徐长卿通过调控过氧化物酶体增殖物激活受体γ改善IgA肾病大鼠肾损伤

The Cynanchi Paniculati Radix et Rhizoma improves renal injury in IgA nephropathy rats by regulating peroxisome proliferator-activated receptor γ

关键词:  IgA肾病  氧化应激  炎症  PPARγ  TLR4/NF-κB通路

Key words:  IgA nephropathy  oxidative stress  inflammation  PPARγ  TLR4/NF-κB pathway

摘要:

摘要：目的 探究过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor γ, PPARγ）在徐长卿治疗IgA肾病大鼠肾损伤中的作用。方法 120只雄性SD大鼠随机分为空白组、模型组、徐长卿低、高剂量组、阳性药物（泼尼松）组、PPARγ激动剂组。收集尿液、血清和肾脏。检测尿蛋白、尿素氮（Blood urea nitrogen, BUN）、肌酐（Serum creatinine, Src）和IgA含量；苏木素-伊红（Hematoxylin-eosin, HE）染色观察肾组织形态学变化；透射电镜观察大鼠肾脏超微结构变化；原位末端转移酶标记法（TdT-mediated dUTP Nick-End Labeling, TUNEL）观察肾脏凋亡阳性细胞比例；酶联免疫吸附法检测白介素（interleukin, IL）-1β、IL-6、肿瘤坏死因子-α（Tumor necrosis factor-alpha, TNF-α）、活性氧（Reactive oxygen species, ROS）水平；比色法检测丙二醛（Malondialdehyde, MDA）、谷胱甘肽过氧化物酶（Glutathione peroxidase , GSH-Px）和超氧化物歧化酶（Superoxide dismutase, SOD）水平；实时荧光定量PCR（quantitative Real-time polymerase chain reaction, qRT-PCR）检测肾脏PPARγ、Toll样受体4（Toll-like receptor 4, TLR4）和核转录因子κB（Nuclear factor kappa-B, NF-κB）mRNA表达；蛋白免疫印迹检测肾脏PPARγ、TLR4、磷酸化NF-κB（p-NF-κB）、剪切的半胱氨酸蛋白酶3（Cleaved Caspase-3）蛋白表达；免疫组化法检测NOD样受体蛋白3（NOD-Like receptor protein 3, NLRP3）蛋白表达；免疫荧光检测剪切的半胱氨酸蛋白酶1（Cleaved caspase-1）蛋白表达。结果 与模型组比较，徐长卿处理能降低IgA肾病大鼠尿蛋白、BUN、Src和IgA含量（P<0.05, P<0.01），肾脏炎性细胞浸润和线粒体损伤明显缓解。徐长卿低、高剂量组肾脏TUNEL阳性细胞数，Cleaved Caspase-3蛋白表达，IL-1β、IL-6和TNF-α含量以及ROS和MDA水平降低，而SOD和GSH-Px活性升高，较模型组差异显著（P<0.05, P<0.01）。与模型组相比，徐长卿低、高剂量组大鼠肾脏PPARγ mRNA表达水平升高，而TLR4和NF-κB mRNA表达水平降低（P<0.05, P<0.01）。此外，徐长卿低、高剂量组大鼠肾脏p-NF-κB 、Cleaved Caspase-3、NLPR3和Cleaved Caspase-1蛋白水平显著降低，较模型组有着显著水平的差异（P<0.01）。结论 徐长卿可能通过调控PPARγ来抑制氧化应激、凋亡和炎症反应，从而缓解IgA肾病大鼠肾损伤。

Abstract:

Abstract: Objective To investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) in the treatment of renal injury in IgA nephropathy rats by Cyperus rotundus. Methods 120 SD rats were randomly divided into blank group, model group, Cynanchi Paniculati Radix et Rhizoma low, high dose group, positive drug (prednisone) group, and PPARγ agonist group. Collect urine, serum, and kidney tissues. Measure urinary protein, blood urea nitrogen (BUN), serum creatinine (Src), and IgA levels.Hematoxylin-eosin (HE) staining was used to observe the histopomorphological changes of renal tissues; Transmission electron microscope was used to observe the ultrastructural damages of rat kidneys; TUNEL staining (TdT-mediated dUTP Nick-End Labeling) was used to observe the proportion of apoptotic positive cells in rat kidneys; Enzyme-linked immunosorbent assay (ELISA) was used to detect the contents IL-1β, IL-6, TNF-α and reactive oxygen species (ROS) in kidneys; Colorimetric method was used to detect the levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and total superoxide dismutase (SOD) in kidney tissue; Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression of PPARγ, TLR4 and NF-κB at the mRNA level in rat kidney tissue; Western blot was used to detect the protein expressions of PPARγ, TLR4, NF-κB and cysteine aspartate protease-3 (Caspase-3) in rat kidney tissue. Immunohistochemical method (IHC) was used to detect the protein expressions of NOD-like receptor protein 3 (NLRP3) and Cleaved Caspase-1. immunofluorescence(IF) was used to detect the protein expression of Cleaved Caspase-1 in rat kidney tissue. Results Compared with the model group, treatment with Cyperus rotundus significantly reduced urinary protein, BUN, Src and IgA levels in rats with IgA nephropathy(P<0.05, P<0.01), and the inflammatory cell infiltration and mitochondrial damage of kidney tissue were significantly alleviated. The number of TUNEL-positive cells, the expression and activity of Caspase-3 protein, the contents of IL-1β, IL-6 and TNF-α, the levels of ROS and MDA were decreased, and the activities of SOD and GSH-Px were increased in Low- and high-dose groups of Cyperus rotundus, which were significantly different from those in model group (P<0.05, P<0.01). In addition, compared with the model group, the mRNA and protein levels of PPARγ in kidney of rats in Low- and high-dose groups of Cyperus rotundus were increased, while the mRNA and protein levels of TLR4 and NF-κB were decreased (P<0.05, P<0.01). In addition, the protein levels of NLPR3 and Cleaved Caspase-1 were significantly decreased in the rats kidney tissue in Cynanchi Paniculati Radix et Rhizoma group, and there were significant differences compared with model group (P<0.01). Conclusion The renoprotective effects of Cyperus rotundus in IgA nephropathy rats may be mediated through the regulation of PPARγ, leading to the suppression of oxidative stress, apoptosis, and inflammation.