基于NF-κB p65通路研究自拟五倍子汤对大鼠肛瘘术后镇痛及炎症抑制的作用机制

Mechanism of Self-made Wubeizi Decoction on Analgesia and Inflammation Inhibition in Rats After Fistula Operation Based on NF-κB p65 Pathway

目的:探讨自拟五倍子汤对大鼠肛瘘(anal fistula,AF)术后镇痛及炎症抑制作用及其可能作用机制。方法:从40只SD大鼠中随机选取10只作为对照组,其余大鼠建立AF模型,造模成功大鼠随机分为模型组、自拟五倍子汤组和阳性对照组,每组10只,五倍子汤组大鼠使用自拟五倍子汤煎剂100 mL擦洗创面15 min,擦洗后给予常规包扎;阳性对照组大鼠使用高锰酸钾稀释液100 mL擦洗创面15 min,擦洗后给予常规包扎;对照组及模型组给予等量生理盐水擦洗创面,擦洗后给予常规包扎,每次1次,连续干预14 d。给药第1、7、14天后,记录各组大鼠首次舔舐肛周潜伏时间、5 min内扭体反应次数和舔舐肛周总时长;ELISA法检测各组大鼠血清肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和IL-6的水平;HE染色观察各组大鼠创面组织病理学变化;Western Blot和RT-PCR检测各组大鼠创面组织核转录因子-κB p65(nuclear transcription factor-κB p65,NF-κB p65)、环氧合酶-2(cyclooxygenase-2,COX-2)蛋白和mRNA表达情况。结果:给药后第1、7、14天,与模型组比较,自拟五倍子汤组大鼠5 min内扭体反应次数及舔舐肛周总时长显著减少(P<0.05),首次舔舐肛周潜伏时间显著增加(P<0.05)。与给药后第1天比较,给药后第7、14天后,自拟五倍子汤组大鼠5 min内扭体反应次数及舔舐肛周总时长显著减少(P<0.05),首次舔舐肛周潜伏时间显著增加(P<0.05)。与对照组比较,模型组大鼠TNF-α、IL-1β、IL-6的水平及NF-κB p65和COX-2蛋白与mRNA表达水平显著升高(P<0.05);与模型组比较,自拟五倍子汤组大鼠TNF-α、IL-1β、IL-6的水平及NF-κB p65和COX-2蛋白与mRNA表达水平显著降低(P<0.05)。HE染色显示:自拟五倍子汤可明显改善大鼠创面组织结构受损,减少胞间渗出液及炎性细胞浸润现象。结论:自拟五倍子汤能明显改善AF大鼠炎症因子水平,在一定程度上抑制术后疼痛反应,其作用可能与调节NF-κB p65信号通路有关。

Objective:To investigate the effect and possible mechanism of self-made Wubeizi Decoction on analgesia and inflammation inhibition after surgery for anal fistula(AF)in rats.Methods:10 of the 40 SD rats were randomly selected as the control group,and the remaining rats were established as AF rat models,and the rats with successful modeling were randomly divided into model group,self-simulated quintuple soup group and positive control group,10 rats in each group,rats in the fivefold soup group were scrubbed with 100 mL of self-made Wubeizi Decoction for 15 min,and routine bandaging was given after scrubbing.Rats in the positive control group were scrubbed with 100 mL of potassium permanganate dilution for 15 min,and routine bandaging was given after scrubging.The control group and the model group were given the same amount of normal saline to scrub the wound,and after scrubbing,routine dressing was given once each time,and continuous intervention was performed for 14 days.After the 1st,7th and 14th days of administration,the incubation time of the first perianal licking,the number of twisting reactions within 5 minutes and the total duration of perianal licking were recorded in each group.The ELISA method detected the levels of serum tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in each group.HE staining observed the histopathological changes of rat wounds in each group.Western Blot and RT-PCR detected the expression of nuclear transcription factor-κB p65(NF-κB p65),cyclooxygenase-2(COX-2)protein and mRNA in wound tissue in rat wound tissue.Results:On the 1st,7th and 14th days after administration,compared with the model group,the number of twisting reactions within 5 minutes and the total time of perianal licking were significantly reduced(P<0.05),and the latent time of the first perianal licking was significantly increased in the self-mode Wubiezi Decoction group(P<0.05).Compared with the first day after administration,after the 7th and 14th days after administration,the number of twisting reactions within 5 minutes and the total time of perianal licking in the rats were significantly reduced(P<0.05),and the latent time of the first licking perianal was significantly increased in the self-mode Wubiezi Decoction group(P<0.05).Compared with the control group,the levels of TNF-α,IL-1β,IL-6,NF-κB p65 and COX-2 proteins and mRNA expression levels in rats in the model group were significantly increased(P<0.05),and compared with the model group,the levels of TNF-α,IL-1β,IL-6 and NF-κB p65 and COX-2 proteins and mRNA in rats in the self-mode Wubiezi Decoction group were significantly reduced(P<0.05).HE staining showed that self-made Wubeizi Decoction could significantly improve the damage of wound tissue structure in rats,reduce intercellular exudate and inflammatory cell infiltration.Conclusion:Self-made Wubeizi Decoction can significantly improve the level of inflammatory factors in AF rats and inhibit postoperative pain response to a certain extent,and its effect may be related to the regulation of NF-κB p65 signaling pathway.