养阴活血方药拮抗环孢素慢性肾毒性的大鼠体内实验研究

In vivo study of Yangyin Huoxue recipe on antagonizing chronic nephrotoxicity of ciclosporine in rats

目的探讨养阴活血方药拮抗环孢素(CsA)慢性肾毒性的作用及机制。方法将50只Sprague-Dawley(SD)大鼠随机分为对照组、依那普利组、中药低剂量组、中药中剂量组、中药高剂量组,每组各10只。采用钠耗竭法制作大鼠CsA肾毒性模型:给予钠缺乏饮食,每日皮下注射CsA12.5mg/(kg·d),连续4周。同时,按组别相应予以蒸馏水、依那普利(依那普利10倍成人剂量)、低剂量中药(养阴活血方药5倍成人剂量)、中剂量中药(养阴活血方药10倍成人剂量)、高剂量中药(养阴活血方药20倍成人剂量)灌胃。建组后14d、28d记录各组大鼠死亡率。建组后7d、14d、21d、28d,测定各组大鼠的血清肌酐(Scr)、尿肌酐、24h尿蛋白定量,计算内生肌酐清除率(Ccr)。建组后28d处死大鼠,取右肾组织进行病理学检查和免疫组织化学染色,观察细胞外基质(extracellular matrix,ECM)成分,包括纤维连接蛋白(fibronectin,FN)、胶原蛋白(collagen,Col)Ⅳ和其调节因子转化生长因子(TGF)-β1的表达;取左肾组织进行逆转录聚合酶链反应(RT-PCR),分别检测FN、ColⅣ、TGF-β1,以及抑制ECM生成基因包括组织型纤溶酶原激活物(tissue-type plasminogen activator,tPA)、尿激酶型纤溶酶原激活物(urokinase-plasminogen activator,uPA)、纤溶酶原激活物抑制剂(plasminogen activator inhibitor,PAI)-1基因的核糖核酸(RNA)表达。结果建组后14d,中药高剂量组的死亡率为20%,依那普利组和对照组各为10%,中药中剂量组、中药低剂量组均无死亡。建组后28d,除中药高剂量组的死亡率为20%、中药低剂量组的死亡率为0外,其余3组死亡率均为10%。建组7d后各组大鼠肾功能逐渐恶化,24h尿蛋白定量增加,于建组后14d达到高峰。建组14d后进入CsA慢性肾毒性期,各治疗组大鼠的肾功能均有所改善、24h尿蛋白定量减少,但对照组仍然继续恶化。建组后28d,中药高剂量组和中药中剂量组的Ccr明显高于对照组(均为P<0.05),中药各剂量组及依那普利组的24h尿蛋白定量均低于对照组(P<0.05或P<0.01)。肾组织病理切片常规苏木素-伊红(HE)染色表现为慢性CsA肾毒性肾间质纤维化,以对照组改变最为严重,依那普利组、中药低剂量组、中药中剂量组及中药高剂量组改变依次减弱。马松(Masson)染色可见肾小管及肾小球周围纤维增生,以对照组最为明显。免疫组织化学染色显示对照组的FN、ColⅣ及TGF-β1表达遍布肾小管皮质和髓质,依那普利组及中药各剂量组表达程度较轻。RT-PCR结果提示,FN基因的信使RNA(messenger RNA,mRNA)表达强度以中药高剂量组最低、对照组最高;ColⅣ基因的mRNA表达强度以中药低剂量组最高,中药高剂量组最低;TGF-β1基因的mRNA表达强度以依那普利组、中药高剂量组最低,中药低剂量组表达最高。作为抑制ECM生成的tPA、uPA和PAI-1基因的mRNA在各组表达强度差异均无统计学意义。结论养阴活血方药有类似依那普利拮抗大鼠CsA慢性肾毒性的作用,但对急性肾毒性无保护作用,其作用机制可能与下调TGF-β1等细胞因子,从而抑制ECM的合成有关。

