Study On Protective Effect And Mechanism Of Polysaccharide From Dog Liver On Liver Fibrosis Induced By Dimethylnitrosamine

Objective: To investigate the protective effect of polysaccharide from dog liver(DCP)on dimethylnitrosamine(DMN)-induced liver fibrosis(HF)in rats and the effects on MAPK and Fas/FasL signaling pathways.Methods: in vivo experiment,SD rats were intraperitoneally injected with DMN to establish HF model.The modeling rats were randomly divided into 5 groups,including DMN group,DMN+ colchicine group,DMN+DCP(100,200,300 mg/kg)group,and control group,with 10 rats in each group.After 6 weeks,the serum of rats was collected to detect the biochemical indexes of liver function.Liver tissue was collected for HE,Masson staining and immunohistochemistry.Rt-pcr was used to detect the expression of inflammatory factors.Western blot was used to detect the expression of MAPK pathway related proteins,observe the preventive effect of DCP on HF,and explore its intervention effect on MAPK pathway.DCP was used to treat TGF-β 1-stimulated HSC-T6 cells in vitro.The survival rate of HSC-T6 cells under different concentrations of DCP was detected by MTT assay,the expression of α-SMA in HSC-T6 cells was detected by immunofluorescence assay,the apoptosis of HSC-T6 cells was observed by Hoechst 33258 fluorescence staining method,and the expressions of Fas,FasL and inflammatory factors were detected by RT-PCR.Western blot was used to detect the expression of MAPK and Fas/FasL pathway related proteins.Results: In vivo experiment,liver function of rats in DMN group was severely impaired,with obvious liver cell injury,inflammatory cell infiltration and increased interstitial fibrosis,suggesting that HF model was successfully prepared.Compared with DMN group,after DCP treatment,the activities of ALT,AST,ALP and TBIL in serum of rats were significantly decreased,and the HA,LN,IVC and PCIII in liver fibrosis were also significantly decreased.The results of section showed that the degree of HF lesion,collagen deposition and the expression of inflammatory factors were also significantly decreased.ELISA results showed that the expression levels of TNF-α,IL-6 and IL-1β decreased,and RT-PCR analysis showed that the m RNA levels of TNF-α,IL-6 and IL-1βdecreased.Western blot showed that the expression of P-JNK,P-P38 and P-ERK proteins in rat liver tissue decreased significantly.In vitro experiments,the α-SMA content of HSC-T6 cells stimulated by TGF-β1was significantly increased compared with that of cells not stimulated,suggesting that cell HF model was successfully prepared.DCP could significantly inhibit the proliferation of HSC-T6 cells stimulated by TGF-β1 and induce apoptosis,significantly reduce the expression of inflammatory factors,down-regulate the expression of MAPK pathway protein to alleviate cell inflammation,and up-regulate the expression of Fas/FasL pathway protein to promote cell apoptosis.Conclusion: DCP can interfere with MAPK signaling pathway to inhibit inflammatory response and relieve HF process in rats,and DCP can interfere with MAPK and Fas/FasL signaling pathway to inhibit inflammatory response of HSC-T6 cells and promote apoptosis of HSC-T6 cells.