四神丸对脾肾阳虚型溃疡性结肠炎模型大鼠结肠组织Toll样受体4及其负性调控因子IRAK-M表达的影响
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摘要
目的:观察Toll样受体4(TLR4)及其负性调控因子白细胞介素-1受体相关激酶M(IRAK-M)在实验性溃疡性结肠炎(UC)模型大鼠结肠黏膜中的表达,并探讨四神丸干预UC的作用机制。方法:将90只Wistar大鼠随机分成6组,即空白组,模型组,柳氮磺胺嘧啶组(0. 36 g·kg~(-1)),四神丸低、中、高剂量组(2. 5,5,10 g·kg~(-1)),每组15只。以三硝基苯磺酸/乙醇溶液法制备UC大鼠模型,苏木素-伊红(HE)染色观察大鼠结肠组织病理学改变;采用放射免疫法测定血清游离三碘甲腺原氨酸(FT3),血清游离甲状腺素(FT4),免疫球蛋白(Ig) E,白细胞介素(IL)-2的含量;采用黄嘌呤氧化法测定大鼠血清超氧化物歧化酶(SOD)活性;采用硫代巴比妥酸(TBA)比色法测定大鼠血清丙二醛(MDA)的活性。采用实时荧光定量PCR(Real-time PCR),免疫组化和蛋白免疫印迹法(Western blot)分别检测TLR4,IRAK-M的mRNA及蛋白表达。结果:与空白组比较,模型组大鼠肠黏膜损伤评分显著增高(P <0. 01),大鼠血清Ig E,MDA含量显著升高(P <0. 01),FT3,FT4,IL-2,SOD表达水平显著下降(P <0. 01),TLR4 mRNA和蛋白表达显著升高(P <0. 01),IRAK-M mRNA和蛋白表达显著降低(P <0. 01);与模型组比较,各治疗组鼠肠黏膜损伤评分均显著降低(P <0. 01),Ig E,MDA含量明显下降(P <0. 05,P <0. 01),FT3,FT4,IL-2,SOD水平明显升高(P <0. 05,P <0. 01),TLR4 mRNA和蛋白表达明显降低(P <0. 05,P <0. 01),IRAK-M mRNA和蛋白表达水平显著升高(P <0. 01)。结论:UC的发病机制与TLR4及其负性调控因子IRAK-M的表达失衡有关,且四神丸可能通过抑制TLR4mRNA和蛋白的表达,促进其负性调控因子IRAK-M的表达,起到有效治疗UC的作用。
Objective: To observe the Toll-like receptor 4( TLR4) and its negative regulating factorInterleukin-1 receptor-associated kinase-M( IRAK-M) in colonic mucosa of rats with experimental ulcerative colitis( UC),and to discuss the mechanism of the Chinese medicine Sishenwan. Method: The 90 Wistar rats were randomly divide into six groups,blank group,model group,sulfasalazine group( 0. 36 g·kg~(-1)),Sishenwan low,medium and high-dose group( 2. 5,5,10 g·kg~(-1)),15 cases in each group. A rat model of UC was prepared by using a solution of trinitrobenzenesulfonic acid/ethano. The histopathological changes of colon were observed by hematoxylin-eosin( HE) staining. The contents of serum free triiodothyroid acid( FT3),serum free thyroxine( FT4),immunoglobulin( Ig) E and interleukin( IL)-2 were determined by radioimmunoassay. The activity of superoxide dismutase( SOD) in rat serum was determined by xanthine oxidation method. The activity of malondialdehyde( MDA) in serum of rats was determined by thiobarbituric acid( TBA) colorimetry. Result:Compared with blank group,intestinal mucosal injury score of rats in model group was significantly increased( P <0. 01),serum IgE and MDA contents were significantly increased( P < 0. 01). The expression levels of FT3,FT4,IL-2 and SOD were significantly decreased( P < 0. 01). The TLR4 mRNA and protein expression in model group increased significantly( P < 0. 01). The expression of IRAK-M mRNA and protein decreased significantly( P <0. 01). Compared with model group,scores of each treatment group were significantly decreased( P < 0. 01),Ig E and MDA contents were significantly decreased( P < 0. 05,P < 0. 01),FT3,FT4,IL-2,and SOD contents were significantly increased( P < 0. 05,P < 0. 01). The expression of TLR4 mRNA and proteinin each treatment group was significantly reduced( P < 0. 05,P < 0. 01),IRAK-M mRNA and protein expression level increased( P <0. 01). Conclusion: The unbalanced expressions of TLR4 and its negative regulating factor IRAK-M are connected with the pathogenesis of UC. Sishenwan can cure UC and control the expression of TLR4 and promote the expression of IRAK-M.
