隔药灸对克罗恩病大鼠结肠NLRP3炎症小体及IL-1?调节作用的实验研究
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摘要
目的通过观察隔药灸对克罗恩病大鼠结肠NLRP3炎症小体(NLRP3、ASC、Caspase-1)及下游炎症因子IL-1?表达的影响,探讨隔药灸治疗克罗恩病的抗炎机制。方法将清洁级雄性SD大鼠随机分为正常组、模型组、隔药灸组和假灸组4组。采用三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid, TNBS)与乙醇混合溶液灌肠制备大鼠克罗恩病模型。造模成功后,隔药灸组取天枢(双)、气海穴进行隔药饼灸治疗;假灸组取穴、操作同隔药灸组,但不点燃艾炷。治疗结束后,记录各组大鼠结肠长度及CMDI评分;采用HE染色,光学显微镜下观察各组大鼠结肠组织形态结构,并应用免疫组织化学技术检测各组大鼠结肠NLRP3、ASC、Caspase-1及IL-1?的表达。结果与正常组比较,模型组大鼠结肠组织损伤严重,表现为裂隙样溃疡,炎症细胞浸润伴水肿,部分大鼠结肠黏膜可见肉芽组织形成,且结肠NLRP3、ASC、Caspase-1、IL-1?蛋白表达显著增加(均P<0.05);与模型组比较,隔药灸组大鼠结肠损伤有所修复,炎症反应减轻,结肠NLRP3、ASC、Caspase-1及IL-1?蛋白表达显著降低(均P<0.05);假灸组大鼠结肠损伤程度、炎症反应与模型组类似,两组结肠NLRP3、ASC、Caspase-1、IL-1?表达比较,差异均无统计学意义(P>0.05)。结论隔药灸能下调克罗恩病大鼠结肠NLRP3、ASC、Caspase-1及IL-1?的蛋白表达,促进结肠炎性损伤的修复。
Objective To explore the action mechanism of moxibustion in treating Crohn's disease(CD) by observing the effect of herbal cake-partitioned moxibustion on the expression of NLRP3 inflammasome(NLRP3, ASC and Caspase-1) and IL-1β. Method Male Sprague Dawley(SD) rats of clean conventional grade were randomized into a normal control group(NG), a model control group(MG), a herbal cake-partitioned moxibustion control group(MoxG), and a sham moxibustion control group(SMG). The CD rat models were developed by using the mixture of 2,4,6-trinitrobenzene sulfonic acid(TNBS) and alcohol via enema. When the models were successfully developed, the MoxG received moxibustion intervention at bilateral Tianshu(ST25) and Qihai(CV6), and the SMG were administered by unlighted moxa cones. At the end of treatment, the length of each rats' colon and the colon macroscopic damage index(CMDI) score were recorded, the histopathological variations of rats' colons were observed by adopting HE staining and light microscope, and the expressions of NLRP3, ASC, Caspase-1 and IL-1β in rats' colons were determined by using immunohistochemical technique. Result Compared to the NG, the MG had its rats' colons present with severe damages, fissured ulcers and inflammatory cell infiltration with edema, and granulomas in submucosa of some colons, and its expressions of NLRP3, ASC, Caspase-1 and IL-1β increased significantly(P<0.05);compared to the MG, the MoxG had rats' colons present with improved structures and reduced intestinal inflammation,and its expressions of NLRP3, ASC, Caspase-1 and IL-1β dropped significantly(P<0.05); the SMG had its rats' colon inflammation present similarly to the MG, and its expressions of NLRP3, ASC, Caspase-1 and IL-1β had no significant difference(P>0.05). Conclusion Herbal Cake-partitioned moxibustion can down-regulate the expressions of NLRP3,ASC, caspase-1 and IL-1β in CD rats' colons to promote the repair of colon damage.
