Study On The Mechanism Of TFF3 Ameliorating Liver Injury Of NASH And The Regulation Of Jiangzhi Granules

Objectives: 1.To study the role of TFF3 on hepatic inflammation,apoptosis and tissue repair in NASH and the related regulatory signaling pathway.2.Based on the role of TFF3,explore the mechanism of Jiangzhi Granules(JZG)in improving liver injury induced by high-fat diet in NASH mice.Methods: 1.In vitro study of the role of TFF3 in NASH and signaling pathways:Hep G2 and Huh7 cells with TFF3 endogenous overexpression were constructed through lentivirus transfection.The effects of endogenous TFF3 on palmitic acid(PA)-induced cell injury,apoptosis and expression of inflammatory cytokines were investigated,and the effects on cell proliferation and repair ability were examined.Recombinant human TFF3(rh TFF3)protein was implicated on Hep G2,Huh7 and mouse normal liver cell AML12 induced by PA to test effect of exogenous TFF3 on injury,inflammation and repair.Western blot was used to detect the activation level of EGFR/AKT to explore the signaling pathway of TFF3.In addition,rh TFF3 was used to intervene in PA and lipopolysaccharide(LPS)-induced macrophage RAW264.7 to study the regulation of TFF3 on macrophage inflammatory response.2.In vivo study of the role of TFF3 in NASH and the intervention mechanism of Jiangzhi Granules:C57BL/6J mice were fed with high fat diet(HFD)to establish NASH model and the control mice were given normal feed.The dynamic change of liver histopathology and TFF3 expression were observed.Since the 18 th week,JZG was intragastric administrated to the NASH mice.At the end of 22 nd week,mice were sacrificed.Serum transaminase levels were measured in each group and pathological changes of liver tissue were observed.The levels of TFF3 in liver tissue and the expression levels of apoptosis and inflammation-related genes were detected by RT-PCR and Western Blot.Results: 1.In vitro study of the role of TFF3 in NASH and signaling pathways: The in vitro experiments demonstrated the difference of biological behavior of cells with TFF3 overexpression compared to scramble after 24 h-incubation with 0.2m M PA: higher cell viability,less ratio of TUNEL positive cells,decreased level of cleaved PARP and Bim protein expression,increased level of anti-apoptotic p-Bcl2 protein,and reduced inflammatory factors IL-1? and IL-6.In addition,cell proliferation was observed increased significantly in TFF3 overexpression cells.In consistent with these results,Hep G2,Huh7 and AML12 cells treated with rh TFF3 displayed higher cell viability,decreased level of cleaved PARP and Bim protein expression,increased level of anti-apoptotic p-Bcl2,p-EGFR and p-AKT protein,and reduced level of p-NF-?B protein and inflammatory factors IL-1? and IL-6 when induced by PA.The proliferation ability of cells also increased significantly under the action of rh TFF3.In addition,RAW264.7 cells treated with rh TFF3 reduced level of p-NF-?B protein and inflammatory factors IL-1? and IL-6 when induced by PA or LPS.2.In vivo study of the role of TFF3 in NASH and the intervention mechanism of Jiangzhi Granules: At the end of 8 week,12 week,16 week,and 22 week of high-fat feeding,the degree of hepatic steatosis in the liver tissue of mice gradually increased.Focal inflammatory cell infiltration was observed since the 16 th week,and hepatocyte ballooning could be found at the 22 nd week,which indicates the development of NASH.The expression of TFF3 showed dynamic changes in the liver of model mice: higher at the 8th and 12 th week,then lower at 16 th and 22 nd week,versus control.At the 22 nd week,compared with control,NASH mice had higher ALT level,lower hepatic expression of TFF3 mRNA and protein,and increased expression of cleaved Caspase3,Bim,IL-1?,IL-6,TNF-? and p-NF-?B in liver tissues and decreased expression of p-EGFR and p-AKT.When compared with the model group,mice with JZG treatment for 4 weeks showed improved hepatosteatosis,imflammation and ballooning in liver tissues,reduced ALT,significantly increased the expression of TFF3 mRNA and protein,and p-EGFR and p-AKT.JZG also ameliorated expression of cleaved Caspase3,Bim,IL-1?,TNF-? and p-NF-?B in liver tissues.Conclusions: 1.TFF3 has the effect of alleviating liver damage induced by fatty acid and endotoxin in NASH,and can promote liver tissue repair.The signaling pathway include promoting AKT activation,and suppressing inflammatory response by inhibiting NF-?B activation.2.Jiangzhi Granules can promote the activation of EGFR/AKT and inhibit the activation of NF-?B through up-regulating the expression of TFF3 in liver tissue of NASH mice,and then reducing liver inflammation and apoptosis,which is part of the mechanism of improving liver injury in NASH mice.