摘要
目的:观察补肾活血方是否对对慢性肾脏病(CKD)血管钙化(VC)大鼠模型B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)相关凋亡蛋白有调控作用,从而抑制CKD大鼠的血管钙化,并探讨其作用机制。方法:将90只大鼠按体质量随机均分为正常组,模型组、骨化三醇组,中药高、中、低剂量组,每组15只。实验第1-4周,模型组及中药各剂量组大鼠均按250mg/kg剂量给予腺嘌呤混悬液灌胃及含1.2%高磷饲料喂养,第5-8周腺嘌呤改为隔日灌胃,同时中药各剂量组用补肾活血方灌胃治疗,正常组灌胃等量0.9%氯化钠溶液,连续给药8周。给药结束后,检测小鼠体质量及肝肾指数。HE染色观察肾组织变化,茜素红染色观察主动脉钙化结节情况。腹主动脉取血检测血清钙(Ca)、磷(P)、血肌酐(CREA)、尿素氮(UREA)。Western blot法检测主动脉α-平滑肌蛋白(α-SMA)、ALP、Bcl-2、Bax、Caspase-3蛋白的表达。结果:模型组肝肾脏指数显著增加(P<0.01),中药中剂量和高剂量组肾脏指数具有明显差异(P<0.01)。HE染色显示中药组较模型组正常肾小球数目增多,肾小管扩张程度降低,炎性浸润减少,腺嘌呤结晶减少。茜素红染色显示中药各剂量组钙化减轻,中、高剂量减轻较为明显。与正常组同期比较,模型组血P、CREA、UREA水平升高显著,血Ca水平显著下降,主动脉ALP蛋白表达明显升高,α-SMA蛋白表达明显下降,抑凋亡蛋白Bcl-2表达显著下调、凋亡蛋白Bax、Caspase-3表达显著上调(P<0.01);与模型组比较,第4周中药中剂量组血P、CREA、UREA水平显著下降(P<0.05),血Ca水平显著升高(P<0.01),第8周中药各剂量组血P、CREA、UREA水平显著下降(P<0.01),血钙显著上升(P<0.01)。骨化三醇组第8周血P显著降低,血钙显著升高(P<0.05)。Western blot结果显示,中药中、高剂量组主动脉ALP、Bax、Caspase-3蛋白表达降低、α-SMA、Bcl-2蛋白表达升高(P<0.05,P<0.01)。结论:补肾活血方可改善CKD大鼠的肾功能及血管钙化程度,其机制可能与对凋亡相关蛋白Bcl-2/Bax的调控有关。
Objective: To explore the regulatory effects of Bushen Huoxue Decoction(BHD) on Bcl-2/Bax-related apoptosis in rats with vascular calcification(VC) and chronic kidney disease(CKD), and to find out further whether vascular calcification of subjects tested could be inhibited as well as mechanism associated. Methods: Ninety SD rats were randomly divided into normal group, model group, calcitriol group, BHD high dose group, BHD middle dose group, and BHD low dose group, with 15 rats in each group. For the rats of model group and BHD group, they were treated with 250mg/kg adenine suspension by intragastric administration and were fed with food containing 1.2% phosphorus from the first to the fourth week; during the fifth to eighth week, the frequency of adenine administration was amended toevery other day. At the same time, BHD group was given BHD by intragastric administration; normal group were treated with equal 0.9% Na Cl solution for eight weeks. After administration, the weight of rats, hepatic, renal functions of subjects were measured, and renal and aorta of rats were observed by HE staining and alizarin red staining, respectively. The content of serum calcium, serum phosphate, serum creatinine and urea nitrogen in abdominal aorta were detected. And the expression of α-smooth muscle actin(α-SMA), alkaline phosphatase(ALP), Bcl-2, Bax and Caspase-3 were detected by Western blot method. Results: Compared with normal group, liver and kidney index of model group was significantly increased(P<0.01). Significant disparity between the kidney index of BHD high dose group BHD middle dose group were found(P<0.01). HE staining showed that in the BHD group the number of glomerular raised compared with the model group, and the expansion degree of renal tubules decreased; the number of inflammatory cells decreased; and the crystallization of adenine decreased in the BHD groups. Alizarin red staining showed similar results as HE staining did. Calcification in BHD groups was all alleviated with more significant outcome in middle-dose group and high-dose group. Compared with the normal group, the serum phosphate, CREA, UREA levels in model group significantly increased, while the serum calcium level significantly decreased(P<0.01). The expression of ALP activity of aorta was significantly increased, while the expression of α-SMA protein was significantly decreased(P<0.01). The expression of Bcl-2 was down-regulated, and the expression of both Caspase-3 and Bax protein were up-regulated(P<0.01). Compared with model group, the forth week serum phosphate level, CREA, UREA level in BHD middle dose group were reduced(P<0.01), while serum calcium level increased(P<0.01). In the eighth week, serum phosphate, CREA, UREA level in BHD groups were reduced(P<0.01), while serum calcium level increased(P<0.01); serum phosphate in calcitriol group was reduced, and serum calcium level increased(P<0.05). For proteins expression, the expression of ALP, Bax, Caspase-3 in BHD high dose group and BHD middle dose group decreased significantly, while the expression of α-SMA and Bcl-2 were increased(P<0.05, P<0.01). Conclusion: BHD is able to improve renal function of CKD rats and alleviate their vascular calcification, where the mechanism involved may have relation with regulation of Bcl-2/Bax.
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2017年第5期2188-2193,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.81273747)~~
关键词
补肾活血方
慢性肾脏病
血管钙化
凋亡
B淋巴细胞瘤-2
Bcl-2相关X蛋白
Bushen Huoxue Decoction
Chronic kidney disease
Vascular calcification
Apoptosis
B lymphocyte tumor-2
Bcl-2 associated X protein
