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ObjectiveThis study aims to investigate the mechanism in which Celastrus orbiculatus extract (COE) affects the proliferation and differentiation of gastric organoids and the expression of Lgr5 and thus reverses the precancerous lesions of gastric cancer (PLGC) by regulating the leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5)/Wingless (Wnt)/β-catenin signaling pathway based on a gastric organoid injury model. MethodGastric organoids were established based on stem cells of the mouse gastric gland. Gastric organoid injury models were constructed by treating gastric organoids with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.02 mg·L-1). Gastric organoid injury models were randomly divided into normal group, model group (0.02 mg·L-1 MNNG), low, medium, and high dose (5, 10, 20 mg·L-1) groups of COE, and Wnt inhibitor Dickkopf-related protein 1 (DKK1) (0.5 mg·L-1) group, and they were treated with respective agents for 24 h. The number and volume of gastric organoids under different drug concentrations were observed under a microscope. The viability of the gastric organoid injury models was detected by Methyl thiazolyl tetrazolium (MTT) assay. The morphology and pathology of gastric organoids were observed using Hematoxylin and Eosin (HE) staining. The expression levels of Lgr5, Mucin2 (MUC2), Mucin5AC (MUC5AC), Mucin6 (MUC6), Wnt, and β-catenin in gastric organoids under different drug concentrations were detected by Western blot (WB). ResultCompared with the normal group, the number, volume, and activity of gastric organoids in the model group were decreased (P<0.01), while the expressions of Lgr5, MUC2, Wnt, and β-catenin were significantly increased (P<0.01). The expressions of MUC5AC and MUC6 were significantly decreased (P<0.01). Compared with the model group, the number and volume of gastric organoids in the low, medium, and high dose groups of COE were all improved (P<0.01), and the vitality of gastric organoids was significantly enhanced (P<0.01). The effect was the most significant at a COE concentration of 20 mg·L-1 (P<0.01). The expressions of Lgr5 and MUC2 in the medium and high dose groups of COE were significantly reduced (P<0.01), while the expression of MUC5AC and MUC6 were significantly increased in the low, medium, and high dose groups of COE (P<0.05, P<0.01). Compared with the model group, Wnt inhibitors could promote the expression of MUC5AC and MUC6 in gastric organoids (P<0.05, P<0.01) and reduce the expression of MUC2, Wnt, and β-catenin. In addition, the combined use of COE at high concentrations and Wnt inhibitors could further promote this trend (P<0.01). ConclusionCOE inhibits the Wnt/β-catenin pathway by inhibiting the expression of Lgr5, MUC2, Wnt, and β-catenin and promoting the expression of MUC5AC and MUC6, thus promoting the proliferation and differentiation of gastric organoids and reversing the PLGC process.
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Objective@#To investigate the effect of Celastrus orbiculatus extract ( COE) on the growth of gastric organoids and the expression of E-cadherin in gastric epithelial cells of mice.@*Methods@#The gastric pylorus of 8-week-old C57 mice was isolated and cultured into gastric organoids.The dynamic changes of gastric organoid formation were observed under light microscope ; the intercellular structure of gastric epithelium was observed by HE staining ; the expression of epithelial-cadherin E-cadherin in gastric epithelial cells was observed by immunofluorescence staining.After passage to the third-generation ,the organoids were treated with different concentrations of COE (0,5 ,10,20 μg/ ml) ,the organoids were collected ,their numbers were counted ,their diameters were measured,their cellular activities were measured by methyl thiazolyl tetrazolium( MTT) colorimetry,and Western blot was used to detect the expression of E-cadherin in organoids after COE treatment. @*Results @#At 24 to 48 h, cystlike structures were formed and three-dimensional cell clusters with cystic gland-like central structure appeared, and gradually budding and forming gastric organoids after 72 h,suggesting that the organoids were successfully constructed.The epithelial cell marker E-cadherin was expressed in the organoid,which further confirmed the formation of organoid.Compared with the control group,the number and diameter of gastric organoids in the COE group significantly increased,cell activity was significantly enhanced (P<0. 05) ,and the expression of E-cadherin increased with the increase of COE dose (P<0. 01) .@*Conclution @#Low dose COE can promote the expression of E-cadherin and the growth and formation of organoids,which may affect the repair of gastric mucosa injury.
