Study On The Mechanism Of INKT Cells In Chronic Inflammation Of Obese Adipose Tissue

ObjectiveIn order to explore the mechanism of iNKT cells in chronic inflammation of obese adipose tissue,(1)we constructed a chronic inflammation model of adipose tissue in obese mice to analyze the relevant indicators and changes in cytokines of successful modeling;(2)We observed the frequency and subsets of iNKT cells in adipose tissue and related tissues at different stages of obesity,and the changes of macrophage frequency and subsets in adipose tissue;(3)Observing the effect of obesity improvement by different injection methods of?-Galcer(100 ng/g body weight);Investigating the effect of activation of different subsets of iNKT cells on macrophage polarization,so as to provide basic research data for the treatment of obesity-related diseases.Methods1.Construction of chronic inflammation model of obese adipose tissue in mice4-5W male C57BL/6J mice were fed with high-fat diet for 4W and 12W respectively.After modeling,healthy mice were taken as control,the changes of body weight,mental state,and organ index of mice were observed and recorded;the GTT and ITT of mice were measured by blood glucose meter;the inflammatory cells infiltration of adipose tissue and liver was observed by hematoxylin-eosin staining(HE)staining;and the level of cytokines in the serum and lymphocyte stimulating supernatant of liver and adipose tissue were measured by Cytometric Bead Array(CBA).2.Analysis of immune function status in different stages of chronic inflammation of obese adipose tissue in miceFlow cytometry(FCM)was used to detect and compare the frequency of CD4~+T,CD8~+T and iNKT cells,the ratio of iNKT1/iNKT2/iNKT10 subset and the change of macrophage frequency and subset in adipose tissue at different stages of obesity;The expressions of T-bet,GATA-3,E4BP4,Arg-1 and iNOS in related tissues and organs were detected by Western blot.3.Mechanisms of intraperitoneal and subcutaneous injection of?-Galcer on chronic inflammation of obese adipose tissue in miceDifferent injection methods of?-Galcer were used to intervene obesity model mice.Mice were divided by intraperitoneal and subcutaneous injection of?-Galcer.Observing the changes of mice body weight,mental status,GTT and ITT in each group;the pathological changes of adipose tissue and liver were observed by HE staining;the frequency of CD4~+T,CD8~+T,iNKT cells,iNKT subsets and the frequency and subsets of macrophages were detected by FCM;The level of cytokines in serum,adipose tissue and liver lymphocyte stimulation supernatant were detected by CBA at different stages of mice in each group;the expressions of related proteins in liver and adipose tissue of mice in each group were detected by Western blot;Through co-culture of iNKT cells and macrophages,the polarization of macrophage subsets was detected by FCM.Result1.Compared with the healthy control group,the weight of the model group increased rapidly(P<0.05),the body size became larger.After 4 weeks of high fat diet,the average body weight of the model mice did not meet the body weight requirement of obese model mice,while after 12 weeks,the weight met the requirement of obese model mice;GTT and ITT tests showed that the average blood sugar level of model mice was always at a high level,but there was no impaired glucose tolerance and insulin resistance;HE observation showed that after 4 weeks of high fat feeding,the volume of adipocytes increased,but the inflammatory cell infiltration did not increase significantly,while the liver showed hepatic steatosis,some hepatocyte balloon-like changes,fat vacuoles.After 12 weeks of high-fat feeding,the volume of adipocytes not only continued to increase,but also the infiltration of inflammatory cells increased significantly,surrounded by flower rings around the fat cells,and the liver began to appear fatty liver and a large number of hepatocyte vacuoles,furthermore,cell steatosis is more serious.The adipose tissue index and morphology of the epididymis was significantly increased in the high-fat feeding group for 4 weeks,and further increased after 12 weeks of modeling(P<0.05),while the high fat feeding group for 4 weeks and 12 weeks,there were no significant changes in spleen and liver index,but there were significant changes in liver morphology;Cytokine levels were analyzed and found that at 4and 12 weeks of high-fat feeding,the level of inflammatory cytokines in the serum and tissue stimulating the supernatant were significantly increased(P<0.05),and the levels of inflammatory cytokines TNF-?and IL-17A were significantly increased in the serum of the high-fat feeding group at 12 weeks,while the levels of IL-6 and IL-2 were significantly decreased.2.The CD4~+T and CD8~+T cells in peripheral blood and adipose tissue were almost unchanged at other time points except that CD4~+T cells were significantly