川黄方对临床剂量三价砷肾毒性大鼠肾小管上皮细胞凋亡的影响

Effect of Chuanhuang Fang on apoptosis of renal tubular epithelial cells in rats with trivalent arsenic nephrotoxicity

目的探讨川黄方对临床剂量三价砷肾毒性的抑制效应及机制。方法将32只雄性Wistar大鼠分为正常组(A组)、模型组(B组)、川黄方组(C组)、N-乙酰半胱氨酸(NAC)组(D组),每组8只;B、C、D组采用亚砷酸钠(NaAsO2)制备三价砷肾毒性大鼠模型,即每日腹腔注射10 mg/kg NaAsO2,共10 d。C组于造模前5 d每日川黄方水煎液灌胃,灌胃剂量为成人标准体质量(60 kg)常规用量50倍;D组于造模前5 d每日腹腔注射150 mg/kg的NAC。造模结束后24 h处死动物,测定肾功能(SCr、BUN)、肝功能(ALT、AST),HE染色观察肾、肝病理改变,扫描电镜观察肾小管上皮细胞凋亡,Western印迹检测肾组织cleaved-caspase-3蛋白表达。结果肾功能:与A组相比,造模后B组SCr、BUN均明显升高(P <0.01);与B组相比,C、D组SCr、BUN明显降低(P <0.05)。肝功能:造模后各组ALT、AST比较差异无统计学意义(P> 0.05)。病理形态学检测:HE染色提示B组大鼠肾组织出现明显肾间质水肿、肾小管上皮细胞胞质空泡样变,肝组织出现局灶性肝细胞水肿、局灶性炎细胞浸润;电镜提示B组肾小管上皮细胞空泡样变、线粒体肿胀和嵴断裂、肾小管上皮细胞凋亡增多;C、D组的肾组织病理改变明显减轻、肾小管和肾间质形态结构相对完整、凋亡细胞减少、肾小管上皮细胞空泡样变区域明显减少;肝组织结构基本正常。Western印迹检测:与A组相比,B组肾组织中cleaved-caspase 3蛋白表达显著增多(P <0.01);与B组相比,C、D组肾组织中cleaved-caspase-3蛋白表达明显回落(P <0.01)。结论川黄方能有效抑制临床剂量三价砷的肾脏毒性,其分子机制与抑制caspase 3参与介导的肾小管上皮细胞凋亡有关。

Objective To investigate the inhibitory effect and mechanisms of Chuanhuang Fang on renal toxicity of trivalent arsenic in clinical dosage. Methods Thirty-two male Wistar rats were divided into normal group(A group), model group(B group), Chuanhuang Fang group(C group), N-acetylcysteine group(D group), with eight rats in each group. Sodium arsenite(NaAsO2) was used to prepare trivalent arsenic nephrotoxicity rat model. 10 mg/kg NaAsO2 was injected intraperitoneally every day for 10 days in group B, C and D. In group C, the decoction of Chuanhuang Fang with clinical relevant dose was administered daily five days before the establishment of the model. In group D, 150 mg/kg NAC was injected intraperitoneally five days before the establishment of the model. 24 hours after the model was established, the animals were killed and the renal function(SCr, BUN), liver function(ALT, AST) were measured. The pathological changes of kidney and liver were observed by HE staining. The apoptosis of renal tubular epithelial cells was observed by scanning electron microscopy, and cleaved-caspase-3 protein expression was deteced by Western blotting. Results Compared with group A, SCr and BUN in group B increased significantly(P<0.01). SCr and BUN in group C and D decreased significantly(P<0.05). There was no significant difference in ALT and AST in each group after modeling(P>0.05). HE staining showed that there were obvious interstitial edema and cytoplasm of renal tubular epithelial cells in group B. Vacuolar degeneration, focal hepatocyte edema and focal inflammatory cell infiltration appeared in liver tissue. Electron microscopy showed vacuolar degeneration, mitochondrial swelling and cristae rupture of renal tubular epithelial cells, and increased apoptosis of renal tubular epithelial cells in group B. In group C and D, pathological changes of renal tissue were significantly alleviated, morphological structure of renal tubules and renal interstitium were relatively intact, and apoptotic cells were reduced. The expression of cleaved-caspase-3 protein in renal tissue of group B was significantly higher than that of group A(P<0.01). Compared with group B, the expression of cleaved-caspase-3 protein in renal tissue of group C and D decreased significantly(P<0.01). Conclusions Chuanhuang Fang can effectively inhibit the renal toxicity of clinical relevant dose of trivalent arsenic, and the potential molecular mechanism is related to the inhibition of caspase 3-mediated apoptosis of renal tubular epithelial cells.