Objective To explore the action mechanism of moxibustion in treating Crohn's disease(CD) by observing the effect of herbal cake-partitioned moxibustion on the expression of NLRP3 inflammasome(NLRP3, ASC and Caspase-1) and IL-1β. Method Male Sprague Dawley(SD) rats of clean conventional grade were randomized into a normal control group(NG), a model control group(MG), a herbal cake-partitioned moxibustion control group(MoxG), and a sham moxibustion control group(SMG). The CD rat models were developed by using the mixture of 2,4,6-trinitrobenzene sulfonic acid(TNBS) and alcohol via enema. When the models were successfully developed, the MoxG received moxibustion intervention at bilateral Tianshu(ST25) and Qihai(CV6), and the SMG were administered by unlighted moxa cones. At the end of treatment, the length of each rats' colon and the colon macroscopic damage index(CMDI) score were recorded, the histopathological variations of rats' colons were observed by adopting HE staining and light microscope, and the expressions of NLRP3, ASC, Caspase-1 and IL-1β in rats' colons were determined by using immunohistochemical technique. Result Compared to the NG, the MG had its rats' colons present with severe damages, fissured ulcers and inflammatory cell infiltration with edema, and granulomas in submucosa of some colons, and its expressions of NLRP3, ASC, Caspase-1 and IL-1β increased significantly(P<0.05);compared to the MG, the MoxG had rats' colons present with improved structures and reduced intestinal inflammation,and its expressions of NLRP3, ASC, Caspase-1 and IL-1β dropped significantly(P<0.05); the SMG had its rats' colon inflammation present similarly to the MG, and its expressions of NLRP3, ASC, Caspase-1 and IL-1β had no significant difference(P>0.05). Conclusion Herbal Cake-partitioned moxibustion can down-regulate the expressions of NLRP3,ASC, caspase-1 and IL-1β in CD rats' colons to promote the repair of colon damage.
引文
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[14]Spinelli A,Correale C,Szabo H,et al.Intestinal fibrosis in Crohn's disease:medical treatment or surgery?[J].Curr Drug Targets,2010,11(2):242-248.
[15]Oshima T,Miwa H.Gastrointestinal mucosal barrier function and diseases[J].J Gastroenterol,2016,51(8):768-778.
[16]Kayagaki N,Warming S,Lamkanfi M,et al.Noncanonical inflammasome activation targets caspase-11[J].Nature,2011,479(7371):117-121.
[17]Liu L,Dong Y,Ye M,et al.The pathogenic role of NLRP3 inflammasome activation in Inflammatory bowel diseases of both mice and humans[J].J Crohns Colitis,2017,11(6):737-750.
[18]Lazaridis LD,Pistiki A,Giamarellos-Bourboulis EJ,et al.Activation of NLRP3 inflammasome in inflammatory bowel disease:differences between Crohn's disease and ulcerative colitis[J].Dig Dis Sci,2017,62(9):2348-2356.
[19]Elia PP,Tolentino YF,Bernardazzi C,et al.The role of innate immunity receptors in the pathogenesis of inflammatory bowel disease[J].Mediators Inflamm,2015:936193.
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[22]Joos S,Brinkhaus B,Maluche C,et al.Acupuncture and moxibustion in the treatment of active Crohn's disease:a randomized controlled study[J].Digestion,2004,69(3):131-139.
[23]包春辉,吴璐一,吴焕淦,等.针灸治疗活动期克罗恩病:随机对照研究[J].中国针灸,2016,36(7):683-688.
[24]Shang HX,Wang AQ,Bao CH,et al.Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients[J].World JGastroenterol,2015,21(16):4986-4996.
[25]Wei K,Zhang D,Hong J,et al.Herb-partitioned moxibustion and the mi RNAs related to Crohn's disease:a study based on rat models[J].Evid Based Complement Alternat Med,2015:265238.
[26]Du X,Chen W,Wang Y,et al.Therapeutic efficacy of carboxyamidotriazole on 2,4,6-trinitrobenzene sulfonic acid-induced colitis model is associated with the inhibition of NLRP3 inflammasome and NF-kappa Bactivation[J].Int Immunopharmacol,2017,45:16-25.
