格列齐特对糖尿病大鼠胰岛素抵抗及胰脏NLRP3炎症小体的影响

Effects of Gliclazide on insulin resistance and NLRP3 inflammasomes in pancreas of diabetes rats

目的探究格列齐特(Gliclazide,GL)对糖尿病大鼠胰岛素抵抗(insulin resistance,IR)及胰脏含NOD样受体家族Pyrin域蛋白3(Nod-like receptor family pyrin domain containing protein 3,NLRP3)炎症小体的影响。方法60只大鼠随机分为对照(Control)组、2型糖尿病(type 2 diabetes mellitus,T2DM)组、GL低剂量(GL low dose,GL-L)组和GL高剂量(GL high dose,GL-H)组,每组15只。采用高糖高脂喂养联合腹腔注射链脲佐菌素(streptozotocin,STZ)诱导T2DM大鼠模型。便携式血糖监测仪检测大鼠空腹血糖(fasting blood glucose,FBG),腹腔葡萄糖耐量试验(intraperitoneal glucose tolerance tests,IPGTT)检测血清胰岛素水平,评估胰岛素抵抗指数(homeostasis model assessment of insulin resistance,HOMA-IR);HE染色观察胰脏组织病理损伤;酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测血清炎性因子白细胞介素(interleukin,IL)-18、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;免疫组化检测胰脏组织NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)、半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate-specific proteinase-1,Caspase-1)水平;Western blot检测胰脏组织炎性因子NLRP3、ASC、Caspase-1水平。结果与Control组相比,T2DM组、GL-L组、GL-H组体重、FBG、IPGTT-AUC、胰岛素水平、HOMA-IR水平明显升高,差异有统计学意义(F=34.48、53.42、47.90、32.89、34.15,P值均<0.05)。HE染色结果显示,GL-L组、GL-H组大鼠较T2DM组炎症程度明显减轻,胰岛细胞形态较规则、胞浆充盈。ELISA结果显示,与Control组相比,T2DM组、GL-L组、GL-H组大鼠血清和胰腺组织炎性因子TNF-α、IL-1β、IL-18水平明显升高,差异有统计学意义(F=27.02、42.12、35.22、44.02、52.82、42.70,P值均<0.05);与T2DM组相比,GL-L组、GL-H组大鼠血清和胰腺组织炎症因子TNF-α、IL-1β、IL-18水平明显降低,差异有统计学意义[血清:pg/mL:(185.34±19.31)比(141.22±11.24)或(111.36±10.56),(268.41±16.34)比(175.67±7.16)或(125.67±7.33),(280.66±18.15)比(197.61±8.55)或(127.43±8.57);胰腺:(1.10±0.08)比(0.81±0.06)或(0.41±0.04),(1.12±0.07)比(0.65±0.07)或(0.23±0.03),(1.11±0.09)比(0.60±0.05)或(0.32±0.03),P值均<0.05]。免疫组化和Western blot结果显示,与Control组相比,T2DM组、GL-L组、GL-H组大鼠胰腺组织NLRP3、ASC、Caspase-1水平明显升高,差异有统计学意义(F=37.58、45.37、34.75、44.51、48.54、38.48,P值均<0.05)。选择1μmol/L胰岛素、48 h作为IR-HepG2细胞模型最佳浓度和作用时间。与Control组相比,IR组、GL-L组、GL-H组细胞葡萄糖消耗量和糖原含量明显降低,差异有统计学意义(F=17.84、15.58,P值均<0.05)。Western blot结果显示,与Control组相比,IR组、GL-L组、GL-H组细胞NLRP3、ASC、Caspase-1水平明显升高,差异有统计学意义(F=47.74、41.21、46.74,P值均<0.05);与IR组相比,GL-L组、GL-H组细胞NLRP3、ASC、Caspase-1水平明显降低,差异有统计学意义[(0.85±0.05)比(0.68±0.04)或(0.20±0.04),(0.60±0.05)比(0.40±0.04)或(0.20±0.03),(0.93±0.06)比(0.72±0.05)或(0.25±0.03),P值均<0.05]。结论GL能够改善T2DM大鼠IR,抑制胰腺NLRP3炎症小体表达。

