| BackgroundNon-alcoholic fatty liver disease(NAFLD)is a common liver metabolic disease that occurs worldwide and in all age groups.In the absence of a large amount of alcohol intake,excess fat is deposited in the liver,resulting in hepatic steatosis.As the disease progresses,NAFLD can progress to non-alcoholic fatty liver inflammation(Non-Alcoholic Steatohepatitis,NASH),accompanied by immune inflammatory response and liver fibrosis,and finally progresses into hepatocytes Liver cancer(hepatocellular carcinoma,HCC).In recent years,the prevalence of NAFLD has been increasing,and the vast majority of patients will be accompanied by hyperlipidemia,obesity,insulin resistance,diabetes and other metabolic diseases,which will bring heavy economic pressure to individuals,families and society.At present,the pathophysiological mechanism of NAFLD is not clear,and there are many hypotheses,involving the combined effects of genetics,immunity,environment and other aspects.The existing diagnostic and therapeutic methods need to be improved in specificity and effectiveness,and more simple and accurate diagnostic methods and targeted therapeutic measures are urgently needed.Senescence plays a key role in the pathogenesis of age-related diseases,such as diabetes,heart failure,and cerebrovascular diseases.Cellular senescence may be associated with replicative senescence,DNA damage accumulation,telomerase shortening and dysfunction,oxidative stress,and mitochondrial dysfunction.Hepatocyte senescence plays an important role in NAFLD,and the molecular mechanism related to hepatocyte senescence has gradually become an important target for the treatment of NAFLD.Copper is the most basic element required by all living organisms.As a cofactor of many metabolic key enzymes,copper can drive a series of physiological processes including mitochondrial respiration,detoxification,and biological structure synthesis.In order to ensure normal physiological functions,the copper content in the body needs to be kept stable within a small range,and a slight increase can lead to cytotoxicity and even cell death.Cuproptosis is a recently discovered regulated cell death(RCD)induced by excess copper ions in the body.Cuproptosis is different from other ways of programmed cell death(PCD)such as apoptosis,ferroptosis and necrosis.Intracellular copper targeting to fatty acylation components plays a role in the tricarboxylic acid cycle(TCA),aggregation of copper-bound fatty acylated mitochondrial proteins and iron-sulfur(Fe-S)clusters reduction induces proteotoxic stress,ultimately leading to cell death.Mesenchymal stem cells(MSCs)are one of the most important human adult stem cells.MSCs can be isolated from umbilical cord,umbilical cord blood,bone marrow,fat and other tissues and organs.Now it is the most promising clinical research direction in the world.In recent years,MSCs have emerged as a highly anticipated cellular therapy in human diseases.Exosomes(Exosomes,EXOs)are extracellular vesicles,mostly between 40-100nm in diameter,containing proteins,DNAs,mRNAs,miRNAs and other components,and play important biological functions.Mesenchymal stem cell-derived exosomes(MSC-EXO)have similar functions to MSCs and have lower immunogenicity and tumorigenicity.Human umbilical cord MSCs(HucMSCs)-derived exosomes(HucMSC-EXO)have been widely used in regenerative medicine and the treatment of various diseases.Studies have shown that exosomes derived from mesenchymal stem cells can alleviate liver injury,liver fibrosis and inflammation,and mineral metabolism is involved in affecting the progression of NAFLD.Therefore,this study aims to explore the effects of exosomes derived from mesenchymal stem cells on lipid metabolism in aging mice and the mechanism of cuproptosis in NAFLD.Part Ⅰ Human umbilical cord mesenchymal stem cell-derived exosomes improve liver lipid metabolism in aging mice through autophagyPurposeTo investigate the effect of exosomes derived from human umbilical cord mesenchymal stem cells on liver lipid metabolism in aging state and whether the autophagy play a role in it MethodsThirty C57 mice were divided into young group,old group and old exosome injection group according to the experimental design.Weight,blood sugar and other indexes were detected during feeding.The young group was fed with normal diet until 12 weeks of age.The rest were fed with high fat to establish the model of hepatocyte steatosis.The old group was fed with high fat to 18-22 months of age.In the aged exosome injection group,each mouse was given 20ug of exosome derived from human umbilical cord mesenchymal stem cells,once every 3 days for 10 weeks.Lipid metabolism,senescence and autophagy were detected in each group after death.Normal liver cells of AML 12 mice were stimulated by Palmitic