Objective To investigate the effect and mechanism of Yangyin Huoxue recipe on antagonizing chronic renal toxicity of ciclosporine(CsA). Methods Fifty Sprague-Dawley(SD) rats were randomly assigned to 5 groups including the control group, enalapril group, low-dose Chinese herbal drugs group, medium-dose Chinese herbal drugs group and high-dose Chinese herbal drugs group, with 10 rats in each. All rats were given salt-deficiency diet and CsA 12.5 mg/(kg·d)everyday for 4 consecutive weeks to build the rat nephrotoxicity of CsA model. And the rats in the 5 groups were fed with distilled water, the enalapril(10 times of adult dosage), low-dose Chinese herbal drugs(5 times of adult dosage), medium-dose Chinese herbal drugs (10 times of adult dosage) and high-dose Chinese herbal drugs (20 times of adult dosage) by intragastric administration respectively at the same time. The death rates of the rats were recorded after day 14, day 28. The serum creatinine(Scr), urine creatinine, 24-hour urine protein were tested on day 7、day 14、day 21、day 28 and calculated the endogenous creatinine clearance rate(Ccr). The right renal tissues of rats were harvested on day 28. Histopathological examination and immunohistochemistry methods were used to observe the component of extracellular matrix(ECM) including the expression of fibronectin(FN), collagen(Col)Ⅳ and regulatory factor transformation growth factor (TGF)-β1. The left renal tissues were harvested to test the FN, ColⅣ, TGF-β1 and the ribonucleic acid (RNA) expression of the suppressor genes of ECM including the tissue-type plasminogen activator(tPA), urokinase-plasminogen activator(uPA), plasminogen activator inhibitor(PAI)-1 by reverse transcription polymerase chain reaction (RT-PCR)method. Results The death rates of the rats were 20%, 10%, 10% on the day 14 in the group of high-dose Chinese herbal drugs group, enalapril group,control group respectively.And no death occurred in the medium-dose and low-dose Chinese herbal drugs group. The death rates were 20% on day 28 in the high-dose Chinese herbal drugs group and 0 in the low-dose Chinese herbal drugs group. The death rates were 10% in the medium-dose Chinese herbal drugs,enalapril and control group respectively. The renal function of rats in the 5 groups decreased from day 7. The 24-hour urine protein increased and topped on day 14. The rats suffered from chronic nephrotoxicity of CsA and the renal function recovered mildly after day 14. The 24-hour urine protein decreased but even worsed in the control group. The Ccr in the high-dose and medium-dose Chinese herbal drugs group were significantly higher than the control group (all in P<0.05). The 24-hour urine protein in all dose Chinese herbal drugs and enalapril groups were lower than the control group on day 28(P<0.05 or P<0.01). The hematoxylin-eosin(HE) stained renal tissues showed fibrosis of renal interstitium weakening in turn from the control group to enalapril, low-dose, medium-dose, high-dose Chinese herbal drugs group. In Masson stained samples, fibroelastosis around glomerular and tubular could be seen,which was the most obvious in the control group.The expression of FN、ColⅣ and TGF-β1 were strong in the cortex and medulla of renal tubules in the control group, while milder in the enalapril and all dose Chinese herbal drugs groups by immunohistochemistry method. RT-PCR suggested that the expression of messenger ribonucleic acid (mRNA) of FN was the least in the high-dose Chinese herbal drugs group, while the strongest in the control group. The expression of mRNA of ColⅣ was the strongest in the low-dose Chinese herbal drugs group while the least in the high-dose Chinese herbal drugs group. The expression of mRNA of TGF-β1 was the least in the enalapril and high-dose Chinese herbal drugs group while the strongest in the low-dose Chinese herbal drugs group. There was no significant difference in the expression of tPA、uPA and PAI-1 mRNA among the 5 groups. Conclusions The Yangyin Huoxue recipe can antagonize chronic nephrotoxicity of CsA in rats like enalapril, but no effect to acute nephrotoxicity. The mechanism of effect may be associated with the down-regulation of TGF-β1 mRNA expression which thereby suppress the generation of ECM.