Preliminary Investigation On The Regulation Of Liver Fibrosis By Blood Ammonia Through Mediating Autophagy In Hepatic Sinusoidal Endothelial Cells

Background and Objective:Background:The etiology of liver fibrosis is complex,which is a common result of chronic liver disease,so it is of great significance to prevent or delay the progress of liver fibrosis.Sinusoidal endothelial cells（Sinusoidal endothelial cells,SECs）are highly specific cells in the liver,which can mediate substance exchange and maintain the homeostasis of the liver immune environment under physiological conditions.Under pathological conditions,SECs appears capillarization,which promotes liver injury and inflammation.Autophagy is a process of energy regulation in eukaryotes.Proper autophagy can promote cell survival,excessive autophagy can lead to cell death,and autophagy is closely related to liver fibrosis.Blood ammonia is the product of amino acid metabolism in organisms.if blood ammonia can not be effectively synthesized and excreted from urea during liver injury,it may lead to hyperammonemia.Studies have shown that high blood ammonia can activate autophagy and lead to a variety of diseases.Objective:To clarify the role of blood ammonia in the process of liver fibrosis,to elucidate its effect on the phenotype of SECs,and to explore the related mechanisms of action.To evaluate therapeutic strategies to control the liver fibrosis process by targeting blood ammonia accumulation and its mediating key molecules.Materials and Methods:Firstly,SECs were treated with different concentrations of NHCL₄ solution,and the effect of the drug on cell proliferation rate was determined by CCK8 method.Next,cellular immunofluorescence and WB were employed to evaluate the phenotypic alterations of hepatic sinusoidal cells,while RT-q PCR and WB revealed the expression of autophagy-related proteins and ERK/JNK/p38MAPKs signaling pathway-related proteins,and then validated by using autophagy inhibitor 3MA or autophagy agonist rapamycin,respectively.Finally,SECs were co-cultured with Hepatic stellate cell（Hepatic stellate cell,HSCs）,and theα-SMA expression and autophagy level of HSCs were detected.Result:1.In vitro experiments,direct exposure of SECs to ammonia stimulation resulted in decreased cell survival of SECs compared to the control group,suggesting that the cell proliferation rate of SECs decreased and showed a dose-dependent.2.RT-q PCR and WB revealed that,when exposed to ammonia,the m RNA and protein expression of surface markers v WF and CD31 was significantly higher than in the control group.Moreover,the m RNA and protein expression of autophagy-related genes LC3,Cav-1,and Atg7 were also significantly higher in the ammonia-stimulated SECs than in the control group;however,these proteins were decreased after concurrent stimulation with the autophagy inhibitor 3MA.Furthermore,the expression levels of these proteins were also significantly higher in the ammonia-stimulated SECs after concurrent stimulation with rapamycin compared to the control group.3.Compared to the control group,WB detection revealed a marked upregulation of proteins in ammonia-stimulated SECs associated with the ERK/JNK/p38MAPKs signaling pathway,such as ERK,p-ERK,JNK,p-JNK,p38,and p-p38.In contrast,the expression levels of these proteins were downregulated in ammonia-stimulated SECs after concurrent intervention with the autophagy inhibitor 3MA compared with the ammonia-stimulated group alone;the expression levels of these proteins were also upregulated in ammonia-stimulated SECs after concurrent rapamycin stimulation compared with the ammonia-stimulated group alone.4.When SECs were co-cultured with HSCs by transwell and detected by WB,the expression levels ofα-SMA,Cav-1,LC3 and Atg7 proteins in HSCs were significantly higher than those in the control group.Conclusion:1.In vitro studies have shown that direct stimulation with ammonia inhibits the cell proliferation rate of SECs and exhibits dose-dependent.2.In vitro studies have shown that ammonia stimulation leads to phenotypic changes in SECs that activate autophagy,and that exogenous activation of autophagy or inhibition of autophagy leads to changes in the level of autophagy in SECs,a process regulated by the ERK/JNK/p38MAPKs signaling pathway.3.Capillarized SECs were co-cultured with HSCs,which were activated with increased levels of autophagy.