Melatonin Alleviates The Mechanism Of Nonalcoholic Fatty Liver Disease By Regulating The Expression Of Ip3r In MAM

Non-alcoholic fatty liver disease(NAFLD)is a disease of lipid accumulation in hepatocytes that is independent of alcohol induction.Simple fatty liver at the beginning can gradually develop into more serious non-alcoholic steatohepatitis,fibrosis and even hepatocellular carcinoma.Mitochondria-associated endoplasmic reticulum membrane(MAM)is a protein complex located between mitochondria and endoplasmic reticulum,and plays an important role in the normal function of mitochondria and endoplasmic reticulum.Among them,inositol-1,4,5-triphosphate receptor(IP3R)protein located on MAM is the main Ca²⁺transport protein and also plays an important role in maintaining cell homeostasis.In addition,melatonin(Mel)is the main neuroendocrine hormone secreted by the pineal gland,which has biological functions such as antioxidant,anti-diabetes,anti-obesity and cardiovascular protection.At present,Mel has been studied in the treatment of NAFLD,but whether Mel can treat NAFLD by regulating the expression of IP3R in MAM has not been studied.In this study,oleic acid(OA)and palmitic acid(PA)were used to induce AML12cells to establish lipid accumulation models in vitro.C57BL/6 mice were induced by Tyloxapol(Ty)and high fat diet(HFD)to establish NAFLD models in vivo.We analyzed the lipid content and lipid metabolism of AML12 cells and mice liver by Triglyceride(TG)kit,oil red O staining,BODIPY staining,Western blot detection of lipid metabolism proteins and fluorescence quantitative detection of lipid metabolic genes.It was found that OA and PA,Ty and HFD under different stimuli caused different lipid accumulation.Further research found that low concentration of OA and PA,low concentration of Ty and 4 weeks of HFD could promote lipid metabolism,while high concentration of OA and PA and 12 weeks HFD could inhibit lipid metabolism.Importantly,through the above experimental methods,we found that Mel could reduce lipid accumulation and promote lipid metabolism.In this study,we next used Western blot,fluorescence quantification and immunohistochemistry to explore the mechanism of Mel promoting autophagy through IP3R.The results showed that low concentrations of OA and PA and 4 weeks HFD could promote the expression of Atg5,Beclin1 and Microtubule-associated protein 1 light chain 3?(Lc3B),and inhibit the expression of b-cell lymphoma-2(Bcl-2)and ubiquitin-binding protein(P62).However,high concentrations of OA and PA and 12 weeks HFD inhibited the expression of Atg5 and promoted the expression of Beclin1,Lc3B,Bcl-2 and P62.What's more,Mel treatment could significantly increase the expressions of Atg5,Beclin1 and Lc3B and inhibite the expressions of Bcl-2 and P62.In addition,low concentrations of OA and PA and 4 weeks HFD could appropriately activate IP3R pathway and intracellular Ca²⁺content,while high concentrations of OA and PA and 12 weeks HFD could significantly activate IP3R pathway and increase intracellular Ca²⁺content,increase the contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and Reactive oxygen species(ROS)in serum and reduce the content of glutathione(GSH)in liver.Through the study of Mel,it was found that Mel could reduce the increase of ALT,AST and ROS in serum,Ca²⁺and IP3R in liver of mice induced by high concentrations of OA and PA and 12 weeks HFD.Through further studies,we found that the reduction of IP3R could inhibit the activation of IP3R pathway and intracellular Ca²⁺content by low concentrations of OA and PA.In addition,in the case of IP3R reduction,low concentrations of OA and PA could reduce the expression of Beclin1,which might reduce the production of Bcl-2 and Beclin1 complex,but increase the expression of Lc3B and P62.Our findings also found that low concentrations of OA and PA could inhibit the activation of autophagy by mammalian target of rapamycin/adenosine5'-monophosphate-activated protein kinase(m TOR/AMPK)pathway in the case of reduced IP3R.We also used oil red O staining,BODIPY staining and Western blot to detect the expression of lipid metabolism proteins,and found that when IP3R decreased,low concentrations of OA and PA urther accelerated lipid accumulation and lipid metabolism disorder.Similarly,si RNA-IP3R was used to verify the effect of IP3R silencing on Mel treatment of high concentrations of OA and PA,and it was found that IP3R silencing reduced the expression of Bcl-2,Beclin1,Lc3B and P62,and inhibited the activation of autophagy by m TOR/AMPK pathway.Moreover,IP3R silencing decreased the ability of Mel to induce lipid accumulation in the treatment of OA and PA.Next,we studied the effect of the increase or decrease of MAM on Mel regulation of IP3R.And found that low concentrations of OA and PA and high concentrations of OA and PA and 12 weeks HFD could slightly and severely increase MAM content,respectively.In addition,we found that Mel could reduce MAM increase induced by high concentrations of OA and PA and 12 weeks HFD.In order to further study whether IP3R is on MAM,it was found by Western blot and laser confocal experiments that IP3R does co-localize with MAM.To investigate whether the increase or decrease of MAM will affect the function of IP3R,we used si RNA-MFN2,si RNA-PACS-2 to decrease MAM and used linker to increase MAM.It was found that the increase or decrease of MAM could affect the regulation of low concentrations of OA and PA on IP3R.Importantly,when MAM increases or decreases,it will also affect the regulation of IP3R by Mel.In conclusion,Mel can alleviate lipid accumulation induced by NAFLD model.Mel alleviates NAFLD by reducing the increase of IP3R and regulating m TOR/AMPK pathway to activate autophagy.In OA and PA-induced AML12 cells,the effect of Mel on IP3R is associated with the increase or decrease of MAM.