 µM lenvatinib-treated GBC-SD and NOZ cells for 48 h, a significant increase was observed in the expression of cleaved caspase-9. The expression of caspase-9, CDK2, CDK4 and cyclin D1 was decreased, while that of CytoC and Bax exhibited no significant changes in GBC-SD cells and Bax exhibited no significant changes in NOZ cells. These data indicated that treatment with lenvatinib significantly induced cell apoptosis and regulated cell cycle at the G0/G1 phase in GBC-SD and NOZ cells by regulating the expression of caspase-9, CDK2, CDK4 and cyclin D1.
Lenvatinib inhibited the migration and invasion of GBC-SD and NOZ cells
To further observe the effect of lenvatinib on GBC-SD and NOZ cell migration and invasion, wound healing and Transwell assays were performed to detect the migration and invasion ability of GBC-SD and NOZ cells. As shown in Fig. 3A, the wound closure ratio in GBC-SD cells and NOZ cells in the NC and lenvatinib groups for 48 h were 0.392±0.030 and 0.177±0.014; 0.618±0.037 and 0.198±0.033, respectively. It is also shown in Fig. 3B that the number of migrated and invasive cells in GBC-SD cells per high-power field in the NC and lenvatinib groups were 212.4±5.9 and 116.8±8.7; 182.7±10.1 and 67.7±13.8, respectively. Furthermore, consistent with the above results, that the number of migrated and invasive cells in NOZ cells per high-power field in the NC and lenvatinib groups were 109.2±10.9 and 69.0±7.3; 83.0±5.4 and 50.0±8.9, respectively (Fig. 3C). To further elucidate the molecular mechanism involved in cell migration and invasion, the protein expression of MMP-2 and CEMIP was detected by western blotting. As shown in Fig. 3D and 3E, the expression of MMP-2 and CEMIP was decreased following treatment with 25 or 50 µM lenvatinib for 48 h. These results indicated that treatment with lenvatinib may significantly reduce the migration and invasion ability of the cells by inhibiting MMP-2 and CEMIP.
Lenvatinib suppressed autophagy of GBC-SD cells
GBC-SD and NOZ cells were treated with 25 µM and 50 µM lenvatinib for 48 h. At the end of treatment, cells were harvested and autophagy relative gene were observed by western blot analysis. It was found that treatment with lenvatinib significantly decreased LC3II/I ratio, whereas the expression of p-62 was increased (Fig. 4A and 4B). These results suggested that treatment with lenvatinib inhibited autophagy.
Lenvatinib suppresses GBC cell growth in vivo by targeting p-AKT
To further explore the effects of lenvatinib on GBC, GBC-SD tumor cells were established using a xenograft mouse model. As shown in Fig. 5A and 5B, the tumor volume and weight in the NC group at the end of the treatments were 959.97±322.63 mm3 and 0.82±0.37 g, whereas those in the group treated with 15 and 30 mg/kg/day lenvatinib were 404.90±48.81 mm3 and 0.25±0.06 g, and 165.69±65.02 mm3 and 0.10±0.06 g, respectively. These data showed a marked, dose-dependent decrease in the tumor growth rate and weight of the lenvatinib treatment group. In addition, the protein expression of p-AKT in tumor tissues was also determined by immunohistochemistry. As shown in Fig. 5C, the expression levels of p-AKT were significantly increased in the tumor tissues from the lenvatinib treatment group, as compared with the control group.
Lenvatinib inhibited the expression of p-AKT and enhanced the sensitivity of GBC-SD and NOZ cells to gemcitabine by inhibiting the expression of p-AKT
To investigate the underlying molecular mechanism of the anticancer effect of lenvatinib, the expression of p-AKT and poly ADP-ribose polymerase 1 (PAPR1) was detected by blotting. As shown in Fig. 6A and 6B, the expression of p-AKT was decreased following treatment with 25uM or 50uM lenvatinib in GBC-SD and NOZ cells, whereas no significant changes in the expression of PAPR1 were observed following treatment with 25 µM lenvatinib in GBC-SD cells. To further identify the anticancer effect of lenvatinib on GEM-mediated cell growth inhibition, cell proliferation was detected by CCK-8 assay in GBC-SD and NOZ cells treated with lenvatinib and GEM. As shown in Fig. 6C and 6D, treatment with lenvatinib enhanced the sensitivity of GBC cells to GEM. To further investigate the underlying molecular mechanism of lenvatinib in enhancing the sensitivity of GBC to GEM, the activation of the PI3K/AKT pathway was also examined. As expected, the expression of p-AKT was suppressed following treatment with lenvatinib or GEM (Fig. 6E and 6F). Combined treatment with lenvatinib and GEM significantly inhibited the expression of p-AKT, as compared with treatment with lenvatinib or GEM alone.
GBC is one of the most common types of bile duct cancer worldwide with high recurrence and metastasis rates, despite complete surgical resection. More than 70% of GBC patients are considered to be unsuitable for surgical treatment. The prognosis of GBC is poor and a severe threat to human health and longevity. GEM is currently the first-line anticancer drug for GBC patients, which acts by suppressing DNA synthesis . However, evidence has demonstrated that the anticancer effect of GEM has been shown to be unsatisfactory, due to its chemoresistance . Therefore, it is urgent to develop new anticancer drugs or underlying resistant genes.
Lenvatinib inhibits migration and invasion in GBC-SD and NOZ cells by downregulating MMP-2 and CEMIP. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h. (A) Influence of lenvatinib on cell migration by wound closure. (B and C) Influence of lenvatinib on cell migration and invasion by Transwell assays. (D and E) Analysis of MMP-2 and CEMIP following treatment with lenvatinib in GBC‑SD and NOZ cells, as assessed by western blotting. β-actin or GAPDH was used as a loading control. Original magnification, x100. *P<0.05 vs. NC. CEMIP, cell migration-inducing protein; Len, lenvatinib. MMP, matrix metalloproteinase; NC, negative control.(Click on the image to enlarge.)
