Mechanism Exploration Of Time-restricted Feeding Alleviating Nonalcoholic Steatohepatitis By Regulating The Circadian Gene Per2

Aim: Non-alcoholic Steatohepatitis(NASH)is a progressive form of Non-alcoholic fatty liver disease(NAFLD).Despite years of exploration,the mechanism of NASH is not completely clear,and the treatment strategy for NASH is also limited.Time-Restricted Feeding(TRF)was proved to alleviate metabolism-related disease without influencing the calorie intake and the amount of exercise,but the efficacy and mechanism of TRF in the treatment of NASH are not clear.Studies reported that TRF might work by regulating circadian genes.Therefore,the aims of this study are exploring the efficacy and specific mechanism of TRF in alleviating NASH by constructing NASH experimental model.Methods: High fat and high fructose diet(HFHFD)was fed to mice for 16 weeks to induce NASH model.1.The exploration of efficacy and mechanism of TRF in alleviating NASH.1.1、C57BL/6J mice were randomly divided into 4 groups:(1)the group with HFHFD available all the day(FA);(2)the group with HFHFD available in limited time(FT);(3)the group with control diet available all the day(NA);(4)the group with control diet available in limited time(NT).The mice in TRF group were given food from 9 p.m.to 7 a.m.The weight of mice was recorded and the blood glucose levels were detected.The levels of blood lipid,the liver fat concentrations and the levels of serum hepatic enzymes were detected by biochemical kit.The degree of liver injury was evaluated by HE,Oil Red O and Sirius Red staining.RT-q PCR was used to detect the indexes of liver inflammation and fibrosis.1.2、RT-q PCR was adopted to measure the expression of circadian genes in liver;the protein levels of Per2 were detected by Western blotting.1.3、Hepatocyte specific Per2 knockout mice(Per2△hep)and their control mice(Per2fl/fl)were constructed and HFHFD was used to induce NASH model.After collecting samples,the indexes which the same as 1.1 were detected.2.Mechanism exploration of TRF alleviating NASH after inhibiting the expression of Per2.2.1、RNA-Seq was performed in Per2△hep and Per2fl/fl mice to detect the expression of different genes.2.2、In NA、NT、FA、FT groups and Per2△hep 、Per2fl/fl mice,the levels of serum iron,liver iron,liver MDA and GSH were detected by biochemical kit.The expression levels of ferroptosis related genes and iron metabolism related genes were detected by RTq PCR and Western blotting.C11-Bodipy staining was used to detect the levels of liver lipid peroxidation(LPO).The morphological changes of mitochondria in cells were observed by transmission electron microscope.3.Exploring the mechanism of ferroptosis participating in NASH.3.1、C57BL/6J mice were divided into two groups: Liproxstatin-1(Lip-1)injected group(FA+Lip-1)and control group(FA+Veh): Lip-1 or the corresponding control were injected intraperitoneally to mice every day from the 13 th week to the 16 th week of HFHFD feeding.The levels of MDA and GSH in liver,the expression levels of ferroptosis related genes and the level of LPO in the liver were detected.The detective methods were the same as 2.1.3.2、To evaluate the effects of ferroptosis on NASH,FA+Lip-1 and FA+Veh mice were used for the following tests: recording the body weight of mice,measuring the metabolic indexes and the levels of serum hepatic enzymes,and evaluating the degree of liver injury of the mice,the methods are the same as 1.1.3.3、RNA-Seq was performed in FA+Lip-1 and FA+Veh mice to detect the expression of different genes;Western blotting was used to detect the protein levels of PPARα、PPARγ in FA+Lip-1 and FA+Veh mice and the protein levels of PPARα in NA、NT、FA、FT groups and Per2△hep 、Per2fl/fl mice.Results(1)TRF effectively alleviated NASH and inhibited the expression of circadian gene Per2,and NASH was improved after the specific knockout of Per2 in hepatocyte.(2)The results of RNA-Seq of Per2△hep and Per2fl/fl mice showed that Per2 significantly affected arachidonic acid metabolism pathway and glutathione metabolism pathway.Further investigation indicated that hepatocyte specific Per2 knockout significantly inhibited ferroptosis in NASH,which was manifested with the decreasing of LPO levels,the obvious remission of mitochondrial morphological changes and the decreasing of expression levels of ferroptosis related genes.(3)The results from NA,NT,FA and FT groups showed that TRF significantly alleviated ferroptosis in NASH.(4)Lip-1 effectively inhibited the ferroptosis in NASH and improved the metabolic indexes、the hepatic enzymes as well as the liver pathological findings of NASH mice.(5)RNA-Seq showed that ferroptosis inhibitor Lip-1 significantly affected the expression of genes in fatty acid metabolism pathways.The further results of showed that both Lip-1、TRF and Per2 knockout increased the expression of PPARα.Conclusions TRF effectively alleviated NASH by inhibiting the expression of circadian gene Per2,and the inhibition of gene Per2 could inhibit the occurrence of ferroptosis,which further promoted the expression of PPARα,and finally alleviated NASH.