| Objective:To investigate the mechanism of Lnc RNA 085865 in regulating macrophage polarization to alleviate the non-alcoholic fatty liver disease(NAFLD)and liver injury in mice.Methods: Part 1:The effect of overexpressed Lnc RNA 085865 on liver function of HFD-induced mice with NAFLD.Construction of myeloid cell-specific knock in mice(MYKI):MYKI mouse was generated by crossing Lyz2-Cre+/-mice with Lnc RNA085865SA26/ROSA26 mice.MYKI mice and WT mice were randomly fed with normal diet(10%fat,ND),or high-fat diet(60%fat,HFD)for 12 weeks,and their weight was measured weekly.After fed with 12 weeks,the serum levels of alanine aminotransferase(ALT)and aspartic aminotransferase(AST)were collected and measured.Liver tissues were collected and weighed,and parts of liver tissues were taken to detect alanine aminotransferase(ALT)and aspartic acid aminotransferase(AST)levels.Part 2:The effect of overexpressed Lnc RNA 085865 on the fatty changes in liver tissue of mice induced by HFD diet.Liver tissue was collected for HE staining to observe the degree of fatty change in liver tissue.Part 3:The effect of overexpressed LncRNA 085865 on the macrophage infiltration in liver tissue of mice induced by HFD diet.F4/80 immunohistochemical staining was used to analyze the aggregation of macrophages in liver tissue.RT-q PCR and Elisa were used to detect inflammatory factors and M1 polarization markers in liver tissue,such as: The expression levels of tumor necrosis factor α(TNF-α),interleukin 1-β(IL-1β)and interleukin 6(IL-6),M2 polarization markers interleukin-10(IL-I0),arginase 1(Arg1)and chitinase 3-like 3(Ym1) expression levels.Part 4:The effect of overexpressed Lnc RNA 085865 on the insulin resistance in liver tissue of mice induced by HFD diet.Intraperitoneal glucose tolerance analysis and fasting insulin tolerance test were performed in mice at 12 weeks,and the expression levels of phosphorylated protein kinase B(p-Akt)and protein kinase B(Akt)were detected by Western blot.Part 5:The effect of overexpressed Lnc RNA 085865 on the liver pyroptosis in liver tissue of mice induced by HFD diet.The expressions of NLRC4,Caspase-1 and Gasdermin D(GSDMD)in liver tissues were detected by WB.The expression level of Caspase-1 in liver tissue was detected by the kit.Liver tissue activated Caspase-1 levels were detected by immunofluorescence.Results:(1)Overexpression of Lnc RNA 085865 alleviated liver injury induced by HFD in mice,which was reflected in the reduction of liver function indexes.(2)Overexpression of Lnc RNA085865 improved liver adiposis induced by HFD diet.(3)The overexpression of Lnc RNA085865 could alleviate the inflammatory response caused by HFD in mice by regulating macrophages.The overexpression of Lnc RNA 085865 could reduce the infiltration of macrophages caused by high fat diet,and also reduce the expression levels of macrophage inflammatory factors TNF-α,IL-1β and IL-6.TNF-α,IL-1β and INOS were also decreased in liver tissue,and the ratio of M1 was decreased and the ratio of M2 was increased.(4)Compared with the control group,Lnc RNA085865 overexpression could alleviate insulin resistance induced by HFD and improve abnormal glucose tolerance in mice.(5)Overexpression of Lnc RNA 085865 could alleviate Caspase-1/ GSDMD-mediated hepatocyte scorch death by reducing the release of NLRC4 inflamomes,thus alleviating hepatocyte scorch death.Conclusion: Lnc RNA 085865 can reduce liver injury in non-alcohol fatty liver,reduce macrophage infiltration,and regulate macrophage polarization,and then reduce the release of macrophage pro-inflammatory factors to improve insulin resistance in mice.Lnc RNA 085865 can also reduce the expression of NLRC4 in inflammatories.The Caspase-1/GSDMD pathway alleviates hepatocyte pyrodeath,thereby alleviating NAFLD. |