Lenvatinib, a new anticancer agent that acts by inhibiting the expression of VEGFR1-3, FGFR1-4, PDGFRα, SCF, RET and KIT, has been widely used for unresectable or metastatic HCC in China . Recent studies have reported that lenvatinib inhibited tumor growth and induced apoptosis in anaplastic thyroid cancer in vitro and in vivo by suppressing the expression of epidermal growth factor receptor, AKT, extracellular signal-regulated kinase 1/2 and VEGF-A . Wang et al. showed that lenvatinib promoted cell apoptosis in HK-1 cells by inducing endoplasmic reticulum stress . However, the effect of lenvatinib on GBC remains unclear. In the present study, lenvatinib exhibited anti-proliferation in vitro and in vivo. In addition, lenvatinib also induced apoptosis by increasing the expression of caspase-9. As expected, lenvatinib also regulated cell cycle arrest progression by decreasing the expression of CDK-2, CDK-4 and cyclin D1 in GBC cells. Here, the different result of cell cycle arrest progression between GBC-SD and NOZ cells may due to different kinds of GBC cells and cell biological characteristics.
Lenvatinib suppressed autophagy in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h. (A and B) Analysis of LC3II/I ration and p-62 following treatment with lenvatinib in GBC‑SD and NOZ cells, as assessed by western blotting. β-actin or GAPDH was used as a loading control. *P<0.05 vs. NC. LC3, microtubules associated proein 1 light chain 3β; NC, negative control.(Click on the image to enlarge.)
Lenvatinib suppresses tumor growth in GBC‑SD xenografts. (A) Growth curves are representative of tumor volumes in BALB/c nude mice in the NC or lenvatinib groups. (B) Tumor weight was detected in the NC and lenvatinib groups at the endpoint of the animal experiment. (C) The protein expression of p-AKT in GBC-SD tumor tissues was measured by immunohistochemistry. *P<0.05 vs. NC. Len, lenvatinib; NC, negative control; IHC, immunohistochemistry.(Click on the image to enlarge.)
A previous study demonstrated that lenvatinib inhibited migration and invasion in non-small cell lung cancer cells . He et al. also revealed that lenvatinib inhibits cell migration and invasion in HCC SMMC7721 and Hep3B cells by suppressing the expression of MMP-1, 2, 7, 9, 10 and 16 and increasing the expression of tissue inhibitor of metalloproteinases (TIMP)-1, 3 and 4 . Lenvatinib significantly inhibited the migration and invasion of nasopharyngeal carcinoma HK-1 cells, as well as the expression of tumor angiogenesis-related proteins, such as VEGF and PDGF . The cell migration-inducing CEMIP gene was first reported in non-syndromic hearing loss in 2006 by He et al. . It was also reported that CEMIP has a regulatory effect on hyaluronan, which results in the occurrence of hyaluronic acid-rich organ diseases . Recent studies have demonstrated that the overexpression of CEMIP leads to tumor cell invasion, migration and carcinogenesis in many types of cancer . In the present study, lenvatinib inhibited cell migration and invasion by inhibiting the expression of MMP-2 and CEMIP in GBC-SD and NOZ cells.
Autophagy is a physiological cleaning process for mediating the degradation of intracellular component sand damaged organelles. Accumulating evidences have shown that autophagy plays an important role in regulating biological processes, including cell proliferation, apoptosis, angiogenesis and chemoresistance . However, the role of lenvatinib in regulating autophagy remains unclear. In the present, we first reported the relationship between lenvatinib and autophagy. Treatment with lenvatinib significantly inhibited autophagy in GBC-SD and NOZ cells.
Lenvatinib inhibits the expression of p-AKT and enhances GEM-mediated cell growth inhibition in GBC-SD and NOZ cells by inactivating the expression of p-AKT. GBC-SD and NOZ cells were pretreated with lenvatinib for 1 h, then treated with or without lenvatinib for 48 h. (A and B) The protein expression levels of p-AKT and PAPR1 were detected by western blotting with β-actin or GAPDH as a loading control. (C and D) Cell growth proliferation was detected by CCK-8. (E and F) The protein expression levels of p-AKT were measured by western blotting in GBC-SD and NOZ cells following treatment with lenvatinib and GEM. *P<0.05 vs. NC. **P<0.05 vs. GEM group. NC, negative control; GEM, gemcitabine; PAPR1, poly ADP-ribose polymerase 1; Len, lenvatinib.(Click on the image to enlarge.)
The PI3K/AKT pathway transmits signals from the cell membrane to the nucleus and plays an important role in oncogenesis. The PI3K/AKT pathway is known to play an important role in chemoresistance, and its activation has been shown to increase the chemoresistance of cancer cells . GEM is currently the first-line chemotherapeutic drug for bile tract and pancreatic cancer. Accumulating evidence has revealed that its benefit to cancer patients and level of and toxicity remain unsatisfactory. A previous study revealed that integrin β1 induced gemcitabine resistance in pancreatic cancer cells through activation of the PI3K signaling pathway . On the contrary, Wang et al. showed that LncRNA ab209630 enhanced the sensitivity of pancreatic ductal adenocarcinoma to gemcitabine by inhibiting the PI3K/AKT signaling pathway . In the present study, lenvatinib decreased the activation of PI3K and lenvatinib increased the sensitivity of GBC-SD and NOZ cells to gemcitabine through inhibiting the activation of PI3K. However, there are several limitations to the present study: lack of further machanism of how lenvatinib inhibited autophagy; the lack of a positive control (for example sorafenib, a known anticancer drug used to treat thyroid cancer).
In conclusion, the results of the present study revealed that lenvatinib inhibited cell growth and colony formation, induced cellular apoptosis by increasing the expression of cleaved caspase-9, and regulated the cell cycle at the G0/G1 phase. Furthermore, lenvatinib decreased cellular migration and invasion by inhibiting the expression of MMP-2 and CEMIP. Further investigation in vivo revealed that lenvatinib suppressed GBC cell growth by targeting p-AKT, which was consistent with the in vitro results. Mechanistically, lenvatinib promoted the sensitivity of GBC-SD and NOZ cells to gemcitabine by targeting the PI3K/AKT pathway. The mechanism of autophagy in mediating autophagy in GBC remains unclear, and it will be investigated further in our future study.
This research was supported Medical Science and Technology Project of Henan Province (SBGJ2018021), Key Scientific Research Project of Colleges and Universities in Henan Province (19B320039, 20A320037, 20A320047). Joint construction project of medical science and technology research plan of Henan Province (LHGJ20190028).
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
JY, LQ, JL, WL and RL performed the experiments, WZ and RF designed the study, KZ and WL analyzed the data. JY and RF prepared and wrote the study.
Ethics approval and consent to participate
All experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University.
The authors have declared that no competing interest exists.