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ObjectiveTo study the inhibitory effect of Celastrus orbiculatus extract (COE) on gastric cancer cells, to clarify the specific mechanism of COE promoting the apoptosis of gastric cancer cells by affecting the mitochondrial structure and function, and to provide an experimental basis for the further development and clinical application of C. orbiculatus. MethodBrdu staining combined with flow cytometry and Annexin V-fluorescein isothiocyanate (AnnexinV-FITC) staining combined with flow cytometry were employed to detect the effects of COE (20, 40, 80 mg·L-1) on the proliferation and apoptosis of gastric cancer cells, respectively. The changes in mitochondrial membrane potential were detected with JC-1 mitochondrial membrane potential assay kit. The expression of apoptosis-associated proteins including B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-xL (Bcl-xL), Bcl-2-associated X (Bax), and cysteine aspartutespecific protease-3 (Caspase-3) in gastric cancer cells was determined by Western blot. Transmission electron microscopy was employed to detect changes in the mitochondrial microstructure of gastric cancer cells exposed to COE. Western blot was employed to measure the expression of mitochondrial marker proteins [superoxide dismutase 1 (SOD1), voltage-dependent anion channel (VDAC), prohibitin 1 (PHB1), and heat shock protein 60 (HSP60)] in gastric cancer cells. ResultCompared with the control group, COE (40, 80 mg·L-1) inhibited the proliferation and promoted the apoptosis of gastric cancer cells (P<0.05). Furthermore, COE reduced the mitochondrial membrane potential of gastric cancer cells. Compared with the control group, COE (20, 40, 80 mg·L-1) up-regulated the expression of Bax and Caspase-3 which promoted apoptosis of gastric cells (P<0.05, P<0.01), and COE at 40 and 80 mg·L-1 down-regulated the expression of Bcl-2 and Bcl-xL which inhibited the apoptosis of gastric cancer cells (P<0.01). The results of transmission electron microscopy showed that COE changed the microstructure of gastric cancer cells, which led to the appearance of vacuoles in the cell membrane and mitochondria and damaged the mitochondrial structure. Compared with the control group, COE (20, 40, 80 mg·L-1) changed the expression of mitochondrial marker proteins. Specifically, it up-regulated the expression of SOD1 involved in stress response (P<0.05, P<0.01) and down-regulated that of VDAC, PHB1, and HSP60 associated with mitochondrial stability and permeability (P<0.01). ConclusionCOE can significantly inhibit the proliferation and promote the apoptosis of gastric cancer cells. It may activate the mitochondrial apoptosis pathway by destroying the mitochondrial structure and function of gastric cancer cells.
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ObjectiveTo study the inhibitory effect of Celastrus orbiculatus extract (COE) on gastric cancer cells, to clarify the specific mechanism of COE promoting the apoptosis of gastric cancer cells by affecting the mitochondrial structure and function, and to provide an experimental basis for the further development and clinical application of C. orbiculatus. MethodBrdu staining combined with flow cytometry and Annexin V-fluorescein isothiocyanate (AnnexinV-FITC) staining combined with flow cytometry were employed to detect the effects of COE (20, 40, 80 mg·L-1) on the proliferation and apoptosis of gastric cancer cells, respectively. The changes in mitochondrial membrane potential were detected with JC-1 mitochondrial membrane potential assay kit. The expression of apoptosis-associated proteins including B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-xL (Bcl-xL), Bcl-2-associated X (Bax), and cysteine aspartutespecific protease-3 (Caspase-3) in gastric cancer cells was determined by Western blot. Transmission electron microscopy was employed to detect changes in the mitochondrial microstructure of gastric cancer cells exposed to COE. Western blot was employed to measure the expression of mitochondrial marker proteins [superoxide dismutase 1 (SOD1), voltage-dependent anion channel (VDAC), prohibitin 1 (PHB1), and heat shock protein 60 (HSP60)] in gastric cancer cells. ResultCompared with the control group, COE (40, 80 mg·L-1) inhibited the proliferation and promoted the apoptosis of gastric cancer cells (P<0.05). Furthermore, COE reduced the mitochondrial membrane potential of gastric cancer cells. Compared with the control group, COE (20, 40, 80 mg·L-1) up-regulated the expression of Bax and Caspase-3 which promoted apoptosis of gastric cells (P<0.05, P<0.01), and COE at 40 and 80 mg·L-1 down-regulated the expression of Bcl-2 and Bcl-xL which inhibited the apoptosis of gastric cancer cells (P<0.01). The results of transmission electron microscopy showed that COE changed the microstructure of gastric cancer cells, which led to the appearance of vacuoles in the cell membrane and mitochondria and damaged the mitochondrial structure. Compared with the control group, COE (20, 40, 80 mg·L-1) changed the expression of mitochondrial marker proteins. Specifically, it up-regulated the expression of SOD1 involved in stress response (P<0.05, P<0.01) and down-regulated that of VDAC, PHB1, and HSP60 associated with mitochondrial stability and permeability (P<0.01). ConclusionCOE can significantly inhibit the proliferation and promote the apoptosis of gastric cancer cells. It may activate the mitochondrial apoptosis pathway by destroying the mitochondrial structure and function of gastric cancer cells.
