The Function Of Mitochondrial Antiviral Signal Protein MAVS In Nonalcoholic Fatty Liver Disease

BackgroundAt present,nonalcoholic fatty liver disease（NAFLD）is becoming a very common chronic liver disease.At the same time,it will also lead to a significant increase in the cost of medical care and huge economic losses,as well as reduce the quality of people’s healthy life.As a clinically and biologically heterogeneous disease,NAFLD includes a series of common liver diseases and a wide range of histological features.Its occurrence and development covers simple steatosis,nonalcoholic steatohepatitis,advanced fibrosis,liver cirrhosis,and ultimately liver cancer.It is estimated that at present,25% of the global population has NAFLD,and its prevalence is increasing rapidly.Although NAFLD is closely related to obesity,NAFLD also occurs in normal weight individuals,especially in Asian populations.The pathogenesis of NAFLD involves many factors,including metabolic factors,environmental factors and genetic factors.In recent decades,the research on the pathogenesis of NAFLD has made major breakthroughs,and the targets and drug development for the treatment of NAFLD have been clarified,but there are still many problems that can not be avoided.Mitochondrial antiviral signal protein（MAVS）is the only connector protein of retinoic acid-induced gene I（RIG-I）protein signal pathway in the innate immune system.It is also the first molecule related to innate immune response found in mitochondria.It plays a pivotal role in the whole signal transduction process of transmitting antiviral signal,regulating the expression of interferon gene and stimulating the expression of inflammatory factors.Two recent studies have shown that MAVS is an intermediary between immune response and metabolic response.In addition,some research results showed that the expression of MAVS protein was significantly down regulated in the liver of NAFLD mice.However,how MAVS participates in the pathogenesis of NAFLD remains unclear.Therefore,it is a worthwhile scientific experiment to study the role of MAVS in the progress of NAFLD.MethodIn order to explore the role of MAVS in NAFLD,we first detected the level of MAVS protein in human and mouse liver tissue samples in the control group and NAFLD group,and analyzed the expression of lipids and MAVS by HE staining and IHC staining.At the same time,we isolated various cell types（primary hepatocytes,Kupffer cells and stellate cells）in liver tissue,and established the cellular NAFLD model stimulated by oleic acid（OA）and lipopolysaccharide（LPS）,The expression of MAVS protein in these cells was detected.In order to further study the role of hepatocyte MAVS in the early stage of NAFLD,we constructed hepatocyte specific MAVS knockout mice（MAVS HKO）,and detected a series of physiological and biochemical indexes such as liver weight,liver weight / body weight,white fat weight,white fat / body weight,alt,AST,TG and TC in serum of control mice（Flox）and MAVS HKO mice.At the same time,transcriptome analysis was used to further reveal the changes of related pathways and genes in MAVS HKO mice.In order to determine the direct target of MAVS signal in regulating mitochondrial metabolism,we analyzed MAVS protein complex by LC-MS / MS.Taking the MAVS mutants transfected with GFP and lacking mitochondrial transmembrane domain as the negative control,compared with the negative control,four mitochondrial proteins were found in the cells transfected with MAVS,but not in the negative control.Then,the four proteins were tested by CO IP to determine whether they interact with MAVS.At the same time,under the stimulation of OA,the protein molecules interacting with MAVS were further detected by CO IP experiment to observe whether the interaction between them and MAVS changed.Then,immunofluorescence co localization,IP analysis and GST pulldown of VDAC2 protein with changed interaction were carried out.The results further confirmed the direct interaction between MAVS and VDAC2.In order to determine the interaction region between MAVS and VDAC2,we verified it by computer simulation prediction and co IP experiment.After determining the direct interaction between MAVS and VDAC2,in order to explore whether the inhibitory effect of MAVS on NAFLD is mediated by VDAC2.Therefore,we detected the m RNA levels of MAVS and VDAC2 in mouse primary hepatocytes of mock,ad MAVS,Si VDAC2 and ad MAVS + Si VDAC2 groups.After stimulating the primary hepatocytes of the above four groups with OA,they were stained with oil red O to detect the lipid droplets.And TG,TC,NEFA of the four groups and the m RNA levels of fat related pathway genes were detected.ResultCompared with the control group without steatosis,the level of MAVS protein in liver tissue of NAFLD group was significantly lower;Compared with the liver of the normal diet（NC）group,the liver of NAFLD mice induced by high-fat diet（HFD）always showed significant down-regulation of MAVS protein;At the same time,he staining and IHC staining of human and mouse liver sections confirmed that the expression of MAVS protein was downregulated;At the same time,compared with the primary hepatocytes of the control group,the level of MAVS protein in OA treated primary hepatocytes was down-regulated.In contrast,the level of MAVS in Kupffer cells and stellate cells was not affected by LPS treatment.These results were also confirmed in human hepatic stellate cell line lx2-7.The above results show that there is a strong correlation between NAFLD and MAVS in hepatocytes.Compared with Flox control mice,MAVS-HKO mice had increased liver weight and liver weight/body weight after 12 weeks of HFD feeding,while white fat weight and white fat/body weight had no significant changes.Furthermore,hepatocyte-specific knockout of MAVS aggravated hepatic steatosis and liver injury,manifested by increased hepatic lipid content,increased histological lipid accumulation,and elevated serum ALT and AST levels.Serum TG and TC levels were also elevated in MAVS-HKO mice compared with Flox mice.Transcriptomic analysis further revealed extensive upregulation of pro-adipogenic pathways and genes in MAVS-HKO mice.The above results indicated the specific and main role of hepatocyte MAVS in protecting fatty liver pathology.Compared with the negative control group,four mitochondrial proteins were found by protein mass spectrometry.Among them,VDAC2,pacs2 and atpif1 were confirmed to interact with MAVS by CO IP experiment.At the same time,under OA stimulation,the interaction between MAVS and VDAC2 was specifically down regulated,while the interaction between MAVS and pacs2 or atpif1 did not change,indicating that there was a direct interaction between VDAC2 and MAVS.Finally,the direct interaction between MAVS and VDAC2 was further confirmed by immunofluorescence co localization,IP analysis and GST pulldown.Computer simulation prediction and co IP experimental verification show that MAVS interacts with the N-terminal extension domain of VDAC2.VDAC2 deletion significantly aggravated lipid accumulation in hepatocytes upon OA stimulation.More importantly,the protective effect of MAVS against excessive lipid accumulation in hepatocytes was largely abolished after knockdown of VDAC2.Furthermore,the downregulation of adipogenesis genes caused by overexpression of MAVS disappeared when VDAC2 was knocked out.These results suggest that VDAC2 is required for MAVS to protect against hepatic steatosis.ConclusionMAVS is significantly down regulated in NAFLD.MAVS protein can block lipid accumulation in hepatocytes.At the same time,hepatocyte specific knockout of MAVS can aggravate hepatic steatosis.VDAC2 is the downstream target of MAVS,and VDAC2 is necessary for MAVS to affect hepatic steatosis.Innovation1.Determine that knockout MAVS aggravates hepatocyte steatosis in the occurrence and development of NAFLD.2.It is found that the effect of MAVS on the progression of NAFLD depends on vdav2.3.It is proposed that MAVS VDAC2 axis can be used as an ideal target to inhibit the progress of NAFLD.