Objective: To observe the Toll-like receptor 4( TLR4) and its negative regulating factorInterleukin-1 receptor-associated kinase-M( IRAK-M) in colonic mucosa of rats with experimental ulcerative colitis( UC),and to discuss the mechanism of the Chinese medicine Sishenwan. Method: The 90 Wistar rats were randomly divide into six groups,blank group,model group,sulfasalazine group( 0. 36 g·kg~(-1)),Sishenwan low,medium and high-dose group( 2. 5,5,10 g·kg~(-1)),15 cases in each group. A rat model of UC was prepared by using a solution of trinitrobenzenesulfonic acid/ethano. The histopathological changes of colon were observed by hematoxylin-eosin( HE) staining. The contents of serum free triiodothyroid acid( FT3),serum free thyroxine( FT4),immunoglobulin( Ig) E and interleukin( IL)-2 were determined by radioimmunoassay. The activity of superoxide dismutase( SOD) in rat serum was determined by xanthine oxidation method. The activity of malondialdehyde( MDA) in serum of rats was determined by thiobarbituric acid( TBA) colorimetry. Result:Compared with blank group,intestinal mucosal injury score of rats in model group was significantly increased( P <0. 01),serum IgE and MDA contents were significantly increased( P < 0. 01). The expression levels of FT3,FT4,IL-2 and SOD were significantly decreased( P < 0. 01). The TLR4 mRNA and protein expression in model group increased significantly( P < 0. 01). The expression of IRAK-M mRNA and protein decreased significantly( P <0. 01). Compared with model group,scores of each treatment group were significantly decreased( P < 0. 01),Ig E and MDA contents were significantly decreased( P < 0. 05,P < 0. 01),FT3,FT4,IL-2,and SOD contents were significantly increased( P < 0. 05,P < 0. 01). The expression of TLR4 mRNA and proteinin each treatment group was significantly reduced( P < 0. 05,P < 0. 01),IRAK-M mRNA and protein expression level increased( P <0. 01). Conclusion: The unbalanced expressions of TLR4 and its negative regulating factor IRAK-M are connected with the pathogenesis of UC. Sishenwan can cure UC and control the expression of TLR4 and promote the expression of IRAK-M.
引文
[1]王移飞,王凤仪,徐兰萍,等.芍药汤经HMGB1调节湿热型溃疡性结肠炎大鼠MyD88和NF-κB的分子机制[J].中国实验方剂学杂志,2018,24(12):86-91.
[2] Iskandar H N,Dhere T,Farraye F A. Ulcerativecolitis:update on medical managemen[J]. Curr Gastroenterol Rep,2015,17(11):44-53.
[3]高鸿亮,王磊,麦麦提图尔荪·阿不杜拉,等.维生素D3对溃疡性结肠炎大鼠肠黏膜上皮细胞Toll样受体表达的调控作用[J].中华实用诊断与治疗杂志,2016,30(12):1181-1183.
[4]于高水,杨玉荣,梁宏德. Toll样受体研究进展[J].细胞生物学杂志,2009,31(3):339-343.
[5]邢陈,黎燕,沈倍奋,等.调节性B细胞的研究进展[J].国际药学研究杂志,2015,42(3):326-330,354.
[6]吴东升,曹晖,张彧,等.芍药汤对溃疡性结肠炎大鼠肠黏膜免疫屏障的干预作用[J].中国实验方剂学杂志,2019,25(9):6-11.
[7]王旭丹,葛东宇,李根茂,等.四神丸和葛根芩连片对DSS诱导的小鼠实验性结肠炎的作用比较[J].中国实验方剂学杂志,2015,21(15):142-146.
[8]刘端勇,徐荣,黄敏芳,等.四神丸调节结肠炎大鼠PP结T细胞亚群及Treg/Th17间平衡的作用机制[J].中国实验方剂学杂志,2016,22(3):107-111.
[9]朱丹,张怡,王毅,等.大鼠溃疡性结肠炎(脾肾阳虚模型)模型实验研究[J].辽宁中医药大学学报,2015,17(5):120-123.
[10]王燚霈,朱莹,王璇.溃疡性结肠炎(脾肾阳虚模证)大鼠模型的实验研究[J].中国中医急症,2015,24(8):1332-1334,1391.
[11]梁茂新,高天舒.《中药新药临床研究指导原则》脏腑诸证考察与分析[J].中国中医基础医学杂志,2008,14(5):330-331,342.
[12]陈小野.脾虚证动物模型的造模思路[J].中医杂志,1995,36(4):239-241.
[13]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2006:18.
[14] Wallace J L,Keenan C M. An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis[J]. AM J Physiol,1990,258(4Pt1):G257-G534.
[15]顾培青,沈洪,朱磊,等.清肠化湿方对溃疡性结肠炎大鼠结肠组织PPAR-γ,NF-κB及MUC2,TFF3的影响[J].中国实验方剂学杂志,2017,23(3):79-85.
[16]杨一新,李桂源. LPS所介导的信号转导通路研究进展[J].中南大学学报:医学版,2006,31(1):141-145.
