Molecular Mechanism Of MIFG-E8 Involved In Diabetes Related Intestinal Inflammation

BackgroundDiabetes mellitus(DM)is a metabolic disease,which is thought to be caused by multiple risk factors such as age,obesity,or an un-healthy lifestyle.High fat or high sugar diet can cause low-grade inflammation and change the composition of intestinal microbiota,so as to promote metabolic endotoxemia and intestinal epithelial damage.The accumulation of advanced glycation end products(AGEs),as an important mechanism in the pathogenesis of diabetes,can cause oxidative stress damage and lead to cell dysfunction or even death,which eventually leads to multiple histiocytic damage in diabetes.When enteroendocrine cells distributed in the intestinal epithelium are continuously exposed to high concentrations of AGEs,the secretion function of incretin is impaired.Incretin(including GLP-1 and GIP)stimulates insulin secretion and improves insulin sensitivity,which is essential for maintaining glucose homeostasis.Decreased GLP-1 secretion and undermined GIP activity are thought to be involved in the pathogenesis of diabetes.Milk fat globule epidermal growth factor VIII(MFG-E8)is a glycoprotein that was originally found in milk and mammary epithelial cell.In addition to the conventional role of enhancing the clearance of apoptotic cells,increasing evidence suggests that MFG-E8 mediates a multifunctional therapeutic role in reducing inflammation,wound healing,arterial remodeling,and also ischemia reperfusion injury.In addition,MFG-E8 also plays an important role in the damage and repair of intestinal mucosal barrier.In mice with sepsis or dextran sodium sulfate(DSS)-induced colitis,rhMFG-E8 appears to be a benefit in maintaining intestinal mucosal homeostasis and repairing intestinal epithelium.Das et al.found that hyperglycemia and exposure to AGEs inactivated MFG-E8,resulting in impaired wound closure in diabetes,while recombinant MFG-E8 resolved inflammation and promoted skin wound healing.It seems that MFG-E8 is beneficial for diabetes-related inflammatory injury,but its mechanisms,especially in the intestine,were not well understood and need to be further explored.Receptor interacting protein kinase(RIPK)is a key molecule for the programmed death pathway and plays an important role in maintaining a healthy intestinal barrier.Recent studies have suggested that,through the release of cellular damage-associated molecular patterns(DAMPs),RIPK1-RIPK3-mixed lineage kinase domain-like(MLKL)pathway may cause many inflammatory diseases through necrosis independent mechanism,which in turn lead to secondary tissue damage.Thus,inhibitors of the RIPKs kinases might be potential therapeutic targets for inflammatory diseases.In the present study,we detected this necroptosis-related RIPKs signaling pathway and established the molecular linkage connecting MFG-E8 with AGE-modified BSA(AGE-BSA)stimulated intestinal L cells.We also used D-pinitol,a natural compound that was suggested to have multi-functional properties,such as anti-inflammatory,anti-hyperglycemic,and antioxidant competence,as a protective agent in this model.We aimed to preliminarily explore the possible molecular mechanism of MFG-E8 in diabetes related intestinal necro-inflammatory and to detect the effect of MFG-E8 on the secretion of GLP-1.ObjectiveThe aim of this study was to investigate the role and molecular mechanism of MFG-E8 in regulating necro-inflammatory response in AGEs induced intestinal injury model,and try to find out the role of RIPK1/RIPK3/MLKL pathway in the progression of intestinal injury,as well as the subsequent influence on the endocrine function of intestinal L cells.Methods1.In vivo study1.1 Twelve-weeks-old senescence-accelerated mice prone 8(SAMP8)and senescenceaccelerated mice resistant 1(SAMR1)male mice were randomly divided into the control group(n=10/group)and the diabetes group(n=12/group).The control group were given sham intraperitoneal injections of 0.1mol/L citrate buffer only.The diabetes group received a single intraperitoneal injection of STZ(50 mg/kg)dissolved in freshly prepared 0.1 mol/L sodium citrate buffer(pH 4.5)for five consecutive days.Only mice with fasting blood glucose(FBG)≥16.7 mmol/L after two week were considered successful in the diabetes model for subsequent experiments.Fasting blood glucose and body weight were measured weekly.1.2 All mice fasted overnight and were sacrificed under pentobarbital anesthesia four weeks later.The peripheral blood and terminal ileum tissues were taken.One intestinal tissue sample was fixed with 4%polymethanol and then paraffin embedded for pathological detection.The others were frozen in-80℃ for Western blot and qPCR detection.Serial sections of ileum tissue,about 4um thick,were stained with hematoxylin-eosin(H&E)staining to observe the morphology of intestinal tissue,immunohistochemical(IHC)staining to observe the expression of MFG-E8 protein in intestinal epithelial.Western blot was to detect the expression of MFG-E8,phospho-MLKL(p-MLKL)and HMGB1 protein,and RT-qPCR was to detect the expression of Gcg and Pcskl mRNA and inflammatory factor(TNF-α.IL-1β,IL-6)in intestinal tissue.2.In vitro study2.1 Intestinal epithelial endocrine cells(STC-1)were cultured in vitro and given different concentrations of AGEs(0,100,200,300,400 μg/ml)or pretreated with different concentrations of D-pinitol(40,60,80 μM)before adding AGEs 200μg/ml for 24-48h.MTT assay,hoechst33342/PI double staining,and Annexin V/PI flow cytometry were used