The Function Of Mitochondrial Iron Carrier SLC25A28 In Mitochondrial Iron Metabolism And Its Roles And Mechanism In NASH Development

Background:Non-alcoholic fatty liver disease(NAFLD)is the most prevalent chronic liver disease with a global incidence of around 25%.As a key cause of end-stage liver diseases and liver transplantation,NAFLD is triggered by lipid deposition in the liver and associated with chronic hepatitis and other hepatocyte damages,which leads to liver fibrosis and,and eventually cirrhosis and hepatocellular carcinoma.Currently,it is suggested that excessive lipid deposition is a contributor to the occurrence and development of NAFLD,but not the direct cause of non-alcoholic steatohepatitis(NASH).Mitochondria are indispensable organelles and energy factories,and play critical roles in maintaining the physiological functions of liver cells.There are enormous evidences that mitochondrial dysfunction is an important cog in NASH progression.The metabolic function and the crucial processes in mitochondria have been well understood.Mitochondria are metabolically active multifunctional organelles,including TCA cycle,fatty acid β-oxidation,oxidative phosphorylation,energy conversion and ion storages.In addition,mitochondria are also involved in intercellular signaling,cell differentiation,apoptosis,and play important roles in the metabolism of cellular materials.In order to transport across the mitochondrial bilayer membrane,most substances inside and outside the mitochondria require transporters.Thus,the transporters on mitochondrial membrane are critical in energy metabolism and substance metabolism.SLC25(solute carrier family 25),located on mitochondrial inner membranes,are a group of vital mitochondrial transport carriers,transporting various substances across mitochondrial inner membranes and maintaining metabolite homeostasis.As a member of SLC25 family,SLC25A28 is widely expressed in human tissues and cells,and located in the mitochondrial inner membrane,encoding Mitoferrin 2 and regulating the iron utilization and storage in mitochondria.The studies on SLC25A28 have been conducted in the nervous system,cardiovascular system and reproductive system,and SLC25A28 has been confirmed to be essential for liver regeneration and cell proliferation in mice.However,the specific influences of SLC25A28 on mitochondrial functions and its physiological function and mechanism in the progression of NASH remain unclear.Aims:To determine the correlation between the expression of SLC25A28 and the occurrence and development of NAFLD,to clarify the effects of SLC25A28 deficiency on liver morphology,physiological function and mitochondrial structure and function of hepatocytes,and to clarify the main mechanism of the influence of SLC25A28 deficiency on the progression of NASH in mice.Methods:(1)Rabbit anti-SLC25A28 polyclonal antibody was prepared using convertional method,and the expression of SLC25A28 was analyzed in the fatty livers of ob/ob mice and the fatty acid-induced Hep G2 cells.(2)Liver specific SLC25A28 knockout mice were generated by mating SLC25A28-floxed mice with Albumin-Cre(Alb-Cre)mice.Under the fed and fasted conditions,the HE staining,Oil Red O staining and TG quantification were used to determine the effect of SLC25A28 deficiency on liver lipid contents.q PCR and Western blot were recruited to analyze the expressions of key molecules in liver lipid metabolism,including CPT1,ACC1 and FASN,in SLC25A28 knockout mice.(3)Ultrastructural alterations in the livers of SLC25A28 knockout mice were observed by transmission electron microscopy(TEM).The mitochondrial functions of SLC25A28 knockout hepatocytes were analyzed by cell staining,Western blot,q PCR and other methods.(4)The mouse model of NASH was established by feeding the methionine-choline deficient(MCD)diet,and the oxidative stress,lipid peroxidation and inflammation were analyzed in the livers of SLC25A28 knockout mice by HE staining,immunohistochemistry,q PCR and other quantitative assays.Result:(1)Rabbit anti-SLC25A28 polyclonal antibody was successfully prepared,and the expressions of SLC25A28 were found to be increased in fatty livers of ob/ob mice and fatty acid-induced Hep G2 cells.(2)In order to determine the roles of SLC25A28 in liver lipid metabolism,we first studied the expression of SLC25A28 in mouse fatty liver,and explored the effect of SLC25A28 deletion on liver lipid metabolism by using SLC25A28 knockout(SLC25A28-LKO)mice.The following results were obtained:(1)No significant difference was found in liver structure and lipid contents in SLC25A28 knockout mice and the wildtype mice under fed condition.(2)Under fasted conditions,the content of lipids in the liver of SLC25A28 mice increased.(3)Under fasted conditions,SLC25A28 deficiency elevated the expression of lipogenic genes(CPT1,ACC1 and FASN),promotes liver lipid synthesis and led to lipid deposition.(3)In order to explore the effect of SLC25A28 deficiency on mitochondria of hepatocytes,the structure and function of mitochondria were assessed.We found that:(1)SLC25A28 deficiency significantly increased the volume of mitochondria of hepatocytes,while the number of mitochondria decreases,accompanied by different degrees of inner and outer membrane separation.(2)SLC25A28 deficiency caused decreased mitochondrial membrane potential.(3)The deficiency of SLC25A28 caused the disorder of iron metabolism and inhibited the utilization of iron by mitochondria,resulting in the decreased synthesis of Fe-S cluster proteins and Heme in mitochondria.As a result,mitochondrial oxidative respiratory chain complex was dysfunctional,and ATP synthesis and oxygen consumption were reduced.(4)In order to make out the underlying mechanism of liver inflammation caused by SLC25A28 deficiency,we adopted NASH mouse model and found that SLC25A28 deficiency led to increased oxidative stress in liver,the production of lipid peroxide,macrophage activation and lymphocyte aggregation,as well as expression of inflammatory factors.Collectively,SLC25A28 deficiency promotes NASH progression.Conclusion:(1)The rabbit anti-SLC25A28 polyclonal antibody was successfully generated.The expression of SLC25A28 was elevated during hepatocyte steatosis.(2)SLC25A28 deficiency affected the process of lipid metabolism and caused lipid deposition in the liver of mice,suggesting that it is closely related to abnormal liver fat metabolism..(3)SLC25A28 deficiency impaired Fe-S cluster and Heme biosynthesis,which resulted in damaged mitochondrial structures and functions.These results indicate that SLC25A28 deficiency leads to mitochondrial iron metabolism disorder,and thus affects mitochondrial function,which is a critical contributor to liver steatosis..(4)SLC25A28 deficiency aggravated mitochondria-related oxidative stress and promoted the progression of NASH in mice,suggesting that mitochondrial iron metabolism disorders play an important role in the occurrence and development of NASH.