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Title: [Effect of electroacupuncture intervention on relieving pain and inflammation by suppressing TLR4/NF-κB signaling in rats with primary dysmenorrhea].
Author: Xue X, Liu Y, Wang SH, Yuan HY, Li J, Pan SA, Yue ZH.
Journal: Zhen Ci Yan Jiu; 2023 Jan 25; 48(1):63-70. PubMed ID: 36734500.
Abstract:
OBJECTIVE: To investigate the mechanism of electroacupuncture(EA) intervention in rats with primary dysmenorrhea(PDM) based on the Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB signaling pathway. METHODS: Forty female SD rats were randomly divided into blank control, model, EA and medication groups, with 10 rats in each group. PDM rat model was established by subcutaneous injection of estradiol benzoate combined with intraperitoneal injection of oxytocin. At the same time of model procedures, EA(50 Hz, dense wave) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao" (SP6) of rats in the EA group, with needles retained for 20 min, for 10 consecutive days. Rats in the medication group received ibuprofen(125 mg/100 mL, 0.8 mL) by gavage for 10 consecutive days. At the 11th day, writhing behavior of rats was assessed. Uterine morphology was observed by eyes and uterine pathological changes were observed after HE staining. Content of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) in serum and uterine tissues was detected by ELISA; NF-κB p65 positive expression in nucleus was detected by immunofluorescence; protein expression levels of TLR4, NF-κB p65, p-NF-κB p65 and inflammatory factors interleukin (IL) -1β and IL-18 were detected by Western blot. RESULTS: After modeling, uterus tissues were congested and edematous, with necrosis of luminal epithelium, severe edema and extensive shedding of endometrium, nuclear pyknosis, fragmentation and disappearance, neutrophils infiltration, and slight expansion of glandular cavity, which was milder in the EA and the medication groups. Compared with the blank control group, writhing times, scores and incubation period, HE pathological scores, PGF2α contents in serum and uterine tissues, ratio of NF-κB p65 positive expression in nucleus, TLR4, NF-κB p65, p-NF-κB p65, IL-1β and IL-18 protein expression levels in uterine tissues of rats in the model group were all significantly increased(P<0.01), while PGE2 contents in serum and uterine tissues were significantly decreased(P<0.01). Compared with the model group, writhing times and scores, HE pathological scores, PGF2α contents in serum and uterine tissues, ratio of NF-κB p65 positive expression in nucleus, TLR4, NF-κB p65, p-NF-κB p65, IL-1β and IL-18 protein expression levels in uterine tissues of rats in the EA and medication group were all significantly decreased(P<0.01), while writhing incubation period, PGE2 contents in serum and uterine tissues were significantly increased(P<0.05, P<0.01). CONCLUSION: EA intervention could relieve inflammatory response and pain in PDM rats, which may be related to its effect in reducing TLR4 expression, inhibiting NF-κB activation and down-regulating inflammatory factors levels of IL-1β and IL-18. 目的：基于Toll样受体4(TLR4)/核转录因子κB(NF-κB)信号通路，探讨电针治疗原发性痛经(PDM)的作用机制。方法：雌性SD大鼠随机分为空白组、模型组、电针组、西药组，每组10只。采用苯甲酸雌二醇皮下注射联合缩宫素腹腔注射法制备PDM大鼠模型。电针组造模同时进行电针“关元”“三阴交”干预，密波，频率50 Hz，留针20 min，1次/d，连续10 d。西药组造模同时予布洛芬灌胃0.8 mL(125 mg/100 mL)，1次/d，连续10 d。实验第11天评估各组大鼠扭体行为学；HE染色法观察大鼠子宫形态；ELISA法检测大鼠血清及子宫组织中前列腺素E2(PGE2)、前列腺素F2α(PGF2α)的含量；免疫荧光法观察大鼠子宫组织中NF-κB p65入核阳性率；Western blot法检测大鼠子宫组织中TLR4、NF-κB p65、磷酸化(p)-NF-κB p65、白细胞介素(IL)-1β、IL-18蛋白相对表达量。结果：HE染色示，空白组子宫黏膜上皮层完整，未见明显上皮细胞变性和坏死，子宫内膜无明显中性粒细胞浸润；模型组有较多的子宫内膜上皮细胞坏死，子宫内膜严重水肿、伴广泛脱落，胞核固缩、碎裂、消失，伴有中性粒细胞浸润，且腺体腔有轻度扩张；电针组有较多的子宫内膜上皮细胞坏死，子宫内膜轻度水肿；西药组子宫内膜未见明显炎性细胞浸润。与空白组比较，模型组大鼠扭体次数、评分升高(P<0.01)，出现扭体潜伏期(P<0.01)，病理损伤评分增加(P<0.01)，血清及子宫组织中PGE2含量降低(P<0.01)，PGF2α含量升高(P<0.01)，NF-κB p65入核阳性率升高(P<0.01)，子宫组织中TLR4、NF-κB p65、p-NF-κB p65、IL-1β、IL-18蛋白表达量升高(P<0.01)；与模型组比较，电针组和西药组大鼠扭体次数、评分均降低(P<0.05，P<0.01)，扭体潜伏期延长(P<0.01)，病理损伤评分降低(P<0.05，P<0.01)，血清及子宫组织中PGE2含量升高(P<0.01)，PGF2α含量降低(P<0.01)，NF-κB p65入核阳性率降低(P<0.01)，子宫组织中TLR4、NF-κB p65、p-NF-κB p65、IL-1β、IL-18蛋白表达量降低(P<0.01)。结论：电针可能是通过降低子宫组织中TLR4表达，抑制NF-κB活化，下调炎性因子IL-1β、IL-18水平，从而改善PDM大鼠子宫组织炎性状态并缓解疼痛的。.

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