摘要
目的探讨棕色脂肪源性神经调节蛋白4(NRG4)对糖尿病肾病(DN)小鼠炎症的保护作用。方法采用高脂饲料喂养联合腹腔注射链脲佐菌素建立DN小鼠模型,造模成功后将野生型(WT)和NRG4基因敲除(KO)小鼠分为WT-DN+绿色荧光蛋白(D-WT-GFP)组、KO-DN+绿色荧光蛋白(D-KO-GFP)组、KO-DN+NRG4(D-KO-NRG4)组,D-WT-GFP组和D-KO-GFP组在小鼠肩胛间区棕色脂肪一次性注射腺相关病毒绿色荧光蛋白,D-KO-NRG4组注射腺相关病毒转录NRG4。另设普通饲料喂养WT小鼠作为对照组(WT-CON)。每组6只。8周实验结束后,检测各组小鼠糖化血红蛋白(HbA1c)、血清甘油三酯(TG)、总胆固醇(TC)、游离脂肪酸(FFA)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、尿白蛋白/肌酐比值(UACR)、血清白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)等指标。采用过碘酸希夫染色和电镜观察足细胞损伤情况及肾组织病理改变。采用免疫组织化学法检测肾组织F4/80表达。采用实时荧光定量聚合酶链反应分析各组小鼠肾组织IL-1β、IL-6、TNF-αmRNA表达量。采用Western blotting法检测D-KO-GFP组与D-KO-NRG4组小鼠各组织NRG4蛋白表达水平。采用单因素方差分析比较组间差异。采用Pearson相关分析法分析血清NRG4水平与血清炎症因子、UACR、肾组织F4/80表达的相关性。结果与D-WT-GFP组相比,D-KO-GFP组小鼠UACR明显升高(P<0.01),肾小球面积增大,系膜基质增生和足细胞损伤,血清IL-1β、IL-6、TNF-α水平显著升高且肾组织中IL-1β、IL-6、TNF-α的mRNA表达也升高(P<0.01),同时肾组织F4/80表达增加(P<0.01)。此外,与D-WT-GFP组相比,D-KO-GFP组小鼠HbA1c、TG、TC、FFA、LDL-C水平升高且体重增加(P<0.01)。而与D-KO-GFP组相比,D-KO-NRG4组小鼠UACR水平降低(P<0.01),肾脏炎症指标F4/80表达降低(P<0.01),HbA1c、TG、TC、FFA、LDL-C水平下降且体重降低(P<0.01)。Pearson相关分析结果显示,血清NRG4与IL-1β、IL-6、TNF-α、UACR、肾组织F4/80表达呈负相关(r=-0.548、-0.637、-0.553、-0.503、-0.554,均P<0.05)。Western blotting结果显示,D-KO-NRG4组小鼠棕色脂肪中NRG4蛋白表达水平较高,而肝脏、肾脏、骨骼肌和白色脂肪中NRG4的表达水平较低(P<0.05)。结论棕色脂肪源性NRG4可以降低DN小鼠抑制肾脏炎症反应,降低蛋白尿,减轻DN肾脏损伤。
Objective To investigate whether brown adipose-derived neuregulin 4(NRG4)can alleviate diabetic nephropathy(DN)through inhibiting renal inflammatory response.Methods DN model in mice was induced by high-fat diet feeding combined with intraperitoneal injection of streptozotocin.After the model establishment,wild-type(WT)and NRG4 knockout(KO)mice were divided into WT-DN+green fluorescent protein group(D-WT-GFP),KO-DN+green fluorescent protein group(D-KO-GFP)and KO-DN+NRG4 group(D-KO-NRG4).Then,adeno-associated virus green fluorescent protein or adeno-associated virus NRG4 transcription was injected into the brown adipose tissue in the interscapular region of mice.WT mice feeding normal chow diet were used as control group(WT-CON)(6 mice in each group).After 8 weeks of the experiment,glycated hemoglobin A1c(HbA1c),triglyceride(TG),total cholesterol(TC),free fatty acid(FFA),low-density lipoprotein cholesterol(LDL-C),the ratio of urinary microalbumin to urine creatinine(UACR),serum interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected.The pathological glomerular changes and podocyte injury were observed by periodic acid Schiff staining and transmission electron microscopy.The expression of F4/80 in renal tissues was measured by immunohistochemistry.The mRNA of IL-1β,IL-6 and TNF-αin renal tissue were analyzed by real-time quantitative polymerase chain reaction.The expression level of NRG4 protein in multiple tissues of D-KO-GFP group and D-KO-NRG4 group was detected by Western blotting.One-way analysis of variance(ANOVA)was used for comparison between groups.Pearson correlation analysis was used to analyze the correlation of serum NRG4 and the expression of serum inflammatory factors,UACR and F4/80 in renal tissue.Results Compared with D-WT-GFP group,UACR was significantly increased in D-KO-GFP group(P<0.01),glomerular volume and mesangial matrix hyperplasia increased and podocyte injury,serum levels of IL-1β,IL-6 and TNF-αwere significantly increased,and mRNA expression of these inflammatory genes in renal tissue was also increased(all P<0.01),and the expression of F4/80 in renal tissue increased(P<0.01).In addition,compared with the D-WT-GFP group,in the D-KO-GFP group,the HbA1c,TG,TC,FFA,LDL-C and body weight increased(all P<0.01).Compared with D-KO-GFP group,D-KO-NRG4 group significantly decreased UACR level(P<0.01)and the expression of renal inflammation index F4/80 was decreased(P<0.01),the HbA1c,TG,TC,FFA,LDL-C and body weight reduced(all P<0.01).Pearson correlation analysis showed that serum NRG4 was negatively correlated with the expression of IL-1β,IL-6,TNF-α,UACR and F4/80 in renal tissue(r=-0.548,-0.637,-0.553,-0.503,-0.554,all P<0.05).Western blotting results showed that the expression level of NRG4 protein in brown fat of D-KO-NRG4 group was higher,but the expression level of NRG4 protein in liver,kidney,skeletal muscle and white fat was lower(all P<0.05).Conclusion Brown adipose-derived NRG4 can reduce renal inflammatory response,proteinuria and renal injury in DN mice.
作者
童佳悦
徐金玲
丁胜
徐晓丽
何凯悦
石凌峰
向光大
Tong Jiayue;Xu Jinling;Ding Sheng;Xu Xiaoli;He Kaiyue;Shi Lingfeng;Xiang Guangda(First School of Clinical Medicine,Southern Medical University,Guangzhou 510515,China;Department of Endocrinology,General Hospital of Central Theater Command of Chinese People′s Liberation Army,Wuhan 430070,China)
出处
《中华糖尿病杂志》
CAS
CSCD
北大核心
2023年第2期152-159,共8页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金(81870573,81570730)。
关键词
糖尿病肾病
炎症
神经调节蛋白4
棕色脂肪
Diabetic nephropathy
Inflammation
Neuregulin 4
Brown adipose tissue
作者简介
通信作者:向光大,Email:Guangda64@hotmail.com。
