| Section 1 Correlation between irisin and newly diagnosed type 2 diabetes mellitusObjective:To investigate the correlation between irisin and newly diagnosed Type 2 diabetes mellitus.Methods:Forty newly diagnosed T2DM patients in the Department of cadre Health Care of the First People’s Hospital of Yunnan Province from July 2019 to January 2020 were collected as the T2DM group.During the same period,40 healthy subjects in the physical examination center of the hospital were collected as the control group(NC).Both groups were measured for physical indexes,metabolic indexes were detected by automatic biochemical analyzer.Serum concentrations of irisin and inflammatory cytokines interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and interferon-y(IFN-y)were detected by Enzyme-linked immunosorbent assay(ELISA).The serum irisin level between the T2DM group and the NC group was compared,and the correlation of irisin with physical indexes,metabolic indexes,inflammatory cytokines and the effect of irisin on the occurrence of T2DM were analyzed.Results:1.Serum irisin level in T2DM group was significantly lower than that in NC group(P<0.05).2.Serum irisin was negatively correlated with fasting plasma glucose(FPG)、2 h plasma glucose(2hPG)、glycated hemoglobin(HbA1c)、homeostasis model assessment-insulin resistance(HOMA-IR)、IL-1β and IFN-γ(P<0.01).3.There was no significant correlation with age,height,weight,blood pressure,lipid,homeostasis model assessment-β(HOMA-β),IL-6 and TNF-α(P>0.05).4.Irisin is an protective factor for the occurrence of T2DM(P<0.01).Conclusion:Serum irisin level decreased in newly diagnosed T2DM patients.Irisin is closely related to glucose metabolism,insulin resistance and inflammation.It may play a protective role in the pathogenesis of T2DM.Section 2 Effects and mechanism of irisin on glucolipid metabolism,pancreatic β cell function and islet inflammatory injury in T2DM miceObjective:Through the intervention of irisin on T2DM model mice in vivo,the effects of irisin on glucolipid metabolism,pancreatic β cell function and islet inflammatory injury were preliminatively elucidated,and the mechanism was explored from the perspective of inflammation.Methods:1.Animal modeling and grouping:Fifty C57BL/6J mice aged 6 weeks were randomly divided into 5 groups with 10 mice in each group:NC group,T2DM group,T2DM+irisin group,T2DM+pcDNA-IL-1RI group and T2DM+irisin+pcDNA-IL-1RI group.T2DM model m ice were established by fed high fat diet(HFD)for 8 weeks and then one time intraperitoneal injection of streptozotocin(STZ)at 35 mg/kg body weight.NC group was fed normal diet for 8 weeks,then intraperitoneal injection with 0.1mol/L sodium citrate buffer at the same volume as STZ.After the successful built of T2DM model mice,an overexpressed IL-1RI vector of lentivirus 50μL with a titer of 1×108TU/mL was injected every other day for 14 days into the caudal vein of T2DM+pcDNA-IL-1RI group and T2DM+irisin+pcDNA-IL-1RI group mice to build a T2DM mice model with overexpression of IL-1RI.The other three groups were injected with same volume normal saline through caudal vein once every other day for 14 days.After 1 week,mice in T2DM+irisin group and T2DM+irisin+pcDNA-IL-1RI group were intraperitoneally injected with irisin at 0.5 mg/kg body weight once a day for 14 days.The other three groups were intraperitoneally injected with same volume normal saline once a day for 14 days.2.Detection of indicators:At the end of the experiment,the body weight of each group was measured by fasting for 12h.Serum samples were collected and the concentrations of irisin,FPG,FINS,TG,TC,IL-1β,IL-6,TNF-α and IFN-γ were detected by ELISA.Intraperitoneal glucose tolerance test was performed to evaluate βcell function.The pancreas were removed after the mice were put to death.The islet tissue structure of mice was observed by hematoxylin-eosin(HE)staining,the expression of insulin in islet was observed by immunohistochemistry.After separation of islet,the expressions of IL-1RI,inflammatory cytokines IL-1β,IL-6,TNF-α,IFN-γin islet were detected by ELISA.Apoptosis-related protein