桃仁承气汤对脓毒症大鼠肠黏膜屏障及TLR9信号通路的影响
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摘要
目的:探讨桃仁承气汤对脓毒症大鼠肠黏膜屏障的保护作用以及作用机制。方法:将大鼠分为假手术组,模型组(盲肠结扎穿孔法复制脓毒症大鼠),桃仁承气汤低、中、高剂量组(2. 85,5. 70,8. 55 g·kg-1),地塞米松组(0. 01 g·kg-1),每组12只。末次给药结束后,处死大鼠,电镜下观察肠黏膜形态变化;检测肠系膜淋巴结、肝、肾、脾组织细菌移位率;酶联免疫吸附测定(ELISA)检测血清肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β)以及小肠组织中二胺氧化酶(DAO),肠型脂肪酸结合蛋白(i-FABP)水平;蛋白免疫印迹法(Western blot),免疫组化法(IHC)检测小肠组织中Toll样受体9(TLR9),髓样分化因子88(MyD88),核转录因子-κB亚基p65(NF-κB p65)蛋白表达。结果:与假手术组比较,模型组大鼠器官细菌移位率,TNF-α,IL-1β水平明显升高,DAO,i-FABP水平,黏膜厚度,绒毛高度明显降低,TLR9,MyD88,细胞核NF-κB p65蛋白表达明显升高(P <0. 05,P <0. 01),细胞质NF-κB p65蛋白表达明显降低(P <0. 05);与模型组比较,桃仁承气汤低、中、高剂量组,地塞米松组器官细菌移位率,TNF-α,IL-1β水平明显降低,DAO,i-FABP水平,黏膜厚度,绒毛高度明显升高,TLR9,MyD88,细胞核NF-κB p65蛋白表达明显降低(P <0. 05),细胞质NF-κB p65蛋白表达明显升高(P <0. 05)。结论:桃仁承气汤对脓毒症大鼠肠黏膜屏障具有一定的保护作用,这可能与抑制TLR9信号通路有关。
Objective: To study the protective effect of Taoren Chengqitang on intestinal mucosal barrier in septic rats and its possible mechanism. Method: Rats were divided into sham operation group,model group( replication of septic rats with cecal ligation and perforation),low,middle and high-dose Taoren Chengqitang groups( 2. 85,5. 70,8. 55 g·kg-1),and dexamethasone group( 0. 01 g·kg-1),with 12 rats in each group.After last administration,rats were put to death,the morphological changes of intestinal mucosa were observed under electron microscope,the bacterial translocation rates in lymphoglandulae mesentericae,liver,kidney and spleen tissues were detected; the levels of serum tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β),and diamine oxidase( DAO) and intestinal fatty acid binding protein( i-FABP) levels in small intestine tissues were detected by enzyme-linked immunosorbent assay( ELISA). Expressions of Toll like receptor 9( TLR9),myeloid differentiation factor 88( MyD88) and nuclear factor-kappa B subunit p65 in small intestine tissues were detected by Western blot and immunohistochemistry. Result: Compared with sham operated group, the bacterial translocation rate,TNF-α and IL-1β levels in model group increased significantly,DAO,i-FABP,mucosal thickness and villus height decreased significantly,and protein expressions of TLR9,MyD88 and nucleus NF-κB p65 increased significantly( P < 0. 05,P < 0. 01),and expression of cytoplasmic NF-κB p65 protein decreased significantly. Compared with model group,the bacterial translocation rate,TNF-α and IL-1β levels in organs of low,medium and high-dose Taoren Chengqitang groups and dexamethasone group decreased significantly,DAO,i-FABP,mucosal thickness and villus height increased significantly,while the protein expressions of TLR9,My D88 and nucleus NF-κB p65 decreased significantly( P < 0. 05),and expression of cytoplasmic NF-κB p65 protein increased significantly. Conclusion: Taoren Chengqitang has a certain protective effect on intestinal mucosal barrier in septic rats,which may be related to the inhibition of TLR9 signaling pathway.
