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首页|期刊导航|针刺研究|夹脊电针对急性脊髓损伤大鼠脊髓组织NOD样受体热蛋白结构域相关蛋白3介导的细胞焦亡的影响

夹脊电针对急性脊髓损伤大鼠脊髓组织NOD样受体热蛋白结构域相关蛋白3介导的细胞焦亡的影响OA北大核心CSTPCD

Effect of electroacupuncture of"Jiaji"(EX-B2)on NOD-like receptor protein 3 mediated pyroptosis in spinal cord tissue of rats with acute spinal cord injury

中文摘要英文摘要

目的:观察夹脊电针对脊髓损伤(SCI)大鼠运动功能、脊髓组织形态、脊髓组织NOD样受体热蛋白结构域相关蛋白3(NLRP3)及活化的半胱氨酸蛋白酶-1(cleaved Caspase-1)、焦孔素 D(GSDMD)-N表达的影响,探讨夹脊电针治疗SCI的作用机制.方法:SD大鼠随机分为假手术组、模型组、电针组、激动剂+电针组,每组分为3、7 d两个亚组,每个时间点6只大鼠.采用改良Allen's法制备急性SCI大鼠模型.电针组大鼠电针双侧胸(T)9、T11"夹脊"治疗,每次30 min,每日1次,分别治疗3、7 d.激动剂+电针组除电针外造模后腹腔注射单钠尿酸盐200 μg/kg.采用BBB评分对各组大鼠运动功能进行评价;HE染色法观察各组大鼠脊髓组织形态结构;免疫组织化学染色法检测各组大鼠脊髓组织焦亡相关因子NLRP3、cleaved Caspase-1、GSDMD-N 阳性表达;Western blot法检测各组大鼠脊髓组织 NLRP3、cleaved Caspase-1、GSDMD-N的蛋白表达水平;免疫荧光双标法检测各组大鼠离子钙接头蛋白1(Iba-1)与GSDMD-N在脊髓组织中的表达及定位情况.结果:与假手术组相比,模型组大鼠造模后及3、7 d时BBB评分降低(P<0.05);大鼠脊髓组织可见明显出血区,组织松散,出现炎性细胞浸润;脊髓组织NLRP3、cleaved Caspase-1、GSDMD-N阳性表达及蛋白表达水平均升高(P<0.05);Iba-1、GSDMD-N荧光共表达强度升高(P<0.05).与模型组比较,电针组3、7 d BBB评分升高(P<0.05);脊髓组织出血、炎性细胞浸润减少,神经元形态结构基本恢复正常;脊髓组织中NLRP3、cleaved Caspase-1、GSDMD-N阳性表达及蛋白表达水平均降低(P<0.05);3、7 d时Iba-1与GSDMD-N荧光共表达强度降低(P<0.05).给予激动剂注射能够部分逆转上述电针作用.结论:夹脊电针能够改善SCI大鼠运动功能,其机制可能与抑制NLRP3/Caspase-1信号通路介导的小胶质细胞焦亡有关.

Objective To observe the effect of electroacupuncture(EA)stimulation of"Jiaji"(EX-B2)on motor function,histomorphology,and expression of NOD-like receptor protein 3(NLRP3)and N-terminal domain of gasdermin D(GSDMD-N)in the spinal cord tissue of rats with spinal cord injury(SCI),so as to explore its mecha-nism underlying improvement of SCI.Methods Forty eight female SD rats were randomly divided into sham surgery(sham),SCI model(model),EA,and NLRP3 agonist(monosodium urate,MSU)combined with Jiaji EA(MSU+EA)groups,with 12 rats in each group which were further divided into 3 d and 7 d subgroups,with 6 rats at each time point.Two EA groups received EA stimulation of EX-B2 with a frequency of 100 Hz,electrical current of 1-2 mA for 30 min,once a day for 3 or 7 days.After 5 min,6 h,and 24 h of modeling,rats of the MSU+EA group received intraperitoneal injection of MSU(200 μg/kg,200 μg/mL).The motor function was evaluated using Basso-Beattie-Bresnahan(BBB)scale,the morphological structure of rat spinal cord tissue was observed by H.E.staining.The expression of pyroptosis related factors NLRP3,cleaved Caspase-1 and GSDMD-N of the spinal cord was observed by using immunohisto-chemistry and Western blot separately,the expression and localization of Iba-1 and GSDMD-N in the spinal cord tissue were observed using immunofluorescence double staining method.Results Compared with the sham group,the BBB scores after modeling and on day 3 and 7 were decreased(P<0.05),while the average OD values(immunoactivity)and expression levels of NLRP3,cleaved Caspase-1 and GSDMD-N proteins,and the immunofluorescence intensity of Iba-1/GSDMD-N(co-expression)of the spinal cord tissues on day 3 and 7 were significantly increased in the model group(P<0.05).In comparison with the model group,the BBB scores on day 3 and 7 were obviously increased(P<0.05),while the immunoactivity and expression levels of NLRP3,cleaved Caspase-1 and GSDMD proteins,and the immunofluorescence intensity of Iba-1/GSDMD-N on day 3 and 7 significantly down-regulated in the EA group(P<0.05)but not in the MSU+EA group(P>0.05),suggesting an elimination of the effects of EA after administration of NLRP3 agonist(MSU).H.E.staining showed obvious bleeding area in the spinal cord tissue,loose tissue and inflammatory cell infiltration on day 3 after modeling,and basic absorption of the bleeding,loose tissue,obvious vacuolar changes of the white matter area,loss and contraction of neurons with infiltration of a large number of inflammatory cells,which was milder in the EA group but not in the MSU+EA group.Conclusion EA of EX-B2 can improve the motor function of SCI rats,which may be related to its functions in inhibiting pyroptosis of microglia mediated by NLRP3/Caspase-1 signaling pathway.

梅继林;杨志新;李晓宁

黑龙江中医药大学,哈尔滨 150040承德医学院,河北承德 067000黑龙江中医药大学附属第二医院,哈尔滨 150001

电针夹脊脊髓损伤NOD样受体热蛋白结构域相关蛋白3细胞焦亡小胶质细胞

ElectroacupunctureJiaji(EX-B2)Spinal cord injuryNLRP3PyroptosisMicroglia

《针刺研究》 2024 (002)

110-118 / 9

黑龙江省头雁团队项目基金(No.2019-9);黑龙江中医药大学校级科技创新研究平台项目(No.2018pt03)

10.13702/j.1000-0607.20230701

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