Effect Of Salvianolic Acid B On Apoptosis Of Fibrosis-HCC Cells Based On Cleaved Caspase-9

Aims1.Preparation of Diethylnitrosamine（DEN）-induced hepatic fibrosis-hepatocellular carcinoma model in mice to simulate the development and progression of hepatic fibrosis-hepatocellular carcinoma.2.To observe the effect of salvianolic acid B（SalB）on the pathological changes of diethylnitrosamine（DEN）-induced hepatic fibrosis in mice during the process of liver cancer3.To observe the effect of salvianolic acid B on the cell apoptosis induced by DEN in the process of liver fibrosis.4.To investigate the effect of salvianolic acid B on Cleaved caspase-9 protein expression levels in DEN induced liver tissues of mice.5.To observe the effect of salvianolic acid B on the apoptosis and cycle of liver fibrosis-liver cancer cells（HepG2/HSC-T₆/SMMC-7721/Bel-7402）intervening with TGF-beta1（Transforming growth factor beta1,TGF-β₁）.6.To observe the effect of salvianolic acid B on the expression level of Cleaved caspase-9 protein in liver fibrosis cells)interfered by TGF-β₁.7.To observe the effect of salvianolic acid B on the expression level of Cleaved caspase-9 protein in liver cancer cells（Bel-7402）interfered by TGF-β₁.Methods1.Preparation of DEN-induced hepatic fibrosis-hepatocellular carcinoma model Sterile saline injections in normal mice,the other four group injected 1.0%DEN solution induced liver fibrosis model of liver cancer,according to the body quality injected 10ml·kg^-1（1 time/week）.On the day of modeling,mice in the SalB group were given 15and 30 mg·kg^-1·d^-11 SalB by gavage,those in the positive group were given 0.2 mg·kg^-1·d^-1 colchicine by gavage,and those in the normal control group were given the corresponding solution by gavage,which continued to be given until the end of the 16th week.Mice were sacrificed in batches at 12 and 16 weeks.2.HE staining method was used to detect pathological changes.Hoechst33258fluorescent staining cells were used to observe the apoptosis distribution in the tissues Corresponding to the same part of HE staining,the conventional method was to use Hoechst33258 fluorescent staining kit to avoid light staining.The fibrosis and apoptosis of cancer cells in the tissue were observed under low and high power fluorescence microscopy,respectively.2.In vitro culture of HepG2/HSC-T6/SMMC-7721/Bel-7402 cellsHepG2/HSC-T₆/SMMC-7721/Bel-7402 cell lines were cultured in vitro,the cells were cultured in six-well plates.The experiment was divided into six groups:Control group,TGF-β1 group,drug group(SalB 25,50,100μmol·L^-1+TGF-β₁ 9 pmol·L^-1)group,and drug Control group(SalB 50 mol·L^-1),when the monolayer spreading area of cells in the six-well plate reaches 70%-80%,change to DMEM culture medium without serum,culture for 24 h,prepare salvianolic acid B solution,and add the corresponding concentration of SalB to drug group and drug Control group.The culture was continued for 24 h,and TGF-β₁(9 pmol·L^-1)was added to the TGF-β₁ group and the drug group6 h before the termination of the culture.Routine Annexin v-FITC double staining kit was used to treat cells.Apoptosis rate was measured by Flow cytometry.4.Western blot analysis of Cleaved caspase-9 protein expression in mouse liver fibrosis-liver precancerous lesions tissuesTotal protein was extracted from liver tissues of mice at 12 and 16 weeks by conventional methods.Expression of Cleaved caspase-9 was detected by goat and rabbit2.Western blot analysis of Cleaved caspase-9 protein expression in liver fibrosis-HCC cells.HSC-T₆ was selected from hepatic stellate cells,Bel-7402 was selected from hepatocellular carcinoma cells,and 50μmol·L^-1 was selected from the salvianolic acid B group.The total protein in HSC-T₆/Bel-7402 was extracted by conventional methods.The expression of Cleaved caspase-9 was detected by goat anti-caspase-9 and rabbit anti-gapdh.Results1.Effect of salvianolic acid B on liver histopathological changes in DEN-induced mice at 12 weeksAt 12 weeks,normal liver tissue structure and clear hepatic lobule structure were observed under light microscope.The degree of hepatic fibrosis was obvious in the model group,the hepatic lobules were separated into pseudo lobules,and the nuclei were stained deeply,suggesting that the liver fibrosis period was 12 weeks.The degree of fibrosis and nuclear heteromorphism in the low and high dose groups of salvianolic acid B were all light.2.Effect of salvianolic acid B on liver histopathological changes induced by DEN in mice at 16 weeksAt 16 weeks,the hepatic lobules of the model group were observed to be disordered under light microscope,and