| BackgroundIt has been known that high alcohol-producing Klebsiella pneumoniae(HiAlc Kpn)is one of causative agents of nonalcoholic fatty liver disease(NAFLD).However,how HiAlc Kpn promotes liver injury remains unclear.Recent findings suggest that DNA methylation might associate with the pathogenesis of NAFLD.Herein,the role of DNA methylation in HiAlc Kpn-induced liver injury was investigated.Murine models of NAFLD were established in C57BL/6N wild-type mice by gavaging HiAlc Kpn for 8 weeks.In addition,it is known that heavy alcohol consumption can induce DNA damage,which causes damage and mutations in hematopoietic stem cells.However,a large amount of endogenous ethanol produced by HiAlc Kpn can enter all tissues and organs of the body along with the blood circulation.The effect of endogenous ethanol produced by HiAlc Kpn on hematopoietic stem cells is still unclear.Here,flow cytometry and RNA-Seq were used to analyze the effect of endogenous ethanol produced by HiAlc Kpn on hematopoietic stem cells.Aim1.To elucidate the mechanism of HiAlc Kpn-induced liver injury.2.To explore the effect of HiAlc Kpn on hematopoietic stem cells.MethodsI Study on the mechanism of HiAlc Kpn-induced liver injury.1.The pathological and biochemical indexes of liver tissue in HiAlc Kpn-induced NAFLD mice model were analyzed.2.The methylation level in liver tissue of HiAlc Kpn-induced mouse was detected.2.1 DNA methylation in hepatic tissue of C57BL/6N mice after 4,6 and 8 weeks of HiAlc Kpn gavage was assessed by using dot bolt of 5-methylcytosine(5-mC).2.2 The 5-mC in HepG2 cells treated with HiAlc Kpn supernatant was also detected by dot blot.2.3 The 5-mC in liver tissue of C57BL/6N mice after 8 weeks of HiAlc Kpn gavage was analyzed by immunohistochemistry.Combined with the above experimental results,changes of methylation levels in HiAlc Kpn-induced mice were determined.3.RNA-Seq was used to analyze the transcriptional changes in HiAlc Kpn-induced mouse liver tissue.3.1 The changes of liver transcriptome in mice after 8 weeks of HiAlc Kpn gavage were analyzed.3.2 The changes of liver transcriptome in EtOH-fed group were analyzed.3.3 Conjoint analysis of the transcriptomic levels of HiAlc Kpn-fed and EtOHfed group,and the expression levels of differentially expressed genes(DEGs)in liver tissues were verified by RT-qPCR.4.Genome-wide bisulfite sequencing(WGBS)and RNA-Seq analysis were used to investigate the role of methylation in liver injury and its possible signaling pathways.4.1 The changes of liver methylation levels and different gene elements in HiAlc Kpn-fed group were analyzed by WGBS technology.4.2 Combined with the RNA-Seq data and the WGBS results,a joint analysis was conducted to explore the DEGs with the methylation and transcription level changes.4.3 Methylation-specific PCR(MSP)and RT-qPCR were used to detect the DEGs in the combined analysis of WGBS and RNA-Seq,respectively,to verify the sequencing results of WGBS and RNA-Seq.Combined with the above experimental results,it was determined that HiAlc Kpn induced changes in methylation level and regulated gene transcription level in vivo,which may be involved in the development of NAFLD.II Study on the effect of HiAlc Kpn on hematopoietic stem cells.1.HiAlc Kpn results in bone marrow hematopoietic stem cell damage.1.1 To evaluate whether the genome was stable,flow cytometry was used to detect the micronucleus erythrocytes in peripheral blood of WT and aldh2-/-mice after4,6 and 8 weeks of HiAlc Kpn gavage,respectively.1.2 Bone marrow stem cell proportion of WT and aldh2-/-mice after 4,6 and 8 weeks of HiAlc Kpn gavage was detected by flow cytometry.1.3 Bone marrow transplantation was used to detect the changes of bone marrow stem cell regeneration in WT and aldh2-/-mice after 6 weeks of HiAlc Kpn gavage.1.4 Pathological changes of tibia and liver of WT and aldh2-/-mice after 4,6 and 8 weeks of HiAlc Kpn gavage were analyzed by H&E.1.5 The biochemical indices of WT and aldh2-/-mice were analyzed after 4,6 and 8 weeks of HiAlc Kpn gavage.2.Transcriptomic sequencing was performed on WT and aldh2-/-mouse bone marrow hematopoietic stem cells after 8 weeks of HiAlc Kpn gavage,and the changes of mRNA expression levels were analyzed to obtain the signal pathway regulating hematopoietic stem cell damage.ResultsⅠ Study on the mechanism of HiAlc Kpn-induced liver injury.1.HiAlc Kpn induced pathological changes of lipid droplet deposition and accumulation of biochemical indexes in mouse liver tissues.2.The 5-mC in liver tissues of mice after 4,6 and 8 weeks of HiAlc Kpn gavage,and in HepG2 cells treated with HiAlc Kpn culture supernatant was decreased.Immunohistochemistry further verified that HiAlc Kpn induced decreased 5-mC in liver tissues of mice.3.RNA-Seq analysis showed that HiAlc Kpn induced enrichment of adipose metabolism and DNA damage related pathways in mouse liver.The accumulation of epigenetic pathways and DNA damage pathways in EtOH-fed mouse liver.4.WGBS analysis showed that HiAlc Kpn induced DNA hypomethylation in liver tissue.5.WGBS and RNA-Seq analysis showed that HiAlc Kpn could induce the change of DNA methylation and gene expression.Ⅱ Study on the effect of HiAlc Kpn on hematopoietic stem cells.1.HiAlc Kpn can induce genomic instability of mouse bone marrow hematopoietic stem cells.2.HiAlc Kpn could induce the decrease of the proportion of bone marrow hematopoietic stem cells in mice.3.HiAlc Kpn induced a decrease in the regenerative capacity of mouse bone marrow hematopoietic stem cells.4.HiAlc Kpn induced bone marrow injury in mice by H&E.5.The DEGs were significantly enriched in the signaling pathways related to hematopoietic stem cells by RNA-Seq analysis.Conclusion1.HiAlc Kpn can induce changes of DNA methylation in liver tissue in vivo to regulate the expression of lipid metabolism-related genes.DNA hypomethylation might play an important role in liver injury of NAFLD induced by HiAlc Kpn.Which possibly provides a new sight for understanding the mechanisms of NAFLD and selecting the potential therapeutic targets.2.The endogenous ethanol produced by HiAlc Kpn can induce the injury of hematopoietic stem cells in mice. |
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