Objective: To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis.
Methods: Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR.
Results: Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68+ and M2-type microglia marked by TMEM119+ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68+ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119+ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention.
Conclusion: The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.
目的：探讨电针“曲池”“足三里”对脑缺血再灌注大鼠的神经保护作用及其与小胶质细胞焦亡相关的作用机制。方法：将60只SD大鼠随机分为假手术组、模型组与电针组，每组20只。模型组和电针组大鼠参照Zea Longa法制备左侧大脑中动脉栓塞再灌注（MCAO/R）模型，电针组造模后次日于右侧“曲池”“足三里”行电针干预，予疏密波，频率4 Hz/20 Hz，电流强度0.2 mA，每次30 min，每日1次，连续干预7 d。采用激光多普勒血流仪检测术中大鼠脑血流下降率；Zea Longa神经行为学评分观察大鼠神经功能；TTC染色法检测大鼠脑梗死体积；免疫荧光法检测缺血侧皮质小胶质细胞阳性表达；透射电镜观察缺血侧皮质小胶质细胞超微结构；实时荧光定量PCR法检测缺血侧皮质组织核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、凋亡相关斑点样蛋白（ASC）、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）及焦孔素-D（GSDMD）mRNA表达。结果：与假手术组比较，模型组大鼠术中脑血流下降率升高（P<0.001），Zea Longa神经行为学评分、脑梗死体积百分比升高（P<0.001），缺血侧皮质CD68+的M1型小胶质细胞及TMEM119+的M2型小胶质细胞数量增多（P<0.001），NLRP3、ASC、Caspase-1及GSDMD mRNA表达升高（P<0.001，P<0.01）；缺血侧皮质小胶质细胞膜结构破坏，较多孔隙形成。与模型组比较，干预后，Zea Longa神经行为学评分、脑梗死体积百分比降低（P<0.05），缺血侧皮质CD68+的M1型小胶质细胞数量减少（P<0.05），TMEM119+的M2型小胶质细胞数量增多（P<0.05），NLRP3、ASC、Caspase-1、GSDMD mRNA表达降低（P<0.01，P<0.05）；缺血侧皮质小胶质细胞膜结构虽然破坏，但较少细胞膜孔隙形成。结论：电针干预可改善脑缺血再灌注大鼠神经功能障碍、减小脑梗死体积，其机制可能与调控NLRP3/Caspase-1/GSDMD轴抑制小胶质细胞焦亡有关。.
Keywords: cerebral ischemia reperfusion; electroacupuncture; neurological function; pyroptosis.