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10. Ogasawara S, Mihara Y, Kondo R, Kusano H, Akiba J, Yano H. Antiproliferative Effect of Lenvatinib on Human Liver Cancer Cell Lines In vitro and In vivo. Anticancer research. 2019;39:5973-82
11. Wang G, Zhuang J, Ni J, Ye Y, He S, Xia W. Combined effects of Lenvatinib and iodine-131 on cell apoptosis in nasopharyngeal carcinoma through inducing endoplasmic reticulum stress. Exp Ther Med. 2018;16:3325-32
12. Lee YS, Kim SM, Kim BW, Chang HJ, Kim SY, Park CS. et al. Anti-cancer Effects of HNHA and Lenvatinib by the Suppression of EMT-Mediated Drug Resistance in Cancer Stem Cells. Neoplasia (New York, NY). 2018;20:197-206
13. Noorolyai S, Shajari N, Baghbani E, Sadreddini S, Baradaran B. The relation between PI3K/AKT signalling pathway and cancer. Gene. 2019;698:120-8
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Corresponding authors: Dr Wenlong Zhai, Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, 1 Jianshe East Road, Zhengzhou, Henan 450052, P.R. China. Prof Ruo Feng, Department of Histology and Embryology, Medical College of Zhengzhou University, No. 100, Science avenue, Gao Xin district, Zhengzhou, Henan 450052, P.R. China. E-mail: fcczhaiwledu.cn (ZWL); fredu.cn (FR).

domain: medical


text:
IMPHAL (Manipur): The Manipur government on Sunday extended the ban on internet services in the state for five more days to prevent any disturbances of peace and public order.
According to an official release, the ban on internet services in the Northeast state, which has been rocked by ethnic clashes and violence, has been extended till 3 pm on June 30 to "prevent any disturbances of the peace and public order in the jurisdiction of the State of Manipur".
The government release informed further that the ban on mobile internet services in the state was extended due to the "persisting unrest".
Director General of Police, Manipur, in a statement, informed that there are still reports of incidents of violence, attacks, and arson of houses and premises including exchange of firing.
"There is apprehension that some anti-social elements might use social media extensively for the transmission of images, hate speech and hate video messages inciting the passions of the public which might have serious repercussions for the law and offer situation in the State of Manipur," it added.
This is the third straight extension of the ban on internet services in the state.
Earlier, on May 3, the Manipur government suspended mobile internet services in the state, amid the spiralling ethnic violence, and the ban has remained in force ever since.
The government extended the ban on June 11, till June 15, and then again, for five more days, from June 20 to June 25.
Manipur has been in the throes of ethnic violence for a month, in the wake of a directive of the high court asking the state government to consider including the Meitei community in the list of Scheduled Tribes (STs).
The Centre had to deploy paramilitary forces as violence escalated in the Northeast state.
Violence gripped Manipur on May 3 after clashes erupted during a rally organised by the All Tribals Students Union (ATSU) to protest against the demand for the inclusion of Meiteis in the list of Scheduled Tribes (STs).

domain: sociology


text:
7 of 7 people found the following review helpful
To boldly go where no cartoon has gone before,
Verified Purchase(What is this?)
This review is from: Star Trek: The Animated Series [DVD] (DVD)
Well, this is indeed an oddity, and not something I'd ever even heard of before seeing it here on Amazon. A follow-up to the classic / okay / past-its-best (delete depending on your point of view) TV show from the 60's, Star Trek: The Animated Series isn't so much a sequel to, as it is a continuation of, that idea. It features the voice talents of all of the original cast (with the exception of Walter Koenig as Chekov, who was left out apparently for budget reasons) as well as similar settings and story lines, and the same old mission to explore strange new worlds, to seek out new life and new civilizations, etc...
The overall quality of the print is excellent and the sound is also first-rate, I would guess virtually as good as the day the show was first aired. There are very few motion artefacts to detract from the viewing experience or to betray the show's age, in fact the only give-away is that the animation is of the classic 70's style, the kind you may well have seen in Hanna-Barbera shows of the same period. And although making a cartoon (I use that term loosely) did allow the makers to explore plots that would have been prohibitively expensive for a live-action show, a lot of the time settings and backgrounds are, understandably, overly colourful but lacking in detail, so don't expect the kind of quality you might see in a big-budget animated movie or even in some of today's cartoons. What it does give you is more of the classic series but in a different form, and rather than it being Star Trek on-the-cheap, I think that you'll quickly see this as nothing less than the original show in slightly different trousers.
However, looking at this with a severely critical eye, at times the show's producers seemed a little constrained by each episode's 20-odd-minute run time, having to squeeze in stories that I feel should have been given the full 45-minute treatment. On a few occasions, events and reasons were rushed through or not explained quite as fully as I felt they should have been, leaving the viewer to make assumptions about why things were happening. The key demographic of the show is also a little uncertain - at first glance I would have said pre-teens; after watching a few of the episodes I would have said teens; a few more and I'm not so sure. Some have story lines with a maturity that rivals those found in the original series and whilst they may be acceptable for today's kids, I fear that much of what was pitched by the writers will be lost on a younger audience. Then again, both are accusations that could be levelled at the original series, which positively serves to promote this as more of the same.
The box houses just the 4 DVDs containing the complete 22-episode run of the show; there's no booklet, no postcards and no posters, even though there are clips for them in the box's interior spine. The episode listings are printed on the inside of the box's inlay card, so are obscured by the DVDs until you take them out. All told, I'm guessing that this is the 'cheap' release made for viewing purposes only, so if you want anything more than the bare minimum you should probably seek out the premium version.
To get the most out of this set I think you have to feel at least a modicum of love for the original Star Trek series; I'm not one of its greatest fans by any means, but I do appreciate it as a science fiction trailblazer and as the father of several (and, in my opinion, much better) Star Trek shows. My concern before buying it had been that it would be nothing more than a low-cost cash-in on the Star Trek brand, but fortunately I was proved wrong and after only a couple of episodes I was drawn in by the familiar storytelling and voice acting, almost forgetting that it was a cartoon. In a nutshell, if at least a part of you enjoyed the original series and you want more of the same, I think you'll get a kick out of this. If you didn't like the original, or if you think that 70's cartoon shows are best left in the past, then it almost certainly won't be for you.

