Aging Exaggerates Alcohol-associated Liver Disease By Inhibiting Neutrophilic Sirtuin1-C/EBPA-miRNA-223 Axis

Alcohol-associated liver disease(ALD)refers to a kind of chronic liver disease caused by long-term heavy drinking.ALD is usually manifested as alcoholic fatty liver(AFL)in the early stage,which can be further developed into alcoholic hepatitis,alcoholic liver fibrosis,alcoholic cirrhosis,and even hepatocellular carcinoma(HCC).The early clinical manifestationof ALD is not typical,and there is a lack of effective diagnostic indicators.It is therefore very urgent and necessary to find new therapeutic targets and developnew drugs.The underlying pathogenesis of ALD is very complex and has not been fully elucidated.Among the many factors that lead to the development of ALD,the activation of innate immune cells plays crucial roles.Moreover,neutrophil infiltration in liver tissue was considered to be a central event that leading to the onset and progression of ALD.In addition,studies have shown that factors that drive cell senescence are also key factors that promote the progression of ALD.We previously showed that aging exaggerates alcohol-related liver injury in mice and humans by increasing neutrophil infiltration.The inflammatory response is very complex,and these immune inflammatory cells play different roles in different triggers and stages of ALD progression.Therefore,it is necessary toexplore the underlying mechanisms of the pathogenesis of ALD.Sirtuin1(SIRT1),a member of the Sirtuin family,is a nicotinamide adenine dinucleotide(NAD⁺)-dependent histone deacetylase that play major roles in the nucleus and cytoplasm.In recent years,SIRT1 plays a beneficial role in the progression of ALD and may become a new therapeutic target for ALD.Although the downregulation of SIRT1 could increase the risk of ALD during the ageing process,previous studies mostly focused on hepatocyte and hepatic stellate cells(HSCs)that separately involved in liver injury and fibrosis progress.The neutrophils are recognized as central immune cell that participatein ethanol-induced inflammation.Howener,the functions and its potential mechanism of SIRT1are not clear.And most importantly,whether neutrophils are also involvedin ALD progression in patients with acute alcoholism,whether SIRT1 expression in neutrophils changed and what specific roles of SIRT1 play,these problems need to be further clarified.Micro RNAs(miRNAs)are a class of non-coding and single-stranded RNAs and can regulate downstream genes expression and function by binding to specific target m RNAs.For the past few years,miRNAs have been widely reported to be closed related to the occurrence and development of ALD.These studies indicate that miRNAs may be a promising biomarker in the treatment of ALD.During the aging process,many neutrophilic miRNAs have changed.The changes of neutrophil-specific miRNAs may play a vital role in regulating ALD function.Therefore,further studiesof miRNAs in neutrophils will help us better understand the ALD pathogenesis of aging aggravated ALD.This study aimed to examine the effects of aging and alcohol consumption on neutrophil SIRT1 and micro RNA-223(miR-223)and their contributions to the pathogenesis of ALD.In our experiments,young and aged mice were subjected to chronic-plus-binge ethanol feeding(E10d+1B).Blood samples from BM and peripheral blood/liver tissue were collected and purified.The effect of aging on Sirt1 expression of neutrophil was analyzed by PCR and western blot(WB)methods.The results showed that the Sirt1m RNA levels in BM and peripheral blood neutrophils of aged mice and the SIRT1protein levels in BM neutrophils of aged mice were lower than those of young mice.By identifying the expression differences of highly abundant micro-RNAs in neutrophils,it was found that neutrophilic pri-miR-223and miR-223 expression were downregulated in aged mice compared to young mice.The expression of Sirt1 m RNA in liver neutrophils of mice fed with E10d+1B was significantly lower than those mice fed with normal diet(including young and aged mice),while the expression of Sirt1 m RNA in BM neutrophils only showed a decrease trend after ethanol feeding.Interestingly,ethanol-fed neutrophilicmiR-223 levels were increased rapidly in young mice,whereas this upregulation was blunted in aged mice.The downregulation of the neutrophilic Sirt1expression in aging process has aroused our attention and been carried out functional research on it.C57BL/6 background mice bred with mice expressing Cre recombinase driven by the lysozyme promotor to generate myeloid cell-specific SIRT1knockout(Sirt1^Mye-/-)and littermate control mice(Sirt1^f/f).Then,Sirt1^Mye-/-mice and Sirt1^f/fmice were subjected to E10d+1B feeding.The deletion of the Sirt1 gene in myeloid cells including neutrophils exacerbated chronic-plus-binge ethanol-induced liver injury and inflammation,liver steatosis and oxidative stress,and downregulated neutrophilic miR-223 expression.These results suggest that neutrophil SIRT1 plays a protective role in the onset and progression of ALD.Furtherly,we explored the underlying mechanisms of the function of neutrophilic Sirt1 gene in ALD.Mechanically,immunoprecipitation experiments revealed that SIRT1 promoted C/EBP?deacetylation by directly interacting with C/EBP?,a key transcription factor that controls miR-223biogenesis,and subsequently elevated miR-223 expression in neutrophils.We analyzed neutrophilic SIRT1 and miR-223 in healthy controls.Firstly,the middle-aged and/or elderly subjects had decreased neutrophilic expression of SIRT1 and miR-223 compared to the young individuals.Furtherly,neutrophilic SIRT1/miR-223 had a negative correlation with the percentage of circulating neutrophils.Remarkably,a positive correlation was observed between neutrophilic SIRT1 and miR-223.In addition,we also analyzed the samples of healthy controls and alcoholic patientswho had a history of chronic alcohol drinking and presented to the Emergency Department with acute intoxication.The results shown that intoxicated patients had an elevation in serum ALT,AST,circulating neutrophils,and serum miR-223,but a reduction of neutrophilic SIRT1compared to healthy controls,which are in agreement with the above results from E10d+1B feeding mouse model.Interestingly,neutrophilic miR-223 levels were lower in the middle-aged group than in the young group in both healthy and intoxicated groups.This study systematically explained the function and mechanism of neutrophilic SIRT1 from multiple dimensions including animal level,cell level and clinical patients.In conclusions,aging increases the susceptibility of alcohol-induced liver injury in mice and humans via the downregulation of the neutrophilic SIRT1-C/EBP?-miR-223 axis.SIRT1would be a novel therapeutic target for the prevention and/or treatment of ALD.