丹参多酚酸配伍三七总皂苷通过调节M1/M2型小胶质细胞极化对大鼠脑缺血再灌注损伤的影响

Effects of salvia polyphenolic acids combined with panax notoginseng saponins on cerebral ischemia-reperfusion injury in rats by regulating M1/M2 microglia polarization

关键词:  丹参多酚酸  三七总皂苷  小胶质细胞  极化  脑缺血再灌注损伤

Key words:  salvia polyphenolic acids  panax notoginseng saponins  microglia  polarization  cerebral ischemia-reperfusion injury

摘要:

[目的] 探究丹参多酚酸配伍三七总皂苷通过调节M1/M2型小胶质细胞极化对大鼠脑缺血再灌注损伤的影响。[方法] 雄性Wistar大鼠60只，采用线栓法制作大脑中动脉阻塞模型，随机分为假手术组（Sham）、缺血再灌注组（I/R）、依达拉奉组（Eda，6 mg/kg）、丹参多酚酸组（SPA，21 mg/kg）、三七总皂苷组（PNS，100 mg/kg）、丹参多酚酸配伍三七总皂苷组（SPA+PNS，21 mg/kg+100 mg/kg），尾静脉注射给药，每日1次，Sham组与I/R注射等容积生理盐水，连续给药5 d后，观察大鼠脑血流变化、神经功能评分，组织形态学变化，Nissl染色及神经元核蛋白（NeuN）免疫荧光染色观察神经元损伤情况，CD16/Iba-1、CD206/Iba-1免疫荧光双染检测缺血半暗带M1型、M2型小胶质细胞表型情况，蛋白免疫印迹（Western-Blot）法检测M1型小胶质细胞标记物白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）的蛋白表达及M2型小胶质细胞标记物CD206、转化生长因子-β（TGF-β）及白细胞介素-10（IL-10）的蛋白表达情况。[结果] 与I/R组比较，SPA组、PNS组、SPA+PNS组在脑血流、神经功能缺损、组织形态、神经元损伤情况均有不同程度改善，并促进M2型小胶质细胞CD206/Iba-1表达，上调CD206、TGF-β、IL-10的蛋白表达；抑制M1型小胶质细胞CD16/Iba-1表达，下调IL-1β、IL-6、TNF-α、iNOS的蛋白表达，且SPA+PNS组改善程度优于SPA组、PNS组。[结论] 丹参多酚酸、三七总皂苷及两者配伍可减轻大鼠脑缺血再灌注损伤，且配伍应用效果优于单独应用，其机制可能与促进小胶质细胞M1型极化向M2型转换有关。

Abstract:

[Objective] To investigate the effect of salvia polyphenolic acids combined with panax notoginseng saponins on cerebral ischemia-reperfusion injury in rats by adjusting the polarization of M1/M2 microglia.[Methods] The occlusion model of middle cerebral artery was established by thread embolism method. Sixty male Wistar rats were randomly divided into sham operation group (Sham),ischemia-reperfusion group (I/R),edaravone group (Eda,6 mg/kg),salvia polyphenolic acids group (SPA,21 mg/kg),panax notoginseng saponins group (PNS,100 mg/kg),salvia polyphenolic acids combined with panax notoginseng saponins group (SPA+PNS,21 mg/kg+100 mg/kg),tail vein injection once a day,Sham group and I/R injection with equal volume of normal saline,after continuous administration for 5 days,the changes of cerebral blood flow,neurological function score and histomorphology of rats were observed. Nissl staining and neuroribonucleoprotein (NeuN) immunofluorescence staining were used to observe neuronal damage. CD16/Iba-1 and CD206/Iba-1 immunofluorescence double staining were used to detect the phenotypes of M1 and M2 microglia in ischemic penumbra. Western-blot assay was used to detect the protein expressions of M1 microglial markers interleukin-1β (IL-1β),interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α),inducible nitric oxide synthase (iNOS),M2 microglial marker CD206,transforming growth factor-β (TGF-β) and interleukin-10 (IL-10).[Results] Compared with I/R group,SPA group,PNS group and SPA+PNS group have improved to varying degrees in cerebral blood flow,nerve function defect,tissue morphology and neuronal damage,and promoted the expression of CD206/Iba-1 in M2 microglial cells,and up-regulated the protein expression of CD206,TGF-β and IL-10. Inhibition of M1 microglia CD16/Iba-1 expression,down-regulation of IL-1β,IL-6,TNF-α,iNOS protein expression,and SPA+PNS group improved better than SPA group,PNS group.[Conclusion] Salvia polyphenolic acids,panax notoginseng saponins and their compatibility can reduce cerebral ischemia-reperfusion injury in rats,and the compatibility application effect is better than that of single application. The mechanism may be related to promoting the conversion of M1 type polarization to M2 type polarization of microglia.