Tong Ting-Ting, Wang Ying, Li Kui-Wu, Zhang Li-da, Wu Xiao-Qing, Wang Jun-Li, Li Cheng-Long, Zhang Guo-Qing, Zhang Jun-Yu, Han Wei
Second College of Clinical Medicine, Anhui University of CM, Hefei 230061, China.
Department of Encephalopathy, 4Department of Geriatrics, Second Affiliated Hospital of Anhui University of CM.
Zhongguo Zhen Jiu. 2023 Jul 12;43(7):783-92. doi: 10.13703/j.0255-2930.20221010-k0004.
To observe the effect of (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI.
A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA.
Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (<0.01), the protein expression of GSDMD-N was reduced (<0.01); and the contents of IL-1β and IL-18 were lower (<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (<0.01, <0.05), the mRNA expression of caspase-1 was reduced (<0.01); and the contents of IL-1β and IL-18 were lower (<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (<0.05, <0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (<0.01), the protein expression of GSDMD-N was reduced (<0.01); and the contents of IL-1β and IL-18 were declined (<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (<0.05, <0.01), the mRNA expression of PPARγ was decreased (<0.01) and the protein expression of GSDMD-N was elevated (<0.05); and the contents of IL-1β and IL-18 were higher (<0.01).
EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.
观察通督调神电针预处理对脑缺血再灌注损伤（CIRI）大鼠大脑皮质中过氧化物酶体增殖物激活受体γ（PPARγ）介导的细胞焦亡的影响，探讨电针防治CIRI的潜在机制。
将110只清洁级雄性SD大鼠随机分为假手术组、模型组、电针组、电针+抑制剂组和激动剂组，每组22只。电针组在造模前，将电针施加于“百会”（GV 20）、“风府”（GV 16）和“大椎”（GV 14），采用疏密波，频率为2 Hz/5 Hz，强度为1至2 mA，持续20分钟；每天1次，连续7天。在电针组干预的基础上，于第7天，电针+抑制剂组腹腔注射PPARγ抑制剂GW9662（10 mg/kg）。激动剂组在第7天腹腔注射PPARγ激动剂盐酸吡格列酮（10 mg/kg）。干预结束时，除假手术组外，其他组大鼠采用改良线栓法建立右侧CIRI模型。采用改良神经功能缺损评分（mNSS）对大鼠神经功能缺损情况进行评价。采用TTC染色检测大鼠相对脑梗死体积，采用TUNEL染色检测大脑皮质神经细胞凋亡，采用透射电子显微镜观察大脑皮质神经细胞焦亡。采用免疫荧光染色检测大脑皮质中PPARγ和核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）的阳性表达。采用蛋白质印迹法检测大脑皮质中PPARγ、NLRP3、半胱天冬酶-1（caspase-1）、gasdermin D（GSDMD）和GSDMD-N端（GSDMD-N）的蛋白表达。采用实时荧光定量PCR检测大脑皮质中PPARγ、NLRP3、caspase-1和GSDMD的mRNA表达。采用ELISA法测定大鼠大脑皮质中白细胞介素（IL）-1β和IL-18的含量。
与假手术组比较，模型组mNSS、相对脑梗死体积和TUNEL阳性细胞率升高（<0.01），细胞焦亡严重，PPARγ、NLRP3、caspase-1和GSDMD的蛋白和mRNA表达水平升高（<0.01）；GSDMD-N蛋白表达及IL-1β和IL-18含量增加（<0.01）。与模型组比较，电针组和激动剂组mNSS、相对脑梗死体积和TUNEL阳性细胞率降低（<0.01），细胞焦亡减轻，PPARγ蛋白和mRNA表达水平升高（<0.01），NLRP3、caspase-1和GSDMD蛋白和mRNA表达水平降低（<0.01），GSDMD-N蛋白表达降低（<0.01）；IL-1β和IL-18含量降低（<0.01）；而电针+抑制剂组PPARγ蛋白表达升高（<0.01），NLRP3和GSDMD蛋白和mRNA表达水平降低（<0.01，<0.05），caspase-1 mRNA表达降低（<0.01）；IL-1β和IL-18含量降低（<0.01）。与电针+抑制剂组比较，电针组mNSS、相对脑梗死体积和TUNEL阳性细胞率降低（<0.05，<0.01），细胞焦亡减轻，PPARγ蛋白和mRNA表达水平升高（<0.01），NLRP3、caspase-1和GSDMD蛋白和mRNA表达水平降低（<0.01），GSDMD-N蛋白表达降低（<0.01）；IL-1β和IL-18含量降低（<0.01）。与激动剂组比较，电针组相对脑梗死体积和TUNEL阳性细胞率升高（<0.05，<0.01），PPARγ mRNA表达降低（<0.01），GSDMD-N蛋白表达升高（<0.05）；IL-1β和IL-18含量升高（<0.01）。
电针预处理可减轻CIRI大鼠的神经功能损伤，其潜在机制与上调PPARγ诱导大鼠大脑皮质中NLRP3的抑制从而影响细胞焦亡有关。
