清化固肾排毒方对UUO大鼠上皮细胞转分化的影响

Effect of “Qinghua Gushen Paidu Decoction”on epithelial mesenchymal transition in UUO rats

目的观察清化固肾排毒方对单侧输尿管结扎(UUO)大鼠肾间质纤维化上皮细胞转分化(EMT)的影响。方法采用单侧输尿管结扎的方法制备肾纤维化大鼠模型。108只SPF级SD大鼠随机分为假手术组(n=18)、UUO模型组(n=18)、洛丁新组(n=18)和清化固肾排毒方高剂量组(n=18)、中剂量组(n=18)、低剂量组(n=18)。6组大鼠给予相应药物治疗,每日1次。观察给药过程中大鼠精神状态、活动度、体毛色泽、大便形状等一般情况和体质量变化。各组分别于第7、14、21天时随机处死6只大鼠,收集大鼠24 h尿液,检测24 h蛋白定量(24 h UP),采集血液检测血清尿素氮(BUN)和肌酐(Scr),留取手术侧肾脏组织进行相关病理检测。采用HE染色评价肾小管损伤程度,免疫组织化学染色检测肾脏组织α平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)的表达。结果与假手术组比较,模型组大鼠一般情况较差,体质量增长缓慢,血Scr、BUN升高(P<0.05),肾小管损伤程度较重(P<0.05),肾组织TGF-β1、α-SMA表达明显增多(P<0.05);随着输尿管梗阻时间延长,各指标程度加重。与模型组比较,中药(高、中、低)组、洛丁新组大鼠的血Scr、BUN下降(P<0.05),小管损伤程度有所缓解(P<0.05),TGF-β1、α-SMA表达降低(P<0.05);与洛丁新组比较,各指标差异无统计学意义(P>0.05);中药高中低剂量组之间比较,差异无统计学意义(P>0.05)。结论清化固肾排毒方可以抑制EMT的发生,其作用机制可能是通过抑制TGF-β1表达,下调α-SMA的表达,从而抑制EMT和ECM的积聚,起到抗肾纤维化的作用。

Objective To investigate the influence and mechanism of ＂Qinghua Gushen Paidu Decoction＂ on renal tubular epithelial mesenchymal transition in unilateral ureteral obstruction （UUO） rats. Methods A rat model of renal fibrosis was prepared by unilateral ureteral obstruction. 108 SD rats were randomly divided into sham operation group （ n = 18 ） , UUO model group （ n = 18 ） , ＂Qinghua Gushen Paidu Decoction＂ high dose （ n = 18 ） , middle dose （ n = 18 ） and low dose （ n = 18 ） groups, and Lotensin treated group （ n = 18 ）. The 6 groups of rats were treated with corresponding drugs, once a day. Mental conditions, activities, hair color, shape of stool, and body weight were observed during administration. At 7, 14, and 21 days, 6 rats in each group were randomly sacrificed. Then 24h urine was collected and 24h urinary protein （24h UP） was detected. Serum blood urea nitrogen（BUN） and serum creatitine（Scr） were also tested. The left surgery renal tissue were collected for pathological examinations. The pathological changes of the obstruction renal tissue were examined by Hematoxylin and eosin staining （HE）. The protein expressions of TGF-131 and et-SMA were detected by immunohistochemical staining. Results Compared with the sham-operation group, the general condition of the rats was poorer in the other groups, their body weight grew slower, and serum levels of BUN, Scr increased （ P 〈 0.05 ）, the renal tubular damaged comparatively serious（ P 〈0.05 ）, protein expression levels of TGF-I31 , ct-SMA increased in the renal tissue （P 〈 0.05 ）. The renal interstitial fibrosis degree was gradually aggravated along with the ureteral obstruction time. Compared with the model group, the serum levels of BUN, Scr decreased （ P 〈0.05 ） in the rats of the TCM groups （ high, middle and low dose groups） and Lotensin treated group, the condition of the damage of the renal tubular get better（ P 〈0.05 ） , protein expression levels of TGF-β1 ,α-SMA decreased（P 〈0.05 ）. Compared with the Lotensin treated group, no significant difference in effect on indexes was observed between TCM groups and Lotensin treated group（ P 〉0.05 ）. No significant difference in effect on indexes was observed between the high, middle and low dose groups（ P 〉0.05 ）. Conclusion ＂ Qinghua Gushen Paidu Decoction＂ may inhibit the EMT by down-regulating the expression of TGF-β1 , α-SMA, can protect the kidney and ameliorate renal interstitial fibrosis.