Objective To explore the effects of Gliclazide(GL)on insulin resistance(IR)and inflammasomes Nod-like receptor family pyrin domain containing protein 3(NLRP3)in pancreas of diabetes rats.Methods Sixty rats were randomly divided into Control group,type 2 diabetes mellitus(T2DM)group,GL low dose(GL-L)group and GL high dose(GL-H)group,fifteen per group.T2DM rats model were induced by intraperitoneal injection of streptozotocin(STZ)in combination with high fat and high glucose.Portable blood glucose monitor was used to detect fasting blood glucose(FBG),intraperitoneal glucose tolerance tests(IPGTT)was used to measure serum insulin level,and homeostasis model assessment of insulin resistance(HOMA-IR)was evaluated.HE staining was used to observe pancreatic pathological damage.Enzyme linked immunosorbent assay(ELISA)was used to detect inflammatory factors interleukin(IL)-18,IL-1β,tumor necrosis factor-α(TNF-α)levels in serum.Immunohistochemistry was used to detect NLRP3,apoptosis-associated speck-like protein containing CARD(ASC),cysteinyl aspartate-specific proteinase-1(Caspase-1)levels in pancreatic tissue.Western blot was used to detect inflammatory factors NLRP3,ASC and Caspase-1 levels in pancreatic tissue.Results Compared with Control group,body weight,FBG,IPGTT-AUC,serum insulin and HOMA-IR levels in T2DM group,GL-L group and GL-H group were significantly increased(F=34.48,53.42,47.90,32.89,34.15,all P values<0.05).HE staining results showed that compared with the T2DM group,the inflammation degree was significantly reduced,and the morphology of pancreatic islet cells was more regular and the cytoplasm was filled in GL-L group and GL-H group rats.ELISA results showed that,compared with Control group,inflammatory factor TNF-α,IL-18,IL-1βlevels in serum and pancreatic tissue in T2DM group,GL-L group and GL-H group were significantly increased(F=27.02,42.12,35.22,44.02,52.82,42.70,44.51,48.54,38.48,all P values<0.05).Compared with T2DM group,inflammatory factor TNF-α,IL-18,IL-1βlevels in serum and pancreatic tissue in GL-L group and GL-H group were significantly decreased[Serum:pg/mL:(185.34±19.31)vs(141.22±11.24)or(111.36±10.56),(268.41±16.34)vs(175.67±7.16)or(125.67±7.33),(280.66±18.15)vs(197.61±8.55)or(127.43±8.57);Pancreas:(1.10±0.08)vs(0.81±0.06)or(0.41±0.04),(1.12±0.07)vs(0.65±0.07)or(0.23±0.03),(1.11±0.09)vs(0.60±0.05)or(0.32±0.03),all P values<0.05].Immunohistochemical and Western blot results showed that,compared with Control group,NLRP3,ASC,Caspase-1 levels in pancreatic tissue in T2DM group,GL-L group and GL-H group were significantly increased(F=37.58,45.37,34.75,44.51,48.54,38.48,all P values<0.05).1μmol/L insulin and 48 h were selected as optimal concentration and duration of action for IR-HepG2 cell model.Compared with Control group,Glucose consumption and glycogen content in IR group,GL-L group and GL-H group were significantly decreased(F=17.84,15.58,both P values<0.05).Western blot results showed that,compared with Control group,NLRP3,ASC and Caspase-1 levels in IR group,GL-L group and GL-H group were significantly increased(F=47.74,41.21,46.74,all P values<0.05).Compared with IR group,NLRP3,ASC and Caspase-1 levels in GL-L group and GL-H group were significantly decreased[(0.85±0.05)vs(0.68±0.04)or(0.20±0.04),(0.60±0.05)vs(0.40±0.04)or(0.20±0.03),(0.93±0.06)vs(0.72±0.05)or(0.25±0.03),all P values<0.05].Conclusion GL can improve the IR of T2DM rats and inhibit the expression of NLRP3 inflammasomes in pancreas.