电针对原发性痛经大鼠子宫组织NLRP3炎性小体和焦亡蛋白GSDMD的影响

Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea

目的:观察电针对原发性痛经(PDM)大鼠核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)及其下游gasderminD(GSDMD)蛋白的影响,探讨电针治疗PDM的可能机制。方法:将40只健康未孕雌性SD大鼠随机分为对照组、模型组、电针组和布洛芬组,各10只。采用苯甲酸雌二醇和缩宫素制备PDM模型,除对照组外,各组大鼠连续皮下注射苯甲酸雌二醇10 d,第11天注射缩宫素。电针组大鼠在造模同时于“关元”“三阴交”行电针干预,予密波,频率50Hz,每天1次,每次20 min,连续干预10d。布洛芬组造模同时进行布洛芬灌胃,每只大鼠灌胃给药0.8 mL(布洛芬溶液浓度为1.25 mg/mL),连续灌胃10 d。造模后,观察大鼠扭体反应;干预后,HE染色法观察大鼠子宫组织形态并评定子宫病理损伤评分,ELISA法检测大鼠血清前列腺素E2(PGE2)、前列腺素F2α(PGF2α)含量及PGF2α/PGE2值,Western blot法检测大鼠子宫组织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)、GSDMD、GSDMD-N和炎性因子[白介素(IL)-1β、IL-18]蛋白表达。结果:模型组大鼠子宫内膜上皮细胞有大量空泡样变性、死亡,固有层大量螺旋小动脉充血,中性粒细胞浸润;电针组大鼠子宫内膜上皮细胞有少量空泡变性、死亡,固有层少量螺旋小动脉充血,少量中性粒细胞浸润;布洛芬组大鼠子宫内膜上皮细胞有非常少量的变性、死亡,固有层未见明显动脉充血,偶见中性粒细胞浸润。与对照组比较,模型组大鼠扭体次数增加(P<0.01),扭体反应评分、血清PGF2α含量和PGF2α/PGE2值升高(P<0.01),PGE2含量降低(P<0.01)。与模型组比较,电针组和布洛芬组大鼠扭体次数减少(P<0.05),扭体潜伏期延长(P<0.01),扭体反应评分、血清PGF2α含量和PGF2α/PGE2值降低(P<0.05,P<0.01),PGE2含量升高(P<0.01)。与对照组比较,模型组大鼠子宫组织NLRP3、ASC、caspase-1、GSDMD、GSDMD-N、IL-1β、IL-18表达升高(P<0.01);与模型组比较,电针组和布洛芬组NLRP3、ASC、caspase-1、GSDMD、GSDMD-N、IL-1β、IL-18表达降低(P<0.01,P<0.05)。电针组和布洛芬组以上指标比较差异无统计学意义(P>0.05)。结论:电针能减轻PDM大鼠疼痛症状,减少子宫组织损伤,其机制可能与电针抑制大鼠子宫组织NLRP3炎性小体的激活,从而抑制焦亡及其炎性因子释放有关。

Objective To observe the effects of electroacupuncture(EA)on NLRP3 inflammasome and its downstream protein gastermin D(GSDMD)in rats with primary dysmenorrhea(PDM),and to explore the potential mechanism of EA on the treatment of PDM.Methods Forty healthy female SD rats without pregnancy were randomly divided into a control group,a model group,an EA group and an ibuprofen group,10 rats in each group.PDM model was prepared by injection of estradiol benzoate and oxytocin.Except the control group,the rats in each group were subcutaneously injected with estradiol benzoate for 10 days,and oxytocin was injected on the 11th day.The rats in the EA group were intervened with EA(dense wave,frequency of 50 Hz)at"Guanyuan"(CV 4)and"Sanyinjiao"(SP 6)at the same time of modeling,once a day,20 min each time,for 10 consecutive days.The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage(concentration of ibuprofen solution was 1.25 mg/m L)for 10 consecutive days.After modeling,the writhing reaction was observed.After intervention,the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus;ELISA method was used to detect the serum levels of prostaglandin E2(PGE2)and prostaglandin F2α(PGF2α);Western blot method was used to detect the protein expression of NLRP3,apoptosis related spot like protein(ASC),caspase-1,GSDMD,GSDMD-N and inflammatory factors(interleukin[IL]-1β,IL-18)in uterine tissue.Results In the model group,a large number of vacuolar degeneration and death of endometrial epithelial cells,spiral arterioles congestion in lamina propria and neutrophil infiltration were observed.In the EA group,there was a small amount of vacuolar degeneration and death of endometrial epithelial cells,a small amount of spiral arterioles congestion in the lamina propria,and a small amount of neutrophils infiltration.In the ibuprofen group,there was very small number of degeneration and death of endometrial epithelial cells,and no obvious arterial congestion was found in lamina propria,and neutrophil infiltration was occasionally seen.Compared with the control group,in the model group the number of writhing was increased(P<0.01),the writhing reaction score and serum level of PGF2αand PGF2α/PGE2 value were increased(P<0.01),the level of PGE2 was decreased(P<0.01).Compared with the model group,in the EA group and the ibuprofen group the number of writhing were decreased(P<0.05),the latency of writhing was prolonged(P<0.01),the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased(P<0.05,P<0.01),the levels of PGE2 were increased(P<0.01).Compared with the control group,the protein expression of NLRP3,ASC,caspase-1,GSDMD,GSDMD-N,IL-1β and IL-18 in the uterine tissues of rats was increased in the model group(P<0.01).Compared with the model group,the protein expression of NLRP3,ASC,caspase-1,GSDMD,GSDMD-N,IL-1β and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group(P<0.01,P<0.05).There was no significant difference between the EA group and the ibuprofen group in the above indexes(P>0.05).Conclusion EA could alleviate pain and uterine tissue injury in rats with PDM.The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues,thereby inhibiting pyroptosis and its inflammatory factors release.