电针“肠病方”对急性溃疡性结肠炎大鼠NLRP3炎性小体和肠黏膜屏障的影响

Effects of electroacupuncture with"intestinal disease prescription"on NLRP3 inflammasome and intestinal mucosal barrier in rats with acute ulcerative colitis

目的:观察电针“肠病方”对葡聚糖硫酸钠(DSS)诱导的急性溃疡性结肠炎(UC)大鼠肠黏膜屏障和核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体的影响,探讨电针“肠病方”治疗UC的作用机制。方法:将32只SPF级健康雄性SD大鼠随机分成空白组、模型组、西药组和电针组,每组8只。除空白组外,大鼠饮用5%DSS溶液7 d制备UC模型。造模后,西药组予美沙拉嗪混悬液(200 mg/kg)灌胃;电针组予针刺双侧“天枢”“上巨虚”和“中脘”穴,同侧“天枢”与“上巨虚”连接电子针疗仪电极,予疏密波,频率10 Hz/50 Hz,每次干预20 min。两组均每日1次,干预3 d。每天观察各组大鼠一般情况。干预后,计算大鼠疾病活动指数(DAI)评分;HE染色法观察大鼠结肠组织形态;ELISA法检测血清促炎性细胞因子[白细胞介素(IL)-18、IL-1β]含量;Western blot法检测大鼠结肠组织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)蛋白表达;免疫荧光法检测结肠组织闭锁连接蛋白-1(ZO-1)、闭锁蛋白(Occludin)阳性表达。结果:与空白组比较,模型组大鼠一般状况较差,体质量增长缓慢,结肠长度缩短(P<0.01),DAI评分与脾脏指数升高(P<0.01),血清IL-18、IL-1β含量与结肠组织NLRP3、ASC、Caspase-1蛋白表达升高(P<0.01),结肠组织ZO-1、Occludin阳性表达减少(P<0.01)。与模型组比较,电针组与西药组大鼠的一般状况改善,体质量增长加快,结肠长度增加(P<0.05),DAI评分与脾脏指数降低(P<0.05),血清IL-18、IL-1β含量与结肠组织NLRP3、ASC、Caspase-1蛋白表达降低(P<0.05),结肠组织ZO-1、Occludin阳性表达增加(P<0.05)。西药组和电针组以上指标比较,差异无统计学意义(P>0.05)。与空白组比较,模型组结肠黏膜形态破坏,腺体排列紊乱,隐窝萎缩,有明显的炎性细胞浸润;与模型组比较,西药组和电针组大鼠结肠黏膜形态相对完好,腺体与隐窝结构得到了恢复与改善,炎性细胞浸润减少。结论:电针“肠病方”对DSS诱导的急性UC具有显著的改善作用,可能是通过调节NLRP3炎性小体和肠黏膜屏障相关蛋白的表达,从而减轻溃疡性结肠炎的症状。

Objective To observe the effects of electroacupuncture(EA)with"intestinal disease prescription"on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium(DSS)-induced acute ulcerative colitis(UC),and explore the underlying mechanism of EA with"intestinal disease prescription"for the treatment of UC.Methods Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group,a model group,a medication group,and an EA group,with 8 rats in each group.Except for the blank group,the UC model was established by administering 5%DSS solution for 7 days.After modeling,the rats in the medication group were treated with mesalazine suspension(200 mg/kg)by gavage,while the rats in the EA group were treated with acupuncture at bilateral"Tianshu"(ST 25),"Shangjuxu"(ST 37)and"Zhongwan"(CV 12),with the ipsilateral"Tianshu"(ST 25)and"Shangjuxu"(ST 37)connected to the electrodes of the EA instrument,using disperse-dense wave,with a frequency of 10 Hz/50 Hz,and each intervention lasted for 20 minutes.Both interventions were performed once daily for 3 days.The general conditions of rats were observed daily.After intervention,the disease activity index(DAI)score was calculated;colon tissue morphology was observed using HE staining;serum levels of pro-inflammatory cytokines(interleukin[IL]-18,IL-1β)were measured by ELISA;protein expression of NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),and Caspase-1 in colon tissues was detected by Western blot;positive expression of zonula occludens-1(ZO-1)and Occludin in colon tissues was examined by immunofluorescence.Results Compared with the blank group,the rats in the model group exhibited poor general conditions,slow body weight gain,shortened colon length(P<0.01),increased DAI score and spleen index(P<0.01),elevated serum IL-18 and IL-1βlevels,and increased protein expression of NLRP3,ASC,and Caspase-1 in colon tissues(P<0.01),along with decreased positive expression of ZO-1 and Occludin in colon tissues(P<0.01).Compared with the model group,the rats in the medication group and the EA group exhibited improved general conditions,accelerated body weight gain,increased colon length(P<0.05),reduced DAI scores and spleen indexes(P<0.05),decreased serum IL-18 and IL-1βlevels,and lower protein expression of NLRP3,ASC and Caspase-1 in colon tissues(P<0.05),as well as increased positive expression of ZO-1 and Occludin in colon tissues(P<0.05).There were no significant differences in the above indexes between the medication group and the EA group(P>0.05).Compared with the blank group,the rats in the model group exhibited disrupted colon mucosal morphology,disordered gland arrangement,and atrophy of crypts,along with significant inflammatory cell infiltration.Compared with the model group,the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology,with restored and improved gland and crypt structures,and reduced inflammatory cell infiltration.Conclusion EA with"intestinal disease prescription"has a significant therapeutic effect on DSS-induced UC,possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier,thereby alleviating symptoms of ulcerative colitis.