Methionine Enkephalin Regulates The Immune Pathogenesis Of Type 2 Diabetes Mellitus Via The IL-33/ST2 Pathway

The incidence and mortality of type 2 diabetes mellitus?T2DM?rank among the top ten worldwide.T2DM often leads to disability from cardia-cerebrovascular diseases,blindness,limb amputation and other effects and poses a serious health and economic challenge worldwide^[1].A survey data shows that the standardized prevalence of diabetes among Chinese adults is 10.9%,among which the proportion of T2DM reached over 90%^[2].Another study shows that there are 98.4 million adults with diabetes in China and the largest number of diabetes patients in China,the number will reach 142.7 million by2035^[3].Thus,strategies designed to control the disease development are urgently needed.The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years,and the role of metabolic-immune imbalance in insulin resistance?IR?and islet?-cell damage in T2DM has gradually received attention^[4,5].Immunemetabolism is a novel therapeutic target to control metabolism in many diseases,including cancer,diabetes and obesity.In addition,an important study elucidated the link between inflammation and glucose metabolism,and T2DM has been widely recognized as a chronic inflammatory disease^[6].T2DM results from the generalized activation of the innate immune system,in which chronic cytokine-mediated inflammation exists^[7,8].Therefore,both the immune system and inflammatory mechanisms are involved in the occurrence and development of T2DM.Based on accumulating evidence,endogenous opioid peptides and opioid binding sites are expressed in the endocrine pancreas of several animal species,including humans^[9,10].Endogenous opioid peptides exert their effects via binding to opioid receptors,which suggesting that these peptides may also play a role in pancreatic secretion and subsequently influence glucose metabolism.Methionine enkephalin?MENK?is an endogenous opioid peptide composed of Tyr-Ala-Ala-Phe-Met that is derived from proenkephalin.MENK is an important mediator of communication between the immune and neuroendocrine systems and modulates both the innate and adaptive immune via binding to opioid receptors:delta?d-,DOR?,kappa?k-,KOR?and mu?m-,MOR?^[11].MENK,as an immune regulatory factor,can activate and regulate the function of dendritic cells,macrophages and CD4⁺T cells,and epithelial and mesenchymal cells[12-16].For several years,the development of autoimmune chronic inflammatory diseases has been attributed to the activity of autoreactive CD4⁺T helper cells^[17,18].Na?ve Th0 cells differentiate into specific Th subsets?Th1,Th2,Th17,Th22,Treg etc.?under the influence of cytokines^[19].Th1 cells,which produce pro-inflammatory cytokines?TNF-a,IL-2,IFN-??,support cell-mediated immunity and consequently promote inflammation,cytotoxicity,and delayed-type hypersensitivity.Th2 cells secrete anti-inflammatory cytokines?IL-4,IL-5,IL-10,and IL-13?,support humoral immunity and ameliorate the inflammatory actions of Th1 cells^[20-22].The Th1/Th2 balance is very important for maintaining the steady-state immunity in the organism.According to a recent study,islet mesenchymal-cell-derived IL-33 orchestrates immunometabolic crosstalk in pancreatic islets.When exposed to diabetic conditions?stimulated by glucose,IL-1?and palmitic acid?,the production of IL-33 by islet mesenchymal cells was significantly enhanced^[23].Group 2 innate lymphoid cells?ILC2s?are considered the first cells to activate the Th2 immune response,serve as a bridge between innate immunity and adaptive immunity,amplify the strength of the body's immune response to damage,and at the same time facilitate the formation of the adaptive immune response^[24].IL-33-responsive cells in islets are resident ILC2s.IL-33 induces Th2 responses by binding to ST2.The IL-33/ST2 pathway prevents an inappropriate,parasite-specific Th1-polarized response and induces IL-4 and IL-13 production.Relevant studies have also confirmed that ILC2s can produce MENK,and in vitro experiments have found that IL-33 can stimulate ILC2s to increase MENK secretion^[24].It has been shown that ST2 may be a potent negative regulator of Toll-like receptor?TLR?signaling,TLR act via MyD88 and TRAF,triggering NF-?B p65 activation,and promoting cytokine secretion and inflammatory responses^[25].The upregulation of ST2on dendritic cells may make them refractory to pro-inflammatory,maturation-inducing stimuli^[26].However,little is known about the influence of MENK on diabetes to date.Objective:To study the effects of MENK on glucose level and insulin secretion from the pancreas of T2DM rats,and on destroyed INS-1 cells induced by hyperglycemia and hyperlipidemia.To further explore the MENK's possible effect mechanism.It is necessary to determine whether MENK exerts its immunomodulatory effect by binding to the opioid receptors,and its influence on immunological Th1 and Th2.To explore the effect of MENK on IL33/ST2 pathway to regulate inflammation,clarify the molecular mechanism of MENK on T2DM and provide novel ideas and theoretical basis for T2DM.This study is divided into the following three parts:Part I:The research on the relationship between T2DM and inflammation,immune abnormalities.Part ?:The intervention effect of MENK on inflammation and immune abnormality in T2DM.Part ?:The role of IL-33/ST2 pathway in the regulation inflammation and immune abnormalities effect of MENK in T2DM.Methods:1.High-glucose and high-fat diet combined intraperitoneal injection of low-dose streptozotocin?STZ?was used to establish the T2DM rat model;2.Rats in the normal group and the T2DM group were randomly subdivided into the control group,MENK group,MENK+naltrexone?NTX?group;3.Body weight of rats were recorded,and tail vein blood glucose was measured by glucometer;4.Rat pancreas pathological changes were observed by hematoxylin-eosin?HE?staining;5.Immunohistochemical?IHC?staining was used to study the expression sites and content of three opioid receptors in pancreatic tissue;6.INS-1 cells were cultured in high-glucose and high-fat medium to induce?