摘要
目的:观察六味地黄汤对慢性肾衰竭大鼠线粒体凋亡途径的影响。方法:将45只雄性SD大鼠随机分为正常组(10只)、假手术组(10只)及手术组(25只),手术组进行5/6肾切除造模,造模成功后再随机分为模型组及治疗组。治疗组予以6.75 g·kg-1六味地黄汤灌胃,其余3组予以等体积蒸馏水灌胃,各组均给药8周。检测各组大鼠血肌酐、血尿素氮及尿蛋白定量水平;苏木素-伊红(HE),马松(Masson)染色观察肾组织形态学变化;电镜观察肾小管上皮细胞中线粒体超微结构变化;原位末端标记法(TUNEL)检测细胞凋亡;蛋白免疫印迹法(Western blot)及实时荧光定量聚合酶链式反应(Real-time PCR)分别检测肾组织B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),细胞色素C(Cyt C),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白及mRNA的表达水平。结果:与正常组比较,模型组大鼠血肌酐、尿素氮及尿蛋白定量水平显著升高(P<0.01),治疗组较模型组上述指标得到改善(P<0.05);正常组及假手术组大鼠肾脏未观察到明显病理变化及线粒体结构破坏,模型组存在不同程度的肾小管萎缩和扩张,间质性炎性细胞浸润和胶原沉积,以及线粒体肿胀和断裂,治疗组较模型组上述病灶明显减轻;与正常组比较,模型组细胞凋亡显著增多(P<0.01),Bax,Caspase-3表达显著升高(P<0.01),Bcl-2表达显著降低(P<0.01);与模型组比较,治疗组细胞凋亡显著减少(P<0.01),Bax,Caspase-3表达显著降低(P<0.01),Bcl-2表达显著升高(P<0.01);正常组及假手术组Cyt C主要定位在线粒体,模型组细胞质中Cyt C表达显著增加(P<0.01),治疗组较模型组细胞质中Cyt C表达显著减少(P<0.01)。结论:六味地黄汤可减轻肾纤维化,延缓肾功能进展,其机制可能是通过保护线粒体结构及调控线粒体凋亡途径相关蛋白的表达减少细胞凋亡。
Objective: To observe the effect of Liuwei Dihuangtang on the mitochondrial apoptotic pathway in rats with chronic renal failure. Method: The 45 male SD rats were randomly divided into normal group(n=10), sham group(n=10) and operation group(n=25). The operation group received 5/6 nephrectomy,and the rats in operation group were randomly divided into the model group and the treatment group after successful modeling. The treatment group received Liuwei Dihuangtang by gavage administration,while the rest of the three groups received equal volume of distilled water by gavage administration,with a treatment course of 8 weeks. The quantitative levels of serum creatinine(SCr),blood urea nitrogen(BUN)and urine protein in each group were determined. Hematoxylin eosin(HE)staining and Masson staining were used to observe the histopathological changes of rat kidney. The structural changes of mitochondria in renal tubular epithelial cells were observed under electron microscope. The apoptotic rate was detected by terminaldeoxynucleoitidyl transferase mediated nick end labeling(TUNEL). The expression of B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cytochrome C(Cyt C)and cysteine aspartic acid protease-3(Caspase-3)were analyzed by Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). Result: As compared with the normal group,the quantitative levels of SCr,BUN and urinary protein in the model group were significantly increased(P<0.01),and the above indicators were improved in the treatment group as compared with the model group(P<0.05). No significant pathological changes and damage of mitochondrial structure were observed in the kidney tissues of the control group and the sham group. In the model group,there were different degrees of renal tubular atrophy and dilatation,interstitial inflammatory cell infiltration and collagen deposition,and mitochondria swelling and breaking,but such lesions were significantly alleviated in the treatment group. As compared with the normal group,apoptotic rate was increased significantly in model group(P<0.01),the expression of Bax and Caspase-3 was significantly increased(P<0.01),and the expression of Bcl-2 was significantly decreased(P<0.01). As compared with the model group,apoptotic rate was significantly reduced in the treatment group(P<0.01),the expression of Bax and Caspase-3 was significantly decreased(P<0.01),and the expression of Bcl-2 was significantly increased(P<0.01). In the normal group and sham group,Cyt C was mainly located in mitochondria. The expression of Cyt C in the cytoplasm of the model group was increased significantly(P<0.01),while that of the treatment group was decreased(P<0.01). Conclusion: Liuwei Dihuangtang can alleviate renal fibrosis and delay the progression of renal function. The mechanism of reducing apoptosis may be associated with protecting mitochondrial structure and regulating the expression of proteins related to mitochondrial apoptotic pathway.
作者
胡爽
何泽云
李旭华
唐群
董翔
HU Shuang;HEZe-yun;LI Xu-hua;TANG Qun;DONG Xiang(Hunan University of Chinese Medicine,Changsha 410208,China;The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第22期26-33,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金青年基金项目(81503442)
研究生创新课题项目(2019CX13)。
关键词
六味地黄汤
慢性肾脏病
肾纤维化
线粒体
细胞凋亡
Liuwei Dihuangtang
chronic kidney disease(CKD)
renal fibrosis
mitochondria
cell apoptosis
