红景天苷调控11β-HSD1/PTEN/PI3K通路对脑缺血大鼠小胶质细胞/巨噬细胞表型转化作用研究

Rhodiola rosea L has long been used for treating ischemic stroke in traditional Chinsese medicine,but the underlying mechanisms of action are poorly understood.Our preliminary data shows that salidroside decreased brain infarct volume after transient middle cerebral artery occlusion (MCAO) with reperfusion in rats, and decreased the expressions of IL-6、IL-1β、11β-HSD1、PTEN、PI3K.We have previously demonstrated that inhabited 11β-HSD1/PTEN/PI3K signaling pathways can reduce inflammation in adipocytes .The purpose of the present study is to confirm whether salidroside reduces cerebral inflammation by transforming M1 macrophages/microglia to M2, and to determine if this occurs through11β -HSD1/PTEN/PI3K signalling. Animals will be treated with salidroside (50mg/kg/d), or placebo, by intraperitoneal injection, for 1d, 3d, 5d,7d or 14d after MCAO with reperfusion to follow the time course of effects of salidroside on neurological deficit scores, infarct volumes, 11β-HSD1 activity, and the levels of PTEN, PKB and markers of M1 and M2 macrophage / microglial phenotypes in brain will be measured. Other groups of rats will be treated similarly except that they will also receive intracerebroventricularly the PTEN inhibitor SF1670, or the PI3K inhibitor wortmannin or placebo, prior to MCAO/reperfusion, and for up to 14 days later, to determine whether the effects of emodin are dependent on PTEN/ PI3K signalling. Finally, we will measure the effects of salidroside on the markers of M1 and M2 phenotypes in LPS- or IL-4-induced primary cultures of rat microglia and human macrophages, and investigate whether depletion of PTEN with target-specific RNAi, or inhibition of PI3K with wortmannin, can alter the effects of emodin. The results of this study will have significant impacts on understanding of the molecular mechanisms of the actions of salidroside in ischemic stroke. It will also contribute to understanding the mechanism transformation of M1 and M2 macrophage / microglial phenotypes.

课题组前期研究表明红景天苷能够降低缺血性脑损伤大鼠的梗死体积，特异地抑制11β-HSD1的活性，降低IL-6和IL-1β的基因表达；结合课题组报道的抑制11β-HSD1能够调节PTEN/PI3K通路，进而对脂肪细胞具有抗炎作用，我们假设，红景天苷通过调节小胶质细胞和巨噬细胞11β-HSD1/PTEN/PI3K通路，减缓M2细胞表型向M1转化，从而抑制脑缺血的炎症反应。本研究通过不同时点腹腔注射给药，检测梗死体积，PTEN，PKB和M1/M2细胞标记物的表达，研究红景天苷对MCAO再灌注大鼠的时效关系。在此基础上，通过侧脑室注射PTEN或PI3K抑制剂后造模，给药，测量以上指标，研究该通路对红景天苷作用的影响。并以LPS或IL-4诱导原代培养小胶质细胞和巨噬细胞为模型，研究红景天苷调节该通路对细胞表型的影响。本项目对认识红景天苷在脑卒中的药理作用有重要意义。

红景天苷是红景天的主要有效成分，本课题研究结果表明，大脑中动脉栓塞再灌注（MCA0）模型大鼠，经红景天苷（50mg/kg/d）连续腹腔注射6天，红景天苷能够降低MCA0模型大鼠脑梗死体积，改善神经功能损伤；红景天苷能够降低脑组织细胞凋亡数目和cleaved caspase-3、Bax、Nogo及其受体NgR的蛋白水平，提高Bcl-xl、NGF、BDNF和NeuN的水平，表明红景天苷能抑制由于缺氧再灌注引起的神经细胞凋亡。红景天苷能够降低BV2小胶质细胞LPS炎症模型IL-6释放量，及iNOS、IL-1β、TNF-α、CD68 mRNA的表达，同时，红景天苷能够降低MCAO大鼠IL-1β、iNOS 、Iba1、CD16、CD11b、CD86的表达水平，这些结果提示红景天苷可选择性抑制小胶质细胞的异常活化并降低炎症水平，减缓小胶质细胞M2表型向M1转化；为进一步探讨红景天苷对MCAO大鼠神经保护的作用机制，本课题研究发现，红景天苷经PI3K/Notch通路能够降低MCAO大鼠CD14, CD44, TNF-α, IL-6, IL-1β, iNOS等炎性基因mRNA表达水平、抑制MCAO大鼠NFκB-p65核转位，以及能够抑制Notch1、RBP JK、Hes1 的表达水平，这些结果提示红景天苷对MCAO大鼠的神经保护及减缓小胶质细胞M2表型向M1转化与PI3K/Notch信号通路相关。综上所述，本项目主要研究并报道红景天苷在脑卒中动物模型的神经保护和减缓小胶质细胞M2表型向M1转化的抗炎作用；对认识传统中药红景天及其有效成分红景天苷在脑卒中的药理作用有重要意义。