胡黄连苷Ⅱ通过抑制调控上皮-间质转化减轻大鼠肾脏缺血再灌注损伤后肾纤维化

Picroside Ⅱ on reducing renal fibrosis after renal ischemia reperfusion injury in rats by inhibiting epithelial-mesenchymal transition

目的建立大鼠肾脏缺血再灌注模型后研究胡黄连苷Ⅱ能否通过抑制上皮-间质转化减轻由肾脏缺血再灌注损伤(IRI)引起的肾组织纤维化。方法健康大鼠30只,随机分为3组,每组10只,包括对照组(假手术组)IRI模型组和药物处理组(胡黄连苷Ⅱ组)。建立大鼠肾脏IRI模型,HE染色观察各组肾脏组织病理学变化;通过免疫组织化学染色检测波形蛋白(vimentin)观察上皮-间质转化程度;通过Masson染色观察各组肾脏组织纤维化程度。Western blot观察上皮-间质转化标志蛋白结缔组织生长因子(CTGF)与纤维连接蛋白(FN)的表达。结果 HE染色结果显示,和假手术组相比,IRI模型组肾小管上皮细胞损伤严重,经胡黄连苷Ⅱ治疗后肾小管上皮细胞损伤程度显著减轻;Massion染色结果显示,与假手术组相比,IRI模型组肾组织纤维化明显,而经药物处理后,肾组织纤维化程度明显减轻;免疫组化结果显示,与假手术组阳性细胞数(5.3±1.53)相比,IRI模型组vimentin阳性细胞数(63.7±4.93)显著增加,差异有统计学意义(t=19.57,P<0.01);而IRI模型组与药物处理组相比,vimentin表达阳性细胞数(23.3±4.51)显著减少,差异有统计学意义(t=11.04,P<0.01)。Western-blot结果显示,与假手术组FN(0.21±0.02)和CTGF(0.63±0.10)蛋白表达相比,IRI模型组FN(1.23±0.09)和CTGF(1.15±0.08)蛋白表达显著增加,差异有统计学意义(t=17.97,7.15,均P<0.01);而药物处理组FN(0.46±0.05)和CTGF(0.79±0.11)蛋白表达与模型组相比,FN、CTFG蛋白表达水平明显下降,差异均有统计学意义(t=12.37,4.75,均P<0.01)。结论胡黄连苷Ⅱ可通过抑制上皮-间质转化,减轻肾脏缺血后肾脏组织的纤维化。

[Objective]To establish a model of renal ischemia reperfusion injury in rats,investigate whether Picroside Ⅱ reduce renal fibrosis caused by ischemia reperfusion injury(IRI) by inhibiting epithelial-mesenchymal transition(EMT).[Methods]30 healthy rats were randomly divided into three groups,10 in each group,named sham-operation group,IRI model group and drug-treated group(Picroside Ⅱ group). After esta blishment of the renal ischemia reperfusion injury model,the pathological changes of renal tissues were tested by HE staining. The protein vimentin was detected by immunohistochemical staining to observe the degree of EMT. The degree of renal fibrosis was observed by Mass’ on staining. The expression levels of Connective Tissue Growth Factor(CTGF) and fibronectin( FN) were tested by Western blot.[Results]HE staining showed that,compared with the sham-operation group,the renal tubular epithelial cells in the IRI model group were severely damaged,and the degree of renal tubular epithelial cell damage was significantly reduced after treatment with Picroside Ⅱ. Massion staining results showed that,compared with the sham-operation group,the renal tissue fibrosis was evident in the IRI model group,and the degree of renal fibrosis was significantly reduced after drug treatment. The results of immunohistochemistry showed that,the number of vimentin-positive cells in the IRI model group(60.02±2.641) was significantly higher than that in the sham-operation group(5.3±1.53),and the difference was statistically significant(t=19.57,P<0.01). Compared with the drug-treated group(22.50±1.73),the number of Vimentin-positive cells was significantly decreased in the IRI model group,and the difference was statistically significant(t=11.04,P<0.01). Western-blot showed that the expression of FN(1.23±0.09) and CTGF(1.15±0.08) in the IRI model group were higher than those in the sham-operation group[FN(90.21 ±0.02),CTGF(0.63 ±0.10)],and the differenced were statistically significant(t =17.97,7.15,both P<0.01). While the expression of FN(0.46±0.05) and CTGF(0.79±0.11)in the drug-treated group were lower than those in the IRI model group,and the differenced were statistically significant(t=12.37,4.75,both P<0.01).[Conclusion]Picroside Ⅱ reduces fibrosis of kidney tissue after renal ischemia injury by inhibiting epithelial-mesenchymal transition.