Acid(PA),and the in vitro steatosis model was constructed to detect lipid metabolism,autophagy and other indexes.All data were represented by mean value and standard error,and the graphs were drawn by GraphPad8.P<0.05 was considered statistically significant.ResultsHuman umbilical cord mesenchymal stem cells were extracted and cultured.Stem cells were identified by flow cytometry,osteogenesis and lipid induction.Exosomes were extracted from supernatant of cultured stem cells and identified by electron microscopy and NTA.The body weight of the aging group was higher than that of the young group,and the body weight of the exosome group was lower than that of the aging group.The glucose tolerance and insulin sensitivity of the aging group were decreased,and the exosome group was better than the aging group.Compared with the aging group,the liver structure of exosome group mice recovered,lipid deposition decreased,glycogen synthesis increased and senescence state decreased.Western blot results showed that compared with the aging group,the lipid synthesis of exosome group was decreased,the lipid decomposition was increased,and the autophagy level was enhanced.AML 12 cells were stimulated by PA in vitro to construct lipid deposition model.The results showed that compared with PA group,exosome group had less lipid deposition,more glycogen synthesis,decreased senescence level,increased lipid metabolism level and enhanced autophagy level.The changes in autophagy levels after exosomes were detected by silencing autophagy related genes with small interfering RNA.The results showed that exosomes could enhance autophagy in liver cells.ConclusionHepatic lipid metabolism was decreased in the aging state.Exosomes derived from human umbilical cord mesenchymal stem cells can improve lipid metabolism in aging liver.Exosomes derived from human umbilical cord mesenchymal stem cells may affect lipid metabolism through autophagy.Part Ⅱ Cuproptosis may lead to the occurrence and development of NAFLD through the key sites of DLD and DLATPurposeTo explore the role of cuproptosis in the pathophysiological mechanism of NAFLD and identifies key targets by conducting bioinformation analysis on the gene and clinical information of NALFD patients in public databases.Clinical samples were collected and the NAFLD mouse model fed with high fat and the lipid deposition model of AML 12 cells induced by PA in vitro were used to verified the findings.MethodsThe differential gene sequence was obtained by comparing the gene expression level data of NAFLD patients with the cuproptosis-related gene expression level in the existing public database.According to the diagnosis and exclusion criteria of the guidelines for the prevention and treatment of NAFLD(updated in 2018),hospitalized patients with NAFLD were selected as the research subjects,and healthy volunteers were recruited as the control group.The basic information,blood samples,and medical history of the patients and healthy volunteers were collected.After extracting RNA from the blood samples of patients and healthy volunteers and obtaining cDNA by reverse transcription,the expression level changes of various differential genes were detected by PCR.And then,the results were verified in high-fat-fed NALFD mouse model and the cell model induced by PA stimulation by PCR,WB and IHC staining.ResultsSix differential expressed cuproptosis related genes(DECRGs),namely NFE2L2,ATP7B,LIAS,DLD,DLAT and PDHB,were analyzed from the gene expression data set of normal subjects and NAFLD patients,and their diagnostic function was verified by ROC.We found that cuproptosis plays an important role in NAFLD through two consensus cluster analyses,and plotted Sankey plots to show the relationship between the two consensus cluster analyses.The results of KEGG enrichment analysis showed that these genes were involved in multiple metabolic processes.In addition,we also conducted GO enrichment analysis,and these genes are closely related to ATP metabolism in mitochondrial inner membrane.We screened DECRGs based on random forest method,identified DLD and DLAT as key DECRGs,and found a close relationship between key DECRGs and clinicopathological factors of NAFLD.We collected serum from healthy controls and patients with NAFLD and extracted total RNA.The expression levels of differential expressed genes were verified by RT-qPCR.In addition,we further verified the results in mouse liver tissue and in vitro cells,both of which obtained the same results.ConclusionCuproptosis related genes DLD and DLAT are closely related to NAFLD.These genes may play a key role in the pathophysiological mechanism of NAFLD and provide a new target for the diagnosis and treatment of NAFLD. |
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