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目的探讨清热化瘀方联合缺氧预处理骨髓间充质干细胞(MSCs)移植对脑缺血再灌注损伤大鼠内皮型一氧化氮合酶(eNOS)mRNA及其蛋白表达的影响。方法将216只雄性SD大鼠随机分为6组:假手术组、模型组、MSCs移植对照组(N-MSCs组)、经缺氧预处理后的MSCs移植组(HP-MSCs组)、MSCs移植联合清热化瘀方组(MSCs+QRHY组)、HP-MSCs移植联合清热化瘀方组(HP-MSCs+QRHY组),每组36只。按移植后取材时间点(7,14,28 d)各组又分3个亚组,每组12只。用线栓法制作大鼠脑缺血再灌注损伤动物模型,给予清热化瘀颗粒灌胃及颈内动脉移植BMSCs。分别于术后7,14,28 d对各组大鼠进行神经功能缺损评分(NSS)。RT-PCR及Western blot检测大鼠缺血半暗带区eNOS mRNA及其蛋白表达变化。结果与模型组比较,各时间点N-MSCs组神经功能评分明显减少(P<0.05),各移植组中以HP+QRHY组神经功能改善最为显著(P<0.05)。假手术组eNOS mRNA及其蛋白呈现低水平表达,模型组及移植组其表达较假手术组明显增加(P<0.05);各组eNOS mRNA及其蛋白的表达均于7 d达到高峰,14,28 d表达逐渐下降;同一时间点组间比较,HP-MSCs组、MSCs+QRHY组及HP-MSCs+QRHY组的表达均明显高于N-MSCs组(P<0.05),而以HP-MSCs+QRHY组增高最为明显(P<0.05)。结论 HP-MSCs移植能够显著提高脑缺血再灌注损伤大鼠eNOS的表达,清热化瘀方能够进一步上调其表达及改善脑缺血大鼠的神经功能,发挥其神经保护作用。
Objective To investigate the effect of Qingre Huayu Recipe combined with hypoxia preconditioning on mesenchymal stem cells (MSCs) transplantation on endothelial nitric oxide synthase (eNOS) mRNA and protein expression in rats with cerebral ischemia-reperfusion injury. Methods Totally 216 male Sprague-Dawley rats were randomly divided into 6 groups: sham operation group, model group, MSCs transplantation control group (N-MSCs group), MSCs transplantation group (HP-MSCs group) MSCs + QRHY group and HP-MSCs transplantation combined with Qingre Huayu Recipe group (HP-MSCs + QRHY group), 36 in each group. According to the time point after transplantation (7, 14, 28 d), each group was divided into 3 subgroups, 12 in each group. The rat model of cerebral ischemia-reperfusion injury was established by thread occlusion, and the BMSCs were delivered by Qingre Huayu Granules and intracarotid artery. Neurological deficit scores (NSS) were performed on the 7th, 28th and 28th day after operation in each group. The expression of eNOS mRNA and protein in ischemic penumbra of rats were detected by RT-PCR and Western blot. Results Compared with the model group, the scores of neurological function in N-MSCs group were significantly decreased at each time point (P <0.05). The improvement of neurological function in HP + QRHY group was the most significant (P <0.05). The expression of eNOS mRNA and protein in sham operation group was low, and the expression of eNOS mRNA and protein in model group and transplantation group was significantly higher than that in sham operation group (P <0.05). The expression of eNOS mRNA and protein in each group reached its peak on day 7, MSCs + QRHY group and HP-MSCs + QRHY group were significantly higher than those in N-MSCs group (P <0.05), while the expression of HP-MSCs + QRHY group increased the most obvious (P <0.05). Conclusion HP-MSCs transplantation can significantly increase the expression of eNOS in rats with cerebral ischemia-reperfusion injury. Qingre Huayu Recipe can up-regulate its expression and improve the neurological function of rats with cerebral ischemia and exert its neuroprotective effects.