穴位埋线调控NOD样受体热蛋白结构域3/半胱氨酸蛋白酶-1信号通路抗溃疡性结肠炎损伤的机制研究

Acupoint catgut embedding relieves colonic inflammatory injury by down-regulating NLRP3/Caspase-1signaling pathway in rats with ulcerative colitis

目的:观察穴位埋线对“虚、瘀”状态下溃疡性结肠炎(UC)大鼠的治疗作用及对NOD样受体热蛋白结构域3(NLRP3)/半胱氨酸蛋白酶-1(Caspase-1)信号通路的调控作用,探讨穴位埋线治疗UC的作用机制。方法:SD大鼠随机分为空白组10只和造模组48只,采用腺嘌呤、番泻叶分阶段灌胃,再予2,4,6-三硝基苯磺酸与乙醇复合物灌肠复制“虚、瘀”状态下UC大鼠模型。造模成功的大鼠按体质量随机分为模型组、柳氮磺胺吡啶(SASP)组、非穴位埋线组和穴位埋线组,每组11只。穴位埋线组选“足三里”“天枢”“肾俞”等穴进行穴位埋线治疗,非穴位埋线组于大鼠臀部肌肉丰厚处进行埋线治疗,每14 d治疗1次,共治疗3次;SASP组予SASP溶液(50 mg·kg^(-1)·d^(-1))灌胃,每日1次,共治疗42 d。观察大鼠一般情况,计算大鼠疾病活动指数(DAI);测量各组大鼠结肠长度;肉眼评估结肠黏膜大体病理学改变,并进行结肠黏膜损伤指数(CMDI)评分;HE染色评估结肠组织病理学改变,进行结肠病理损伤(TDI)评分;ELISA法检测各组大鼠血清NLRP3、白细胞介素(IL)-1β和IL-18含量;荧光定量PCR法检测各组大鼠结肠组织NLRP3、Caspase-1、凋亡相关斑点样蛋白(ASC)、IL-1β及IL-18 mRNA水平;Western blot法检测各组大鼠结肠组织NLRP3、Caspase-1蛋白表达水平;免疫组织化学染色法检测结肠组织ASC阳性表达。结果:与空白组比较,模型组大鼠体质量降低(P<0.01),结肠长度明显缩短(P<0.01),结肠黏膜形成明显溃疡面,隐窝破坏明显,大量炎性细胞浸润,DAI、CMDI及TDI评分升高(P<0.01),血清中NLRP3、IL-1β及IL-18含量升高(P<0.01),结肠组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18 mRNA水平和Caspase-1、ASC、NLRP3蛋白表达水平明显升高(P<0.01)。与模型组比较,穴位埋线组和SASP组体质量升高(P<0.01),大鼠结肠长度增长(P<0.01),结肠黏膜病理损伤明显减轻,DAI、CMDI及TDI评分显著降低(P<0.01),血清中NLRP3、IL-1β及IL-18含量显著降低(P<0.01),结肠组织内NLRP3、ASC、Caspase-1、IL-1β、IL-18 mRNA和Caspase-1、ASC、NLRP3蛋白表达水平明显降低(P<0.01)。穴位埋线组各指标较非穴位埋线组均有明显改善(P<0.01)。结论:穴位埋线可通过调控NLRP3/Caspase-1信号通路抑制炎性小体活化,缓解UC大鼠的炎性反应。

Objective To observe the effect of acupoint catgut embedding(CE)on Nod-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway in“deficiency-stasis”syndrome type ulcerative colitis(UC)rats,so as to explore its mechanisms underlying improvement of UC.Methods A total of 58 male SD rats were randomly divided into control group(10 rats)and model group(48 rats).The“deficiency-stasis”type UC model was established by gavage of adenine and folium sennae solution for 4 weeks,followed by clycter of mixture solution of 5%trinitro-benzene-sulfonic acid and 50%ethanol.A total of 44 UC rats were randomized into model,salicylazosulfapyridine(SASP),non-acupoint CE,and acupoint CE groups(11 rats in each group).The catgut embedment was applied to bilateral“Zusanli”(ST36),“Shenshu”(BL23),“Pishu”(BL20),“Dachangshu”(BL25),“Geshu”(BL17)and“Tianshu”(ST25),or non-acupoints(the fat muscles of the buttocks),separately,once every two weeks,3 times altogether.Rats of the SASP group received gavage of SASP solution,and those of the other groups received gavage of same amount of normal saline,once daily for 42 days.The rat's general conditions and the colon length were recorded,the disease activity index(DAI,0 to 4 points)and colonic mucosal damage index(CMDI,0 to 5 points)were calculated.Histopathological changes of the colonic mucosa tissue were observed after HE staining,and the tissue damage index(TDI,0 to 6 points)was given.The levels of serum NLRP3,interleukin(IL)-1βand IL-18 were measured by ELISA,and the expression levels of NLRP3,Caspase-1,apoptosis-associated speck-like protein(ASC),IL-1βand IL-18 mRNAs were measured by fluorescence quantitative PCR.The expression levels of NLRP3 and Caspase-1 proteins in the colon tissues were measured by Western blot,and the immunoactivity of colonic ASC was detected by immunohistochemistry.Results Compared with the control group,the rats'body mass and colonic length were significantly decreased(P<0.01),and DAI score,CMDI score,TDI score,contents of serum NLRP3,IL-1βand IL-18,expression levels of colonic NLRP3,ASC,Caspase-1,IL-1βand IL-18 mRNAs,and NLRP3 and Caspase-1 proteins as well as colonic ASC immunoactivity were significantly up-regulated in the model group(P<0.01).Compared with the model group,both SASP and acupoint CE groups had a significant increase in body mass and colonic length(P<0.01),and a marked decrease in DAI score,CMDI score,TDI score,contents of serum NLRP3,IL-1βand IL-18,expression levels of NLRP3,ASC,Caspase-1,IL-1βand IL-18 mRNAs and NLRP3 and Caspase-1 proteins and ASC immunoactivity(P<0.01).The above indexes were improved in the acupoint CE group in relevant to those of the non-acupoint CE group(P<0.01).HE staining of colonic mucosal tissue showed obvious ulcerative surface,destroyed recess,disordered arrangement of glands,mucosal edema and congestion,infiltration of a large number of inflammatory cells in the model group,which was obviously milder in both SASP and acupoint CE groups.Conclusion Acupoint embedding can alleviate colonic injury and inhibit inflammatory reaction in rats with“deficiency-stasis”type UC by down-regulating colonic NLRP3/Caspase-1 signaling.