益肾活血方通过miR-126/VEGF-Notch信号通路改善肾纤维化

Research on the intervention of Yishen Huoxue prescription to renal fibrosis through the signal regulation by microRNA-126 to VEGF-Notch

目的 探讨益肾活血方通过调控微小RNA-126/血管内皮生长因子- Notch（miR-126/VEGF-Notch）信号通路延缓肾纤维化发展的分子机制.方法 按照随机数字表法将90只雄性SD大鼠平均分为假手术（Sham）组、单侧输尿管结扎术（UUO）模型组、氯沙坦治疗组（50 mg·kg^-1·d^-1）以及高、中、低剂量益肾活血方治疗组（25.2、12.6、6.3 mL/kg）.各治疗组于UUO制模结束当日开始灌胃给药,直至实验结束.术后7、14、21 d取大鼠左侧肾脏,用苏木素-伊红（HE）和Masson染色于镜下观察肾组织病理学改变,并计算肾纤维化评分;用反转录-聚合酶链反应（RT-PCR）检测肾组织miR-126、血管内皮生长因子A（VEGFA）、血管内皮生长因子受体2 （VEGFR-2）、Notch1的mRNA表达.结果 ① 病理结果：Sham组肾组织形态正常.UUO模型组肾小管扩张,肾间质炎性细胞增多,术后7 d即可见肾小管间质纤维化,并随时间延长程度逐渐加重;术后各时间点肾纤维化评分较Sham组明显增高.各治疗组肾脏肿胀程度、肾实质萎缩等均较UUO模型组有所改善;中、高剂量益肾活血方组和氯沙坦组术后7 d肾纤维化评分即明显低于UUO模型组（分：1.00±1.00、0.91±0.58、1.01±0.58比2.00±0.00,均P＜0.01）,而低剂量益肾活血方组肾纤维化评分与UUO模型组无明显差异.② RT-PCR结果：UUO模型组术后各时间点肾组织miR-126、VEGFA、VEGFR-2及Notch1的mRNA表达量均较Sham组明显升高.与UUO模型组比较,中、高剂量益肾活血方组和氯沙坦组术后7 d肾组织miR-126、VEGFA、VEGFR-2及Notch1的mRNA表达量即明显升高〔miR-126（2-ΔΔCt）：0.465±0.067、0.639±0.092、0.404±0.069比0.132± 0.021,VEGFA（2-ΔΔCt）：0.024±0.005、0.027±0.007、0.023±0.006 比 0.014±0.006,VEGFR-2（2-ΔΔCt）：0.021±0.007、0.023±0.008、0.019±0.007 比 0.012±0.004,Notch1（2-ΔΔCt）：0.017±0.004、0.020±0.005、0.018±0.005比0.007±0.004,均P＜0.05〕,且中、高剂量益肾活血方组各指标基因表达量较氯沙坦组有一定上升趋势（但P＞0.05）;而低剂量益肾活血方组各指标基因表达量与UUO模型组比较差异无统计学意义.结论中、高剂量益肾活血方可以有效拮抗肾纤维化,其机制可能是通过上调miR-126/VEGF-Notch信号通路,从而介导肾脏微血管新生,最终改善肾微血管损伤和延缓肾纤维化进程.

Objective To explore the molecular mechanism of Yishen Huoxue prescription in delaying the development of renal fibrosis by regulating the microRNA-126/vascular endothelial growth factor-Notch （miR-126/VEGF-Notch） signaling pathway. Methods Ninety male Sprague-Dawley （SD） rats were randomly divided into sham operation group （sham group）, unilateral ureteral obstruction （UUO） model group, losartan group （50 mg·kg^-1·d^-1） and high, medium and low doses Yishen Huoxue prescription group （25.2, 12.6, 6.3 mL/kg）. Each treatment group began to administer drugs by gavage on the day when UUO modeling was finished until the end of the experiment. Left renal tissues of rats were harvested after 7, 14 and 21 days postoperatively. The pathological changes of renal tissue were observed after hematoxylin and eosin （HE） and Masson staining under the microscope, and the renal fibrosis score was calculated. The mRNA expressions of renal tissues miR-126, VEGFA, vascular endothelial growth factor receptor-2 （VEGFR-2）, Notch1 were detected by reverse transcription-polymerase chain reaction （RT-PCR）. Results① Pathology results： the kidney tissue of sham group was normal. In UUO model group, renal tubules expanded and inflammatory cells in renal interstitium increased; renal tubulointerstitial fibrosis could be seen 7 days after operation, and the degree of fibrosis was gradually increased with time. The renal fibrosis score at each time point after operation in UUO model group was significantly higher than that in sham group. Compared with UUO model group, each treatment group were improved the degree of renal swelling and atrophy of renal parenchyma; the score of renal fibrosis were significantly decreased in the middle and high doses Yishen Huoxue prescription group and losartan group at the 7th day after operation （1.00±1.00, 0.91±0.58, 1.01±0.58 vs. 2.00±0.00,all P 〈 0.01）; but there was no significant difference between low dose Yishen Huoxue prescription group and UUO model group. ② RT-PCR results： Compared with sham group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissues were significantly increased at each time point after operation in UUO model group. Compared with the UUO model group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissue of 7 days postoperatively in the middle and high doses Yishen Huoxue prescription group and the losartan group were significantly increased [miR-126（2-ΔΔCt）：0.465±0.067, 0.639±0.092, 0.404±0.069 vs. 0.132±0.021; VEGFA（2-ΔΔCt）： 0.024±0.005, 0.027±0.007, 0.023±0.006 vs. 0.014±0.006; VEGFR-2（2-ΔΔCt）：0.021±0.007, 0.023±0.008, 0.019±0.007 vs. 0.012±0.004; Notch1（2-ΔΔCt）：0.017±0.004, 0.020±0.005, 0.018±0.005 vs. 0.007±0.004; all P 〈 0.05]; compared with the losartan group, the mRNA expressions of each index in the middle and high doses Yishen Huoxue prescription group were increased at all the time points, but there was no significant difference between them （all P 〉 0.05）. There was no significant difference in mRNA expression of each index between low dose Yishen Huoxue prescription group and UUO model group. Conclusions The medium and high doses of Yishen Huoxue prescription can effectively antagonize renal fibrosis. Yishen Huoxue prescription may use up-regulation the signaling pathways of miR-126/VEGF-Notch to mediate renal microvascular newly born, eventually to improve renal microvascular damage and delay the development of renal fibrosis progression.