domain: entertainment


text:
- Research article
- Open Access
Network pharmacology and in vitro studies reveal the pharmacological effects and molecular mechanisms of Shenzhi Jiannao prescription against vascular dementia
BMC Complementary Medicine and Therapies volume 22, Article number: 33 (2022)
Shenzhi Jiannao (SZJN) prescription is a type of herbal formula adopted in the management of cognitive impairment and related disorders. However, its effects and related regulatory mechanisms on vascular dementia (VD) are elusive. Herein, network pharmacology prediction was employed to explore the pharmacological effects and molecular mechanisms of SZJN prescription on VD using network pharmacology prediction, and validated the results through in vitro experiments.
Through a search in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, chemical composition and targets for SZJN prescription were retrieved. The potential targets for VD were then obtained from the GeneCards and DisGeNET databases. The network was constructed that depicted the interactions between putative SZJN prescription and known therapeutic targets for VD using Cytoscape 3.7.1. Analysis of protein-protein interaction was achieved via STRING 11.0 software, followed by Gene Ontology (GO) functional enrichment and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analyses. To validate the computer-predicted results, in vitro experiments based on an excitotoxic injury model were designed using glutamate-exposed PC12 cells, and treated with varying concentrations (low, 0.05; medium, 0.1 and high, 0.2 mg/mL) of SZJN prescription. Cell viability and cell death were detected using the IncuCyte imaging system. Moreover, the expression profiles of Caspase-3 were analyzed through qRT-PCR.
Twenty-eight potentially active ingredients for SZJN prescription, including stigmasterol, beta-sitosterol, and kaempferol, plus 21 therapeutic targets for VD, including PTGS2, PTGS1, and PGR were revealed. The protein-protein interaction network was employed for the analysis of 20 target proteins, including CASP3, JUN, and AChE. The enrichment analysis demonstrated candidate targets of SZJN prescription were more frequently involved in neuroactive ligand-receptor interaction, calcium, apoptosis, and cholinergic synaptic signaling pathways. In vitro experiments revealed that SZJN prescription could significantly reverse glutamate-induced cell viability loss and cell death, and lower the levels of Caspase-3 mRNA in glutamate-induced PC12 cells.
Collectively, this study demonstrated that SZJN prescription exerted the effect of treating VD by regulating multi-targets and multi-channels with multi-components through the method of network pharmacology. Furthermore, in vitro results confirmed that SZJN prescription attenuated glutamate-induced neurotoxicity.
Vascular dementia (VD), as the main type of dementia in Asia, is mainly the result of vascular risk factors such as atherosclerosis, hypertension and micro-strokes . VD accounts for about 30% of dementia in Asia, and the incidence rate in China is 1.1 to 3% [2,3,4]. With aging, the incidence of VD is doubled every 5.3 years, which brings about a large burden on society and patients’ families [2, 5]. The currently used primary preventive medications, including cholinesterase inhibitors and excitatory amino acid receptor antagonists against vascular cognitive impairment have very limited efficacy. Thus far, there are no curative treatments available and therefore, development of therapy for VD has become an essential but unmet need. A large number of experimental studies and clinical observations have confirmed that Chinese medicine has unique advantages in treating VD. Various empirical prescriptions and active ingredient extracts have shown clear neuroprotective effects on VD [6,7,8].
Shenzhi Jiannao (SZJN) prescription, the national new formula in China (ZL201010249665.8), is a classical herbal formula for the treatment of VD based on the clinical practice. This prescription is mainly composed of Panax ginseng C.A.Mey. (Ginseng), Anemarrhena asphodeloides Bunge (Anemarrhenae), and Paeonia veitchii Lynch (Paeoniae rubra). In previous pharmacological study, it has been found that SZJN prescription improved cerebral ischemia in VD rats, reduced the levels of glutamate and γ - aminobutyric acid in the anterior cortex, and increased acetylcholine content . Moreover, SZJN prescription was associated with significantly low expression of N-methyl-D-aspartate receptor 1 (NMDAR1) in the brain of rats with vascular dementia. Furthermore, administration of this prescription impeded the loss of hippocampal neurons, neuronal damage, degeneration, and necrosis; this consequently improved cognitive function [10, 11]. The effect of SZJN prescription on the central cholinergic system improves the cognitive ability of mice with acquired memory disorder through inhibition of AChE activity . However, reports about the therapeutic effects and mechanisms of action of SZJN prescription on VD are scanty. The molecular mechanism of SZJN prescription has not been certainly clear.
Network pharmacology approaches have been proven to be a powerful way for mechanistic exploration of Chinese medicine from the perspective of multicomponents, multitargets, and multichannels, which is consistent with the holistic view of Chinese medicine [13, 14]. The aim of this study was to explore the underlying molecular mechanisms of SZJN prescription in treating vascular dementia by using network pharmacology approaches and its protective effect on glutamate-induced neurotoxicity by in vitro experiment based on network pharmacology prediction. These findings provide a new scientific basis for the future development of SZJN prescription. A flowchart of the experimental procedures of this study is outlined in Fig. 1.
Collection, retrieval, and screening of main chemical components of SZJN prescription
To determine and collect the chemical components of the three herbs contained in SZJN prescription, a search was performed from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database (TCMSP, http://ibts.hkbu.edu.hk/lsp/tcmsp.php) manually. Subsequently, compound names and their molecular structures were verified in the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Oral bioavailability (OB) ≥ 30% and drug-like (DL) ≥ 0.18 parameters were incorporated in the TCMSP database as drug screening conditions for the compounds.
Predicting SZJN prescription chemical components targets, VD significant targets and SZJN prescription therapeutic targets
The main chemical components in SZJN prescription were determined and individually matched with potential targets according to TCMSP. First, names of the predicted target proteins were transformed into gene names via the UniProt database (http://www.uniprot.org/), then target species were selected as “humans”. Targets related to VD were collected from two databases. One was the GeneCards (https://www.genecards.org/), which is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes . The other database was the DisGeNET (http://www.disgenet.org/), which is a comprehensive gene-disease association (GDA) database that provides the current knowledge of human genetic diseases . A general search for all genes was conducted using the keywords “vascular dementia”, then retrieved from the GeneCards and DisGeNET databases. An intersection between SZJN prescription targets and VD significant targets was made to obtain the therapeutic targets for SZJN prescription for subsequent studies.
Network construction and analysis
Using the Cytoscape 3.7.1 software, prediction results of potential targets for SZJN prescription were acquired, and a network of herbs-putative active components-putative therapeutic targets was constructed. The network topology analysis was achieved using a plugin in Cytoscape 3.7.1, after which a protein-protein interaction (PPI) network of the SZJN prescription was constructed via the STRING 10.5 (https://string-db.org) software.