-cell injury in vitro,and cells were cultured in indifferent media which added MENK and NTX;7.Glucose stimulated insulin secretion?GSIS?was used to evaluate the functional damage of INS-1 cells stimulated by high glucose and high fat in vitro;8.Enzyme Linked Immunosorbent Assay?ELISA?was used to detect the content of insulin,TNF-a,Interleukin?IL?-2,IL-5 and IL-10 in serum of rats and in INS-1 cell culture supernatant;9.Real-time quantitative reverse transcription polymerase chain reaction?real-time RT-PCR?was used to detect the opioid receptors?MOR,DOR and KOR?,IL33/ST2 and TLR pathway related molecule-MyD88,TRAF6 and NF-?B p65 mRNA expression in the rat pancreatic tissue and in the INS-1 cells;10.Western blot was used to detect the expression levels of the opioid receptors?MOR,DOR and KOR?,IL33/ST2 and TLR pathway related molecule-MyD88,TRAF6 and NF-?B p65 proteins expression in rat pancreatic tissue and INS-1 cells.Results:1.Effects of MENK on rats1.1 High-glucose and high-fat diet combined low dose of STZ intraperitoneal injection successfully established the T2DM rat model.The plasma glucose of rats increased to meet the model standard.The typical diabetic symptoms of polyuria,polydipsia,polyphagia and weight loss were obvious,and the serum insulin level decreased in T2DM rats compared with that in the normal rats.Intraperitoneal injection of MENK with different doses reduced the plasma glucose in T2DM rats.At the same time,MENK improved the weight loss caused by T2DM and increased the serum insulin level in T2DM rats,the difference was statistically significant.1.2 The effects of MENK on morphological changes in rat pancreatic tissue:based on the results of HE staining of pancreatic tissues,islets in the normal group contained spherical endocrine cells with a uniform size,clear nuclear chromatin,cytoplasm boundaries and connective tissue.Compared with those in the normal group,the islet?-cells in the T2DM rats were sparse and displayed a disorderly arrangement.Fewer?-cells with a different morphology and unclear cytoplasm boundaries were observed.After MENK intervention,the number of cells significantly increased and the edges were rounder,the cytoplasm was slightly uneven,the nucleoli were clear and the cells were arranged evenly.Thus,MENK intervention improved the morphology of pancreatic tissue and maintained its normal function.1.3 The expression level of opioid receptors in the pancreas of rats:according to the results of immunohistochemical staining of opioid receptors,the opioid receptors?DOR,MOR and KOR?all expressed in the pancreatic tissue,mainly in the cell membrane,in which the KOR expression level was very low.Combined with results of immunohistochemical staining,real-time RT-PCR and western blot showed that the expression level of opioid receptor significantly decreased in T2DM rats than that in the normal group.MENK significantly increased the expression level of opioid receptor in T2DM rats,and the difference was statistically significant.1.4 The mRNA and protein expression levels of IL-33/ST2 pathway and TLR pathway related molecules-MyD88,TRAF6 and NF-?B p65 increased in the pancreas of T2DM rats than those in the control group.MENK down-regulated the mRNA and protein expressions of what is said above molecules in the pancreas of T2DM rats.1.5 Obvious disorder of Th1/Th2 cytokines balance were observed in the serum of T2DM rats,as Th1 cytokines TNF-a and IL-2 increased meanwhile Th2 cytokines IL-5and IL-10 decreased.MENK regulated the balance of serum Th1/Th2 cytokines,reduced the contents of TNF-a and IL-2,and increased the contents of IL-5 and IL-10.2.Effects of MENK on INS-1 cells2.1 The INS-1 cells were cultured in high-glucose and high-fat media showed significant glycolipidtoxicity,the number of cells decreased and the secreted insulin decreased.After combined MENK culture,MENK significantly increased the number of cells,improved cell morphology,and increased the level of insulin.The GSIS assay also confirmed that MENK improved INS-1 cell insulin secretion disorder caused by glycolipidtoxicity.2.2 MENK increased the expression level of opioid receptors in INS-1 cells cultured with high-glucose and high-fat.MENK also decreased the mRNA and protein expression levels of IL-33/ST2 pathway and TLR pathway related molecules-MyD88,TRAF6 and NF-?B p65.MENK significantly regulated the balance of Th1/Th2 cytokines secreted by INS-1 cells.These results were consistent with the results in vivo.Conclusion:MENK reduced plasma glucose level and stimulated insulin secretion from the pancreas by regulating the immune response and inhibiting the inflammatory response after the combination with opioid receptors.MENK may play a potential clinical application as a novel method to treat T2DM and assist other hypoglycemic therapies.