PKM2调控的巨噬细胞糖酵解在糖尿病肾病炎症中的作用  

Role of pyruvate kinase M20-regulated glycolysis in inflammatory activation of macrophages in diabetic nephropathy

作　　者：谢姗 徐兴欣[2] XIE Shan;XU Xingxin(Department of Nephrology,Fourth Affiliated Hospital of Anhui Medical University,Anhui,Hefei Province,230031,China;Department of Nephrology,First Affiliated Hospital of Anhui Medical University,Anhui,Hefei Province,230031,China)

机构地区：[1]安徽医科大学第四附属医院肾内科,合肥230031 [2]安徽医科大学第一附属医院肾内科,合肥230031

出　　处：《第三军医大学学报》2020年第24期2394-2402,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81600568);安徽省自然科学基金青年项目(1708085QH178)。

摘　　要：目的探究在糖尿病肾病(diabetic nephropathy,DN)中M2型丙酮酸激酶(pyruvate kinase M2,PKM2)调控的糖酵解对Nod样受体蛋白3(nod-like receptor protein 3,NLRP3)和黑色素瘤缺乏因子2(absentin melanoma 2,AIM2)介导的巨噬细胞炎症激活的影响。方法(1)收取30例DN患者的血清和肾穿刺活检标本作为DN组,20例健康人血清标本和20例癌旁正常肾组织标本作为正常(Normal)组,通过免疫组化检测糖酵解标志物PKM2、巨噬细胞标志物CD68、炎症因子TNF-α的表达变化,ELISA检测血清TNF-α、IL-1β、MCP-1水平,激光共聚焦显微镜观察肾组织CD68与PKM2共表达。(2)选取小鼠骨髓来源巨噬细胞分组培养,通过简单随机化的方法分为甘露醇对照组、siRNA对照组、低糖对照组、高糖刺激组、高糖刺激+PKM2 siRNA组、高糖刺激+糖酵解抑制剂组;Western blot检测各组细胞PKM2、p-EIF2AK2、EIF2AK2、NLRP3、AIM2表达,ELISA及RT-PCR检测各组细胞MCP-1、IL-1β、TNF-α分泌及mRNA表达,乳酸测定试剂盒及葡萄糖吸收试剂盒检测各组细胞乳酸含量和葡萄糖吸收。结果与Normal组相比,DN患者的肾组织中PKM2表达增加(P<0.01),巨噬细胞浸润增多及炎症因子表达增多(P<0.01)。与Control组相比,高糖可以上调巨噬细胞PKM2、p-EIF2AK2、NLRP3、AIM2蛋白表达(P<0.01)及MCP-1、IL-1β、TNF-αmRNA的表达(P<0.01),增加细胞上清液中MCP-1、IL-1β、TNF-α、乳酸及葡萄糖水平(P<0.01),而PKM2 siRNA可以抑制高糖诱导的炎症。结论高糖可以刺激巨噬细胞的糖酵解水平增加,并促进炎症小体激活及炎症因子的释放,其机制与PKM2-EIF2AK2信号通路激活有关。Objective To explore the role of pyruvate kinase M20(PKM2)-regulated glycolysis on nod-like receptor protein 3(NLRP3)-and absentin melanoma 2(AIM2)-mediated inflammatory activation of macrophages in diabetic nephropathy(DN).Methods Renal biopsy samples from 30 DN patients and tumor-adjacent renal tissue samples from 20 renal cancer patients were collected for detection of the expressions of the glycolytic marker PKM2,the macrophage infiltration marker CD68 and tumor necrosis factor-α(TNF-α)using immunohistochemistry.The levels of TNF-α,interleukin-1β(IL-1β)and monocyte chemoattractant protein-1(MCP-1)in serum samples from the DN patients and 20 healthy volunteers were detected by ELISA.In cultured mouse bone marrow-derived macrophages,the effects of mannitol,high-glucose exposure,high-glucose exposure plus PKM2 siRNA,and high-glucose exposure plus glycolysis inhibitor on the expression of PKM2,p-EIF2 AK2,EIF2 AK2,NLRP3 and AIM2 were detected using Western blotting;the secretion and mRNA expression of TNF-α,IL-1βand MCP-1 were detected by ELISA and RT-PCR,and the lactate content and glucose absorption of the cells were assayed using commercial detection kits.Results Compared with the tumor-adjacent renal tissues,the renal tissues from DN patients showed significantly increased expression of PKM2,CD68 and TNF-α(P<0.01).In the cell cultures of macrophages,high glucose exposure obviously upregulated the cellular expression of PKM2,p-EIF2 AK2,NLRP3 and AIM2 proteins(P<0.01)and the mRNA expression of TNF-α,IL-1βand MCP-1(P<0.01)and increased the levels of TNF-α,IL-1β,MCP-1,lactic acid and glucose in the cell supernatant(P<0.01).Transfection of the cells with PKM2 siRNA significantly inhibited inflammatory responses of the macrophages induced by high-glucose exposure.Conclusion High glucose stimulation can enhance the level of glycolysis in macrophages and promote the activation of inflammasomes and release of inflammatory cytokines through a mechanism involving the activation of PKM2-EIF2 AK2 signaling path

关 键 词：糖尿病肾病 巨噬细胞 糖酵解 M2型丙酮酸激酶 炎性小体 

分 类 号：R392.32[医药卫生—免疫学] R587.2[医药卫生—基础医学]