Objective: To study the protective effect of Taoren Chengqitang on intestinal mucosal barrier in septic rats and its possible mechanism. Method: Rats were divided into sham operation group,model group( replication of septic rats with cecal ligation and perforation),low,middle and high-dose Taoren Chengqitang groups( 2. 85,5. 70,8. 55 g·kg-1),and dexamethasone group( 0. 01 g·kg-1),with 12 rats in each group.After last administration,rats were put to death,the morphological changes of intestinal mucosa were observed under electron microscope,the bacterial translocation rates in lymphoglandulae mesentericae,liver,kidney and spleen tissues were detected; the levels of serum tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β),and diamine oxidase( DAO) and intestinal fatty acid binding protein( i-FABP) levels in small intestine tissues were detected by enzyme-linked immunosorbent assay( ELISA). Expressions of Toll like receptor 9( TLR9),myeloid differentiation factor 88( MyD88) and nuclear factor-kappa B subunit p65 in small intestine tissues were detected by Western blot and immunohistochemistry. Result: Compared with sham operated group, the bacterial translocation rate,TNF-α and IL-1β levels in model group increased significantly,DAO,i-FABP,mucosal thickness and villus height decreased significantly,and protein expressions of TLR9,MyD88 and nucleus NF-κB p65 increased significantly( P < 0. 05,P < 0. 01),and expression of cytoplasmic NF-κB p65 protein decreased significantly. Compared with model group,the bacterial translocation rate,TNF-α and IL-1β levels in organs of low,medium and high-dose Taoren Chengqitang groups and dexamethasone group decreased significantly,DAO,i-FABP,mucosal thickness and villus height increased significantly,while the protein expressions of TLR9,My D88 and nucleus NF-κB p65 decreased significantly( P < 0. 05),and expression of cytoplasmic NF-κB p65 protein increased significantly. Conclusion: Taoren Chengqitang has a certain protective effect on intestinal mucosal barrier in septic rats,which may be related to the inhibition of TLR9 signaling pathway.
引文
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[10]任钰鑫,刘小军,连永红,等.清瘟败毒饮合凉隔散加减辅助血必净注射液治疗脓毒症热毒炽盛证的多靶点效应[J].中国实验方剂学杂志,2017,23(10):189-194.
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[14] KUO C K,LI W J,Mauck R L,et al. Cartilage tissue engineering:its potential and uses[J]. Curr Opin Rheumatol,2006,18(1):64-73.
[15]沈海平,周云,何苏丽,等.新加黄龙汤对脓毒症休克患者胃肠道功能的临床研究[J].中药药理与临床,2016,32(6):187-189.
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[18] LING X,LING L P,WEI X D,et al. Protective effects of bifidobacterium on intestinal barrier function in LPSinduced enterocyte barrier injury of caco-2 monolayers and in a rat NEC model[J]. PLo S One,2016,11(8):161635-161642.
[19]王桂华,兰涛,吴向军,等.桃仁提取物对急性胰腺炎大鼠肠道屏障功能及炎性反应的影响[J].中药材,2014,37(12):2267-2270.
[20]兰涛,李志娟,付立平,等.不同剂量桃仁提取物对急性胰腺炎大鼠肠道黏膜屏障功能及免疫功能的作用[J].中国免疫学杂志,2015,11(3):339-343,353.
[21]郜智慧,王倩青.加味承气汤对保留盆腔自主神经的广泛性子宫切除术后胃肠功能恢复的影响[J].中药药理与临床,2015,31(4):220-222.
[22] HUANG C,PAN L,LIN F,et al. Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88 and NF-κB dependent signaling[J]. Int J Mol Med,2017,39(3):693-700.
[23] YAN Y,LU B,LI P,et al. NOD receptor and TLR9modulation in severe acute pancreatitis-induced intestinal injury[J]. Mol Med Rep,2017,16(6):8471-8480.
[24] Latorre E,Layunta E,Grasa L,et al. Intestinal serotonin transporter inhibition by Toll-like receptor 2 activation a feedback modulation[J]. PLo S One,2016,11(12):169303-169312.
[25] HOU Y,LU X,ZHANG Y. IRAK inhibitor protects the intestinal tract of necrotizing enterocolitis by inhibiting the Toll-Like receptor(TLR)inflammatory signaling pathway in rats[J]. Med Sci Monit,2018,24(5):3366-3373.
[26]苏伟,欧娅,杨自力,等. Toll样受体9信号通路在重症急性胰腺炎大鼠肠黏膜屏障功能障碍中的作用[J].广东医学,2014,35(23):3609-3612.
[2] Delano M J,Ward P A. Sepsis-induced immune dysfunction:can immune therapies reduce mortality?[J]. J Clin Invest,2016,126(1):23-31.
[3]王亦君,冯舒涵,程锦堂,等.大黄蒽醌类化学成分和药理作用研究进展[J].中国实验方剂学杂志,2018,24(13):227-234.