to detect cell viability and cell necrosis.The expression of MFG-E8 protein was detected by Western blot.The expression of inflammatory factor(TNF-α,IL-1β,IL-6),damage-associated molecular patterns(HMGB1),and GLP-1 in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA).2.2 By constructing MFG-E8 silencing and overexpression transgenic strains,we used Western blot,ELISA,flow cytometry,immunocytochemical staining and other techniques to detect the expression of inflammatory factors and GLP-1 secretion in the cell supernatant of each group,and to detect the expression of RIPK1/RIPK3/MLKL pathway related proteins.3.Statistical AnalysisResults were expressed as means ± standard deviation(SD)or standard error of the mean(SEM).One-way Aanalysis of variance(ANOVA)was used for multiple comparisons and Student’s t test for comparison between groups.SPSS 20.0 and Graphpad Prism 8 software were used for data analysis and histogram plotting.All tests were bilateral tests.P value less than 0.05 was considered statistically significant.Results1.Results of in vivo study1.1 Body weight,FBG,and H&E staining in the intestinal tissues of mice STZ induced SAMR1/SAMP8 diabetes mice showed elevated blood glucose,weight loss,polydipsia,overeating,and polyuria.Compared with the control group,the structure of intestinal epithelial villi in SAMR1/SAMP8 diabetes mice was disordered.The villus became blunt and shorter,and the villus height/crypt depth ratio was evidently decreased.In addition,the expression of incretin related genes(Gcg and pcskl mRNA)were down-regulated and inflammatory factor related genes(TNF-α,IL-1β,and IL-6)were up-regulated in intestinal epithelium of diabetes mice compared with the control group(P<0.05).1.2 Histological findings and the expression of MFG-E8,p-MLKL and HMGB1 in the intestinal tissuesWe then performed immunohistochemistry and Western blot to explore the localization and expression of MFG-E8.The positive cells appeared brown in color and were mostly located on the surface of the intestinal villus and crypts of normal intestinal tissues.Compared with controls,diabetic mice demonstrated a significant decrease in MFG-E8-positive rate,with expression mainly reduced in the base of villus and crypts.Western blotting analysis showed that the protein expression of MFG-E8 was abundant in intestinal tissues of SAMR1/SAMP8 normal control mice,while significantly reduced in groups of STZ-induced diabetic mice(P<0.01).Since diabetic mice exhibited marked impairment in the intestinal epithelium,we measured the expression of p-MLKL and HMGB1,and found that necroptosis-related hallmarks of p-MLKL and HMGB1 were both elevated in diabetic SAMR1/SAMP8 mice(P<0.05).2.Results of in vitro study2.1 The expression of MFG-E8 in glycosylation injury of STC-1 cells and the protective effect of D-pinitolMTT assay was used to detect the effect of AGEs on the viability of STC-1 cells and detect the protective effect of D-pinitol.The results showed that the cell viability was decreased in a dose-dependent manner when STC-1 was exposed to AGEs(0,100,200,300,400μg/ml).Pretreatment with D-pinitol alleviated AGEs-induced decrease in cell viability.Results of Hoechst33342/PI staining and flow cytometry showed an increase in cell necrosis with AGEs stimulation,while pretreatment with different concentrations of D-pinitol attenuated the increase in cell necrosis.The protein expression of MFG-E8 was significantly decreased after AGEs stimulation,and D-pinitol pretreatment led to the upregulation of MFG-E8 compared with that in the normal control group and AGEs-treated group(P<0.05).HMGB1,as a critical cytokine of DAMPs,was significantly increased in supernatant of AGEs-treated STC-1 cells,accompanied by the increase in pro-inflammatory cytokines of TNF-α,IL-1β,and IL-6,while D-pinitol pretreatment decreased these levels(P<0.05).2.2 Molecular mechanism of MFG-E8 in inflammatory injury of STC-1 cells In this part,MFG-E8 gene silencing and overexpression constructs were performed to detect the possible mechanism of MFG-E8 in necrosis related inflammatory injury in STC-1 cells.The results showed that after MFG-E8 silencing,the viability of STC-1 cells decreased,accompanied by increase in necroptotic cells,HMGB1 and inflammatory factors(TNF-α,IL-1β,and IL-6),In addition,the overexpression of MFG-E8 attenuated AGEs-induced necrosis and release of pro-inflammatory factors.The secretion of GLP-1 in the supernatant was opposite to that of pro-inflammatory factors.ELISA results showed that MLKL pathway was involved in regulating the expression of inflammatory factors.Further detection in RIPK1/RIPK3/MLKL pathway revealed that MFG-E8 silencing or AGEs treatment activated RIPKs pathway,while overexpression of MFG-E8 attenuated the activation.The results indicate that MFG-E8 may regulate the necro-inflammatory response through the RIPKs pathway and then regulate the release of GLP-1 in STC-1 cells.Conclusions1.High glucose induced injury of the intestinal epithelium and low expression of MFG-E8 in diabetic mice,which contributed to the activation of MLKL pathways and the impairment of GLP-1 secretion function.2.AGEs could damage the function of intestinal endocrine cells through necrosis-related mechanisms in STC-1 cells,while D-pinitol had a protective effect.3.MFG-E8 may inhibit the necro-inflammatory response induced by AGEs through RIPK1/RIPK3/MLKL pathway in intestinal endocrine L cells,and then may have a protective effect on intestinal epithelial endocrine function...