基于NLRP3/Caspase-1/GSDMD通路探讨电针“四神聪”“风池”改善血管性痴呆大鼠认知功能障碍的作用机制  

Study on the mechanism of electroacupuncture“Sishencong”and“Fengchi”to improve cognitive dysfunction in rats with vascular dementia based on NLRP3/Caspase⁃1/GSDMD pathway

作　　者：孙玮 陈英华[2] 宋元毓 王浩宇 秦瑞琦 李俊峰 苏晓庆 吴彤 赵红旭 韩玉生[3] 苗悦 SUN Wei;CHEN Yinghua;SONG Yuanyu;WANG Haoyu;QIN Ruiqi;LI Junfeng;SU Xiaoqing;WU Tong;ZHAO Hongxu;HAN Yusheng;MIAO Yue(Heilongjiang University of Chinese Medicine,Harbin 150040,China;The Fifth Department of Acupuncture,The First Affiliated Hospital of Heilongjiang University of Chinese Medicine,Harbin 150040,China;Laboratory Center,Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江中医药大学,黑龙江哈尔滨150040 [2]黑龙江中医药大学附属第一医院针灸五科,黑龙江哈尔滨150040 [3]黑龙江中医药大学实验中心,黑龙江哈尔滨150040

出　　处：《海南医学院学报》2024年第21期1607-1616,共10页Journal of Hainan Medical University

基　　金：国家自然科学基金项目(82074530);黑龙江省自然科学基金项目(ZD2021H007);黑龙江省中医药科研项目(ZHY2023-189,ZHY2023-209,ZHY2024-090);黑龙江中医药大学研究生创新科研项目(2024yjscx001)。

摘　　要：目的:基于NOD样受体热蛋白结构域相关蛋白3(NLRP3)/胱天蛋白酶-1(Caspase-1)/焦孔素D通路(GSDMD),探讨电针“四神聪”、“风池”改善血管性痴呆(VD)大鼠认知功能障碍的可能作用机制。方法:将64只Morris水迷宫筛选后的雄性SD大鼠随机分为空白组12只、假手术组12只、造模组40只。采用四血管阻断法(4-VO)建立VD大鼠模型,造模成功的24只大鼠随机分为模型组、电针组各12只,电针组取“四神聪”、“风池”穴(连续波,频率2 Hz,强度1 mA),每日1次,30 min/次,连续21 d。假手术组和模型组相同抓取固定,不予任何治疗。Morris水迷宫试验、新物体认知试验评估造模前后及干预后大鼠的学习记忆能力;HE染色观察海马组织病理变化;ELISA法检测海马组织及血清中白细胞介素1β(IL-1β)、白细胞介素18(IL-18)水平;免疫组化检测海马组织中NLRP3、凋亡相关斑点样蛋白(ASC)、Caspase-1和GSDMD蛋白表达;蛋白质印迹法检测海马组织中NLRP3、ASC、pro-Caspase-1、Caspase-1 p20、GSDMD和GSDMD-N蛋白表达。结果:与假手术组对比,模型组大鼠逃避潜伏期延长及穿越平台次数减少、新物体识别指数(RI)降低(P<0.01);模型组大鼠海马神经元排列紊乱,细胞结构不同程度受损;IL-1β、IL-18水平升高(P<0.01);NLRP3、ASC、pro-Caspase-1、Caspase-1 p20、GSDMD和GSDMD-N蛋白表达上调(P<0.01,P<0.05)。与模型组对比,电针组大鼠逃避潜伏期显著缩短及穿越平台次数增加、新物体识别指数(RI)升高(P<0.01);电针组海马神经元整体形态较规则,核固缩、凋亡小体和炎性细胞数量减少;IL-1β、IL-18水平降低(P<0.01);NLRP3、ASC、pro-Caspase-1、Caspase-1 p20、GSDMD和GSDMD-N蛋白表达下调(P<0.01,P<0.05)。结论:电针“四神聪”、“风池”可改善VD大鼠认知功能障碍,其作用机制可能与抑制NLRP3/Caspase-1/GSDMD通路,从而改善炎症,减轻神经细胞焦亡相关。Objective:To investigate the possible mechanism of electroacupuncture of“Si Shen Cong”(EX-HN 1)and“Feng Chi”(GB 20)in improving cognitive impairment in vascular dementia(VD)rats,based on the NOD-like receptor thermoprotein structural domain-related protein 3(NLRP3)/cysteinyl aspartate specific proteinase 1(Caspase-1)/Pyroglottoconjugate D pathway(GSDMD)pathway.Methods:64 Morris water maze-screened male SD rats were randomly divided into the blank group(n=12),sham-operated(n=12),and modeling group(n=40).Four-vessel blockade(4-VO)was used to establish a VD rat model,and the 24 rats that were successfully modeled were divided randomly into the model group(n=12)and the electroacupuncture group(n=12).In the electroacupuncture group,the points o“f Si Shen Cong”(EX-HN 1)and“Feng Chi”(GB 20)were taken(continuous wave with a frequency of 2 Hz and an intensity of 1 mA),once a day for 30 min/times.Electroacupuncture was performed once a day for 30 min/time for 21 d.The sham-operated group was fixed in the same grip as the model group without any treatment.Morris water maze test and new object recognition test were performed to evaluate learning memory ability in rats pre and post modelling and post intervention;Histopathological changes in the hippocampus were observed by HE staining;ELISA was used to detect the levels of interleukin 1β(IL-1β)and interleukin 18(IL-18)in hippocampal tissues and serum;Immunohistochemistry was performed to detect NLRP3,apoptosis-associated speck-like protein(ASC),Caspase-1,and GSDMD protein expression in hippocampal tissues;and Western blot was performed to detect NLRP3,ASC,pro-Caspase-1 in hippocampal tissues,Caspase-1 p20,GSDMD and GSDMD-N protein expression in hippocampal tissue.Results:Compared with the sham-operated group,rats in the model group had prolonged evasion latency and reduced number of crossing platforms,and lowered new object recognition index(RI)(P<0.01);hippocampal neurons in the model group were disarranged and cellular structure was damaged to varying deg

关 键 词：血管性痴呆 电针 NLRP3/Caspase-1/GSDMD通路 细胞焦亡 炎症 

分 类 号：R245[医药卫生—针灸推拿学]