萝卜硫素通过调节ALOX5/NF-κB信号通路调控巨噬细胞糖酵解抑制糖尿病肾病进展  

Sulforaphane regulates macrophage glycolysis and inhibits the progression of diabetic nephropathy by modulating the ALOX5/NF-κB signaling pathway

作　　者：乌日娜[1] 丁海东[1] 常宏[1] 孙娜娜[1] 张磊[1] Wu Rina;Ding Haidong;Chang Hong;Sun Nana;Zhang Lei(Dept of Endocrinology,Affiliated Hospital of Inner Mongolia University for Nationalities,Tongliao City,Inner Mongolia 028000)

机构地区：[1]内蒙古民族大学附属医院内分泌科,通辽028000

出　　处：《安徽医科大学学报》2024年第3期390-397,共8页Acta Universitatis Medicinalis Anhui

基　　金：2023年度内蒙古自治区高校科研项目(编号:NJZY23104)。

摘　　要：目的探讨萝卜硫素(SFN)调节花生四烯酸5-脂氧合酶基因(arachidonic acid 5-lipoxygenase,ALOX5)/核因子kappa B(NF-κB)信号通路调节巨噬细胞糖酵解对糖尿病肾病(DN)进展的影响。方法生物信息学分析SFN治疗DN的靶基因。使用30 mmol/L高葡萄糖(HG)处理人近端肾小管上皮细胞系(HK-2细胞)诱导体外DN模型。将HK-2细胞分为如下组:正常糖(NG)组、HG组、HG+SFN(3 mmol/L)组、HG+ALOX5组、HG+SFN(3 mmol/L)+ALOX5组、HG处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)处理的巨噬细胞+HK-2细胞组、HG+ALOX5转染处理的巨噬细胞+HK-2细胞组、HG+SFN(3 mmol/L)+ALOX5转染处理的巨噬细胞+HK-2细胞组。CCK-8检测细胞活力,原位末端脱氧核苷酸转移酶标记(TUNEL)法检测细胞凋亡;葡萄糖和乳酸试剂盒检测各组细胞中葡萄糖和乳酸水平;Western blot检测各组细胞中ALOX5、NF-κB以及糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达;使用链脲佐菌素(STZ)构建DN小鼠模型,DN小鼠给与SFN(0.5 mg/kg)治疗;检测小鼠各项生化指标,HE染色检测肾组织病理变化;Western blot检测小鼠肾脏巨噬细胞中糖酵解相关蛋白己糖激酶-2(HK2)、丙酮酸激酶M2(PKM2)、葡萄糖转运蛋白1(GLUT1)的表达。结果生物信息学分析结果显示ALOX5是SFN治疗DN的靶基因。与HG组相比,SFN处理增强HK-2细胞活力并抑制细胞凋亡(P<0.05);同时,SFN处理抑制HG诱导的巨噬细胞糖酵解相关蛋白的表达,减弱巨噬细胞介导的HK-2细胞损伤(P<0.05);Western blot结果表明SFN抑制ALOX5和NF-κB的表达(P<0.05);小鼠实验结果显示,SFN治疗改善DN小鼠肾功能和肾组织病理学改变,抑制肾组织中巨噬细胞糖酵解相关蛋白的表达(P<0.05)。结论SFN通过抑制ALOX5/NF-κB信号通路抑制巨噬细胞糖酵解从而改善DN进展。Objective To investigate the effects of sulforaphane(SFN)in regulating the macrophage glycolysis via the arachidonate 5-lipoxygenase(ALOX5)/nuclear factor kappa B(NF-κB)signaling pathway on the progression of diabetic nephropathy(DN).Methods Bioinformatics analysis was used to identify the target genes of SFN in the treatment of DN.Human proximal tubular epithelial cell line(HK-2 cells)was induced with 30 mmol/L high glucose(HG)to create an in vitro model of DN.HK-2 cells were divided into the following groups:normal glucose(NG)group,HG group,HG+SFN(3 mmol/L)group,HG+ALOX5 group,HG+SFN(3 mmol/L)+ALOX5 group,HG-treated macrophages+HK-2 group,HG+SFN(3 mmol/L)treated macrophages s+HK-2 group,HG+ALOX5 transfection treated macrophages+HK-2 group,HG+SFN(3 mmol/L)+ALOX5 transfection treated macrophages+HK-2 group.CCK-8 assay was used to detect cell viability,Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)method was used to detect cell apoptosis;glucose and lactate levels in the cells were measured using assay kits;Western blot was performed to detect the expression of ALOX5,NF-κB,and glycolysis-related proteins hexokinase-2(HK2),pyruvate kinase M2(PKM2),glucose transporter 1(GLUT1)in each group.Diabetic nephropathy(DN)mouse models were established using streptozotocin(STZ)and treated with SFN(0.5 mg/kg).Various biochemical parameters were measured in the mice,and kidney tissue pathology was examined using H&E staining.Western blot was used to detect the expression of glycolysis-related proteins(HK2,PKM2,GLUT1)in kidney macrophages.Results Bioinformatics analysis revealed ALOX5 as the target gene of SFN in treating DN.Compared to the HG group,SFN treatment enhanced HK-2 cell viability and inhibited apoptosis(P<0.05);concurrently,SFN treatment suppressed HG-induced macrophage glycolysis-related protein and attenuated macrophage-mediated HK-2 cellular injury(P<0.05).Western blot results showed that SFN inhibited the expression of ALOX5 and NF-κB(P<0.05).The mouse experiment results showed th

关 键 词：萝卜硫素 糖尿病肾病 巨噬细胞 糖酵解 花生四烯酸5-脂氧合酶 NF-ΚB信号通路 

分 类 号：R587.2[医药卫生—内分泌]