B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2 assaciated X protein(Bax),Caspase-3,signaling pathway protein myeloid differentiation factors 88(MyD88),and the levels of nuclear factor kappa B(NF-κB),c-Jun N-terminal kinase(JNK),mitogen-activated protein kinase(MAPK)P38,extracellular signal-regulated kinase(ERK)phosphorylation in islet were detected by Western blot.Results:1.Compared with the NC group,the serum irisin concentration of T2DM group was significantly reduced(P<0.001).The serum irisin concentration in T2DM+pcDNA-IL-1RI group was further decreased(P<0.05).2.Exogenous administration of irisin significantly decreased body weight,FPG,TG,TC,serum concentrations of inflammatory cytokines IL-1β,IL-6,TNF-α,IFN-γ and increased FINS,improve intraperitoneal glucose tolerance,reduce the area under the IPGTT curve in T2DM mice and T2DM mice with overexpression of IL-1RI(P<0.05).These results suggested that irisin can improve the disorder of glucolipid metabolism,alleviate systemic inflammation,improve the function of pancreatic β cells and promote insulin secretion in T2DM mice and T2DM mice with overexpression of IL-1RI.HE staining observed the islet structure,and it was found that exogenous administration of irisin could reduce the islet damage and restore the islet morphology,increase the number of islets and reduce the infiltration of inflammatory cells in T2DM mice and T2DM mice with overexpression of IL-1RI.In addition,immunohistochemistry showed that irisin could increase the expression of insulin in islets of T2DM mice and T2DM mice with overexpression of IL-1RI(P<0.01),which suggested that irisin can improve islet injury and promote insulin secretion in T2DM mice and T2DM mice with overexpression of IL-1RI.4.Exogenous administration of irisin significantly reduced the level of IL-1RI,IL-1β,IL-6,TNF-α,and IFN-y,increased the expression of Bcl-2,decreased the expression of Bax and Caspase-3 in islets of T2DM mice and T2DM mice with overexpression of IL-1RI(P<0.05),which suggested that irisin can reduce the inflammation of islet and inhibit apoptosis in T2DM mice and T2DM mice with overexpression of IL-1RI.5.Exogenous administration of irisin decreased the expression levels of MyD88,P-NF-κB,P-JNK,P-P38 MAPK and P-ERK in islet of T2DM mice and T2DM mice with overexpression of IL-1RI(P<0.05).Compared with the T2DM+irisin+pcDNA-IL-1RI group and the T2DM+irisin group,the beneficial effects of irisin on glucolipid metabolism,systemic inflammatory,β cell function and suppression of islet inflammation in T2DM mice were reduced by the overexpression of IL-1RI(P<0.05).It is suggested that irisin may play a role in further downregulating MyD88-NF-κB/JNK/P38 MAPK/ERK signaling pathway by downregulating IL-1RI expression in islet.Conclusion:Irisin can reduce the disorder of glucolipid metabolism,systemic inflammatory response and improve the function of pancreatic β cells in T2DM mice.The mechanism may be through downregulating the expression of IL-1RI in islet of T2DM mice and then further inhibiting IL-1RI-MyD 88-NF-κB/JNK/P3 8 MAPK/ERK signaling pathway to alleviate the inflammatory response of islet,inhibit cell apoptosis and promote insulin secretion.Section 3 The effect and mechanism of irisin on the inflammatory injury of pancreatic β-cell induced by hyperglycemicObjective:To explore the effect and mechanism of irisin on inflammatory injury of pancreatic β cells induced by hyperglycemic.Methods:1.The effects of irisin on IL-1RI expression and cell viability:Human pancreatic β cell line 1.1 B4 cells were treated with different concentrations of irisin for different times before cultured under the effect of high glucose(HG).The expression of IL-1RI in cells was detected by Western-blot to determined the effect of irisin on IL-1RI expression.The dose-effect relationship and time-effect relationship of irisin were observed.The proliferation activity of cells was detected by CCK-8 to determine the effect of irisin on cell proliferation.2.Cell grouping and treatment:The cells were divided into NC group,HG group,HG+irisin group,HG+pcDNA-IL-1RI group and