[17]赵保胜,霍海如,姜廷良. Toll样受体4的研究及现状[J].中国临床药理学与治疗学,2007,12(1):19-22.
[18]武志娟,张志敏,张大鹏,等.附子理中汤对腹泻型肠易激大鼠模型结肠组织中TLR4 mRNA表达水平的影响[J].中国实验方剂学杂志,2013,19(15):218-221.
[19]郑佳佳,万敬员. IRAK-M在TLRs信号转导中负性调节作用的研究进展[J].生命科学,2009,21(3):430-433.
[20]余烁.白细胞介素1受体相关激酶(IRAK)家族在基于Toll样受体4信号通路介导的急性前葡萄膜炎发病中所起作用及红芪多糖干预机制的研究[D].北京:首都医科大学,2017.
[21] DENG J C,CHENG G H,Newstead M W,et al. Sepsisinduced suppression of lung innate immunity is mediated by IRAK-M[J]. J Clin Invest,2006,116(9):2532-2542.
[22]张文兰,胡同平,王慧,等.系统性红斑狼疮患者单核细胞中TLR-4、IRAK-M的表达及意义[J].免疫学杂志,2013,29(6):510-513.
[2] Iskandar H N,Dhere T,Farraye F A. Ulcerativecolitis:update on medical managemen[J]. Curr Gastroenterol Rep,2015,17(11):44-53.
[3]高鸿亮,王磊,麦麦提图尔荪·阿不杜拉,等.维生素D3对溃疡性结肠炎大鼠肠黏膜上皮细胞Toll样受体表达的调控作用[J].中华实用诊断与治疗杂志,2016,30(12):1181-1183.
[4]于高水,杨玉荣,梁宏德. Toll样受体研究进展[J].细胞生物学杂志,2009,31(3):339-343.
[5]邢陈,黎燕,沈倍奋,等.调节性B细胞的研究进展[J].国际药学研究杂志,2015,42(3):326-330,354.
[6]吴东升,曹晖,张彧,等.芍药汤对溃疡性结肠炎大鼠肠黏膜免疫屏障的干预作用[J].中国实验方剂学杂志,2019,25(9):6-11.
[7]王旭丹,葛东宇,李根茂,等.四神丸和葛根芩连片对DSS诱导的小鼠实验性结肠炎的作用比较[J].中国实验方剂学杂志,2015,21(15):142-146.
[8]刘端勇,徐荣,黄敏芳,等.四神丸调节结肠炎大鼠PP结T细胞亚群及Treg/Th17间平衡的作用机制[J].中国实验方剂学杂志,2016,22(3):107-111.
[9]朱丹,张怡,王毅,等.大鼠溃疡性结肠炎(脾肾阳虚模型)模型实验研究[J].辽宁中医药大学学报,2015,17(5):120-123.
[10]王燚霈,朱莹,王璇.溃疡性结肠炎(脾肾阳虚模证)大鼠模型的实验研究[J].中国中医急症,2015,24(8):1332-1334,1391.
[11]梁茂新,高天舒.《中药新药临床研究指导原则》脏腑诸证考察与分析[J].中国中医基础医学杂志,2008,14(5):330-331,342.
[12]陈小野.脾虚证动物模型的造模思路[J].中医杂志,1995,36(4):239-241.
[13]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2006:18.
[14] Wallace J L,Keenan C M. An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis[J]. AM J Physiol,1990,258(4Pt1):G257-G534.
[15]顾培青,沈洪,朱磊,等.清肠化湿方对溃疡性结肠炎大鼠结肠组织PPAR-γ,NF-κB及MUC2,TFF3的影响[J].中国实验方剂学杂志,2017,23(3):79-85.
[16]杨一新,李桂源. LPS所介导的信号转导通路研究进展[J].中南大学学报:医学版,2006,31(1):141-145.
[17]赵保胜,霍海如,姜廷良. Toll样受体4的研究及现状[J].中国临床药理学与治疗学,2007,12(1):19-22.
[18]武志娟,张志敏,张大鹏,等.附子理中汤对腹泻型肠易激大鼠模型结肠组织中TLR4 mRNA表达水平的影响[J].中国实验方剂学杂志,2013,19(15):218-221.
[19]郑佳佳,万敬员. IRAK-M在TLRs信号转导中负性调节作用的研究进展[J].生命科学,2009,21(3):430-433.
[20]余烁.白细胞介素1受体相关激酶(IRAK)家族在基于Toll样受体4信号通路介导的急性前葡萄膜炎发病中所起作用及红芪多糖干预机制的研究[D].北京:首都医科大学,2017.
[21] DENG J C,CHENG G H,Newstead M W,et al. Sepsisinduced suppression of lung innate immunity is mediated by IRAK-M[J]. J Clin Invest,2006,116(9):2532-2542.
[22]张文兰,胡同平,王慧,等.系统性红斑狼疮患者单核细胞中TLR-4、IRAK-M的表达及意义[J].免疫学杂志,2013,29(6):510-513.