Analysis of Gene Ontology (GO) function enrichment and Kyoto Genes and Genomes Encyclopedia (KEGG) pathway enrichment
GO functional enrichment and KEGG pathway enrichment analyses were conducted using the David v 6.8 database (https://david.ncifcrf.gov/) to explore gene functions and uncover potential therapeutic targets of the active components of SZJN prescription. Analysis of the pathways provided scientific connotation for SZJN prescription.
Materials and reagents for biological experiments
Differentiated PC12 cells, RPMI-1640 complete culture medium (containing 10% FBS, 100 U/mL penicillin, and 100 U/mL of streptomycin) and RPMI-1640 basic culture medium were purchased from Procell Life Science and Technology Co. Ltd. (Wuhan, China); Caspase-3 primers were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China);. L-glutamic acid (glutamate) was purchased from Sigma (USA); SZJN prescription, constituted from Ginseng, Anemarrhenae, and Paeoniae rubra, was acquired from the Chinese Medicine Preparation Department of China-Japan Friendship Hospital; Memantine hydrochloride (MH) was purchased from H. Lundbeck A/S (Denmark). SZJN prescription and glutamate were dissolved in RPMI 1640 complete medium, while MH was dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of less than 0.5% for use in the experiments.
Cell culture and modeling intervention
PC12 cells were cultured in RPMI-1640 complete medium at 95% humidity, 5% carbon dioxide (CO2), and a temperature of 37 °C. Cells were treated with or without SZJN prescription (0.05, 0.1 and 0.2 mg/mL), MH (10 μM), and glutamate (22.5 mM) for 24 h. Each experiment was performed in triplicate.
Cell viability and cell death assays
Real-time examination of cell viability and cell death was achieved using the live-cell Essen Bioscience IncuCyte imaging system, which recorded both phase-contrast and fluorescent images over time. Briefly, cells (30,000 cells/well) were seeded into Corning 96-well tissue culture plates and then placed on the IncuCyte instrument. Results for cell viability and cell death were recorded for over 36 h. Dead cells were identified using fluorescent nuclear dye YOYO-1(Thermo Fisher Scientific, USA), a cell impermeable dye that enters cells via compromised membranes. To calculate the average percentage of cell death, the green fluorescent area (YOYO-1 positive) was divided by the total phase area.
Cell apoptosis using flow cytometry (FCM)
Apoptotic cells were quantitated using the Annexin V-FITC/propidium iodide (PI) apoptosis assay kit (KeyGEN BioTECH, China). Briefly, PC12 cells were digested with 0.25% trypsin, centrifuged, and resuspended in 200 μL 1 × binding buffer. Then, cells were stained with 5 μL Annexin V-FITC in the dark for 10 min. This was followed by another staining with 5 μL PI for 5 min. Samples were analyzed using a FACScan flow cytometer (BD, USA). At least 10,000 events were recorded. Apoptotic cells were expressed as a percentage of the total number of cells.
Effect of SZJN prescription on the mRNA expression of Caspase-3 in glutamate-treated PC12 cells
The effect of SZJN prescription on Caspase-3 was established through quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Briefly, total RNA from cells was extracted using the RNA Extraction Kit (KURABO, Japan) according to the manufacturer’s instructions. The concentration and purity of the extracted RNA were determined using a Q5000 nucleic acid protein analyzer (Quawell, USA) . Then a reverse transcription kit (Thermo Fisher Scientific, USA) was used to reverse transcribed the RNA to cDNA. qRT-PCR was performed using the SYBR Green Master Mix on a CFX Connect Real-Time PCR System (Bio-Rad, USA), with Caspase-3: Forward: 5′-GTACAGAGCTGGACTGCGGTATTG-3′; Reverse: 5′-AGTCGGCCTCCACTGGTATCTTC − 3′ and β-actin: Forward: 5′-GCAGTTGGTTGGAGCAA-3′; Reverse: 5′-ATGCCGTGGATACTTGGA-3′ primer pairs. qRT-PCR conditions were as follows: Initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. β-actin served as the internal reference gene. The relative expression levels of target genes were calculated using the 2−∆∆CT method.
All statistical analyses were performed via SPSS version 25.0. Data were presented as means ± standard deviations (SD). Statistical comparisons were achieved using one-way analysis of variance (ANOVA) for multiple groups with variance depicting homogeneous normal distribution data. Least—Significant Difference (LSD) test was applied for mean separations to reveal statistically significant differences, P < 0.05.
Potential active components of SZJN prescription
Based on the set conditions in the database, 45 potentially active SZJN prescription components were retrieved and screened, including 17, 15, and 13 for Ginseng, Anemarrhenae, and Paeoniae rubra, respectively (Table 1).
Predicted targets for SZJN prescription chemical components, VD significant and therapeutic targets
Names of component targets in SZJN prescription were corrected using the UniProt database, after which repeated targets were eliminated. Eventually, 61, 90, and 71 potential targets for Ginseng, Anemarrhenae, and Paeoniae rubra respectively, were obtained, respectively. From the GeneCards and DisGeNET databases successfully revealed all genes related to VD were revealed; 3077 target genes were retrieved. Integrating potential targets of chemically active components in SZJN prescription with those of VD revealed exactly 21 therapeutic targets (Fig. 2).
Network construction and analysis
A network of SZJN prescription-active compounds-VD targets was successfully constructed via Cytoscape 3.7.1 for topological analysis (Fig. 3). It comprised 52 nodes and 183 edges; a dark blue node represented single herb (3), a red node depicted active compounds (28), and a light blue node denoted therapeutic targets (21).
Twenty-one therapeutic SZJN prescription targets were input into the STRING software and identified 20 target proteins under the set conditions. These results were saved as a TSV file. Network topology analysis and calculations were performed via Cytoscape 3.7.1, for the construction of a PPI network (Fig. 4a). Based on the degree of the nodes, a bar graph depicting the relationship between all protein nodes and the targets was generated (Fig. 4b).
GO and KEGG pathway enrichment analyses
Through GO enrichment analysis of the 20 target proteins in the PPI network, 106 GO items were revealed (P < 0.05), including 70 items related to biological processes, such as drug reaction, transcription initiation of RNA polymerase II promoter, and steroid mediated signaling pathway; 15 items related to cell composition, such as nuclear and plasma membrane components, and membrane raft; 21 items related to molecular function, (steroid receptor, sequence-specific DNA binding, and enzyme binding) were identified (Fig. 5a-c).