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Aim To explore the effeet of the extract of Celastrus orbiculatus extract on the proliferation anrl ap- optosis of multiple myeloma eells (U-1996) and its molecular mechanism.Methods The U-1996 eells were divided into normal control ( NC ) group, the southern snake vine extraet group, si-NC group, si- LINC00472 group, pcDNA-NC group, pcDNA- LINC00472 group, southern snake vine extraet + si-NC group,and southern snake vine extraet + si-LINC00472 group.Heal-time quantitative PCR was used to deteet LINC00472 expression, CCK-8 and flow cytometry to deteet eell proliferation and apoptosis, Western blot to deteet the expression levels of phosphorvlated phos- phatidvlinositol-3 -hydroxykinase (p-PI3K) and phos- phorylated protein kinase B ( p-Akt).Results After treated with Celastrus orbieulatus extract, the viability of U-1996 eells and the expression of p-PI3K and p- Akt were significantly reduced, the apoptotie rate and the expression of LINC0047 were significantly raised ( P < 0.05 ).After inhibiting the expression of LINC00472, the viability of U-1996 eells and the ex¬pression of p-PBK and p-Akt significantly inereased, the apoptotie rate and the expression of LINC0047 sig¬nifieantly deereased ( P <0.05 ).After overexpression of LINC00472, the viability of U-1996 eells and the ex¬pression of p-PI3K and p-Akt were signifieantly re- dueed, the apoptotie rate and the expression of LINC0047 significantly inereased ( P <0.05 ).Inhibi¬ting the expression of LINC0047 can reverse the effects of Celastrus orbiculatus extract on proliferation , apopto- sis and PI3K/AKT signaling pathway of U-1996 cells (P < 0.05 ).Conclusions Celastrus orbiculatus ex¬tract can inhibit the proliferation of multiple myeloma cells and induce apoptosis through up-regulating LINC00472 to inhibit PI3K/Akt signaling pathway.
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Objective: To investigate the effects of the combination of Celastrus orbiculatus extracts and miR-302 on proliferation, invasion and migration of human esophageal cancer cells and the regulation of PI3K/Akt signaling pathway. Methods Quantitative RT-PCR was used to detect the expression of miR-302 in human normal esophageal epithelial cells Het-1A and different esophageal cancer cell lines. The miR-302 mimic and negative control mimic control were transfected into human esophageal cancer TE-1 cells, and qPCR was used to detect the expression of miR-302 in TE-1 cells after plasmid transfection. TE-1 cells were treated with C. orbiculatus extracts alone and combination treatment. The proliferation of TE-1 cells was detected by CCK-8 assay. The invasion and migration of TE-1 cells were detected by Transwell assay. Western blot analysis of the expression of related protein in the PI3K/Akt signaling pathway was carried out. Results: The expression of miR-302 in esophageal cancer cell lines was significantly lower than that in esophageal epithelial cells (P < 0.05). Transfection of miR-302 mimic could effectively increase the expression of miR-302 in TE-1 cells (P < 0.05). The use of C. orbiculatus extracts alone or overexpression of miR-302 inhibited proliferation, invasion and migration of esophageal cancer TE-1 cells (P < 0.05), down-regulated PI3K and p-Akt protein expression; Combination treatment had more significant effect on inhibiting proliferation, invasion and migration of TE-1 cells and down-regulating protein expression of PI3K and p-Akt (P < 0.05). Conclusion: Celastrus orbiculatus extracts combined with miR-302 can synergistically inhibit the proliferation, invasion, and migration of esophageal cancer TE-1 cells, and its mechanism may be related to the inhibition of PI3K/Akt signaling pathway activation.
