STEAP4 Mediates The Regulatory Mechanism Of NAMPT On Oxidative Stress Caused By Obesityinduced NAFLD

Obesity is a key risk factor for nonalcoholic fatty liver disease.In recent years,with the rapid expansion of obesity in the world,nonalcoholic fatty liver disease（NAFLD）has developed into the most common liver metabolic disease in most countries around the world.However,the pathogenesis and therapeutic targets of NAFLD have not been thoroughly studied despite many years of research.Therefore,genes and proteins associated with metabolic diseases have been the focus of research.The study of the relationship and regulation mechanism between these genes and proteins is of great significance to thoroughly explore the pathogenesis of NAFLD.Nicotinamide phosphate ribose transferase（NAMPT）,as a key enzyme in NAD synthesis,is closely related to metabolism and oxidative stress,but the regulatory mechanism involved in the pathogenesis of NAFLD has not been clearly explained.Six transmembrane antigens for the prostate epithelium 4（STEAP4）has been shown to regulate both inflammation and metabolism.STEAP4 also plays an important role in the liver.In this study,the in vivo NAFLD model was established through inducing obesity in mice with high fat diet（HFD）to explore the role of NAMPT and SIRT1 in the development of NAFLD.Meanwhile,we use the overexpression,inhibitors and FFA treatment in cells to explore the regulatory relationship between NAMPT and STEAP4 and the regulatory mechanism in the development of NAFLD.The main results are as follows:1.The expression of NAMPT in mice was inhibited by intraperitoneal injection of FK866 while mice were fed with high-fat diet.It was found that inhibition of NAMPT promoted obesity and lipid accumulation in liver of mice.Moreover,obesity and FK866also decreased the expression of NRF2,a key antioxidant transcription factor,and its downstream protein.The hepatic level of MDA increased.On the contrary,SOD activity decreased.In summary,Inhibition of NAMPT may accelerate the development of obesity-induced NAFLD.2.Obesity significantly reduced the expression of STEAP4 in liver.Meanwhile,inhibition of NAMPT also significantly reduced the expression of STEAP4.This suggests that NAMPT can regulate the expression of STEAP4 in liver.3.NAFLD model was established by inducing obesity in Sirt1^+/-mice with HFD.The results showed that Sirt1 knockdown significantly promoted obesity,insulin resistance and hepatic injury in mice.Moreover,Sirt1 knockdown also significantly reduced the expression levels of NRF2 and its downstream antioxidant protein,suggesting that Sirt1knockdown aggravated liver oxidative stress.In conclusion,Sirt1 knockdown promotes the development of NAFLD in obese mice.4.Sirt1 knockdown also reduced the expression of STEAP4 in the liver of mice.This suggests that SIRT1 can also regulate the expression of STEAP4 in liver.5.The expression levels of SIRT1 and STEAP4 increased in NAMPT overexpressed Hepa1-6 cells.However,the expression of STEAP4 did not increase when SIRT1 was inhibited by inhibitors in the presence of NAMPT overexpression.These results suggest that NAMPT relies on SIRT1 to regulate STEAP4.After inhibition of SIRT1 activity,the expression of C/EBPβ,a transcription factor regulating STEAP4 transcription,decreased.Moreover,SIRT1 was interacted with C/EBPβ.SIRT1 deacetylated C/EBPβ.SIRT1regulates STEAP4 transcription by deacetylating C/EBPβ.6.Hepa1-6 cells overexpressing STEAP4 were treated with PA.The results showed that overexpression of STEAP4 could reduce lipid accumulation and ROS level in PA-treated Hepa1-6 cells,while maintaining the normal physiological state of mitochondria.However,after NAMPT was inhibited,STEAP4 lost its function.PA can promote the accumulation of NRF2 into the nucleus,but also reduce the total expression of NRF2.Overexpression of STEAP4 can increase the total amount and nuclear expression of NRF2and restore the normal antioxidant capacity of cells.However,when NAMPT was inhibited,STEAP4 could no longer regulate NRF2.In summary,NAMPT regulates the activity of SIRT1 by regulating NAD synthesis,and SIRT1 deacetylatation modifies C/EBPβto promote STEAP4 transcription,thereby increasing NRF2 expression and gathering in the nucleus,regulating the transcription of downstream antioxidant enzymes.Maintain the operation of the cell antioxidant system so that the cells survive in the stress of oxidation.