肾衰胶囊调控Keap1/Nrf2/HO-1 通路抑制 SHR 肾脏氧化应激的机制研究 ?? [ ]

Mechanistic Study of Shen-Shuai Capsule Alleviating Renal Oxidative Stress in SHR via Modulation of the Keap1-Nrf2-HO-1 Pathway

关键词:  肾衰胶囊  高血压肾病  自发性高血压大鼠  尿蛋白  氧化应激  

Key words:  shenshuai capsule  hypertensive nephropathy  urinary protein  oxidative stress

摘要:

目的:探讨肾衰胶囊对自发性高血压大鼠(SHR)抗氧化应激通路Keap1-Nrf2-HO-1影响及肾脏保护的作用。方法:选取12只16周龄 Wistar Kyoto(WKY)大鼠及72只 SHR 适应性喂养1周,WKY 大鼠作为空白组,72只SHR随机为模型组、模型组、缬沙坦组(0.0072g.kg-1)、海昆肾喜胶囊组(0.1188g.kg-1)、肾衰胶囊高剂量组(0.216g.kg-1)、中剂量组(0.108g.kg-1)、低剂量组(0.054g.kg-1),空白组和模型组大鼠用等容积0.5%羧甲基纤维素钠(CMC-Na)溶液灌胃。药物干预期间观察各组大鼠一般情况并分别于给药前及给药后每隔2周特定时间监测大鼠血压。药物连续干预8w后,全自动生化分析仪测定尿蛋白(albumin,ALB)、尿肌酐(creatinine,Cr),计算尿白蛋白/肌酐比值(ALB/Cr)；ELISA法检测血清检测血浆中丙二醛(MDA)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4(NOX4)和还原型谷胱甘肽(GSH)水平；HE染色观察肾组织病理改变,Western Blot 检测肾组织 Keap1、Nrf2、HO-1蛋白水平。荧光定量PCR测定 Keap1、Nrf2、HO-1mRNA水平,免疫荧光法评估 Keap1、Nrf2、HO-1蛋白细胞定位与分布情况；结果:与空白组相比,模型组尿(ALB/Cr)水平升高,血清MDA、NOX4升高和GSH水平下降,肾脏结构受到破坏,肾小球局灶性硬化,肾小管浑浊肿胀,肾间质纤维化明显；肾组织Keap1蛋白表达及mRNA升高、蛋白荧光强度增加,Nrf2 、HO-1蛋白表达、mRNA降低及蛋白的荧光强度减弱,与模型组比较,缬沙坦胶囊、肾衰胶囊各剂量组、海昆肾喜胶囊组尿ALB/Cr水平均有下降(P<0. 05 ),缬沙坦胶囊、肾衰胶囊高、低剂量组、海昆肾喜胶囊组血清MDA、NOX降低和肾衰高剂量组GSH水平升高(P<0. 05 )；肾组织损伤程度减轻,Western Blot结果显示:缬沙坦组、海昆肾喜组及肾衰中剂量组Keap1表达被显著抑制(P<0.05),缬沙坦组、肾衰中、低剂量Nrf2蛋白水平蛋白水平上调(P<0. 05 )；缬沙坦组、肾衰高、低剂量组HO-1蛋白水平上调(P<0. 05 )。PCR测定结果显示:缬沙坦组、海昆肾喜组及肾衰不同剂量组均能显著下调Keap1 mRNA,上调Nrf2及其下游靶基因HO-1的mRNA表达(P<0.05,P<0.01)。免疫荧光结果提示:与模型组比较,缬沙坦组、海昆肾喜组及肾衰不同剂量组Keap1蛋白表达均显著降低(P<0.05,P<0.01)；缬沙坦组与肾衰不同剂量组HO-1蛋白表达显著升高(P<0.05)；缬沙坦组、海昆肾喜组及肾衰高、中剂量组Nrf2蛋白表达显著升高(P<0.05,P<0.01)。此外,免疫荧光定位观察发现,药物干预能促进Nrf2从细胞质向细胞核内聚集。结论:肾衰胶囊可能通过调控Keap1/Nrf2/HO-1通路,抑制Keap1表达,促进Nrf2核转位,上调HO-1,减少氧化应激(MDA、NOX4),减少尿ALB/Cr,具有抗氧化,抑制肾脏纤维化作用,进一步保护SHR肾脏。

