肾蕨对乙酸致小鼠肛门溃疡的作用及NF-κB,TNF-α,IL-1β和COX-2表达的影响
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摘要
目的:研究肾蕨叶水提取物(NLD)对小鼠肛门溃疡模型的作用及机制。方法:以20%乙酸(HAc)0.5 m L涂抹小鼠肛门,制备肛门溃疡模型,将50只昆明小鼠随机分为正常组、模型组、NLD低、中、高剂量(0.45,0.9,1.8 g·kg-1)组,每组10只。各组小鼠自外涂给NLD计时,第10天处死。采集肛门、直肠组织,实时荧光定量逆转录聚合酶链式反应(Real-time PCR)检测各组小鼠肛门、直肠组织中核转录因子(NF)-κB,肿瘤坏死因子(TNF)-α,白细胞介素(IL)-1β和环氧合酶(COX)-2mRNA表达;蛋白免疫印迹法(Western blot)检测各组小鼠肛门、直肠组织中NF-κB,TNF-α,IL-1β和COX-2蛋白表达;苏木素-伊红(HE)染色观察小鼠肛门、直肠组织形态学变化。结果:NLD可恢复小鼠体质量增长趋势,降低小鼠肛门脏器指数,下调小鼠肛门、直肠组织中NF-κB,TNF-α,IL-1β和COX-2 mRNA(P<0.01),降低小鼠肛门、直肠组织中NF-κB,TNF-α,IL-1β和COX-2蛋白表达(P<0.01);NLD 0.9,1.8 g·kg-1剂量组可抑制炎性细胞的浸润及黏膜下层血管扩张,改善相应炎症反应。结论:NLD对肛门溃疡模型小鼠的肛门溃疡有明显的改善作用,其机制可能与下调NF-κB mRNA和蛋白的表达,进而下调其下游因子TNF-α,IL-1β与COX-2的基因表达有关。
Objective: To investigate the therapeutic effect of Nephrolepis cordifolia leaf decoction(NLD)on anal ulcer model in mice and explore its mechanism. Method: The anal ulcer model was prepared by applying0. 5 m L of 20% acetic acid(HAc) into the anus of mice. The 50 Kunming mice were randomly divided into normal group,model group,low,middle and high dose NLD(0. 45,0. 9 and 1. 8 g·kg-1) groups,10 mice in each group. All the mice were killed on the 10 thday after NLD application. Their anal and rectal tissues were collected and the mRNA expression levels of nuclear transcription factor-κB(NF-κB),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β) and cyclooxygenase-2(COX-2) in the anal and rectal tissues of each group were detected by Real-time fluorescence quantitative reverse transcription polymerase chain reaction(Real-time PCR). Western blot was used to detect the protein expression levels of NF-κB,TNF-α,IL-1β and COX-2 in anal and rectal tissues of mice in each group. Morphological changes of anal and rectal tissues in mice were observed by hematoxylin-eosin(HE) staining. Result: NLD could restore the growth of mice body mass,reduce the rectoanal coefficient and down-regulate NF-κB,TNF-α,IL-1β and COX-2 mRNA expression in the anal and rectal tissues of mice(P < 0. 01),inhibit the activity of NF-κB,TNF-α,IL-1β and COX-2 protein expression in the anal and rectal tissues of mice(P < 0. 01). NLD 0. 9 and 1. 8 g·kg-1 could inhibit the infiltration of inflammatory cells and submucosa vasodilation and improve the corresponding inflammatory response. Conclusion: NLD has protective effect on the inflammatory response of acetic acid-induced anal ulcer in mice,and the mechanism may be related to inhibiting NF-κB mRNA and protein expression and down-regulating the gene expression of downstream inflammatory factors TNF-α,IL-1β and COX-2.
Objective: To investigate the therapeutic effect of Nephrolepis cordifolia leaf decoction(NLD)on anal ulcer model in mice and explore its mechanism. Method: The anal ulcer model was prepared by applying0. 5 m L of 20% acetic acid(HAc) into the anus of mice. The 50 Kunming mice were randomly divided into normal group,model group,low,middle and high dose NLD(0. 45,0. 9 and 1. 8 g·kg-1) groups,10 mice in each group. All the mice were killed on the 10 thday after NLD application. Their anal and rectal tissues were collected and the mRNA expression levels of nuclear transcription factor-κB(NF-κB),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β) and cyclooxygenase-2(COX-2) in the anal and rectal tissues of each group were detected by Real-time fluorescence quantitative reverse transcription polymerase chain reaction(Real-time PCR). Western blot was used to detect the protein expression levels of NF-κB,TNF-α,IL-1β and COX-2 in anal and rectal tissues of mice in each group. Morphological changes of anal and rectal tissues in mice were observed by hematoxylin-eosin(HE) staining. Result: NLD could restore the growth of mice body mass,reduce the rectoanal coefficient and down-regulate NF-κB,TNF-α,IL-1β and COX-2 mRNA expression in the anal and rectal tissues of mice(P < 0. 01),inhibit the activity of NF-κB,TNF-α,IL-1β and COX-2 protein expression in the anal and rectal tissues of mice(P < 0. 01). NLD 0. 9 and 1. 8 g·kg-1 could inhibit the infiltration of inflammatory cells and submucosa vasodilation and improve the corresponding inflammatory response. Conclusion: NLD has protective effect on the inflammatory response of acetic acid-induced anal ulcer in mice,and the mechanism may be related to inhibiting NF-κB mRNA and protein expression and down-regulating the gene expression of downstream inflammatory factors TNF-α,IL-1β and COX-2.
