The Inhibitory Effect With Its Mechanism Of Cryptotanshinone To The Hepa1-6 Cells And Subcutaneously Translanted Tumor Of Hepa1-6 In Mice

Objective To study the inhibitory effect of cryptotanshinone on liver cancer Hepa1-6 cells,establish Hepa1-6 cell subcutaneous xenograft model in mice,and use intraperitoneal injection to detect the inhibitory effect of cryptotanshinone on liver cancer xenografts.In vitro,Hepa1-6 cells were cultured,and the effects of cryptotanshinone on the proliferation and apoptosis of the cells were detected by flow cytometry,Western blotting and RT-PCR.Further explore the related mechanism of cryptotanshinone’s anti-hepatoma effect.Methods1.CCK-8 method was used to detect the effect of cryptotanshinone on the proliferation and viability of Hepa1-6 cells;flow cytometry was used to analyze the effect of cryptotanshinone on the apoptosis and cycle of Hepa1-6 cells;western blot was used to detect the activation of cryptotanshinone on Hepa1-6 cells Of the expression of polyadenylated diphosphate ribose polymerase（cleaved-PARP）,Cyclin D1,P21 and P53.RT-PCR was used to detect the expression of apoptosis-related genes P53,P21 and Caspase9.2.Hepa1-6 cell lines were cultured in a 5%CO₂ incubator at 37℃.Eighteen C57BL/6 mice were subcutaneously inoculated with Hepa1-6 tumor cells subcutaneously on the right back,and a mouse liver cancer subcutaneous xenograft model was established and randomly divided into 3 groups of 6 mice each.They were model group,cryptotanshinone 40mg/kg group and cryptotanshinone 20mg/kg group.It was started on the first day after modeling,and was administered once every other day.The administration method was intraperitoneal injection,continuous administration for 2 weeks,and weight was measured every 3 days.After 2 weeks,the mice were sacrificed to remove tumor tissue.The tumor tissue was weighed and the tumor inhibition rate was calculated.Western blot was used to detect the expression of Cleaved PARP,PARP,Bcl-2 and Caspase9 protein;The apoptosis,proliferation and expression of Bcl-2 gene protein in tumor tissues were observed by immunohistochemistry.Results1.Effect of cryptotanshinone on Hepa1-6 cell proliferation and apoptosis in vitro:Compared with the control group,after 24h,48h,and 72h administration,cryptotanshinone significantly reduced the proliferation viability of Hepa1-6 cells in a dose-and time-dependent manner.The apoptotic rate increased with the increase of cryptotanshinone concentration,and the cell cycle was arrested in the G1 phase;it could significantly inhibit the expression of cyclin D1 and significantly promote the expression of apoptotic proteins cleaved-PARP,p21 and p53.2.Effect of cryptotanshinone on subcutaneous xenografts in mice:On the seventh day of administration,it was observed that the mice in the model group started to lose weight compared with the administration group,the hair color was dim,the degree of tumor growth on the right back was higher than that in the administration group,and the mobility sensitivity decreased until the end of the experiment.The average body weight gradually decreased.The average weight of mice in the drug group was significantly increased compared with that before administration;the difference between the model group,the cryptotanshinone 40mg/kg group,and the cryptotanshinone 20mg/kg group was statistically significant（p<0.05）.They were 55.83%and 27.91%.The results of Western blotting showed that compared with the model group,cryptotanshinone significantly increased the apoptosis-related proteins Cleaved PARP,PARP,and Caspase 9,and down-regulated the anti-apoptosis related protein Bcl-2.HE staining showed that the tanshinone40mg/kg administration group could significantly induce tissue necrosis;immunohistochemical experiments showed that TUNEL-positive cells increased significantly,the tanshinone 40mg/kg group could induce Hepa1-6 cell apoptosis;Ki67-positive cells significantly Reduced,the cryptotanshinone 40mg/kg group can significantly inhibit Hepa1-6 cell proliferation;the number of Bcl-2 positive cells is significantly less than that of the model group,and the cryptotanshinone 40mg/kg group can down-regulate the apoptosis gene Bcl-2 expression.ConclusionCryptotanshinone can inhibit the growth of Hepa1-6 cells in subcutaneously transplanted tumors in mice.Cryptotanshinone reduces the proliferation activity of Hepa1-6 cells and induces apoptosis by disrupting the cell cycle.The possible mechanism of the apoptosis pathway is closely related to Bcl-2、P53、P21 and Caspase9,Bcl-2 gene Inhibiting cell apoptosis,three other genes promote cell apoptosis:in vivo experiments have shown that cryptotanshinone can inhibit tumor growth.