Mechanism Of Gly-tRF In Non-alcoholic Steatohepatitis In C57BL/6J Mice

Background and purposeNonalcoholic steatohepatitis(non-alcoholicsteatohepatitis,NASH)refers to inflammatory fatty liver disease with the exception of drinking factors or other definite liver injury factors.It is a common clinical disease.NASH has the characteristics of hepatocyte swelling,steatosis and inflammatory injury,can be accompanied by liver fibrosis and liver cirrhosis,and can develop into liver cancer and even liver failure.With the improvement of living standard and the change of life style,the incidence of NASH is getting higher and higher.There is no obvious clinical manifestation in the early stage of NASH.Due to the invasiveness of the diagnostic method(pathological biopsy),it is difficult for most patients to accept that the detection of NASH is limited.When NASH is diagnosed,it has often progressed to liver cirrhosis,liver cancer and other advanced liver diseases.At present,there is no drug approved by FDA to treat NASH,so it is particularly important to find an effective way to prevent and treat NASH.Glycine transport RNA derivative fragment(glycinetransfer(t)RNA-derivedfragments,Gly-t RF)is a non-coding small RNA.It is a fragment produced by specific cleavage of t RNA under pathological stress.Some studies have shown that t RF can cause disease by inhibiting protein synthesis or competing with m RNA for ribosome binding sites.We found that the expression of Gly-t RF was elevated in the mouse model of nonalcoholic steatohepatitis induced by western diet,but the role and mechanism of Gly-t RF in the progression of nonalcoholic steatohepatitis in mice is not clear.The purpose of this study is to explore the function and molecular mechanism of Gly-t RF in NASH,and to find potential therapeutic targets for NASH.MethodsFirst,C57BL/6 mice were fed with western diet for 12 weeks to establish a(NASH)model of nonalcoholic steatohepatitis.The fatty degeneration and lipid deposition of hepatocytes were analyzed by H&E and Oil Red O staining,and the expression of AST and ALT in serum and TG and CHO in serum and liver tissue of mice were detected;The expression of Gly-t RF in liver tissue of mice was detected by quantitative RT-PCR.Then the lipid deposition model of AML-12 cells in vitro was established by stimulation with oleic acid and palmitic acid,and the expression of Gly-t RF was detected.In the hepatocyte lipid stasis model in vitro,AML-12 cells were treated with Gly-t RF antisense inhibitor and its control substance.The pathological changes of AML-12 cells were observed by oil red O staining,the content of TG in cells was measured,and the expression of genes related to lipid synthesis was detected by quantitative RT-PCR.In the mouse model of nonalcoholic steatohepatitis,the mice were treated with chemically modified Gly-t RF antisense inhibitor and its control substance,and the related indexes were detected,such as H&E and Oil Red O staining,serum liver function,the content of TG and CHO in liver tissue,and the expression of lipid synthesis related genes and inflammatory factors at m RNA and protein levels,in order to explore the role of Gly-t RF in the progression of nonalcoholic steatohepatitis in mice.Finally,the liver tissues of mice treated with antisense Gly-t RF inhibitors were performed gene profile sequencing,the differentially expressed genes were analyzed,and the possible pathways were predicted by GO analysis and verified,so as to explore the role and molecular mechanism of Gly-t RF in the progression of nonalcoholic steatohepatitis in mice.Results1.Expression of Gly-t RF in liver tissue of mice with nonalcoholic steatohepatitis:(1)Compared with the control group,the expression of Gly-t RF was increased in western diet-induced mouse NASH model and hepatocyte lipid stasis model in vitro.(2)In the model of lipid accumulation of hepatocytes in vitro,compared with the control group,the lipid deposition of AML-12 cells was reduced by treatment with inhibitor,the TG decreased and the expression of genes related to lipid synthesis was down-regulated compared with the control group.2.The role of Gly-t RF in nonalcoholic steatohepatitis in mice:(1)In the mouse NASH model,compared with the normal mice,the results of Hype and Oil Red O staining in the liver tissue of mice treated with Gly-t RF inhibitor showed that the number of lipid droplets in the liver decreased and the steatosis of hepatocytes alleviated.(2)the levels of ALT in serum and TG and CHO in liver tissue decreased significantly.(3)the key enzymes related to lipid synthesis decreased significantly in m RNA and protein levels.(4)The expression of inflammatory factors in m RNA level was also significantly decreased.3.Molecular mechanism of inhibiting Gly-t RF in reducing nonalcoholic steatohepatitis in mice:(1)After high-throughput sequencing of the liver tissues of the NASH model of mice with Gly-t RF inhibitor and control group,it was found that most of the differentially expressed genes related to lipid metabolism were concentrated in the PPAR-γ signal pathway,while the differentially expressed genes related to inflammation were mostly concentrated in the Inflammatory mediator regulation of TRP channels pathway.(2)Some key genes in the pathway were verified,and it was found that the expression of PPAR-γ,CYP4a10,CYP2E1,ACAA1 b and IL-1β decreased.ConclusionThe expression of Gly-t RF in liver tissue of NASH mice was increased,and inhibition of Gly-t RF alleviated steatosis and inflammation of liver tissue by regulating PPAR-γ and Inflammatory mediator regulation of TRP channels signal pathways.Therefore,Gly-t RF inhibitor is expected to become a new target for the treatment of NASH.