Also, KEGG pathway analysis of the 20 target proteins in the PPI network was performed (at P < 0.05), and the unrelated pathways including those for cancer and hepatitis B were eliminated. Results demonstrated that the targets of SZJN prescription for treating VD treatment were mainly associated with neuroactive ligand-receptor interaction, calcium, cholinergic synapse, cardiac myocyte adrenergic signal transduction, adipocyte lipolysis regulatory, apoptosis, cAMP, salivary secretion, tumor necrosis factor, 5-hydroxy color Aminergic synaptic, neurotrophin and AMPK signaling pathways (Fig. 6). The SZJN prescription-related targets and pathways associated with apoptosis-mediated VD progression have been presented in Fig. 7; the pathway targets are marked in white, whereas the targets of SZJN prescription for VD treatment are marked in purple. It was suggested that SZJN prescription potentially regulates apoptosis, therefore, is crucial in VD treatment.
Validation of the neuroprotective effects of SZJN prescription against glutamate-induced PC12 cell death
The results from the network pharmacological analysis demonstrated that cell proliferation, gene transcription, and cell-cell signaling, and apoptosis were associated with multiple synergies of SZJN prescription on VD treatment. The cytotoxicity of glutamate in PC12 cells was evaluated using YOYO-1 and then the neuroprotective effects of SZJN prescription against glutamate-induced cell death were validated. Cells treated with 22.5 mM glutamate exhibited low viability (Fig. 8a) and showed a large total green fluorescence area within 4 h post-treatment (hpt). The fluorescence intensity increased almost 10 times by 36 hpt (Fig. 8b). Similarly, the number of dead cells cultured with glutamate increased by 4 hpt. Death cell counts increased to over 400/image by 24 hpt (Fig. 8c). The average percent cell death recorded every 2 h over 36 h of monitoring for PC12 cells injury is presented in Fig. 8d. Glutamate-induced cells exhibited a significantly high death rate over the time course, approximately 20% after 36 h of treatment. For injured cells, with complete media, less than 5% cell death was reported over the monitoring time. Exposure to glutamate (22.5 mM) triggered cell death, however, this effect was reversed by SZJN prescription (0.05, 0.1, and 0.2 mg/mL). Moreover, SZJN prescription reduced the total green fluorescence, lowered the number of dead cells and the average percentage of cell death, and recovered cell viability (Fig. 8a-d). Microscopic images were analyzed for positive cell nuclei based on the size and fluorescence intensity of YOYO-1. Representative images of YOYO-1 positive cells at 0, 12, 24, 36 hpt are presented in Fig. 8e.
SZJN prescription prevented glutamate-induced PC12 cell death through apoptosis
Annexin V-FITC/PI double staining was employed to examine apoptotic cells and verify the preventive effects of SZJN prescription on glutamate-induced neurotoxicity through inhibition of cell apoptosis (Fig. 9a). In Fig. 9b, 35.23% ± 17.03% of cells treated with glutamate for 24 h were detected in the apoptotic stage and were significantly higher than the control group (8.53% ± 0.21%, P < 0.01). However, SZJN prescription treatment (0.05, 0.1, and 0.2 mg/mL) and MH significantly decreased the number of apoptotic cells to 15.87% ± 6.37%, 16.47% ± 4.13%, 9.47% ± 0.67% and 12.30% ± 1.67%, respectively (P < 0.05). The results demonstrated that SZJN prescription inhibited glutamate-induced apoptosis of PC12 cells.
Expression profiles of Caspase-3 mRNA were detected using qRT-PCR. Notably, the relative levels of Caspase-3 mRNA were significantly higher after treatment with glutamate (4.05 ± 0.55) compared to the control group (1.00 ± 0.00, P < 0.01). Caspase-3 expression in the SZJN prescription group showed significantly varying profiles as follows: low (SZJN-L, 1.37 ± 0.33, P < 0.01), middle (SZJN-M, 2.13 ± 0.53, P < 0.01) and high (SZJN-H, 2.11 ± 0.61, P < 0.01) relative to the glutamate group. The expression levels in the SZJN-L group exhibited the highest decreasing trend, whereas the expression of Caspase-3 mRNA in the MH group decreased significantly (1.33 ± 0.24, P < 0.01) (Fig. 9c).
According to the theory of traditional Chinese medicine (TCM), VD is described as “dementia” or “stroke combined with dementia”. The pathogenesis of VD in TCM is related to “a deficiency of essential Qi with toxin damaging brain collaterals”. Since VD lasts longer, blockage of the Qi and blood may induce cognitive impairment because brain neurons get limited nutrients . Therefore, approaches that improve the essential energy are vital in the treatment of VD. They can also, detoxify and unblock the collaterals. Consequently, the Qi and blood eliminate the accumulated toxigenic products thereby, establishing SZJN prescription.
Previous studies have revealed that several TCM components exert their therapeutic effects by acting on multiple targets [18,19,20,21]. For instance, Ginseng was found to improve intelligence and relaxation of the nerves, while ginsenosides have the potential to prevent cerebral ischemia and repair neuron damage, which consequently improves vascular cognitive impairment [22, 23]. Similarly, studies have reported that Anemarrhenae-derived saponins improve cerebral ischemia and hypoxia; this subsequently enhances neurotrophic cognitive ability [24, 25]. Additional reports have demonstrated that Paeoniae rubra potentially protects neurons from hypoxia- or neurotoxicity-derived injury, and reduces inflammatory response [26, 27]. A combination of various herbs in SZJN prescription restore blood collaterals by eliminating phlegm, blood stasis, and collateral toxins. Herbal combinations can strengthen the vital energy and tonify the deficiency, detoxify and unblock collaterals, reduce turbidity and benefit wisdom. In the present study, 28 active compounds in SZJN prescription, 222 compound-related targets, and 3077 VD-related targets were revealed from the public databases. In details, the degree of these three herbs was as follows: Ginseng (12), Anemarrhenae (11), and Paeoniae rubra (8), consistent with their compatibility with SZJN prescription. Ginseng is regarded as the sovereign medicine, whereas Anemarrhenae and Paeoniae rubra are considered minister and assistant medicines, respectively . Thus, it may be speculated that Ginseng had a promising potential in the treatment of VD.
