SIRT1-PGC-1α-NRF2通路探讨金樱子多糖介导氧化应激改善糖尿病肾脏病的作用机制

Exploring the Mechanisms of Rosa laevigata polysaccharides in Treating Diabetic Kidney Disease Based on the SIRT1-PGC-1α-NRF2 Signaling Pathway through Oxidative Stress

关键词:  糖尿病肾脏病  金樱子多糖  沉默调节蛋白1  过氧化物酶体增殖物激活受体γ共激活因子1α  核因子类红细胞相关因子2  氧化应激

Key words:  diabetic kidney disease  Rosa laevigata polysaccharides  silencing regulatory protein 1  peroxisome proliferator-activated receptor gamma coactivator 1alpha  nuclear factor-like erythroid-associated factor 2  oxidative stress

摘要:

[目的] 研究金樱子多糖（RLP）治疗糖尿病肾脏病（DKD）的效果并从沉默调节蛋白1-过氧化物酶体增殖物激活受体γ共激活因子1α-核因子类红细胞相关因子2（SIRT1-PGC-1α-NRF2）信号通路的角度探究其作用机制。[方法] 60只C57BL/6J小鼠适应性喂养1周，除正常组（C组）10只基础饲料喂养外，其余均予高糖高脂饮食8周，之后链脲佐菌素（STZ）造模并随机平均分为模型组（M组）、SIRT1激动剂组（S组）、低剂量RLP组（RL组）、中剂量RLP组（RM组）、高剂量RLP组（RH组）。连续灌胃8周，每2周分别测定各组小鼠空腹血糖（FBG）及体质量。治疗8周后，收集小鼠24 h尿液样本、血液样本、肾脏进行分析测定。采用试剂盒测定小鼠24 h尿蛋白定量（24 h-UPQ），血清中的肌酐（Cr）和尿素氮（BUN），及肾组织氧化抗氧化指标超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA），评估其肾功能与氧化应激水平。对肾脏同一部位进行苏木精-伊红（HE）染色，观察其病理学变化。采用实时荧光定量PCR（RT-qPCR）检测沉默调节蛋白1（Sirt1）、过氧化物酶体增殖物激活受体γ共激活因子1α（Pgc-1alpha）、核因子类红细胞相关因子2（Nrf2）、血红素加氧酶1（HO-1）、NAD（P）H：醌氧化还原酶1（Nqo1）的mRNA表达，采用蛋白质免疫印迹（Western blot）检测SIRT1、PGC-1α、NRF2、HO-1、NQO1蛋白表达。[结果] 与模型组相比，SRT1720与RLP治疗后，DKD小鼠体质量下降程度减缓，FBG无差异，24 h-UPQ，Cr、BUN有不同程度降低，SOD、GSH-Px活性有不同程度增强，MDA含量下降，肾组织病理损伤有不同程度的好转，肾组织SIRT1、PGC-1α、NRF2、HO-1、NQO1的蛋白水平和对应基因的表达水平有不同程度升高。[结论] RLP可能通过激活SIRT1-PGC-1α-NRF2信号通路，抑制氧化应激来改善DKD小鼠肾损伤。

Abstract:

[Objective] To investigate the effect of Rosa laevigata polysaccharides(RLP) in the treatment of diabetic kidney disease(DKD) and to explore its mechanism of action from the perspective of Silent regulator protein 1-peroxisome proliferator-activated receptor gamma coactivator 1 alpha-nuclear factor-like erythroid-associated factor 2(SIRT1-PGC-1α-NRF2) signaling pathway.[Methods] Sixty C57BL/6J mice were acclimatized and fed for 1 week, and all of them were given a high-sugar and high-fat diet for 8 weeks, except for 10 mice in the Control group(C), which were fed with basal chow, after which they were modeled with streptozotocin(STZ) and randomly and equally divided into a model group(M), a SIRT1 agonist group(S), a low-dose RLP group(RL), a medium-dose RLP group(RM), and a high-dose RLP group(RH). The mice were gavaged continuously for 8 weeks, and Fasting blood-glucose(FBG) and body weight were measured every 2 weeks in each group. After 8 weeks of treatment, 24h urine samples, blood samples, and kidneys of mice were collected for analysis and determination. Kits were used to determine 24h urine protein quantification(24 h-UPQ), Serum creatinine(Cr), blood urea nitrogen(BUN), and oxidative and antioxidant indices of renal tissues, superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and malondialdehyde(MDA), to assess the renal function and oxidative stress level in mice. Hematoxylin-eosin(HE) staining was performed on the same part of the kidneys to observe the pathological changes. Real-time fluorescence quantitative PCR(RT-qPCR) was used to detect silencing regulatory protein 1(Sirt1), peroxisome proliferator-activated receptor gamma coactivator 1alpha(Pgc-1alpha), nuclear factor-like erythroid-associated factor 2(Nrf2), heme oxygenase 1(HO-1), and NAD(P)H:quinone oxidoreductase 1(Nqo1) mRNA Expression. Western blotting detects SIRT1, PGC-1α, NRF2, HO-1, NQO1 protein expression.[Results] Compared with the model group, after treatment with SRT1720 and RLP, DKD mice showed a slower decrease in body mass, no difference in FBG, different degrees of reduction in 24 h-UPQ, Cr, BUN, different degrees of enhancement of SOD and GSH-Px activities, decrease in MDA content, different degrees of improvement in renal histopathological injury, and different degrees of increase in gene and protein expression levels of renal tissues SIRT1, PGC-1α, NRF2, HO-1, NQO1 protein expression levels corresponding gene were elevated to different degrees.[Conclusion] RLP may ameliorate kidney injury in DKD mice by activating the SIRT1-PGC-1α-NRF2 signaling pathway and inhibiting oxidative stress.