基于miR21/VEGF/Akt信号通路探讨豨莶草对脑缺血再灌注损伤大鼠及HUVECs细胞的保护作用

Study on the Mechanism of the Effect of Euphorbia Extract on Brain Ischemia-Reperfusion Injury in Rats and HUVECs Cells Based on the miR-21/VEGF Signaling Pathway

关键词:  豨莶草  脑缺血再灌注损伤  miR-21  血管生成  VEGF

Key words:  Siegesbeckia orientalis Granules  cerebral ischemia reperfusion injury  miR-21/VEGF signaling pathway  Angiogenesis.

摘要:

目的：基于miR-21/VEGF/Akt信号通路探究豨莶草对脑缺血再灌注损伤大鼠及HUVECs细胞的保护作用。方法：30只大鼠随机分为假手术组、模型组、豨莶草低、高剂量组、阳性对照组,除假手术组外,其他组采用线栓法制备大鼠脑缺血再灌注损伤模型。造模后豨莶草组灌胃不同剂量豨莶草配方颗粒,空白对照组与模型组灌胃等体积生理盐水,阳性对照组灌胃6mg/kg尼莫地平,连续给药7天。采用改良神经功能缺损评分(modified neurological severity score,mNSS)评价各组大鼠神经功能恢复情况；采用Western Blot检测缺血侧脑组织VEGFA、PI3K、AKT等蛋白表达；Real-Time PCR法检测缺血侧脑组织miR-21、CD31、VEGFA等基因表达。同时建立HUVECs氧糖剥夺/复氧复糖(OGD/R)模型,随机分为空白对照组、模型组、豨莶草组、豨莶草+miR-210a-5p抑制剂组、豨莶草+抑制剂阴性对照组,CCK-8法检测细胞活性；管腔形成实验检测促血管生成情况；Real-Time PCR法检测miR-21等基因表达,Western Blot法检测PI3K等蛋白表达,并采用双荧光素酶实验验证VEGFA是miR-21的靶基因。结果：与模型组相比,豨莶草可以促进脑缺血大鼠神经功能恢复,上调脑缺血大鼠miR21/VEGF通路关键信号分子的表达；并且能够增强OGD/R模型HUVECs的细胞活性,上调HUVECs细胞中miR-21表达,激活VEGF通路,提高HUVECs细胞PI3K、p-AKT、AKT蛋白表达,促进血管生成。结论：豨莶草颗粒通过miR21上调VEGF/Akt信号通路促进血管生成,从而改善脑缺血再灌注损伤。

Abstract:

Objective: To explore the protective effect of Herba Siegesbeckiae on cerebral ischemia-reperfusion injury rats and HUVECs cells based on miR-21/VEGF/Akt signaling pathway. Methods: 30 rats were randomly divided into sham operation group, model group, low dose Herba Siegesbeckiae group, high dose herba Siegesbeckiae group and positive control group. The cerebral ischemia-reperfusion injury model of rats was prepared by thread embolization method in other groups except sham operation group. After the model herba Siegesbeckiae group was administrated with different doses of formula granules of Herba Siegesbeckiae. The blank control group and model group were administrated with equal volume normal saline, and the positive control group was administrated with 6mg/kg nimodipine for 7 consecutive days. modified neurological severity score (mNSS) was used to evaluate the neurological recovery in each group. The expressions of VEGFA, PI3K and AKT were detected by Western Blot. The expressions of miR-21, CD31 and VEGFA in ischemic brain were detected by Real-Time PCR. At the same time, HUVECs OGD/R model was established and randomly divided into blank control group, model group, Herba Siegesbeckiae group, Herba Siegesbeckiae +miR-210a-5p inhibitor group and Herba Siegesbeckiae + inhibitor negative control group. The cell activity was detected by CCK-8 method. Lumen formation test was performed to detect the angiogenesis. Real-Time PCR was used to detect the expression of miR-21 and other genes, and Western Blot was used to detect the expression of PI3K and other proteins, and double luciferase assay was used to verify that VEGFA was the target gene of miR-21. Results: Compared with model group, Herba Siegesbeckiae could promote the recovery of nerve function and up-regulate the expression of key signal molecules of miR21/VEGF pathway in ischemic rats. Moreover, it can enhance the cellular activity of HUVECs in OGD/R model, up-regulate the expression of miR-21 in HUVECs cells, activate VEGF pathway, improve the expression of PI3K, p-AKT and AKT protein in HUVECs cells, and promote angiogenesis. Conclusion: Herba Siegesbeckiae granules promote angiogenesis through miR21 up-regulation of VEGF/Akt signaling pathway, thereby improving cerebral ischemia-reperfusion injury.