HG+irisin+pcDNA-IL-1RI group.NC group:1.1 B4 cells were cultured in DMEM medium for 24h.HG group:1.1 B4 cells were cultured in DMEM HG medium(25 mmol/L glucose)for 24 h.HG+irisin group:1.1 B4 cells were pretreated with 100 ng/mL irisin for 24h and then cultured in DMEM HG medium for 24h.HG+pcDNA-IL-1RI group:Lentivirus vector overexpressing IL-1RI infected 1.1 B4 cells and 1.1B4 cells overexpressing IL-1RI were built and then cultured in DMEM HG medium for 24 h.HG+irisin+pcDNA-IL-1RI group:The 1.1B4 cells overexpressing IL-1RI were pretreated with 100 ng/mL irisin for 24h and then cultured in DMEM HG medium for 24h.3.Detection of indicators:Western blot was used to detect the expression levels of IL-1RI,IL-1β;CCK-8 was used to detect cell proliferation activity;Annexin-V/PI was used to detect cell apoptosis rate;ELISA was used to detect glucose stimulated insulin secretion(GSIS);MyD88 and the phosphorylation level of NF-κB,JNK,P38 MAPK,ERK in each group were measured by western blot.4.The role of MyD88 in IL-1RI mediated signaling pathway:Cells that overexpressed IL-1RI were treated with ST2825,an inhibitor of MyD88,and then cultured in DMEM HG medium for 24h.The expression levels of IL-1β were detected by Western-blot,the proliferation activity of cells was detected by CCK-8,and the apoptosis level was detected by Annexin V/PI,GSIS was detected by ELISA in HG group,HG+pcDNA-IL-1RI group and HG+ST2825+pcDNA-IL-1RI group.Results:1.The expression of IL-1RI in HG group was significantly higher than that in NC group(P<0.01).Irisin inhibited the expression of IL-1RI in 1.1 B4 cell under HG.Irisin treatment with 100 ng/mL for 24h had the most significant inhibitory effect on IL-1RI(P<0.01),the inhibition rate was 65%.Irisin promoted the proliferation of 1.1 B4 cells under HG.Irisin treatment at 100 ng/mL for 24h had the most significant effect on promoting cell proliferation(P<0.001).2.Compared with NC group,the expression of IL-1β was significantly increased(P<0.001),cell proliferation was significantly decreased(P<0.05),apoptosis was increased(P<0.01)and GSIS was decreased(P<0.01)of HG group.Irisin treatment could reverse the changes induced by HG.Compared with HG group,the.expression level of IL-1RI and IL-1β in HG+pcDNA-IL-1RI group was significantly increased(P<0.05),cell proliferation were significantly decreased(P<0.05),cell apoptosis level was significantly increased(P<0.05),GSIS were significantly decreased(P<0.01).Irisin treatment significantly down-regulated the expression of IL-1RI(P<0.05),and reduced the promotion of IL-1β expression,inhibition of proliferation,promotion of apoptosis,inhibition of GSIS by overexpression of IL-1RI(P<0.05).Meanwhile,the inhibitory effect on IL-1β expression and protective effect on cells of irisin were decreased due to the overexpression of IL-1RI(P<0.05).These results suggested that irisin inhibits the expression of IL-1β,promotes cell proliferation,inhibits cell apoptosis and promotes GSIS by down-regulating IL-1RI in cells.3.The expression of MyD88 protein,p-NF-κB,p-JNK,p-p38 MAPK and p-ERK were increased in HG group(P<0.01).Irisin significantly reduced the promotion effect of HG on MyD88,p-NF-κB,p-JNK,p-p38 MAPK and p-ERK(P<0.05).In addition,irisin inhibited the expression of MyD88,p-NF-κB,p-JNK,p-P38 MAPK and p-ERK induced by IL-1RI overexpression(P<0.05).The MyD88 inhibitor ST2825 significantly reduced the promotion of IL-1β expression,the inhibition of proliferation,promotion of apoptosis,inhibition of GSIS cells induced by overexpression of IL-1RI(P<0.05).These results suggested that irisin may play a protective role in cells by inhibiting the expression of IL-1RI in β cells,and then further inhibiting the activation of IL-1RI-MyD88-NF-κB/JNK/P38 MAPK/ERK signaling pathway.Conclusion:By down-regulating the expression of IL-1RI in pancreatic β cells,irisin can further inhibit IL-1RI-MyD88-NF-κB/JNK/p38 MAPK/ERK signaling pathway,reduce the inflammation of cells,promote cell proliferation,inhibit cell apoptosis,increase GSIS,play a protective role on β cells. |
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