hyperplastic hepatocytes with abnormal characteristics of cancer cells were observed,which were manifested as increased cell volume,inconsistent size,and darker nuclear staining.It was suggested that 16 weeks was the precancerous stage.The degree of fibrosis,hepatocyte necrosis and nuclear atypia in the low and high dose groups of salvianolic acid B were all lower than those in the model group.3.Effect of salvianolic acid B on apoptosis in DEN-induced liver fibrosis in miceThe normal group showed uniform blue fluorescence and normal nuclear morphology.Compared with the model group at 12 weeks,the low-dose and high-dose groups of salvianolic acid B had more fragmented bright spots,indicating an increase in apoptotic cells.4.Effect of salvianolic acid B on DEN induced apoptosis in mouse precancerous tissues The normal group showed uniform blue fluorescence and normal nuclear morphology.Compared with the model group at 16 weeks,the low-dose and high-dose groups of salvianolic acid B presented large areas of dense and concentrated fluorescence highlights in the precancerous and fibrotic areas,and the nuclei became smaller and rounded,indicating that there were significantly more apoptotic cells in the lesion areas.5.Effect of salvianolic acid B on Cleaved caspase-9 protein expression in liver tissues of den-induced fibrosis miceThe expression level of Cleaved caspase-9 protein was increased in the low-dose group of salvianolic acid B,suggesting that the expression level of Cleaved caspase-9 was increased when salvianolic acid B promoted cell apoptosis.6.Effect of salvianolic acid B on Cleaved caspase-9 protein expression in DEN-induced liver tissues of mice in precancerous lesionsThe expressionof Cleaved caspase-9 protein was increased in the low-dose and high-dose groups of salvianolic acid B,suggesting that the expression level of Cleaved caspase-9was increased when salvianolic acid B promoted cell apoptosis.7.Effect of salvianolic acid B on hepatic fibrosis-hepatocellular carcinoma（HepG2/HSC-T₆/SMMC-7721/Bel-7402）after TGF-β₁ interventionCompared with the Control group,the apoptosis rate of TGF-β₁ group decreased,suggesting that TGF-β₁ had an intervention effect on the apoptosis of HepG₂/HSC-T₆/SMMC-7721/Bel-7402,and inhibited its apoptosis.Compared with the TGF-β₁ group,the apoptosis rate of the drug group(SalB 25,50,100 mol·L^-1+TGF-β₁ 9 pmol·L^-1)was significantly increased,suggesting that salvianolic acid B can promote the apoptosis of HepG2/HSC-T₆/SMMC-7721/Bel-7402 after TGF-β₁ intervention.8.Effect of salvianolic acid B on hepatic fibrosis-hepatocellular carcinoma（HepG₂/HSC-T₆/SMMC-7721/Bel-7402）cycle after TGF-β₁ interventionCompared with the Control group,the G0/G1phase ratio in the TGF-β₁ group was decreased,suggesting that TGF-β₁ promotes DNA synthesis and thereby promotes mitosis.Compared with TGF-β₁ group,SalB 25/50/100 mol·L^-1+TGF-β₁ 9 pmol·L^-1group had significantly higher proportion of G0/G1 phase,suggesting that SalB could inhibit cell cycle in HepG2/HSC-T₆/SMMC-7721/Bel-7402 cells after TGF-β₁intervention.9.Effect of salvianolic acid B on Cleaved caspase-9 protein expression in HSC-T₆/Bel-7402cells after TGF-β1 interventionCompared with the Control group,the expression level of Cleaved caspase-9 was decreased in the TGF-β1 group,suggesting that TGF-β₁ inhibited the apoptosis of HSC-T6/Bel-7402,reducing the expression level of Cleaved caspase-9.Compared with the TGF-β₁ group,the histone expression level of the drug was significantly increased,suggesting that,with the intervention of TGF-β₁,salvianolic acid B promoted the expression level of Cleaved caspase-9 in the apoptosis of HSC-T₆/Bel-7402 Conclusions.Conclusion1.The DEN-induced hepatic fibrosis-hepatocellular carcinoma model in mice showed fibrosis lesion stage at 12 weeks and precancerous lesion stage at 16 weeks2.Salvianolic acid B promoted DEN induced fibrosis and cell apoptosis in the liver tissue of mice before cancer.3.Salvianolic acid B can up-regulate the expression levels of Cleaved caspase-9 protein in liver fibrosis-liver cancer tissues and cells4.TGF-β₁ inhibits apoptosis,and salvianolic acid B promotes the apoptosis of liver fibrosis-liver cancer cells after TGF-β1 intervention5.When TGF-β₁ down-regulates the expression level of Cleaved caspase-9 protein,salvianolic acid B can up-regulate the expression level of Cleaved caspase-9 protein in TGF-β₁ intervened liver fibrosis-liver cancer cells6.The mechanism of salvianolic acid B inhibiting liver fibrosis and delaying liver cancer progression may be related to the up-regulation of Cleaved caspase-9 protein.