decreased in adipose tissue after 12 weeks of modeling;The frequency of iNKT cells in adipose tissue and liver of model mice decreased significantly(p<0.05),but the frequency of iNKT cells in peripheral blood,spleen and thymus did not differ significantly whether fed with high fat for4 or 12 weeks,in addition,the frequency of iNKT cells in adipose tissue of 12-week group was lower than that of 4-week group,but increased in liver(p<0.05);The frequency of iNKT1 cells in thymus increased and iNKT10 cells in adipose tissue decreased significantly after 4-week high-fat feeding(p<0.05);The frequency of iNKT1 cells in thymus,spleen and adipose tissue increased significantly after 12-week high-fat feeding,while iNKT2 in thymus and iNKT10 cells in adipose tissue decreased significantly(p<0.05).It was found that the frequency of macrophages and the proportion of M1 in adipose tissue increased significantly(p<0.05),while the proportion of M2 decreased significantly(p<0.05)after 4 weeks and12 weeks of high-fat diet.Compared with the group fed with high fat for 4 weeks,the frequency of macrophages and M2 subset in adipose tissue of the group fed with high fat for12 weeks did not change significantly,but the frequency of M1 increased significantly(p<0.05).After 4 and 12 weeks fed with high fat,the expressions of E4BP4 and Arg-1 protein in adipose tissue decreased significantly,iNOS protein increased significantly,and the expression of GATA-3 protein in liver decreased significantly(p<0.05),T-bet protein increased significantly(p<0.05).3.Intraperitoneal injection of?-Galcer can significantly reduce the body weight of obese mice and improve the liver pathology,while subcutaneous injection of?-Galcer can increase the body weight of mice,and can improve the pathology of adipose tissue;Neither of the two methods of?-Galcer injection can significantly improve the overall glucose tolerance curve and insulin tolerance curve;intraperitoneal injection of?-Galcer significantly increased spleen volume and spleen index(P<0.05),and improved liver color and greasy feeling,while subcutaneous injection?-Galcer had no significant effect on the epididymal fat,spleen and liver index;intraperitoneal injection of?-Galcer could significantly increase the frequency of iNKT cells in peripheral blood,spleen and adipose tissue(P<0.05),but had no effect on the frequency of iNKT cells in thymus.Subcutaneous injection of?-Galcer can only activate adipose tissue iNKT cells and reduce the frequency of liver iNKT cells,but has no activation effect on iNKT cells in other organs.Intraperitoneal injection of?-Galcer mainly activated spleen and liver iNKT2,while subcutaneous injection of?-Galcer mainly activated thymus iNKT2 and adipose tissue iNKT10 cells,and can significantly increase the proportion of M2and reduce the frequency of macrophages in adipose tissue.After intraperitoneal injection of?-Galcer,the inflammatory cytokines in serum and liver lymphocytes stimulated supernatant were decreased,while the anti-inflammatory cytokines increased significantly.After subcutaneous injection of?-Galcer,the anti-inflammatory cytokines in the adipose tissue lymphocytes stimulated supernatant increased significantly,but inflammatory cytokines were significantly reduced.After subcutaneous injection of?-Galcer,the protein levels of E4BP4and Arg-1 in adipose tissue increased significantly,iNOS decreased significantly,while intraperitoneal injection of?-Galcer could significantly decrease the expression of T-bet and increase GATA-3 protein in liver;After co-culture of iNKT cells and macrophages,the M2subset was significantly increased and M1 decreased significantly,in addition,the anti-inflammatory cytokines in co-culture supernatant increased significantly,while the pro-inflammatory cytokines decreased significantly.Conclusion1.After 4 weeks of high fat feeding,although the body mass level did not meet the obesity requirement,the inflammatory factors increased.After 12 weeks,the body mass reached the obesity standard,and there was a low level of inflammation.2.The decrease of iNKT cell frequency in adipose tissue and imbalance of iNKT subsets(iNKT1?,iNKT10?)may be involved in the occurrence of chronic inflammation in adipose tissue.3.There were differences in the activation of iNKT cell subsets in different tissues by different injection methods of?-Galcer.Intraperitoneal injection of?-Galcer mainly activated iNKT2 cells subset in the spleen and liver,while subcutaneous injection of?-Galcer mainly activated thymic iNKT2 and adipose tissue iNKT10.4.Adipose tissue iNKT10 cells were activated by subcutaneous injection of?-Galcer,which caused polarization of M2 macrophages and improved chronic inflammation in adipose tissue.5.Intraperitoneal injection of?-Galcer activated hepatic iNKT2 cell subsets and effectively improved hepatocyte fat accumulation caused by obesity.