Mechanism Of Palmitic Acid Promoting IP3R1 Protein Stability-induced Mitochondrial Dysfunction In Hepatocytes

Objective:Non-alcoholic fatty liver disease?NAFLD?is a liver manifestation of metabolic syndrome.It is a chronic metabolic disease characterized by steatosis and lipid deposition of liver cells.The prevalence of NAFLD in the general population around the world is approximately 25.24%.The clinical pathological scope of NAFLD includes non-alcoholic fatty liver?NAFL?or simple fatty liver,non-alcoholic steatohepatitis?NASH?,liver fibrosis,and cirrhosis.Palmitic acid?PA?-induced apoptosis of liver cells is essential in the progression of non-alcoholic fatty liver disease?NAFLD?.Inositol1,4,5-triphosphate type 1 receptor?IP3R1?is an intracellular Ca²⁺release channel that is involved in PA-induced hepatocyte apoptosis.Although the expression of IP3R1 protein in NAFLD patients and PA-treated hepatocytes was significantly increased,it is unclear how PA promotes IP3R1 expression.Therefore,this study intends to explore the cause of the increased expression of IP3R1 protein after PA treatment of hepatocytes,and to preliminary explore its molecular mechanism.Methods:Part 1:Hepatocytes treated with different concentrations of PA were tested for mitochondrial membrane potential,apoptotic protein cleaved-caspase3,and flow cytometry to determine the optimal concentration to induce mitochondrial damage and apoptosis in hepatocytes.MitoSOX staining was used to detect mitochondrial superoxide accumulation at the optimal PA concentration.Fluo-4 and mito-tracker fluorescence staining were used to detect mitochondrial calcium overload.Intracellular calcium was inhibited by toxic carotene in ER.Fluo-4 was used to label intracellular Ca²⁺.A microplate reader was used to detect the rate and number of calcium ions released from ER.In addition,hepatocytes were treated with the IP3R inhibitor 2APB.Fluo-4and mito-tracker fluorescent staining were used to observe the effect of2APB on mitochondrial calcium overload;JC-1 staining was used to observe the effect of 2APB on mitochondrial membrane potential;MitoSOX staining was used to observe the effect of 2APB on intracellular ultrafiltration Effects of oxide accumulation.Part 2:After PA treatment of hepatocytes,Western blot was used to detect the expression of IP3R1 protein;real-time fluorescence quantitative PCR?RT-PCR?was used to detect the mRNA level of IP3R1;treated with CHX?CHX group?,CHX and PA?CHX+PA group?Hepatocytes were detected for IP3R1 protein degradation by Western blot.Part 3:After PA treatment of hepatocytes,Western blot was used to detect the phosphorylation of Tyr353 site of IP3R1 protein;Western blot was used to detect the expression level of fyn and the phosphorylation level of src.After using the src kinase family inhibitor su6656,Western blot was used to detect the effect of su6656 on the phosphorylation of Tyr353 of IP3R1 protein;combined use of CHX to observe the effect of su6656 on the stability of IP3R1 protein;Fluo-4 and mito-tracker fluorescence staining to observe The effect of PA on mitochondrial calcium overload;JC-1 staining to observe the effect of su6656 on the reduction of mitochondrial membrane potential caused by PA;MitoSOX staining to observe the effect of su6656 on intracellular superoxide accumulation caused by PA.Results:The first part:JC-1 staining results showed that the mitochondrial membrane potential of hepatocytes and gradually decreased with increasing PA concentration;Western blot and flow cytometry showed that apoptosis of hepatocytes with PA concentration Increased and increased;these results suggest that the effect produced by hepatocytes is most significant under 0.8mM PA treatment.Compared with the Ctrl group,MitoSOX staining results showed that the red fluorescence of the PA-treated group was significantly enhanced?P<0.001?,the yellow fluorescence produced by the co-localization of Ca²⁺and mitochondria was also significantly enhanced?P<0.001?,and the peak time of calcium release from ER Obviously shortened,the area under the curve increased significantly?P<0.01?.After 2APB treatment,compared with the PA treatment group,the yellow fluorescence produced by the colocalization of Ca²⁺and mitochondria in the PA+2APB group was significantly reduced?P<0.001?,the mitochondrial membrane potential was significantly increased?P<0.001?,and the red fluorescence of MitoSOX staining was significantly Reduced?P<0.001?.Part 2:Compared with the Ctrl group,Western blot results showed that PA treatment significantly increased the expression of IP3R1 protein?P<0.01?.RT-PCR results showed that compared with the control group,PA treatment did not increase the mRNA level of IP3R1 protein,and the mRNA level of IP3R1 in the PA treatment group was only about 40%of the Ctrl group?P<0.001?.After CHX inhibited the synthesis of IP3R1protein,Western blot results showed that PA significantly slowed the degradation of IP3R1 protein?P<0.05?.Part 3:Western blot results showed that compared with the control group,PA treatment significantly increased the phosphorylation of the Tyr353 site of IP3R1 protein?P<0.01?,and increased the phosphorylation of src?P<0.05?,while fyn protein The expression has not changed significantly.After using su6656,the co-treatment results of CHX showed that su6656 reduced the stability of IP3R1 protein caused by PA?P<0.05?;and Western blot results showed that the phosphorylation of Tyr353 site of IP3R1 protein was also reduced?P<0.05?).Compared with the PA treatment group,PA+su6656 treatment significantly reduced the colocalization of calcium ions and mitochondria?P<0.001?,increased the mitochondrial membrane potential drop caused by PA treatment?P<0.001?,and reduced the PA treatment Caused by superoxide accumulation and apoptosis?P<0.05?.CONCLUSION:PA promotes Tyr353 phosphorylation of IP3R1 by activating the src pathway,leading to an increase in the stability of IP3R1protein,leading to mitochondrial calcium overload and mitochondrial dysfunction in liver cells.Our results also suggest that inhibition of the src/IP3R1 pathway?such as the use of Su6656 or 2APB?may be a new potential treatment for NAFLD.