Effects Of Solamargine Extracted From Solanum Nigrumon On Growth And Metastasis On The Mechanism Of Cholangiocarcinoma Cells

Background:Cholangiocarcinoma have accounted for 3%of the tumors of the digestive system.And incidence of the disease has been increasing in recent years.Because of the early symptoms of cholangiocarcinoma is not obvious,most of the diagnosis is already late.Because early diagnosis and chemotherapy of cholangiocarcinoma is still poor and unclear.Therefore,seeking new treatment methods and drugs from the perspective of traditional Chinese medicine and traditional Chinese medicine has aroused wide attention of scholars.The Solanaceae nightshade(Solanum nigrumL),has the clear solution heat,detumescence,anti-inflammatory and diuretic effects of traditional Chinese medicine.In recent years,Solanum nigrum in treatment of malignant tumor has become a research focus for clinical,Solanum nigrum has anti-tumor activity,so it is listed as one of the ten herbs in anti tumor.Many studies show that active constituents of Solanum nigrum contains a variety of anti-tumor effects,including solamargine as the main component of the anticancer activity of Solanum nigrum.And the anti-cancer effect has been affirmed.However,the mechanism of its function is still not very clear.Based on the antitumor effect of Solanum nigrum L to explore the effect and mechanism of solamargine on proliferation,apoptosis,metastasis of cholangiocarcinoma cells from the aspect of cell biology,in order to explain the mechanisms of Solanum nigrum anti cholangiocarcinoma,and provide theoretical and experimental basis for clinical application.Objective:Study the solamargine from Solanum nigrum to promote bile duct cancer cell apoptosis,inhibit proliferation and metastasis and its mechanism.Method:① Treated with 0,2,4,6,8,10 μM concentrations of solamargine in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,the number and morphology of cells were observed by optical microscope;the inhibition rate of cell proliferation was detected by MTT;the optimal drug action time and drug concentration were determined.It is to investigate the effect of solanine on the proliferation of human cholangiocarcinoma QBC939 cells.②Treated with upon concentrations of solamargine in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,Cell apoptosis distribution and apoptosis rate was detected by flow cytometry(FCM).It is to investigate the effect of solanine on the apoptosis of human cholangiocarcinoma QBC939 cells.③Treated with upon concentrations of solamargine in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,the change of mitochondrial membrane potential induced by solamargine in QBC939 cells after being stained by JC-1 staining buffer and detected by flow cytometry;Q-PCR was used to detect the expression of mRNA of Bcl-2,Bcl-XL,Bax and XIAP;Western blot was used to detect the expression of protein of Bcl-2,Bax,Caspase-3,XIAP and RARP.It is to investigate the mechanism of solamargine to promote the apoptosis of cholangiocarcinoma cell line QBC939.④Treated with upon concentrations of solamargine in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,the ability of migration and invasion was detected by scratch and transwell assay;Q-PCR and Western blot was used to detect the expression of mRNA and protein about E-cadherin、N-cadherin、Vimentin and Snail.And the expression of pathway of PI3K/AKT was detected by Western blot.Result:①In vitro cell experiments,solamargine treatment could lead to the emergence of cell shrink,irregularity and apoptosis body observed by light microscope.Further MTT assay showed that solamargine inhibited the cell proliferation and viability of QBC939 cells in the dose-dependent manner and the value of IC50 is 9.81μM.②Our study showed that solamargine induced apoptosis of QBC939 cells significantly in dose-dependent manner detected by flow cytometry.There was a significant concentration dependence(especially concentration>6 mu M),and the apoptosis rate in early and late stages of the cells increased,with early apoptosis as the main factor.③In order to investigating the underlying mechanism of solamargine induced apoptosis.The down regulationof mitochondrial membrane potential induced by solamargine in QBC939 cells after being stained by JC-1 staining buffer and detected by flow cytometry.And the change of apoptosis related gene and protein expression was detected by Q-PCR and Western blot experience.The results indicated that solamargine increased the expression of Bax,Caspase3,Cleaved caspase3,Caspase7 and Cleaved PARP,and decreased the expression of Bcl-2,Bcl-xL,XIAP and PARP.④The results of scratch and Transwell assay showed that the cell migration and invasion could be inhibited in dose-dependent manner.Further detection of its mechanism suggests that solanine is expressed by inhibiting the PI3K/AKT signaling pathway,inhibition the expression of transcription factor Snail,increased the expression E-cadherin,and decreased the expression of N-cadherin and Vimentin.Conclusion:Solamargine extracted from Solanum Nigrumon can inhibit the proliferation and viability of human cholangiocarcinoma QBC939 cells.And it can reduce the expression of mitochondrial membrane potential in tumor cells and up regulation of pro-apoptotic proteins and down regulated expression of anti-apoptosis protein induced apoptosis of tumor cells,which may be the mechanism of apoptosis of cholangiocarcinoma QBC939 cells induced by solamargine.The results of scratch and Transwell assay are that solamargine can inhibit metastasis by inhibiting the PI3K/AKT signaling pathway,decreased the expression of transcription factor Snail,increasing the expression E-cadherin,and decreasing the expression of N-cadherin and Vimentin.Solamargine can inhibit the growth and metastasis of cholangiocarcinoma QBC939 cells,suggesting that solamargine may be one of the effective drugs to treat cholangiocarcinoma.