电针调控PRG5/RhoA信号通路对脑梗死远隔损害及神经再生作用机制研究

Secondary remote damage of acute cerebral infarction(ACI) is the key reason for its serious damage, high morbidity, neural functional recovery and treatment difficulty.In the early，we found that RhoA inhibit signaling pathways involved in remote damage of ACI and inhibit eNSCs proliferation，however，electroacupuncture at Baihui and Dazhui acupoints could inhibit RhoA signal transmission across the membrane and reduce the secondary damage of thalamus,substantia nigra and hippocampus.But the key problem that how electroacupuncture reduce secondary damage,regulate PRG5 to mediate RhoA signal transmission across the membrane,and Promote remote damage regeneration is not clear.We set up hypertension cerebral infarction model：① make PRG5 gene silence by mediation of shRNA，and detect PRG5,Cdc42,LPA/LPA-R protein of ACI hematogenously，to make clear how PRG5 inhibit RhoA pathway and the effect of acusector；② detect eNSCs differentiation related Notch signal pathway protein，and make clear the mechanism of acusector mediate PRG5/RhoA to promote eNSCs differentiation；③ observe dynamically the effect of acusector to infarct and hematogenously nerve fiber bundles, filiform pseudopodia and axon growth and nerve repair，to make clear that acusector regulate PRG5 to mediate RhoA signal so that promote eNSCs proliferation in remote damage is the new target for therapy.Then provide new theory basis for electric acupuncture could reduce ACI remote damage.

急性脑梗死（ACI）继发远隔损害是导致病损严重、致残率高和神经功能恢复困难的重要原因。我们既往研究发现RhoA抑制信号通路参与ACI远隔损害并阻遏eNSCs增殖，电针百会、大椎穴可抑制RhoA信号跨膜传导，减轻丘脑、黑质、海马继发损害。但电针减轻远隔损害与PRG5调控RhoA抑制信号跨膜传导、促进远隔部位神经再生等关键问题不清楚。本课题建立高血压脑梗死模型：①用shRNA介导沉默PRG5基因，检测ACI远隔部位PRG5、Cdc42、LPA/LPA-R蛋白，明确PRG5抑制RhoA通路及电针作用；②检测eNSCs分化相关Notch信号通路蛋白，明确电针调控PRG5/RhoA促进eNSCs分化机制；③动态观察电针对梗死灶及远隔部位神经纤维、丝状伪足和轴突生长及神经修复影响，明确电针介导PRG5调控RhoA通路促进远隔损害部位eNSCs增殖为治疗新靶点，为电针减轻ACI远隔损害提供新的理论依据。

电针可刺激缺血性脑卒中动物模型的神经发生，但其相关机制尚不清楚。本研究旨在评价电针介导脑缺血再灌注损伤后神经发生因子5（PRG5）调控勿动蛋白-A（Nogo-A）/NgR与LPA/LPA-R抑制信号传导通路的相关机制，及不同时间点Nestin和Neuron特异性核蛋白抗原(Neun)表达的影响。随机数字表分为假手术组（sham）、模型组（MCAO）、电针组（MCAO+EA）。采用大脑中动脉闭塞(MCAO)模型制作大鼠脑缺血再灌注模型。电针治疗Gv 20(百会)和gv 14(大椎)穴，每日30 min，在MCAO后进行。通过神经功能缺损评分量表评定神经功能，三氯化苯四氮唑(TTC)染色确定梗死范围，蛋白免疫印迹法（Western blot）、免疫组化法、免疫荧光法等观察Sham组、MCAO组和MCAO+EA组溴脱氧尿苷(Brdu)/巢蛋白(Nestin-)、Brdu/Doublecortin(DCX)阳性细胞的数量，及Nogo-A、Ras同源基因家族成员A（RhoA）、溶血磷脂酸（LPA）蛋白等PRG5/RhoA信号通路蛋白及其基因的表达。结果表明：1.MCAO大鼠神经功能缺损评分明显高于同时间段的MCAO+EA组，后者明显高于sham组。2.脑梗死体积TTC检测显示MCAO+EA组神经功能损伤核心明显低于MCAO组；3.脑梗死组远隔区域（如海马、颈髓等）Nogo-A、RhoA及LPA 蛋白的表达均显著高于假手术组，MCAO+EA组远隔区域RhoA、Nogo-A 及 LPA 蛋白的表达均显著低于同时间点脑梗死组。4.免疫染色结果表明，电针组大鼠的Brdu/Nestin-和Brdu/DCX阳性细胞较MCAO组明显增加，电针可显著提高PRG5的蛋白表达水平，显著降低Nogo-A、LPA和RhoA等抑制分子的蛋白表达水平。这些结果提示电针可通过PRG5调控Nogo-A和LPA介导的RhoA信号通路，降低脑梗死远隔损害部位神经元死亡数目、诱导脑缺血再灌注时内源性神经干细胞(ENCS)的增殖和分化，以减轻脑梗死远隔损害，对神经功能的恢复具有重要意义。