清热化瘀方联合缺氧预处理MSCs移植对脑缺血再灌注损伤大鼠eNOS表达的影响

Effect of Qingre Huayu Prescription Combined with HP-MSCs Transplantation on the Expression of eNOS after Cerebal Ischemia-reperfusion Injury in Rats

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 observe the effect of Qingre Huayu Prescription combined with hypoxic preconditionend（HP） bone marrow mesenchymal stem cells （MSCs） transplantation on the expression of eNOS mRNA and protein in rats after cerebral ischemia-reperfusion. Methods Two hundred and sixteen male SD rats were randomly divided into 6 groups including Sham（SO）, middle cerebral artery occlusion group（MCAO）, MSCs transplantation group（N-MSCs）, hypoxic preconditioning MSCs transplantation （HP-MSCs）, Qingre Huayu prescription combined with MSCs transplantation group （MSCs＋QRHY）, and hypoxic preconditionend MSCs transplantation combined with Qingre Huayu Prescription group（ HP-MSCs＋QRHY ）, 36 rats in each. Each group was further divided into 7 days group, 14 days, 28 days groups, 12 for every subgroup. The MCAO models were made in order to evaluate neurological impairment scores at different time points after ischemia and reperfusion by the modified Neurological Severity Scores （mNSS） and to observe the expression changes of mRNA and protein of eNOS after garaging with Qingre Huayu prescription and injecting MSCs or HP-MSCs into rats＇ internal carotid artery for different groups. Results Compared with the model group, the Neurological Severity Scores of transplantion groups were significantly lower（P 〈 0.05）, and the improvement of neurologic function of HP-MSCs＋QRHY group was the most significant（P 〈 0.05）, at all time points. The expressions of eNOS mRNA and protein in the penumbra region all reached the peak at 7 d, then decreased gradually with the time of 14 d and 28 d. In HP-MSCs, MSCs＋ QRHY and HP-MSCs＋QRHY, comparison between groups at the same time point, all increased significantly compared with N-MSCs（P 〈 0.01 or P 〈 0.05）, and the changing trend of HP-MSCs-QRHY group was the most obvious. Conclusion Qingre Huayu prescription combined with HP-MSCs can improve the neurological function of ischemia reperfusion injury in rats, which may be related to up-regulating the expression of eNOS.