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OBJECTIVE@#To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms.@*METHODS@#The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively.@*RESULTS@#COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells.@*CONCLUSIONS@#COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.
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Humans , Biomarkers, Tumor , Metabolism , Carcinoma, Hepatocellular , Drug Therapy , Pathology , Celastrus , Chemistry , Cell Hypoxia , Cell Proliferation , Cell Shape , Cobalt , Down-Regulation , Epithelial-Mesenchymal Transition , Hep G2 Cells , Liver Neoplasms , Drug Therapy , Pathology , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Proteins , Metabolism , Plant Extracts , Pharmacology , Therapeutic Uses , Signal TransductionABSTRACT
OBJECTIVE@#To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb. extracts (COE).@*METHODS@#The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin (mTOR) knockdown expressed (HepG2/mTOR) were constructed using molecular biological technology. In vitro, the HepG2/mTOR cells were treated with COE at various concentrations (10, 20, 40, 80, 160 and 320 µ g/mL). Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. According to the half-maximal inhibitory concentration (IC) value (140 mg/L), the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity. The HepG2/mTOR cells were divided into 5 groups: negative control (untreated), COE treatment groups (40, 80, 120 mg/L COE) and positive control group (cisplatin, DDP, 2 mg/L), respectively. Wild-type HepG2 cells were used as a blank control. The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy (TEM), respectively. The protein expression levels of mTOR signal pathways were determined by Western blotting. In vivo, HepG2/mTOR cells (2 × 10 cell/mice) were subcutaneously injected into the right flank of nude mice. Thirty-six female nude mice were randomly assigned to 6 groups according to body weight (6 mice per group) as follows: solvent vehicle control, Banmao Capsule treated group (BM, 195 mg/kg), Tegafur, Gimeracil and Oteracil Potassium Capsules (10 mg/kg) treated group, and different dosages of COE (10, 20, 40 mg/kg) groups. Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.@*RESULTS@#COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR cells (P<0.01). COE significantly induced the apoptosis of HepG2/mTOR cells (P<0.01), and the apoptotic bodies can be observed under TEM. COE significantly inhibits the proteins expression of mTOR-related signal pathways. In vivo, COE significantly inhibited tumor growth in nude mice (P<0.01). Moreover, the results showed that COE down-regulated the expression of Bcl-2 and Bcl-xL, and up-regulated the levels of Bax and caspase-3 protein (P<0.01).@*CONCLUSION@#COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway.
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Objective To investigate the molecular mechanisms of Celastrus orbiculatus extracts (COE) of the invasion and metastasis inhibition in human hepatocellular carcinoma HepG2 cells by targeting mTOR. Methods The HepG2/mTOR- cells with mTOR knockout expression were constructed by using siRNA technology. The effect of COE on the proliferation of the HepG2/mTOR- cells was also studied. The HepG2/mTOR- cells were treated with COE in different concentrations (20, 40, 80, 160, and 320 mg/L) for 24 h. The cell reproductive capability of HepG2/mTOR- cells was detected by MTT. The effect of COE on the metastatic ability of HepG2/mTOR- cells in vitro was investigated by scratch assay and Transwell migration assay. The expression levels of molecular mechanisms related proteins MMP-2 and MMP-9 were assessed by Western blotting. Results The HepG2/mTOR- cells with mTOR knockout expression were successfully constructed. COE significantly inhibited the proliferation of HepG2/mTOR- cells in a concentration-dependent manner (P < 0.05). COE decreased the invasion and migration of HepG2/mTOR- cells. The results of Transwell experiment indicated that COE (80 mg/L) significantly reduced the number of transmembrane cells (P < 0.05). And the expression levels of MMP2 and MMP9 protein were significantly reduced in the HepG2/mTOR- cells after the treatment of COE. Conclusion COE can significantly inhibit the proliferation, invasion, and migration in the HepG2/mTOR- cells. Our data reveal that COE is a potential chemotherapeutic drug in human hepatocellular carcinoma treatments via targeting mTOR signal pathway.