[27]Castro J,Ocampo Y,Franco L.Cape Gooseberry[Physalis peruviana L.]Calyces Ameliorate TNBS Acidinduced Colitis in Rats[J].J Crohns Colitis,2015,9(11):1004-1015.
[28]Bauer C,Duewell P,Mayer C,et al.Colitis induced in mice with dextran sulfate sodium(DSS)is mediated by the NLRP3 inflammasome[J].Gut,2010,59(9):1192-1199.
[29]De Santis S,Kunde D,Galleggiante V,et al.TNFalpha deficiency results in increased IL-1beta in an early onset of spontaneous murine colitis[J].Cell Death Dis,2017,8(8):e2993.
[30]Ogawa M,Osada H,Hasegawa A,et al.Effect of interleukin-1beta on occludin m RNA expression in the duodenal and colonic mucosa of dogs with inflammatory bowel disease[J].J Vet Intern Med,2018,32(3):1019-1025.
[31]张璐,王诗怡,范一宏,等.克罗恩病患者小肠细菌过生长的监测及意义研究[J].浙江临床医学,2017,19(6):1029-1032.
[32]吴璐一,翁志军,吴焕淦,等.Th17细胞因子在CD大鼠结肠不同炎性时期的差异表达[J].世界华人消化杂志,2016,24(14):2161-2169.
[2]Feuerstein JD,Cheifetz AS.Crohn Disease:Epidemiology,Diagnosis,and Management[J].Mayo Clin Proc,2017,92(7):1088-1103.
[3]Vavricka SR,Schoepfer A,Scharl M,et al.Extraintestinal manifestations of inflammatory bowel disease[J].Inflamm Bowel Dis,2015,21(8):1982-1992.
[4]Baumgart DC,Sandborn WJ.Crohn's disease[J].Lancet,2012,380(9853):1590-2605.
[5]魏凯,张丹,窦传字,等.艾灸对克罗恩病大鼠结肠NF-?B p65及TNF-?、IL-1?调节作用的研究[J].世界中医药,2013,8(8):862-866.
[6]Morris GP,Beck PL,Herridge MS,et al.Hapten-induced model of chronic inflammation and ulceration in the rat colon[J].Gastroenterology,1989,96(2):795-803.
[7]Paiotti AP,Miszputen SJ,Oshima CT,et al.Etanercept attenuates TNBS-induced experimental colitis:role of TNF-αexpression[J].J Mol Histol,2011,42(5):443-450.
[8]Cheifetz AS.Management of active Crohn disease[J].Jama,2013,309(20):2150-2158.
[9]Zaki MH,Vogel P,Body-Malapel M,et al.IL-18production downstream of the Nlrp3 inflammasome confers protection against colorectal tumor formation[J].J Immunol,2010,185(8):4912-4920.
[10]Ng SC,Shi HY,Hamidi N,et al.Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century:a systematic review of population-based studies[J].Lancet,2018,390(10114):2769-2778.
[11]Molodecky NA,Soon IS,Rabi DM,et al.Increasing incidence and prevalence of the inflammatory bowel diseases with time,based on systematic review[J].Gastroenterology,2012,142(1):46-54.e42;quiz e30.
[12]Sirois FM.Health-related self-perceptions over time and provider-based Complementary and Alternative Medicine(CAM)use in people with inflammatory bowel disease or arthritis[J].Complement Ther Med,2014,22(4):701-709.
[13]Opheim R,Bernklev T,Fagermoen MS,et al.Use of complementary and alternative medicine in patients with inflammatory bowel disease:results of a cross-sectional study in Norway[J].Scand J Gastroenterol,2012,47(12):1436-1447.
[14]Spinelli A,Correale C,Szabo H,et al.Intestinal fibrosis in Crohn's disease:medical treatment or surgery?[J].Curr Drug Targets,2010,11(2):242-248.
