右归丸通过AOPPs调控RAGE/ROS/NF-κB轴及Wnt/β-catenin信号通路对阿霉素诱导肾病综合征大鼠的保护机制

Protective Mechanism of Youguiwan through AOPPs Regulating RAGE/ROS/NF-κB Axis and Wnt/β-catenin Signal in Rats with Adriamycin-induced Nephrotic Syndrome

目的:探究右归丸对阿霉素诱导的肾病综合征(NS)大鼠的影响及其作用机制。方法:SD大鼠随机分为正常组、模型组、右归丸低、中、高剂量组和泼尼松组。模型组大鼠尾静脉注射阿霉素构建NS模型,右归丸低、中、高剂量组分别按生药2.8、5.6、11.2 g·kg^(-1)·d^(-1)灌胃,泼尼松组以醋酸泼尼松6.3 mg·kg^(-1)·d^(-1)灌胃。各用药组连续给药6周,正常组及模型组灌服等体积生理盐水。BCA法检测24 h尿蛋白(24 h UP);全自动生化分析仪检测血清尿素氮(BUN)、肌酐(SCr)、白蛋白(ALB)、总胆固醇(TC)、甘油三酯(TG)水平;苏木素-伊红(HE)染色观察肾组织病理形态变化;试剂盒检测血清晚期氧化蛋白产物(AOPPs)、活性氧(ROS);蛋白免疫印迹法(Western blot)检测肾组织晚期糖基化终产物受体(RAGE)、核转录因子-κB(NF-κB)磷酸化水平、Wnt及β-连环蛋白(β-catenin)蛋白表达。结果:与正常组比较,模型组大鼠24 h UP、血清BUN、SCr、TC、TG、AOPPs、ROS水平均显著升高(P<0.01),ALB显著降低(P<0.01),且肾组织存在典型的病理性损伤,RAGE、磷酸化(p)-NF-κB、Wnt1及β-catenin蛋白表达均显著升高(P<0.01)。与模型组比较,右归丸低、中、高剂量组大鼠24 h UP、血清BUN、SCr、TC、TG、AOPPs、ROS水平均显著降低(P<0.01),ALB明显升高(P<0.01),肾组织损伤减轻,RAGE、p-NF-κB、Wnt1及β-catenin蛋白表达均显著降低(P<0.01),且呈剂量相关。结论:右归丸能够改善阿霉素诱导的NS大鼠肾损伤,其机制可能与降低AOPPs水平,抑制RAGE/ROS/NF-κB轴及Wnt/β-catenin信号活化相关。

Objective: To explore the effect of Youguiwan on the rats with adriamycin-induced nephrotic syndrome (NS) and its mechanism.Method: SD rats were randomly divided into a normal group, a model group, three Youguiwan low, medium, and high-dose groups, and a prednisone group. Rats in the model group were intravenously injected with adriamycin in the tail vein to induce the NS model. Rats in the Youguiwan low, medium, and high-dose groups were given 2.8, 5.6, 11.2 g·kg^(-1)·d^(-1) of crude drugs, respectively, and rats in the prednisone group were given 6.3 mg·kg^(-1)·d^(-1) of prednisone acetate. Each administration group was given continuous medicine for 6 weeks, and the normal group and model group were given an equal volume of normal saline. Bicinchoninic acid (BCA) assay was used to detect 24 h urine protein (24 h UP). Automatic biochemical analyzer was used to detect serum urea nitrogen (BUN), creatinine (SCr), albumin (ALB), total cholesterol (TC), and triglyceride (TG) levels. Hematoxylin-eosin (HE) staining was used to observe renal tissue morphology, and kit was used to detect serum advanced oxidized protein products (AOPPs) and reactive oxygen species (ROS). Western blot was used to detect the receptor of advanced glycation endproducts (RAGE) of renal tissue, nuclear factor-κB (NF-κB) phosphorylation levels, Wnt, and β-catenin protein expression.Result: As compared with the normal group, 24 h UP, serum BUN, SCr, TC, TG, AOPPs, and ROS levels in the model group increased significantly (P<0.01), whereas ALB decreased (P<0.01). There were typical pathological injuries in the renal tissue, and the expressions of RAGE, phosphorylation(p)-NF-κB, Wnt1, and β-catenin protein were significantly increased (P<0.01). As compared with the model group, the 24 h UP, serum BUN, SCr, TC, TG, AOPPs, and ROS levels of rats in the Youguiwan low, medium, and high-dose groups significantly reduced (P<0.01), and ALB significantly increased (P<0.01). The renal tissue damage was reduced, and the expressions of RAGE, p-NF-κB, Wnt1, and β-catenin protein were significantly decreased (P<0.01) in a dose-dependent manner.Conclusion: Youguiwan improves the kidney injury of rats with adriamycin-induced NS. The mechanism may be related to the reduction of AOPPs level, inhibition of RAGE/ROS/NF-κB axis, and activation of Wnt/β-catenin signal.