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Objective: To study the effect and the mechanism of Celastrus orbiculatus extract (COE) on inhibiting the invasion and metastasis of human gastric cancer SGC-7901 cells. Methods: The groups of different cells were set up, such as negative control group, COE with different concentration groups, and positive control group. The effects of COE on the cytotoxicity and proliferation of human gastric cancer SGC-7901 cells were determined by MTT assay. The effect of COE on the metastatic ability of human gastric cancer SGC-7901 cells in vitro was observed by invasion assay and migration assay. The effects of COE on the expression of matrix metalloproteinases (MMPs) and their inhibitors in human gastric cancer SGC-7901 cells were examined by Western blotting. After the treatment in different groups, mRNAs of MMP2, MMP9, tissue inhibitors of metalloproteinases (TIMPs), TIMP1, and TIMP3 in cells were detected by RT-PCR. Results: Compared with the negative control group, human gastric cancer SGC-7901 cells treated with COE at different concentration exhibited a dose-dependent growth inhibition. COE obviously reduced the migration and invasion potential of human gastric cancer SGC-7901 cells. In addition, the expression levels of MMP2 and MMP9 were dramatically suppressed by COE in a concentration-dependent manner, while the expression levels of TIMP1 and TIMP3 were raised. RT-PCR showed that mRNAs of TIMP1 and TIMP3 were raised with the increase of drug concentration. Conclusion: COE could significantly inhibit the invasion and metastasis of human gastric cancer SGC-7901 cells via an up-regulation of the expression levels of TIMP1 and TIMP3 and down-regulation of the expression levels of MMP-2 and MMP-9.
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This study aimed at investigating the effects of the extract of Chinese herb,Nansheteng (C.orbiculatus Thunb.),on human HepG2 cells through the overexpression of mTOR.The GV238-mTOR recombinant plasmids were transfected into HepG2 cells using molecular biological technology.The expression level of mTOR was evaluated by means of relative activity of luciferase and western blot.Human hepatic carcinoma HepG2/mTOR++ cells were treated with C.orbiculatus extract in different concentrations (20,40,80,160 and 320 tg·mL-1) for 24 h.The mTOR protein expression was detected by western blot.It was found that the protein expression of mTOR in transfected HepG2 cells was significantly enhanced.C.orbiculatus extract significantly inhibited the proliferation of HepG2/mTOR++ cells.Simultaneously,C.orbiculatus extract inhibited mTOR at its protein level in a dose-dependent manner.In conclusion,we successfully constructed recombinant mTOR cloning vectors,and established the stable HepG2 cell line with the overexpression of mTOR.Besides,C.orbiculatus extract significantly inhibited mTOR protein expression in human hepatic carcinoma HepG2 cells.
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To explore the effects of Celastrus orbiculatus extract (COE) on the expressions of apoptosis-related proteins and growth of human gastric adenocarcinoma SGC-7901 cell xenografts in nude mice. Thirty-five mice with subcutaneous xenografts of a gastric cancer cell line (SGC-7901) were randomly divided into five groups: a negative control group, a positive control group (xeloda), and low-dose, medium-dose, and high-dose COE groups (10, 20, and 40 mg/kg, respectively). There were seven mice in each group. Tumor volume, tumor weight, and body weight were measured to draw up tumor growth curves and the tumor weight inhibition rates were calculated. The apoptosis rates were measured by TUNEL staining method and the expressions of p53, Bcl-2, and Bax proteins were detected by immunohistochemical staining and Western blotting assay. The COE could inhibit the growth of SGC-7901 xenografts in a dose-dependent manner. The tumor inhibitory rates were 33.3%, 46.8%, and 57.7% when treated with 10, 20, and 40 mg/kg COE, respectively. TUNEL assay showed that COE presented with apparently more apoptosis than the negative control group. The results of immunohistochemical staining showed the expressions of p53 and Bax proteins had a trend of up-regulation, while the expression of Bcl-2 protein had a trend of down-regulation in nude mice after treatment with COE. Compared with the negative control group, the expressions of p53 and Bax were significantly up-regulated, while the expression of Bcl-2 was down-regulated in the high-dose COE group. Western blotting analysis showed the similar results to immunohistochemical staining. COE could significantly inhibit the growth of SGC-7901 cell xenografts in nude mice, which might be related with the up-regulation of the expressions of p53 and Bax and down-regulation of the expression of Bcl-2.