[15]Oshima T,Miwa H.Gastrointestinal mucosal barrier function and diseases[J].J Gastroenterol,2016,51(8):768-778.
[16]Kayagaki N,Warming S,Lamkanfi M,et al.Noncanonical inflammasome activation targets caspase-11[J].Nature,2011,479(7371):117-121.
[17]Liu L,Dong Y,Ye M,et al.The pathogenic role of NLRP3 inflammasome activation in Inflammatory bowel diseases of both mice and humans[J].J Crohns Colitis,2017,11(6):737-750.
[18]Lazaridis LD,Pistiki A,Giamarellos-Bourboulis EJ,et al.Activation of NLRP3 inflammasome in inflammatory bowel disease:differences between Crohn's disease and ulcerative colitis[J].Dig Dis Sci,2017,62(9):2348-2356.
[19]Elia PP,Tolentino YF,Bernardazzi C,et al.The role of innate immunity receptors in the pathogenesis of inflammatory bowel disease[J].Mediators Inflamm,2015:936193.
[20]Schoultz I,Verma D,Halfvarsson J,et al.Combined polymorphisms in genes encoding the inflammasome components NALP3 and CARD8 confer susceptibility to Crohn's disease in Swedish men[J].Am J Gastroenterol,2009,104(5):1180-1188.
[21]Villani AC,Lemire M,Fortin G,et al.Common variants in the NLRP3 region contribute to Crohn's disease susceptibility[J].Nat Genet,2009,41(1):71-76.
[22]Joos S,Brinkhaus B,Maluche C,et al.Acupuncture and moxibustion in the treatment of active Crohn's disease:a randomized controlled study[J].Digestion,2004,69(3):131-139.
[23]包春辉,吴璐一,吴焕淦,等.针灸治疗活动期克罗恩病:随机对照研究[J].中国针灸,2016,36(7):683-688.
[24]Shang HX,Wang AQ,Bao CH,et al.Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients[J].World JGastroenterol,2015,21(16):4986-4996.
[25]Wei K,Zhang D,Hong J,et al.Herb-partitioned moxibustion and the mi RNAs related to Crohn's disease:a study based on rat models[J].Evid Based Complement Alternat Med,2015:265238.
[26]Du X,Chen W,Wang Y,et al.Therapeutic efficacy of carboxyamidotriazole on 2,4,6-trinitrobenzene sulfonic acid-induced colitis model is associated with the inhibition of NLRP3 inflammasome and NF-kappa Bactivation[J].Int Immunopharmacol,2017,45:16-25.
[27]Castro J,Ocampo Y,Franco L.Cape Gooseberry[Physalis peruviana L.]Calyces Ameliorate TNBS Acidinduced Colitis in Rats[J].J Crohns Colitis,2015,9(11):1004-1015.
[28]Bauer C,Duewell P,Mayer C,et al.Colitis induced in mice with dextran sulfate sodium(DSS)is mediated by the NLRP3 inflammasome[J].Gut,2010,59(9):1192-1199.
[29]De Santis S,Kunde D,Galleggiante V,et al.TNFalpha deficiency results in increased IL-1beta in an early onset of spontaneous murine colitis[J].Cell Death Dis,2017,8(8):e2993.
[30]Ogawa M,Osada H,Hasegawa A,et al.Effect of interleukin-1beta on occludin m RNA expression in the duodenal and colonic mucosa of dogs with inflammatory bowel disease[J].J Vet Intern Med,2018,32(3):1019-1025.
[31]张璐,王诗怡,范一宏,等.克罗恩病患者小肠细菌过生长的监测及意义研究[J].浙江临床医学,2017,19(6):1029-1032.
[32]吴璐一,翁志军,吴焕淦,等.Th17细胞因子在CD大鼠结肠不同炎性时期的差异表达[J].世界华人消化杂志,2016,24(14):2161-2169.