Aging Related Autophagy Deficiency Aggravates Liver Injury By Regulating Macrophage Reprogramming

Part Ⅰ ATG5 deficiency exacerbates liver injury during agingObjective:The maintenance of liver homeostasis is disrupted,thereby aggravating liver damage after being attacked by pathogenic factors during aging.Macrophages are involved in regulating the progression and outcome of liver injury,and their immune properties are regulated by autophagy.However,how aging drives the transition of macrophage immune phenotype through autophagy,and its role and mechanism in liver injury remains unclear.Therefore,this part aims to explore the relationship between aging-related macrophage phenotype changes and autophagy characteristics,and to further clarify the role and mechanism of these links in liver injury.Methods:A model of thioacetamide（TAA）-induced liver injury（TAA-induced liver injury,TAA-LI）was constructed in young and aged mice,and the serological indicators of liver injury（ALT,AST）and liver tissue sections were analyzed.Hematoxylin-eosin（H＆E）staining was used to evaluate the severity of liver injury,and TUNEL（Td T-mediated d UTP Nick-End Labeling）staining and Western Blotting（WB）experiments were used to detect the apoptosis of liver cells in young and aged mice.The infiltration of neutrophils and macrophages in the livers of mice in each group was observed by immunohistochemistry（IHC）.The effects of aging on autophagy-related protein molecules and inflammatory phenotypes in macrophages were investigated in vitro.Then autophagy-related protein 5（ATG5）was used to restore autophagy to reprogram macrophages derived from aging mice to observe the changes in immunophenotype and their role in aging-related liver injury.Further adoptive transplantation of macrophages with different treatments into mice was performed to explore the role of macrophages with different phenotypes in TAA-LI.Results:Aged mice had more severe liver damage and increased apoptotic and necrotic liver cells after TAA-LI,accompanied by an increase in the senescence-associated secretory phenotype（SASP）.In addition,autophagy levels in aged mouse-derived macrophages were significantly decreased,which was associated with a decrease in ATG5 in senescent cells.Downregulation of autophagy in senescent macrophages promotes their classic-type polarization（M1）to produce more pro-inflammatory cytokines.Notably,rescuing ATG5 expression in aged mouse-derived macrophages enhanced autophagy,reduced the pro-inflammatory phenotype of macrophages,and reduced senescence-associatedβ-galactosidase（SA-β-gal）staining.Finally,we found that aged mouse-derived macrophages that restored ATG5 expression had a similar protective effect on liver injury as young mouse-derived macrophages,reduced the inflammatory response after liver injury in aged mice,and enhanced the effect of high-dose TAA lower survival rate.Conclusions:The study confirmed that the effect of aging on macrophage polarization is an important factor in the aggravation of liver injury,and the recovery of autophagy in macrophages by restoring ATG5 expression can alleviate the aging phenotype and alleviate aging-related deterioration of TAA-LI injury.Part Ⅱ Aging aggravates liver injury through enhanced C/EBPα-BMP9 signalingObjective:The relationship between aging and autophagy is complex,and how autophagy changes in aged macrophages are still unclear.In the first part,we demonstrated that ATG5 downregulation in senescent macrophages is an important factor leading to impaired autophagy,and thus the hepatic inflammatory disorder is an important factor in the aggravation of liver injury.How aging drives changes in autophagy-related core proteins remains unclear.Bone morphogenetic protein 9（BMP9）is a member of the transforming growth factor-β（TGF-β）superfamily.Previous studies have shown that it is almost exclusively produced in the liver and reaches the body as a secreted protein Function everywhere.However,the role and mechanism of BMP9 in aging-related liver injury and inflammation repair are still unclear.Methods:Firstly,the acetaminophen（APAP）-induced liver injury model（APAP-induced liver injury,APAP-LI）was established in young and aged mice under fasted and unfasted conditions,and the degree of liver injury in each group was analyzed.Expression of BMP9 and CCAAT/enhancer binding proteinsα（C/EBPα）was detected in the livers of young and old mice,as well as in hepatocytes and macrophages derived from the liver.The mouse BMP9 recombinant protein（Rm-BMP9）recombinant protein was used in cultured hepatocytes to study the injury of BMP9 to hepatocytes under the action of APAP,as well as the regulation of autophagy,apoptosis and senescence.The localization of BMP9 expression in the liver was analyzed by immunofluorescence（IF）of BMP9 and F4/80,and the expression and secretion pattern of BMP9 in macrophages was analyzed.Shifts in macrophage autophagy,immune properties,and senescence characterization under the treatment by BMP9 were investigated in cultured macrophages.APAP-LI was established in young and aged Bmp9^-/-mice to analyze the effect of BMP9 on liver injury.Then,in order to explore the upstream regulatory mechanism of BMP9,the regulatory effect of Cebpa expression on BMP9 was investigated in cultured hepatocytes and macrophages.The expression of BMP9 and its effect on liver injury and inflammation were further examined by overexpressing Cebpa in the liver using adeno-associated virus AAV8.Results:APAP-LI was more severe in aged mice in both fasted and unfasted conditions.Significant increase in BMP9 expression was detected in liver tissue of aged mice.In vitro experiments confirmed that BMP9 down-regulated the expression of ATG3 and ATG7 in hepatocytes,down-regulated the level of autophagy,increased the death of hepatocytes with the treatment of APAP,and increased the SA-β-gal staining induced by etoposide.In the resting liver,BMP9 is mainly localized to macrophages,and macrophages produce more BMP9 than hepatocytes when liver injury occurs.Then,we found that BMP9 similarly down-regulated ATG3 and ATG7 in macrophages,inhibiting autophagic flux.Knockdown of BMP9 significantly down-regulated the pro-inflammatory activation of macrophages induced by etoposide and/or APAP-treated hepatocyte culture supernatant;Rm-BMP9 promoted the pro-inflammatory polarization of macrophages and increased etoposide-induced SA-β-gal staining.In animal experiments,Bmp9 knockout significantly reduced APAP-induced liver injury and neutrophil infiltration,and reduced hepatocyte apoptosis.In addition,high expression of BMP9 and C/EBPαwas detected in both senescent macrophages and hepatocytes.BMP9expression in cultured hepatocytes and macrophages was positively regulated by Cebpa gene expression.Furthermore,liver injury was aggravated after overexpression of Cebpa in the liver,and high expression of BMP9 and activation of SMAD1/5/9（Drosophila mothers against decapentaplegic protein 1/5/9）signaling were detected.At the same time,down-regulation of ATG3 and ATG7 protein levels,block of autophagy,and increase of pro-inflammatory cytokines and chemokines were detected after hepatic Cebpa overexpression.Conclusions:BMP9 promotes the apoptosis and necrosis of liver cells during liver injury,and can regulate the immune behavior of macrophages in the liver,promoting the inflammatory response and liver injury.In the induction of senescence in hepatocytes and macrophages in vitro,BMP9 downregulated autophagy levels through ATG3 and ATG7,increasing the features of cellular senescence,suggesting that it may play an important role in the aging process by regulating autophagy and inflammation.Furthermore,BMP9release in the livers of aged mice is associated with upregulation of C/EBPα,possibly aggravating liver injury and inflammation through the transduction of C/EBPα-BMP9signaling.