中乌宁通过BAX/BCL-2/Caspase 3信号通路抑制细胞凋亡改善慢性肾衰竭大鼠肾纤维化

Mesaconine Inhibits Apoptosis through the BAX/BCL-2/Caspase 3 Signaling Pathway to Improve Renal Fibrosis in Rats with Chronic Renal Failure

目的:本研究旨在探讨中乌宁是否通过BAX/BCL-2/Caspase 3信号通路抑制细胞凋亡,改善慢性肾衰竭(Chronic renal failure,CRF)大鼠肾纤维化。方法:雄性SD大鼠建立5/6肾切除致CRF模型,随机分为模型对照组、中乌宁2、5 mg/kg组及依那普利2 mg/kg组,另取大鼠作为正常对照组及假手术对照组。术后1 w按组别灌胃给药,连续8 w,末次给药后收集24 h尿液测定尿量及尿蛋白含量,取血及肾脏,检测血清中肾损伤标志物肌酐(CREA)、尿素氮(BUN)含量及肾脏纤维化标志物转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)含量;HE染色及Masson染色观察组织病变及纤维化程度;TUNEL染色测定肾组织细胞凋亡率;Western blot法测定细胞凋亡相关蛋白BAX、BCL-2及Cleaved-caspase 3的蛋白表达。结果:与正常对照组或假手术对照组相比,模型对照组大鼠尿量明显减小,尿蛋白、血清CREA、BUN、TGF-β1、α-SMA含量、肾组织损伤病理评分、肾组织纤维化程度及肾组织细胞凋亡率明显增加,肾组织中BAX及Cleaved-caspase 3蛋白表达明显上调,BCL-2蛋白表达明显下调(P<0.05或P<0.01);与模型对照组相比,中乌宁5 mg/kg组大鼠尿量明显增加,各给药组大鼠尿蛋白、血清CREA、BUN、TGF-β1、α-SMA含量、肾组织损伤病理评分、肾组织纤维化程度及肾组织细胞凋亡率明显降低,肾组织中BAX及Cleaved-caspase 3蛋白表达均明显下调,BCL-2蛋白表达明显上调(P<0.05或P<0.01)。结论:中乌宁可有效改善5/6肾切除CRF模型大鼠的肾脏功能。这种肾保护作用与其调控BAX/BCL-2/Caspase 3信号通路,抑制细胞凋亡,从而减轻5/6肾切除CRF模型大鼠的肾纤维化有关。

Objective:To investigate whether mesaconine inhibits apoptosis through the Bax/Bcl-2/caspase 3 signaling pathway to improve renal fibrosis in rats with chronic renal failure(CRF).Methods:Male SD rats were used to establish CRF model by 5/6 nephrectomy,and the rats were randomly divided into model group,mesaconine 2 mg/kg group,mesaconine 5 mg/kg group and enalapril 2 mg/kg group.Other rats were taken as blank group and sham operation group.One week after operation,the rats were given drugs by gavage for 8 weeks.After the last drug administration,24-h urine was collected to detect urine volume and protein content.Blood and renal tissue were collected to determine the level of serum renal injury markers,including creatinine(CREA)and blood urea nitrogen(BUN),as well as the level of renal fibrosis markers,including transforming growth factor-β1(TGF-β1)andα-smooth muscle actin(α-SMA).HE staining and Masson staining were performed to observe the pathological changes and renal fibrosis.TUNEL staining was conducted to observe the apoptosis rate of renal cells.Western blot method was used to detect the expressions of apoptosis-related proteins,such as Bax,Bcl-2 and cleaved-caspase 3.Results:Compared with the blank group or sham operation group,the rats in the model group showed significantly lower urine volume,as well as significantly increased urine protein,serum contents of CREA,BUN,TGF-β1 andα-SMA,pathological score of kidney histological damage,fibrosis degree and apoptosis rate of renal cells.The protein expressions of Bax and cleaved-caspase 3 were significantly upregulated,while the expression level of Bcl-2 was significantly downregulated(P<0.05 or P<0.01).Compared with the model group,the rats in the mesaconine 5 mg/kg group exhibited remarkably elevated urine volume.The urinary protein level,serum contents of CREA,BUN,TGF-β1 andα-SMA,pathological score of renal tissue injury,degree of fibrosis,and apoptosis rate of renal cells were significantly decreased in all treatment groups.The expressions of Bax and cleaved-caspase 3 protein in renal tissue were significantly downregulated,while the protein expression of Bcl-2 was significantly upregulated(P<0.05 or P<0.01).Conclusion:Mesaconine effectively improves renal function in 5/6 nephrectomy-induced CRF rat model.Its renal protective effect is potentially associated with the modulation of the Bax/Bcl-2/caspase 3 signaling pathway to inhibit apoptosis,thereby reducing renal fibrosis in the CRF rat model.