Effect of electroacupuncture on NF-κB and NLRP3 inflammasome in uterine tissues of rats with primary dysmenorrhea

Abstract

Objective

To observe the effect of electroacupuncture (EA) on nuclear factor kappa B (NF-κB) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in uterine tissues of rats with primary dysmenorrhea (PD), thus to explore the possible mechanism of EA for PD.

Methods

Fifty female Sprague-Dawley (SD) rats were randomly divided into a normal group, a model group, an EA at non-acupoint group, an EA at acupoint group and a Western medicine group, with 10 rats in each group. Except for the normal group, rats in the other four groups were treated with estradiol benzoate combined with oxytocin for 11 d to establish PD rat models. From day 1 of the modeling, rats in the normal group and the model group were only properly grasped without any intervention; Guanyuan (CV 4) and Sanyinjiao (SP 6) were selected for EA treatment in the EA at acupoint group; rats in the EA at non-acupoint group were treated with EA at 5 mm away from the acupoints selected above; rats in the Western medicine group were treated with ibuprofen via gavage. Rats in each group were treated for 10-day successively. On the 11th day, except for the normal group, rats in the other groups were intraperitoneally injected with oxytocin (2 U/rat), and the writhing number within 30 min in each group was compared; the pathological changes in rat uteruses were observed by hematoxylin-eosin (HE) staining, and the pathological damage scores were evaluated. Protein expression levels of NF-κB p65, phospho-NF-κB p65, NLRP3, cysteine aspastic acid-specific protease 1 (caspase-1), interleukin (IL)-1β and IL-18 were detected by Western blot.

Results

Compared with the normal group, the writhing number increased significantly (P<0.05), and the extensive exfoliation of the endometrium, severe edema, and histopathological score all increased significantly in the model group (P<0.05) as well as the protein levels of NLRP3, caspase-1, IL-1β and IL-18, and the ratio of phospho-NF-κB p65/NF-κB p65 in rat uterine tissues (all P<0.05); compared with the model group, the numbers of writhing reaction decreased within 30 min (P<0.05), the endometrial exfoliation was rare, the edema degree was mild, and the histopathological scores decreased significantly (all P<0.05) in the EA at acupoint group and the Western medicine group; compared with the model group, the phospho-NF-κB p65/NF-κB p65 ratio and the NLRP3, caspase-1, IL-1β and IL-18 protein levels of rat uterine tissues in the EA at acupoint group were significantly lower (Ps <0.05); compared with the model group, the caspase-1, IL-1β and IL-18 protein levels of the rat uterine tissues decreased significantly (all P<0.05), and the differences in the NLRP3 and phospho-NF-κB p65/NF-κB p65 levels were statistically insignificant (all P>0.05) in the Western medicine group; compared with the Western medicine group, the phospho-NF-κB p65/NF-κB p65 ratio, also the NLRP3, IL-1β and IL-18 protein levels of the uterine tissues decreased significantly in the EA at acupoint group (all P<0.05), while the difference in the caspase-1 level was statistically insignificant (P>0.05); there were no significant differences between the EA at non-acupoint group and the model group in any indicators (all P>0.05).

Conclusion

EA at acupoints significantly improves the pain and pathological damages of PD rats. The mechanism may be related to the reduced uterine inflammation via inhibiting NF-κB phosphorylation and NLRP3 activation in uteruses of PD rats.

摘要

目的

观察电针对原发性痛经(PD)大鼠子宫组织核转录因子κB(NF-κB)和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体的影响, 探讨电针治疗 PD 的可能机制。

方法

将 50 只Sprague-Dawley(SD)雌性大鼠随机分为正常组、 模型组、 电针非穴位组、 电针穴位组和西药组, 每组 10 只。 除正常组以外, 其余四组采用苯甲酸雌二醇联合缩宫素建立 PD 大鼠模型, 造模持续 11 d。 自造模第 1 天起, 正常组和模型组只适度抓取, 不作任何干预; 电针穴位组取大鼠关元和三阴交行电针治疗; 电针非穴位组选取上述两穴旁开 5 mm 非经非穴位处行电针治疗; 西药组予布洛芬灌胃治疗。 各组均连续治疗 10 d。 于第 11 天, 除正常组以外, 其余各组大鼠腹腔注射缩宫素(2 U/只)后比较各组大鼠 30 min 内扭体次数; 苏木精-伊红(HE)染色法观察大鼠子宫病理形态学的变化, 并进行病理损伤评分; Western blot 法检测子宫组织核转录因子 κB p65(NF-κB p65)、 磷酸化 NF-κB(phospho-NF-κB p65)、 NLRP3、 半胱氨酸天冬氨酸特异性蛋白酶1(caspase-1)、 白细胞介素(IL)-1β 和 IL-18 的蛋白表达水平。

结果

与正常组相比, 模型组扭体次数明显增多(P<0.05)、 子宫内膜大范围剥脱及较严重的水肿、 组织病理评分明显上升(P<0.05), 大鼠子宫组织中NLRP3、 caspase-1、 IL-1β 和 IL-18 蛋白水平及 phospho-NF-κB p65/NF-κB p65 水平明显升高(均P<0.05); 与模型组相比, 电针穴位组和西药组 30 min 内扭体反应次数均减少(均P<0.05), 子宫内膜剥脱少见且水肿程度较轻, 组织病理评分显著降低(均P<0.05); 与模型组相比, 电针穴位组大鼠子宫组织 pospho-NF-κB p65/NF-κB p65 水平及 NLRP3、 caspase-1、 IL-1β 和 IL-18 蛋白水平明显降低(均P<0.05); 与模型组相比, 西药组大鼠子宫组织 caspase-1、 IL-1β 和 IL-18 蛋白水平明显降低(均P<0.05), NLRP3 和 phospho-NF-κB p65/NF-κB p65 水平差异无统计学意义(均P>0.05); 与西药组相比, 电针穴位组大鼠子宫组织 phospho-NF-κB p65/NF-κB p65 水平及 NLRP3、 IL-1β 和 IL-18 蛋白水平明显降低(均P<0.05), caspase-1 水平差异无统计学意义(P>0.05); 电针非穴位组与模型组相比, 各项指标差异无统计学意义(均P>0.05)。

结论

电针穴位可明显改善 PD 大鼠疼痛症状和病理损伤情况, 其作用机制可能与抑制 PD 大鼠子宫组织中 NF-κB 磷酸化和 NLRP3 活化, 减轻子宫炎症有关。