| Objective: In this study,hUCMSCs transfected with HGF were transplanted into rat model of liver fibrosis induced by CCl4 through tail vein to investigate the effect of hUCMSCs on liver fibrosis and NF-κB signaling pathway,and to explore its mechanism of inhibiting liver fibrosis,which will bring a new idea for the treatment of liver fibrosis.Methods: HUCMSCs were obtained by tissue block attachment method,and hUCMSCs transfected with HGF or LV5-NC were retained in the laboratory.The rat model of liver fibrosis was established by intraperitoneal injection of CCl4 combined with high-fat diet for 8 weeks.During this period,diet and activity of rats were observed,weight and other indexes were recorded,too.Then,serum biochemical indexes such as AST,ALT,ALP,ALB and liver pathological changes were detected to verify the success of the model at the end of 8th week.The successfully modeled rats were randomly divided into CCl4 group,hUCMSC group,LV5-NC group and HGF group,and then transplanted with 1× 106 cells/0.5 ml/rat through tail vein,once a week for four weeks.After transplantion,content of PⅢNP and HA in serum were tested by automatic serum biochemical analyzer,the expression of IL-1 and TNF-α in liver and serum were detected by ELISA;the pathological changes of liver were detected by HE and Masson trichrome staining;the positive expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and p65 in liver were detected by IHC;the protein expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ,p65,IκBα and p-IκBα were detected by Wb;and the m RNA expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and p65 were detected by RT-q PCR.Results: 1.HUCMSCs were obtained by tissue block adhesion method.There was no significant difference in hUCMSCs transfected with HGF or LV5-NC compared with hUCMSCs.2.After intraperitoneal injection of CCl4 combined with high-fat diet for 8 weeks,levels of AST,ALT and ALP in serum of the model group were significantly increased(P<0.05)but ALB was significantly decreased(P<0.01)compared with the control group.The results of HE and Masson staining of liver showed that: the structure of liver lobules was complete,the morphology of hepatocytes was normal and radially arranged around the central vein,the structure of hepatic sinuses was normal in the control group.Moreover,there was no inflammatory cell infiltration but a few collagen fibers can be seen in portal area.Nevertheless,the structure of liver lobules was destroyed together with steatosis hepatocytes.Furthermore,inflammatory cell infiltration,massive collagen fibers even pseudolobules were formed in the model group.These results showed that the rat model of liver fibrosis was successfully established in this experiment.3.After cell transplantation,PⅢNP and HA in serum of hUCMSC,LV5-NC and HGF groups were significantly decreased(P<0.05)compared with CCl4 group;HGF group was also significantly decreased(P<0.05)compared with hUCMSC and LV5-NC groups;and there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).ELISA results also showed that the levels of IL-1 and TNF-α in liver and serum of hUCMSC,LV5-NC and HGF groups were significantly decreased(P<0.05)compared with CCl4 group;HGF group was also significantly decreased(P<0.05)compared with hUCMSC and LV5-NC groups;there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).Besides,HE and Masson staining results showed that compared with CCl4 group,necrotic and steatosis hepatocytes,the degree of inflammatory cell infiltration and fibrosis of hUCMSC,LV5-NC and HGF groups were significantly reduced(P<0.05);Compared with hUCMSC and LV5-NC groups,the degree of fibrosis in HGF group was significantly reduced;and there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).Furthermore,the results of positive expression α-SMA,Collagen Ⅰ,Collagen Ⅲ and p65 detected by IHC in hUCMSC,LV5-NC and HGF groups were significantly decreased(P<0.05)compared with CCl4 group;HGF group was also significantly decreased(P<0.05)compared with hUCMSC and LV5-NC groups;there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).Wb results showed that compared with CCl4 group,protein expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ,p65 and p-IκBα were significantly decreased(P<0.05),but protein expression of IκBα was significantly increased(P<0.05)in hUCMSC,LV5-NC and HGF groups;except for IκBα was significantly increased(P<0.05),other proteins in HGF group were significantly decreased(P<0.05)compared with hUCMSC and LV5-NC groups;and there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).Finally,RT-q PCR results showed that,compared with CCl4 group,m RNA expression of Collagen Ⅰ,Collagen Ⅲ and p65 in hUCMSC,LV5-NC group and HGF groups were significantly decreased(P<0.05)compared with CCl4 group;and all of them were also significantly increased(P<0.05)in HGF group compared with hUCMSC and LV5-NC groups;and there was no significant difference between hUCMSC group and LV5-NC group(P>0.05).Conclusion: HGF-transfected hUCMSCs could inhibit the activation of HSCs,reduce collagen deposition,alleviate inflammatory response,and then ameliorate liver fibrosis of rats,which may be related to the inhibition of the activation of NF-κB signaling pathway. |
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