Abstract:

OBJECTIVE To investigate the effect of Shen-Shuai Capsule on the Keap1-Nrf2-HO-1 antioxidant stress pathway and its renal protective role in spontaneously hypertensive rats (SHR).METHODS Twelve 16-week-old Wistar Kyoto (WKY) rats and 72 SHR rats were acclimated for one week. WKY rats served as the blank control group, while 72 SHR rats were randomly divided into the model group, valsartan group (0.0072 g·kg?1), Hai-Kun Shen-Xi capsule group (0.1188 g·kg?1), and SSC high-dose (0.216 g·kg?1), medium-dose (0.108 g·kg?1), and low-dose (0.054 g·kg?1) groups. The blank and model groups were administered an equivalent volume of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution by gavage. General conditions were observed throughout the intervention, and blood pressure was monitored every two weeks. After 8 weeks of continuous treatment, urinary albumin (ALB) and creatinine (Cr) levels were measured using an automatic biochemical analyzer to calculate the urinary albumin-to-creatinine ratio (ALB/Cr). Serum levels of malondialdehyde (MDA), NADPH oxidase 4 (NOX4), and reduced glutathione (GSH) were determined by ELISA. Renal histopathological changes were observed via HE staining. Western blot was used to detect renal protein levels of Keap1, Nrf2, and HO-1. Real-time PCR was employed to measure mRNA expression of Keap1, Nrf2, and HO-1. Immunofluorescence was used to assess the cellular localization and distribution of Keap1, Nrf2, and HO-1 proteins.t. RESULTS Compared with the blank group, the model group exhibited increased urinary ALB/Cr, elevated serum MDA and NOX4 levels, decreased GSH levels, and significant renal structural damage, including focal glomerulosclerosis, tubular cloudy swelling, and interstitial fibrosis. Renal Keap1 protein and mRNA expression were increased with enhanced fluorescence intensity, while Nrf2 and HO-1 protein and mRNA levels were decreased with reduced fluorescence intensity. Compared with the model group, valsartan, SSC at all doses, and Hai-Kun Shen-Xi capsule significantly reduced urinary ALB/Cr (P < 0.05). Valsartan, SSC high and low doses, and Hai-Kun Shen-Xi capsule significantly decreased serum MDA and NOX4 levels, and SSC high dose increased GSH levels (P < 0.05). Renal tissue damage was alleviated in all treatment groups. Western blot results showed that valsartan, Hai-Kun Shen-Xi, and SSC medium dose significantly inhibited Keap1 expression (P < 0.05); valsartan and SSC medium and low doses upregulated Nrf2 protein levels (P < 0.05); valsartan and SSC high and low doses upregulated HO-1 protein levels (P < 0.05). PCR results indicated that valsartan, Hai-Kun Shen-Xi, and all SSC dose groups significantly downregulated Keap1 mRNA and upregulated Nrf2 and HO-1 mRNA expression (P < 0.05, P < 0.01). Immunofluorescence results showed that valsartan, Hai-Kun Shen-Xi, and all SSC dose groups significantly reduced Keap1 protein expression (P < 0.05, P < 0.01); valsartan and all SSC dose groups significantly increased HO-1 protein expression (P < 0.05); valsartan, Hai-Kun Shen-Xi, and SSC high and medium dose groups significantly increased Nrf2 protein expression (P < 0.05, P < 0.01). Additionally, immunofluorescence localization showed that drug interventions promoted Nrf2 translocation from the cytoplasm to the nucleus.. CONCLUSION Shen-Shuai Capsule exerts renal protective effects in SHR by modulating the Keap1-Nrf2-HO-1 antioxidant stress pathway, inhibiting oxidative damage, and alleviating renal fibrosis, suggesting its potential as an effective intervention for hypertensive nephropathy.