Study On The Transformation Of HSC-T6 Cells Into Fibroblasts Induced By Oleanolic Acid

Objective: Investigation of the mechanism of hepatic fibrosis caused by oleanolic acid in HSC-T6 cells.Methods: 1.The effects of OA on HSC-T6 cells.After selection by MTT method,Control group,low dose group(10 μM),middle dose group(20 μM)and high dose group(40 μM)were seted and α-SMA,SHH,TGF-β1,Smad2 and intranuclear Nrf2 and Gli2 protein expression were determined by WB method and collagen content was detected by ELISA method in cells after incubated with oleanolic acid for 24 h.2.Transfection and overexpression of Nrf2,silence of HH pathway and TGF pathway.(1)This Paper disposes HSC-T6 cell by adopting siRNA method and detects Nrf2 downstream HO-1,NQO1,GCLC,GCLM mRNA expression by RT-PCR method and detects intranuclear Nrf2 protein expression by WB method;(2)after disposing HSC-T6 cell for 24 h by 5 μM SFN,this Paper detects Nrf2 downstream HO-1,NQO1,GCLC and GCLM mRNA expression and intranuclear Nrf2 protein expression;(3)after disposing HSC-T6 cell with 8 μM Gant-61 for 24 h,this Paper detects intranuclear Gli2 protein expression;(4)after common incubation of 1 μM pirfenidone and HSC-T6 cell for 24 h,this Paper detects horizontal and protein expression of TGF-β1 mRNA.3.The effect of Nrf2 pathway,HH pathway and TGF pathway on liver fibrosis induced by OA.(1)Implements co-incubation by 0,10,20 and 40 μM oleanolic acid and normal si Nrf2 HSC-T6 cell;(2)administration of oleanolic acid in 0,10,20 and 40 μM and at the same time,and combined administration of 5 μM SFN and oleanolic acid in 0,10,20 and 40 μM;(3)administrate 0,10,20 and 40 μM oleanolic acid and at the same time sets parallel group and administrate 8 μM Gant-61 at the same time of administration of oleanolic acid and detect 8 groups of α-SMA protein expression.Detect α-SMA protein expression and collagen I content after 24 h;(4)sets blank group,low dose OA group,high dose OA group,pirfenidone group,pirfenidone + low dose OA group,pirfenidone + middle dose OA group,pirfenidone + high dose OA group.Results: 1.After administration of oleanolic acid for 24 h,α-SMA、collagen Ⅰ content in middle and high dose groups increases obviously,which shows that large dose OA can lead to activation of HSC-T6.Compared with blank group,intranuclear Nrf2 protein expression of middle and high dose groups improves and SHH and Gli2 protein expression improves and TGF-β1 and Smad2 protein expression in high dose group improves.2.PCR results show that Nrf2 downstream HO-1,NQO1,GCLC and GCLM mRNA level decreases obviously after siRNA disposal and WB results show that siRNA method can decrease intranuclear Nrf2 protein expression obviously,which shows that siRNA silence Nrf2 succeeds;after adopting siRNA silence Nrf2 for blank group and low dose group,α-SMA protein expression improves and Collagen I content increases;after high dose group adopts siRNA silence Nrf2,α-SMA protein expression decreases and Collagen I content reduces.3.After administration of SFN,mRNA level of Nrf2 downstream gene increases obviously,intranuclear Nrf2 protein expression increases obviously,which shows that SFN can motivate Nrf2 protein expression;after combined drug therapy of SFN and OA,it is found out that SFN can make HSC-T6 cell α-SMA protein expression decrease and make Collagen I content decrease.4.Pirfenidone can reduce TGF-β1 mRNA level and protein expression obviously,which shows that pirfenidone is valid suppressant of TGF-β1;compared with all OA dose groups,α-SMA protein expression of HSC-T6 cell decreases and Collagen I content reduces after administration of pirfenidone.5.After administration of Gant-61,intranuclear Gli2 protein expression reduces obviously,which shows that Gant-61 can effectively suppress Hedgehog(HH)passage;after combined drug therapy with OA,α-SMA protein expression decreases obviously and Collagen I content reduces.Conclusion: Large dose oleanolic acid can lead to HSC-T6 cell activation and Nrf2 passage plays role of protection in the process of liver fibrosis caused by oleanolic acid and oleanolic acid may lead to liver fibrosis by HH and TGF-β passage.