电针原发性高血压模型大鼠太冲、曲池穴观察microRNA-9对其下游靶点P2X7受体、炎性因子、血管紧张素Ⅱ的影响

Novel mechanism of lowering blood pressure in SHR by electroacupuncting QuChi(LI11)and TaiChong(LR3):MicroRNA9

目的通过观察microRNA-9对其下游靶点P2X7受体、炎性因子、血管紧张素Ⅱ的影响,分析电针降压机制。方法选择原发性高血压模型大鼠(spontaneous hypertension rat,SHR)作为高血压动物模型,将SHR随机分为4组:假手术组、miR9抑制剂+电针组、电针组、模型组,Wistar大鼠作为空白组(共5组,每组10只)。其中,miR9抑制剂+电针组在SHR室旁核内行双极插管植入术后注射microRNA-9抑制剂(每单侧100 nL/天),假手术组、电针组注射等剂量脑脊液。术后5天进行电针干预,穴取双侧太冲、曲池。在电针前和电针后3、7、10、14天测量收缩压,连续电针治疗2周后取材,通过实时荧光定量多聚核苷酸链式反应(real-time quantitative polymerase chain reaction,RT-qPCR)对各组大鼠下丘脑microRNA-9的表达进行定量检测,利用qPCR和蛋白免疫印迹法(western blot,WB)检测各组大鼠P2X7受体的mRNA和蛋白水平,使用酶联免疫吸附法(enzyme linked immunosorbent assay,Elisa)检测下丘脑室旁核和血浆中炎性因子白介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α和血管紧张素Ⅱ(angiotensinⅡ,ANGⅡ)的含量。最后使用抗体Iba1进行免疫组化染色,评估下丘脑室旁核内小胶质细胞的活化程度。结果(1)与假手术组相比,电针组降低了高血压大鼠的收缩压(P<0.01),与电针组相比,miRNA9抑制剂+电针组的降压效果下降(P<0.05);(2)电针组与miRNA9抑制剂+电针组、假手术组相比,miRNA9水平明显升高(P<0.01);(3)与miRNA9抑制剂+电针组相比,电针组室旁核、血清中的IL-6含量更低(P<0.01,P<0.01),与miRNA9抑制剂+电针组相比,电针组室旁核、血清中的TNF-α含量更低(P<0.05,P<0.05);(4)与假手术组相比,电针组的ANGⅡ含量下降明显(P<0.01),但电针组与miR-9抑制剂+电针组ANGII的含量相比无统计学差异(P>0.05);(5)与假手术组相比,电针组降低了P2X7受体的蛋白及mRNA表达(P<0.01,P<0.01),与电针组相比,miRNA9抑制剂+电针组中P2X7受体的蛋白及mRNA表达明显较高(P<0.01,P<0.01);(6)免疫组化染色结果显示,电针组与miRNA9抑制剂+电针组、假手术组相比,室旁核小胶质细胞激活程度明显降低(P<0.01,P<0.01)。结论电针干预后影响了SHR下丘脑室旁核内miRNA-9的表达,并可能通过抑制P2X7受体的表达、炎症因子IL-6、TNF-α的释放、小胶质细胞的激活,从而降低血压。

Objective The research target of this study was to verify P2RX7 as one direct target of miRNA-9,which triggers the release of ANGⅡ and PICs(TNF-α,IL-6)in the PVN.The system of miRNA-9/P2RX7 might be a novel mechanism of lowering blood pressure.Methods Spontaneous Hypertension Rat(SHR)model was selected as the animal model of hypertension,and SHR was randomly divided into 4 groups:the Sham operation group,the miR9 inhibitor+EA group,the EA group,the model group,Wistar rats(WKY,Wistar Kyoto)were used as the blank group(5 groups,10 rats in each group).microRNA-9 inhibitor(100 nL/d per side)was injected after bipolar intubation in the paraventricular nucleus of SHR in the inhibitor+EA group,and the same dose of cerebrospinal fluid was injected in the sham-operation group.Electroacupuncture intervention was performed 5 days after operation,and bilateral Taichong and Quchi points were taken.Systolic blood pressure was measured before and 3,7,10,and 14 days after EA,and samples were collected after continuous EA for 2 weeks.Real-time Quantitative polymerase chain reaction(RT-PCR)was used to quantitatively detect the expression of microRNA-9 in the hypothalamus of rats in each group.The mRNA and protein levels of P2X7 receptor in each group were detected by RT-PCR and Western blotting.The inflammatory factors interleukin(IL)-6 and tumor necrosis factor(TNF)in paraventricular nucleus and plasma of hypothalamus were detected by enzyme-linked immunosorbent assay(Elisa)factor,TNF-αand Angiotensin II(ANG II).Finally,the activation of microglia in paraventricular nucleus of hypothalamus was evaluated by immunohistochemical staining with antibody Iba1.Results Compared with the sham operation group,the systolic blood pressure of hypertensive rats was decreased(P<0.01)in the electroacupuncture group,and hypotensive effect was decreased in the miRNA9 inhibitor+electroacupuncture group compared with the electroacupuncture group(P<0.05);the miRNA9 level in the electroacupuncture group was significantly increased compared with that in the miRNA9 inhibitor+electroacupuncture group and the sham operation group(P<0.01).Compared with the sham operation group,the protein and mRNA contents of P2X7 receptor in the electroacupuncture group were decreased(P<0.01),and the protein and mRNA contents of P2X7 receptor in the miRNA9 inhibitor+electroacupuncture group were significantly increased(P<0.01).Compared with the miRNA9 inhibitor+electroacupuncture group,the content of IL-6 in the paraventricular nucleus and serum in the electroacupuncture group was decreased(P<0.01,P<0.01),the content of TNF-αin the paraventricular nucleus and serum in the electroacupuncture group was decreased(P<0.05,P<0.05),and the content of TNF-αin the paraventricular nucleus and serum in the electroacupuncture group was lower than that in the sham operation group.ANGII content in the electroacupuncture group was decreased significantly(P<0.01),but there was no statistical difference in ANGII content between the electroacupuncture group and the MiRNA-9 inhibitor+electroacupuncture group(P>0.05).Immunohistochemical staining showed that paraventricular microglia activation was significantly decreased in the electroacupuncture group compared with the miRNA9 inhibitor+electroacupuncture group and the sham group(P<0.01).Conclusion After EA intervention,the expression of miRNA-9 in the paraventricular nucleus of the hypothalamus in SHR is affected,and it may reduce blood pressure by inhibiting the expression of P2X7 receptor,the release of inflammatory factors IL-6 and TNF-α,and the activation of microglia.