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Objective: To establish an HPLC method for the determination of kaempferitrin in Celastri Orbiculati Fructus and to compare the quality of samples from different habitats in northeast China. Methods: The extracts were obtained by methanol ultrasonic method and kaempferitrin was determinated by HPLC. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) at 30℃ and the mobile phase was a mixture of methanol- 0.01 mol/L solution of potassium dihydrogenphosphate -glacial acetic acid (40:60:15). The flow rate was 1.0 mL/min and the detection wavelength was 254 nm. Results: The linearity range was 0.092 7-0.926 7 μg (r = 0.999 8), average recovery was 100.8%, and RSD = 1.1% (n = 6). Conclusion: This method is validated in terms of selectivity, linearity, precision, and accuracy. It can be used as the effective method for quality control. The determination results show that the longer the fruit sits followed by picking, the less contents it has.
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Celastrus orbiculatus Thunb .(Celastraceae) was widely distributed in China and more in the south area of the Yangtze River .Research showed the main chemical components including terpenoids ,lipids ,glycosides and flavonoids .Its pharmacological effects were extensive ,which were including antitumor ,anti-inflammatory ,immunomodulatory ,anti-fibrosis and anti-fertility etc .The separation of active compositions and advances in the pharmacological effects were reviewed in recent years ,which would be helpful in providing reference and thinking for the research and development of Celastrus orbiculatus .
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This study was aimed to investigate whether the extracts of Celastrus orbiculatus enhanced the invasion function of maspin tumor inhibitor gene through the construction of maspin overexpression human gastric carcinoma MGC803 cell line. Maspin was cloned into plasmid GV208-EGFP eukaryotic expression vector. And then, the recombinant plasmid GV208-maspin-EGFP was transfected into human gastric carcinoma MGC803 cells. After the maspin overexpression MGC803 cell were treated with Celastrus orbiculatus extracts in different concentrations (10, 20, 40 μg·mL-1), the invasion effects were detected by Transwell chamber assay. The results showed that after the successful construction of maspin overexpression cell line, the number of cells invading through Matrigel was obviously decreased in the Transwell chamber assay. It also showed drug concentration dependency. It was concluded that maspin gene can inhibit invasion of gastric carcinoma MGC803 cells. Simultaneously, the extracts of Celastrus orbiculatus can enhance the function of maspin gene.
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This study was aimed to investigate the effect of Nan-She-Teng (Celastrus orbiculatus) extract on epithe-lial-mesenchymal transition(EMT) in HepG2 cells. Except the control group, human hepatocellular carcinoma HepG2 cells in other groups were treated with Celastrus Orbiculatus extract in different concentrations (10, 20, 40, 80, and 160 μg·mL-1). The protein expression levels related to EMT were detected by western blotting. At 48 h after fertiliza-tion, the zebrafish embryos were randomly assigned to 7 groups as follows: untreated control group (E3 buffer), DMSO group (E3 buffer with 1% DMSO), and different dosages treatment of C.orbiculatus extract (10, 20, 40, 80, and 160μg·mL-1) for 24 h. The protein expressions of mTOR signaling pathways were detected by western blotting. The re-sults showed that compared with the control group, C.orbiculatus extract significantly increased E-cadherin protein expression. Simultaneously, C.orbiculatus extract inhibited vimentin and mTOR signaling pathways at protein levels. It was concluded that to a certain extent, C.orbiculatus extract prevented EMT in HepG2 cells by modulating the mTOR signaling pathway. Therefore, it suggested that mTOR can be chosen as a new therapeutic target for clinical treatment of hepatic carcinoma.
