Effect And Mechanism Of MANF On Non-alcoholic Fatty Liver Disease

PART ? THE EXPRESSION OF MANF IN NAFLD ANIMAL AND CELL MODELSObjective:To explore the expression changes of MANF in NAFLD animal and cell models.Methods: 8-week-old male WT mice were randomly divided into the normal control chow diet group(HFD 0W,n=3)and the high fat diet group.The high fat group was further divided into high fat diet feeding for 8 weeks(HFD 8W),12 weeks(HFD 12W)and 16 weeks(HFD16W),with each group containing 3 mice.At the end of feeding,liver tissues of each group were collected and were used to detect MANF mRNA and protein expression changes.To construct hepatocellular steatosis model in vitro,HepG2 cells was treated with 0.5 mM FFAs for different time points(24h,48 h,72h),and cells treated with fatty-free Bovine serum albumin(BSA)served as controls.Cells were collected after stimulation with corresponding time points and were used to detect MANF expression.Results: Compared with the normal control chow diet group,the hepatic MANF expression level of mice fed with HFD for 8 weeks was significantly elevated(P<0.05),while the hepatic MANF expression level of mice fed with HFD for 12 weeks and 16 weeks was gradually decreased with the extension of feeding time(P<0.05 or P<0.01).Furthermore,MANF expression was increased in HepG2 cells during the first 24 h of stimulation by FFAs overload(P<0.01 or P<0.001)and then gradually decreased at the 48 h and 72 h time points(P<0.05 or P<0.01).Conclusion: In NAFLD animal and cell models,the MANF expression was increased temporarily in the early stage,and gradually decreased with the extension of stimulation time.This expression changes suggest that the role of MANF on NAFLD should be further explored.PART? MANF IMPROVES THE DEVELOPMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE INDUCED BY HIGH FAT DIETObjective:To investigate the effect of MANF on NAFLD induced by high fat diet.Methods: Foutry six 8-week-old male WT mice were randomly divided into the normal control chow diet group(NCD,n=22)and the high fat diet group(HFD,n=24).After 16 weeks of feeding,the NCD group was further divided into the control NCD group(AAV-GFP/NCD group,n=5),the MANF overexpression NCD group(AAV-MANF/NCD group,n=5),the negative control NCD group(AAV-NC/NCD,n=6)and the MANF knockdown NCD group(AAV-MANF sh RNA/NCD,n=6);the HFD group was divided into the control HFD group(AAV-GFP/HFD group,n=6),the MANF overexpression HFD group(AAV-MANF /HFD group,n=6),the negative control HFD group(AAV-NC/HFD,n=6)and the MANF knockdown HFD group(AAV-MANF sh RNA/HFD,n=6).We performed tail-vein injection of adeno-associated virus to NCD-or-HFD-fed mice to interfere hepatic MANF expression,and the mice in each group were fed for 6 weeks.The body weight was measured every two weeks and the fasting blood glucose was measured every four weeks during feeding.IPGTT and IPITT were performed at the 5 and 6 weeks after tail vein injection.After the tolerance experiment,mice in each group were fed for 1 week and their blood was collected to determine the plasma TG and TC levels.The liver tissues were stained with HE and Oil red O staining,and the hepatic content of TG,TC,ALT,AST and the expression of genes involved in fatty acid metabolism were determined.Results: Hepatic MANF expression was significantly increased after the injection of AAV-MANF(P<0.01),but the expression of MANF in the liver was not down-regulated after the injection of AAV-MANF sh RNA,so only the results of the overexpression group were analyzed in the follow-up.The body weight and fasting blood glucose in the HFD group were significantly increased compared with those in NCD group,MANF overexpression slightly decreased the body weight and fasting blood glucose after HFD feeding,but there was no statistical difference.The area under the curve of IPGTT in AAV-MANF/HFD group was significantly decreased than that in AAV-GFP/HFD group(P<0.05),while there was no difference in NCD group.The results of HE staining and Oil red O staining showed that the mice fed with HFD developed serious hepatic steatosis,and MANF overexpression significantly alleviated the steatosis.In addition,compared with the AAV-GFP/HFD group,the serum TG and TC content and the liver TG level in the AAV-MANF/HFD group was obviously decreased(P<0.05 or P<0.01),but the liver ALT and AST levels did not change.The m RNA level of genes involved in fatty acid uptake(CD36,FABP-1)in the AAV-MANF/HFD group was decreased while the m RNA expression of genes related to fatty acid ?-oxidation(PPAR??CPT-1?)was increased than that in the AAV-GFP/HFD group(P<0.01 or P<0.001).Conclusion: MANF overexpression could inhibit lipogenesis,reduce serum TG and TC content and ameliorate hepatic steatosis induced by high fat diet,which plays a potential protective role in the occurrence and development of NAFLD.PART ? MANF SUPPRESSES HEPATOCELLULAR STEATOSIS INDUCED BY FREE FATTY ACIDS AND ITS POTENTIAL MECHANISMObjective:To investigate the role and mechanism of MANF in hepatocellular steatosis induced by FFAs.Methods: Hep G2 cells was treated with 0.5m M FFAs to induce hepatocellular steatosis.MANF overexpression(Lv-MANF)and MANF down-regulated(Lv-sh MANF)stably transfected cell line were constructed to interfere the expression of MANF in vitro,and to explore the role of MANF in FFAs-induced hepatocellular steatosis.Transcriptome sequencing was used to explore potential mechanism.The intracellular TG and TC levels and genes related to fatty acid metabolism in each group were detected.Results: Oil red O staining showed that the Hep G2 cell steatosis model was successfully constructed.The results of transcriptome sequencing analysis showed that MANF gene was closely related to cholesterol metabolism,gluconeogenesis,non-alcoholic fatty liver disease and other metabolic processes.MANF overexpression alleviated hepatocellular steatosis,reduced intracellular TG and TC content and significantly down-regulated the m RNA level of genes related to fatty-acid and cholesterol synthesis(FAS,ACC?,SREBP-1C,HMGCR)and fatty-acid uptake(CD36,FABP-1)with the treatment of FFAs(P<0.05 or P<0.01).Meanwhile,MANF deficiency up-regulated the m RNA level of above genes and increased intracellular TG level(P<0.05 or P<0.01).Compared with the Lv-GFP+FFAs group,MANF overexpression down-regulated the m RNA expression level of IRE1?,XBP1 and XBP1 s while MANF deficiency upregulated the expression level of IRE1?,XBP1 and XBP1s(P<0.05 or P<0.01).Conclusion: Under the stimulation of FFAs in vitro,MANF could alleviate hepatocellular steatosis and inhibit the lipid accumulation and lipogenesis,and the effect of MANF may depend on the IRE1?/XBP1 signaling pathway.