Regulatory Effect And Mechanism Of INKT Cells On Non-alcoholic Fatty Liver Disease

Background With the change of human life style,non-alcoholic fatty liver disease(NAFLD)has become one of the main diseases that endanger human health,and there is still a lack of specific therapeutic drugs in the current state.Studies have shown that the development and deterioration of NAFLD is related to the disorder of liver immune environment,and iNKT cells,as the central cells of immune regulation,the exact role and mechanism of iNKT in NAFLD are still unclear.Therefore,this project aims to explore the regulatory effect of iNKT cells on non-alcoholic fatty liver disease and related mechanisms,and to provide new therapeutic strategies for the immunological prevention and treatment of NAFLD targeting iNKT cells.Methods High-fat diet(HFD)and methionine/choline deficiency diet(MCD)were fed C57BL/6J to establish NAFL and NASH models,and α-Galcer was injected intraperitoneally to interfere.At the same time,immunomagnetic bead sorting technology(MACS)was used to isolate and purify spleen iNKT2 cells and adoptive infusion into the NASH mice by tail vein.Determine the effect of α-Galcer intervention by mouse appearance,weight,blood biochemical indicators(TG,TCH,HDL,LDL,ALT,AST),liver index,liver tissue H&E.Flow cytometry was used to detect the frequency of hepatic iNKT and subpopulations(iNKT1/iNKT2)and peripheral blood iNKT cell.Western Blot was used to detect the expression of related proteins such as SREBP-1c,PPAR-α,NF-kB in liver tissue.Observation of the effect of IL-4 on HepG2 cell steatosis model in vitro.To investigate the mechanism of α-Galcer and adoptive infusion of iNKT2 cells in regulating liver lipid metabolism and inflammation.Results 1.NAFL mice induced by HFD gained weight without producing glucose tolerance and insulin resistance.Serum TCH,HDL,LDL,and ALT levels were elevated.Liver index was not significantly changed compared to control mice,and large areas of steatosis appeared in the liver.Intraperitoneal injection of α-Galcer had no significant effect on body weight,glucose tolerance,and insulin resistance,but decreased serum TCH,HDL,LDL levels and liver fatty deformation in NAFL mice.NASH mice induced by MCD lost weight and did not develop glucose tolerance and insulin resistance,but increased sensitivity to insulin.Serum TCH,TG,LDL levels decreased while ALT and AST levels increased,and liver index increased.The liver has a large area of steatosis and has a large amount of inflammatory cell infiltration.Intraperitoneal injection of α-GalCer has no effect on the body weight,insulin sensitivity,serum TCH,TG,HDL,LDL levels and liver index of NASH mice,which increases the levels of ALT and AST in serum and reduces the degree of liver steatosis in NASH.However,there was no significant change in liver inflammatory cell infiltration.2.The frequencies of hepatic iNKT cells in NAFL mice were lower than those in control mice,among them,iNKT1 subgroups were predominant.Intraperitoneal injection of α-GalCer increased the frequency of hepatic iNKT cells,mainly the iNKT2 subgroup.The frequency of hepatic iNKT cells increased in NASH mice.Intraperitoneal injection of α-GalCer decreased the frequency of hepatic iNKT cells in NASH mice,and the frequency of iNKT2 cells also increased,but the iNKT1 subgroup was still the dominant type.The frequencies of iNKT cells in peripheral blood of NAFL mice were not changed compared with control mice.Intraperitoneal injection of α-GalCer had no effect on the frequencies of iNKT cells in NAFL mice.The frequency of iNKT cells in peripheral blood of NASH mice was lower than that of control mice.Intraperitoneal injection of α-GalCer increased the frequency of iNKT cells in peripheral blood of NASH mice.3.Expression of SREBP-1c and PPAR-α of the liver in NAFL mice was higher than that of control mice,and the level of NF-κB protein phosphorylation was increased.Intraperitoneal injection of α-GalCer down-regulates liver SREBP-1c protein expression,up-regulates PPAR-α protein expression,but does not change phosphorylation level of NF-κB protein.Expression of SREBP-1c and PPAR-α protein of liver in NASH mice was up-regulated,and the level of NF-κB phosphorylation increased.The injection of α-GalCer down-regulated the expression of liver SREBP-1c protein,and the level of NF-κB protein decreased,while there was no change in PPAR-α protein expression.4 Adoptive infusion of iNKT2 cells had no effect on body weight,blood biochemical indicators,and liver lipid accumulation in NASH mice,while liver inflammatory cell infiltration increased.The frequency of hepatic iNKT cells in adoptive infusion mice was increased,and both iNKT1 and iNKT2 subpopulations were increased,but the iNKT1 subpopulation was still the dominant one.The frequency of iNKT cells in peripheral blood remained unchanged.5.Oil red O staining results show that intervention of IL-4 has no significant effect on HepG2 cell liver fat deformation Conclusion 1.Intraperitoneal injection of α-GalCer activates liver iNKT2 cells,which improve liver steatosis in the stages of NAFL and NASH by down-regulating the expression of SREBP-1c,up-regulating the expression level of PPAR-α protein.2.Intraperitoneal injection of α-GalCer activates hepatic iNKT2 cells in NASH mice,which reduce NASH liver inflammation by inhibiting phosphorylation of NF-κB protein.3.Under the pathological state of NASH,the inflammatory microenvironment of the liver may induce the transformation of iNKT2 cells to iNKT1 cells,and adoptive infusion of iNKT2 cells may aggravate the liver inflammation in NASH mice.4.IL-4 may not be the main factor affecting liver lipid metabolism in HepG2 cells.