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Objective To study the effects of Celastrus orbiculatus extracts (COE) on the expression of VEGF and CECs of Hepal-6 tumor bearing mice.Methods Animal Models of Hepal-6 portability liver tumor bearing mice were established and randomly assigned to six groups (eight mice per group) as follows:untreated control group,solvent vehicle control (DMSO) group,physiological saline control group,cisplatin-treated group,and different dosages COE-treated groups (40 mg/kg,20 mg/kg,10 mg/kg).To approach the effects of COE on the expression of VEGF and CECs in Hepal-6 toumor bearing mice.Results The ELISA and flow cytometer showed that COE could significantly inhibit the expression of VEGF(45.24±21.36) pg/ml and CECs (0.83 ± 0.10) % compared with the physiological saline control group (P< 0.01 ) with the dose of 20mg/kg.Conclusion COE could significantly down-regulate the expression of VEGF and CECs.It may be related with the effect of COE in inhibiting angiogenesis of tumor.
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Objective: To investigate the effect of Celastrus orbiculatus extracts (COE) on xenograft tumor growth of HepA1-6 hepatoma in mice, and to explore its possible mechanism. Methods: HepA1-6 hepatoma model was established in mice, and then these mice were randomly divided into blank control group, solvent control group (1% DMSO), negative control group (0.9% NaCl solution), positive control group (cisplatin), and low-, medium- and high-dose COE groups (10, 20 and 40 mg/kg, respectively). The body weight, tumor growth inhibitory rate, liver index, spleen index and thymus gland index were calculated, and the expression levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as microvessel density (MVD) were detected by immunohistochemistry. The apoptosis rate was determined by TUNEL assay. Results: COE could significantly inhibit the tumor growth, increase the apoptosis rate, and down-regulate the expression levels of VEGF, bFGF and MVD of HepA1-6 hepatoma in the liver of mice, as compared with the negative control group (P 0.05). Conclusion: COE can inhibit the xenograft tumor growth of HepA1-6 hepatoma in mice. This effect may be associated with the enhancement of apoptosis and the inhibition of angiogenesis in tumors. Copyright© 2011 by TUMOR.
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Objective To examine the immunoregulation of Celastrus orbiculatus extracts(COE),a traditional Chinese medicine,on maturation and function of dendritic cells(DCs)in vitro and in vivo.Methods In vitro,after treated with COE indifferent nontoxic concentrations(0,10,20,40,80,and 160 μg/mL)for 5 d,the surface immunological molecules andcytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis(FACS)and enzyme linked immunosorbent assay(ELISA),respectively.In vivo,mouse hepatoma cells(Hepal-6,1 ×106)were injected sc and were treated with different dosages of COE(10,20 or 40 mg/kg/d).Effects on tumor growth were determined by tumor volume and histology analysis after 28 d administration of COE.The relative proportions ofmature DCs and CD8+ T cells were measured in mononuclear cells that had been isolated from spleen by FACS.Results COE stimulated IL-2 and IFN-γ secretion of DCs,simultaneously enhanced the maturation of DCs byenhancing immunological molecule(CD40,CDS0,CD86,H-2Kb,and I-Ab)expression in a dose-dependent manner.Furthermore,the chcmotactic responses of DCs were significantly higher in COE-treated than untreated DCs,in association with higher chcmokine receptor 7 expression.Furthermore,COE increased DCs produce IFN-γ and IL-2 ina dose-dependent manner when the concentration of COE less than 40 μg/mL,decreased DCs produce IL-10 and IL-4also in a dose-dependent manner.In in vivo studies,COE can not only suppress growth of malignant hepatocellularcarcinomas but also stimulate maturation of DCs,associated with strongly enhanced CD8+ CTL responses.ConclusionThese data provide new insight into the mechanism of action of COE and indicate that the stimulation of maturation andfunction of DCs by COE contributes to its immunoregulatory effects.
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Objective To discuss the antitumor mechanism preliminarily by observing effects of Celastrus orbiculatus extracts (COE) on vascular endothelial growth factor (VEGF) mRNA and protein expression in hepatoma (Hepal-6) cells of mice.Methods Hepa1-6 cells were treated with COE at different nontoxic concentration (0, 10, 20, 40, 80, and 160 μg/mL) for 16 h. The mRNA and protein expressions of VEGF were detected by reverse transcription-PCR and Western Blotting,respectively. Results COE significantly inhibited VEGF expression at both mRNA and protein levels in a dose-dependent manner. Conclusion COE can inhibit VEGF expression in Hepa1-6 cells, therefore suggest that VEGF could be chosen as an therapeutic target for COE in the context of cancer chemoprevention and anticancer therapy.