A total of 28 active compounds after eliminating the duplicates were identified, particularly, stigmasterol, beta-sitosterol (β-sitosterol), and kaempferol exhibited obvious bioactivities. Pharmacological studies have confirmed that stigmasterol, β-sitosterol and kaempferol are active components of Ginseng [29, 30]. Moreover, these compounds have been revealed as chemical components of Anemarrhenae and Paeoniae rubra [31, 32]. Stigmasterol can significantly relieve the vascular inflammation and nerve damage through reducing NO levels, COX-2 expression and apoptotic responses [33, 34]. In particular, stigmasterol and β-sitosterol were reported to significantly improve vanadium-induced cognitive ability in mice and were associated with improved motor coordination potential and a reduction in anti-oxygen free radicals and oxidative stress . It has been found that β-sitosterol can lower the cholesterol levels, enhance the production of plasminogen activators, and prevent endothelial dysfunction . β-sitosterol plays a vital role in the direct endothelial protective and antiatherogenic effects against oxidized low-density lipoprotein (ox-LDL) . Besides, β-sitosterol was found to potentially reduce neuron demyelination . Kaempferol has been reported to alleviate ox-LDL-induced apoptosis of human endothelial cells and potentiate angiogenic functions in cultured endothelial cells [39, 40]. Moreover, kaempferol was revealed to improve the damage of striatal neurons and increase levels of tyrosine hydroxylase (TH) and postsynaptic density protein 95 (PSD95) in mice striata. This mechanism aided in anti-neuroinflammation and maintenance of the integrity of the blood-brain barrier (BBB) . These findings demonstrate that the aforementioned compounds are associated with several important activities for VD treatment. Thus, they provide a basis for in-depth research on their active ingredients in SZJN prescription.
With regards to target points, PTGS2, PTGS1, PGR, ADRB2, ADRA1B, and CHRM1 recorded degrees≥10. However, PTGS2 had a maximum degree of 17, an implication that it is a potential SZJN prescription target vital in the management of VD. PTGS2, also known as cyclooxygenase-2 (COX-2), is a subtype of prostaglandin G/H synthetase (PGHS), and a vital enzyme in fatty acid metabolism . PTGS2 overexpression results in endothelial apoptosis, thus mediating vascular endothelial injury . Downregulation of PTGS2 expression could inhibit apoptosis and promote proliferation, migration and angiogenesis of endothelial progenitor cells (EPCs) . Similarly, PTGS1 (COX-1), another subtype of prostaglandin G/H synthetase, has been described as an important potential target for VD treatment. This enzyme protects gastrointestinal mucosa, promotes platelet aggregation, and induces peripheral vascular resistance through regulation of prostacyclin synthesis, closely associated with thrombosis [45,46,47]. PTGS1 has clear vascular protective roles associated with the anti-thrombotic potential, vasoconstriction and the development of atherosclerosis and vascular inflammation . On the other hand, the steroid hormone PGR, potentially reduces oxidative stress and neuroinflammation, regulates mitochondrial function, and promotes myelination by triggering several signaling pathways, such as the MAPK and NF-κ B signaling pathways. Therefore, it exerts a therapeutic effect on nervous system injury and neurodegenerative diseases . ADRB2, a transmembrane β-adrenergic receptor, interacts with adrenaline (a hormone and neurotransmitter) to mediate muscle and nerve function through downstream interaction with L-type calcium channels . Moreover, it has been demonstrated that activation of ADRB2 significantly increased EPCs bioactivities including cell proliferation, migration, and tube formation abilities . However, downregulation of ADRB2 could induce the dysfunction of EPCs. There are reports on the critical role of CHRM1 in memory impairment, and a decrease in its density is associated with learning and memory disorder, and cognition dysfunction . Researchers speculate that SZJN prescription may target the aforementioned proteins by regulating their expression levels to promote VD treatment. However, information on this relationship remains scarce and warrants further exploration.
The topology of the PPI network demonstrated that CASP3, JUN, ACHE, PPARG, PTGS2, ADRB2, CHRM1, ADRA1B, AR, PGR (degree≥4.7) are potentially important targets for SZJN prescription in VD treatment. This is based on its multi-target, multi-path, and multi-system characteristics. However, all the targets in the network, except for PTGS2, were consistent with the PPI network. This controversy in results may be attributed to the scarcity of information regarding whether the herbs were processed or decocted, as well as the specificity of target action. It was found that CASP3 recorded the largest degree (8), therefore it acted as the main target for SZJN prescription. Caspase-3, a member of the cysteine protease family, is a downstream protein in the apoptosis pathway and an apoptotic enzyme that functions through the death receptor and mitochondrial pathways . The apoptosis process may be attributed to the delayed neuronal degeneration of the central nervous system following injury to the distal axon . Previous studies found a significant increase in Caspase-3 expression in the hippocampus of VD rats [55,56,57,58], which demonstrated that Caspase-3-mediated apoptosis may be an important molecular mechanism of VD [59, 60]. Herein, the treatment with SZJN prescription showed protective activity against glutamate-induced PC12 cell apoptosis. Similar reports have previously been published, whereby ginsenosides in SZJN prescription protected neural cells against glutamate-mediated oxidative stress and neuronal cell death [61, 62]. Moreover, a significant increase in the relative expression of Caspase-3 mRNA in PC12 cells was found after glutamate injury, consistent with reports from previous studies [63, 64]. This expression was, however, down-regulated with low, middle and high doses of SZJN prescription. Besides, MH significantly reduced the levels of Caspase-3 expression; this consequently inhibited cell apoptosis and protected the nerve cells. With these findings, SZJN prescription potentially lowers Caspase-3 expression and the apoptosis of neurons as it targets the Caspase-3. This is considered to be a vital step in the treatment of VD. Other targets, include JUN, a transcription factor for AP-1 that is closely linked to neuronal death caused by a β amyloid protein , this factor potentially activates the downstream apoptotic pathway.
In the present study, following GO functional enrichment analyses, it was revealed that target genes were associated with a drug reaction, transcription initiation of RNA polymerase II promoter, cell signal transduction, and cell proliferation, among others. These observations demonstrated that SZJN prescription may promote cell proliferation and reduce cell apoptosis. Results from related experiments indicate that SZJN prescription mediates the repair of damaged nerve cells via different mechanisms [66, 67].