[4]刘承远,陈海鸣. IL-10、TNF-α在脓毒症小鼠小肠黏膜中的表达及其意义[J].江西医药,2015,50(11):1173-1175.
[5] Hsiao H B,Chou A H,LIN S I,et al. Toll-like receptor9-mediated protection of enterovirus 71 infection in mice is due to the release of ganger-associated molecular patterns[J]. J Virol,2014,88(20):11658-11670.
[6] Sardi C,Luchini P,Emanuelli A,et al. Three months of Western diet induces small intestinal mucosa alteration in TLR KO mice[J]. Microsc Res Tech,2017,80(6):563-569.
[7]宋慧荣,罗亚敏,李冉,等.桃核承气汤与桃仁承气汤差异探析[J].河南中医,2018,38(6):824-827.
[8]张磊.加味桃仁承气汤治疗胸腰椎压缩性骨折后腹胀便秘的疗效观察[J].成都中医药大学学报,2014,37(3):82-83,115.
[9]胡玉杰.桃仁承气汤治疗急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)的实验研究[D].石家庄:河北医科大学,2014.
[10]任钰鑫,刘小军,连永红,等.清瘟败毒饮合凉隔散加减辅助血必净注射液治疗脓毒症热毒炽盛证的多靶点效应[J].中国实验方剂学杂志,2017,23(10):189-194.
[11]李锦灵,黄树武,李舸,等.大鼠脓毒症模型的凝血功能研究[J].中国实验动物学报,2018,26(2):224-229.
[12] Hubbard W J,Choudhry M,Schwacha M G,et al. Cecal ligation and puncture[J]. Shock,2005,24(1):52-57.
[13] Buras J A,Holzmann B,Sitkovsky M. Animal models of sepsis:setting the stage[J]. Nat Rev Drug Discov,2005,4(10):854-865.
[14] KUO C K,LI W J,Mauck R L,et al. Cartilage tissue engineering:its potential and uses[J]. Curr Opin Rheumatol,2006,18(1):64-73.
[15]沈海平,周云,何苏丽,等.新加黄龙汤对脓毒症休克患者胃肠道功能的临床研究[J].中药药理与临床,2016,32(6):187-189.
[16]张俊亮,曾其毅.脓毒症与胃肠道功能损伤的关系及研究进展[J].实用医学杂志,2016,32(17):2925-2927.
[17] LI Z,ZHANG X,ZHOU H,et al. Exogenous snitrosoglutathione attenuates inflammatory response and intestinal epithelial barrier injury in endotoxemic rats[J]. J Trauma Acute Care Surg,2016,80(6):977-986.
[18] LING X,LING L P,WEI X D,et al. Protective effects of bifidobacterium on intestinal barrier function in LPSinduced enterocyte barrier injury of caco-2 monolayers and in a rat NEC model[J]. PLo S One,2016,11(8):161635-161642.
[19]王桂华,兰涛,吴向军,等.桃仁提取物对急性胰腺炎大鼠肠道屏障功能及炎性反应的影响[J].中药材,2014,37(12):2267-2270.
[20]兰涛,李志娟,付立平,等.不同剂量桃仁提取物对急性胰腺炎大鼠肠道黏膜屏障功能及免疫功能的作用[J].中国免疫学杂志,2015,11(3):339-343,353.
[21]郜智慧,王倩青.加味承气汤对保留盆腔自主神经的广泛性子宫切除术后胃肠功能恢复的影响[J].中药药理与临床,2015,31(4):220-222.
[22] HUANG C,PAN L,LIN F,et al. Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88 and NF-κB dependent signaling[J]. Int J Mol Med,2017,39(3):693-700.
[23] YAN Y,LU B,LI P,et al. NOD receptor and TLR9modulation in severe acute pancreatitis-induced intestinal injury[J]. Mol Med Rep,2017,16(6):8471-8480.
[24] Latorre E,Layunta E,Grasa L,et al. Intestinal serotonin transporter inhibition by Toll-like receptor 2 activation a feedback modulation[J]. PLo S One,2016,11(12):169303-169312.
[25] HOU Y,LU X,ZHANG Y. IRAK inhibitor protects the intestinal tract of necrotizing enterocolitis by inhibiting the Toll-Like receptor(TLR)inflammatory signaling pathway in rats[J]. Med Sci Monit,2018,24(5):3366-3373.
[26]苏伟,欧娅,杨自力,等. Toll样受体9信号通路在重症急性胰腺炎大鼠肠黏膜屏障功能障碍中的作用[J].广东医学,2014,35(23):3609-3612.