清热化瘀Ⅱ号方对大鼠脑缺血再灌注损伤TLR4、TRIF mRNA表达的影响

Effects of Qingre Huayu Formula Ⅱ on the expression of TLR4 and TRIF mRNA in rat model of cerebral ischemia-reperfusion injury

目的:观察清热化瘀Ⅱ号方对脑缺血再灌注损伤大鼠TLR4、TRIF mRNA表达的影响。方法:将成年SD大鼠随机分为两大组,每组再分为:空白对照组(n=6)、假手术组(n=30)、模型组(n=30)、清热化瘀Ⅱ号方组(n=30)。采用线栓法阻断大脑中动脉制备I/R模型,除空白对照组外,每组按照缺血2h后再灌注3、6、12、24、48h 5个时间点分为5个亚组。取材后将一组脑组织行常规HE染色并在光学显微镜下观察其病理形态学改变,另一大组取材后采用实时荧光定量PCR法观察各个时间点TLR4、TRIF mRNA的表达变化。结果:1HE染色结果:光镜下观察清热化瘀Ⅱ号方组与模型组的脑组织病理形态学比较,在各个时间点神经元受损明显减轻。2荧光定量PCR反应结果:TLR4、TRIF mRNA在空白对照组、假手术组均有少量表达,模型组缺血各时间点其表达含量较其他组均明显增多(P<0.05);模型组大鼠随再灌注时间的延长TLR4、TRIF mRNA的表达含量逐渐升高,脑缺血再灌注24h后其mRNA表达达高峰,48h后开始下降;清热化瘀Ⅱ号方组较模型组各时间点其表达含量显著降低,差异均有统计学意义(P<0.05)。结论:清热化瘀Ⅱ号方可通过降低TLR4、TRIF mRNA的表达,以减少神经元的凋亡、炎性反应及脑组织受损程度进而改善脑内炎症环境,促进脑缺血再灌注损伤后的神经功能恢复。

Objective:To investigate the effects of Qingre Huayu Formula Ⅱ(QRHY Ⅱ) on the expression of TLR4 and TRIF mRNA in rat model of cerebral ischemia-reperfusion injury.Methods:SD rats were randomly divided into two groups.In each group, rats were divided into 4 subgroups:the normal control group(group A, n=6), the sham operation group(group B, n=30), the model group(group C, n=30) and Qingre Huayu Formula I group(group D, n=30).The rat model of cerebral ischemia were established by middle cerebral artery occlusion(MCAO).The rats in the group B, C and D experienced the reperfusion after 3h, 6h, 12 h, 24 h, and 48 h of cerebral ischemia injury.The pathological changes of brain were observed by the HE staining.The expression of TLR4 and TRIF mRNA were quantitatively measured by real-time PCR.Results:1HE staining showed:the treatment of QRHY Ⅱ significantly reduced the neurons damage when compared with the C group at each time point.2The result of real-time PCR showed:TLR4 and TRIF mRNA were expressed at low levels in group A and B.The expression levels of group C were significantly higher than other groups at each time point(P<0.05).The expression of TLR4 and TRIF mRNA was increased gradually in the group C with the prolongation of I/R duration and reaching the heights after 24 h.It was decreased after 48 h.Compared with group C, the expression of 12 h, 24 h and 48 h after operation in group D decreased significantly(P<0.05).Conclusion:QRHY I could reduce the neuronal apoptosis, the inflammatory response and the damage degree of brain tissue and improved the inflammatory environment in the brain to promote the recovery of neurological function after cerebral ischemia reperfusion injury in rats by reducing the expression of TLR4 and TRIF mRNA.