Moreover, the KEGG metabolic pathway analysis revealed that the targets were associated with neuroactive ligand-receptor interaction, calcium, and cholinergic synapse signaling pathways. Of note, the neuroactive ligand-receptor interaction signaling pathway incorporates all receptors and ligands related to intracellular and extracellular signaling pathways in the plasma membrane. These include adrenergic, muscarinic acetylcholine receptor (mAChR), and dopamine receptors, among others . Studies have attributed the abnormal expression of various receptor genes in the signaling pathway of receptor interaction in neuroactive ligands to a decline in learning and memory ability, and cognitive dysfunction [69,70,71]. Many subtypes of the adrenergic receptors, such as ADRB2, ADRA1A, ADRA1B, have been described, whereby a combination of these receptors and norepinephrine were found to regulate cell energy metabolism, glutamate transport, and neuroinflammatory response. These reports suggest that the adrenergic receptors represent a vital pharmacological target for the recovery and enhancement of cognitive function . More studies have shown that muscarinic acetylcholine receptor (mAChR), an important neurodegenerative disease drug target, is linked to neuronal excitability, synaptic plasticity, acetylcholine release, and cognitive function, and possesses anti-apoptosis characteristics . In this study, ADRB2, ADRA1A, ADRA1B, and CHRM1 were considered as SZJN prescription targets, and may exert a pharmacological effect in the management of VD through the interaction between ligands and receptors.
Due to the close relationship exists between the Ca2+ signaling pathway and signal transmission of neuron synapses, it was found that an open voltage-gated Ca2+ channel could activate NMDA receptors and drive the release of neurotransmitters. Also, an imbalance in the Ca2+ signaling pathway potentially activated apoptosis signaling molecules, such as Caspase-3 and Bax. This process inhibits the expression of Bcl-2 and consequently activates the apoptosis pathway . Another report revealed significantly high calcium concentration in the primary hippocampal neurons following ischemia-reperfusion injury. This led to calcium overload, and subsequently activation of the apoptosis signaling pathway, which eventually induced neuron apoptosis . In the present study, Caspase-3, Bax, and Bcl-2 were targets of SZJN prescription, therefore, had an association with the calcium signaling pathway. SZJN prescription exerted potential regulatory effects on the 3 targets via the calcium signaling pathway; this could be vital in the pharmacological treatment of VD.
Furthermore, the central cholinergic system coordinates synaptic plasticity, learning, and memory ability. Acetylcholine (ACh) is a neurotransmitter of the cholinergic pathway, and functions as a nicotinic (N) or muscarinic cholinergic receptor (CHRM1–5). Notably, excess hydrolysis of ACh by AChE has been implicated in the loss of cholinergic neurons, prolonged synaptic signal transmission disorder, and cognitive dysfunction . Inhibition of AChE and activation of the cholinergic system have been reported to alleviate the central inflammatory response and repair the synaptic function . This study reported AChE, CHRM1, and CHRM2 as targets for SZJN prescription, as they exerted potential effects via the cholinergic system. Therefore, it was hypothesized that SZJN prescription may regulate AChE, CHRM1, and CHRM2 via the cholinergic signaling pathway, which is critical in the pharmacological treatment of VD. Several other genes and metabolic pathways, as revealed by GO functional enrichment and KEGG metabolic pathways, may also be targets for active components of SZJN prescription. An in-depth analysis of these factors is needed to elucidate their potential molecular mechanisms in SZJN prescription.
However, there are still some limitations for this research. For example, the network pharmacology research relies more on various existing databases, but the screening criteria of compounds in databases may not be accurate enough. Moreover, the screened active ingredients might be inconsistent with the ingredients actually absorbed in blood of the patients with VD. As the accuracy and reliability of data has a great impact on the prediction results, it is necessary to further verify the reliability and quality of the predicted results, optimize the screening criteria of TCM databases, and improve the network pharmacology research. Therefore, further experimental and clinical verification of the efficacy of SZJN prescription on cerebrovascular microenvironment and neuroprotection is demanded to validate theoretical predictions.
In this study, a systematic analysis for the characteristic of “multicomponents, multitargets, and multichannels” of SZJN prescription in treating VD was successfully performed through the method of network pharmacology and in vitro experiments. The experimental results were in good accordance with the results predicted from the network analysis of the SZJN prescription, especially the anti-apoptosis properties of the prescription. In the future, more experimental evidence should be provided for the neuroprotective and cerebral vascular protective effects of SZJN prescription against VD according to the results of network pharmacology research.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Adenosine 5′-monophosphate (AMP)-activated protein kinase
Adrenergic receptor α 1A
Adrenergic receptor α 1B
Adrenoceptor Beta 2
Muscarinic cholinergic receptor M1
Muscarinic cholinergic receptor M2
Cyclic adenosine monophosphate
Transcription factor AP-1
Kyoto Enrichment of Genes and Genomes
N-methyl-d-aspartic acid receptor 1
- PPI network:
Protein-Protein Interaction network
Prostaglandin-endoperoxide synthase 2
Prostaglandin-endoperoxide synthase 2
Peroxisome proliferator-activated receptor-gamma
Quantitative real-time polymerase chain reaction
- SZJN prescription:
Shenzhi Jiannao prescription
Traditional Chinese Medicine Systems Pharmacology
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We would like to extend our gratitude to Professor Zhen-Yun Han from the Shenzhen Hospital, Beijing University of Chinese Medicine (Longgang), Beijing, China, for providing valuable suggestions and revising the manuscript.
This study was supported by the National Natural Science Foundation of China with grant number 81673910. The funding body had no role in designing the study, collection, analysis, and interpretation of data and in writing the manuscript.
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Tian, D., Gao, Q., Chang, Z. et al. Network pharmacology and in vitro studies reveal the pharmacological effects and molecular mechanisms of Shenzhi Jiannao prescription against vascular dementia. BMC Complement Med Ther 22, 33 (2022). https://doi.org/10.1186/s12906-021-03465-1
- Shenzhi Jiannao prescription
- Network pharmacology
- Vascular dementia
- Pharmacological effects and molecular mechanisms
- In vitro studies

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Scroll down to view all of Joshua McHugh’s Piano Publications
Billions of Penguins.
For Solo Harp
Learn to Play & Sight Read Piano Music.
(In four volumes)
Preludes in Every Mode of Every Key
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Clockworks & Dovetails. Opus 13.
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Festival in the Woods. Opus 3 No. 3
Hayley of the Lake. Opus 23.
Etude in B Minor. Opus 3 No. 5

domain: artistic


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