引文
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[20]于舒雁,魏荣锐,苗明三.不同品种威灵仙水煎剂外用对大鼠痔疮、佐剂性关节炎模型的影响[J].中国现代应用药学,2014,31(4):391-397.
[21]HU W S,LIN C L.Hemorrhoid is associated with increased risk of peripheral artery occlusive disease:a nationwide cohort study[J].J Epidemiol,2017,27(12):574-577.
[22]姬培震,张怡,李雪萍,等.附子理中汤灌肠对脾肾阳虚型溃疡性结肠炎大鼠NF-κB,TNF-α,IL-1β表达的影响[J].中国实验方剂学杂志,2015,21(14):124-128.
[2]韩炜,王振军,赵博,等.痔组织弹性纤维退变和血管生成的机制及其意义[J].中华胃肠外科杂志,2005,8(1):56-59.
[3]周小莉.DH001栓剂的药效学研究[D].苏州:苏州大学,2013.
[4]Lohsiriwat V.Hemorrhoids:from basic pathophysiology to clinical management[J].World J Gastroenterol,2012,18(17):2009-2017.
[5]李汉隆,欧阳莎.中重度痔疮采用吻合器痔上黏膜环切术联合硬化剂注射术治疗的临床效果分析[J].中华临床医师杂志,2016,10(8):1120-1123.
[6]徐伟,倪平,吴春珠.塞来昔布在痔疮手术中的超前镇痛作用[J].中国新药与临床杂志,2015,34(6):464-466.
[7]刘史佳,申龙树,戴国梁,等.IL-17、IL-6、TNF-α细胞因子在痔疮患者中的表达[J].药学与临床研究,2016,24(3):201-204.
[8]南京中医药大学.中药大辞典(修订本)(上)[M].2版.上海:上海科学技术出版社,2014:1629.
[9]王国强.全国中草药汇编.第2卷[M].3版.北京:人民卫生出版社,2014:625.
[10]王小青,李爽,赵涵,等.壮药肾蕨的研究进展[J].承德医学院学报,2017,34(5):399-401.
[11]谢凤凤,黎理,马雯芳,等.肾蕨多糖提取工艺优化及其含量测定研究[J].临床医药文献电子杂志,2017,4(41):7933-7935.
[12]赵莉,杨文枉.蕨类植物的活性成分研究进展[J].中药材,2004,27(6):452.
[13]陈晓清,苏育才,李晓晶,等.抗菌肾蕨多糖的提取与分离[J].漳州师范学院学报:自然科学版,2006,4(54):112-115.
[14]王音音,李峰,王霆.红衫醇在植物种类中的分布[J].今日药学,2008,17(1):70-74.
[15]马宗国,江南,余梦瑶,等.痔疮动物模型的研究进展[J].中国实验动物学报,2010,18(5):446-450.
[16]徐进,宫毅,杨阳,等.四黄痔疮膏对大鼠痔相近模型局部炎症及组织病理学的影响[J].中国肛肠病杂志,2015,35(10):7-12.
[17]马雅鸽,赵声兰,阮苑,等.地木耳菌粉对2型糖尿病大鼠血糖的影响[J].中国实验方剂学杂志,2015,21(2):111-114.
[18]韦婷,王天虹,曾勇,等.藏族药五味甘露药浴散加减方对佐剂关节炎大鼠的药效及其对大鼠血清NF-κB,TNF-α,IL-1,EGF的影响[J].中国实验方剂学杂志,2016,22(10):134-137.
[19]Azeemuddin M,Viswanatha G L,Rafiq M,et al.An improved experimental model of hemorrhoids in rats:evaluation of antihemorrhoidal activity of an Herbal Formulation[J].ISRN Pharmacol,2014,2014(3):1-7.
[20]于舒雁,魏荣锐,苗明三.不同品种威灵仙水煎剂外用对大鼠痔疮、佐剂性关节炎模型的影响[J].中国现代应用药学,2014,31(4):391-397.
[21]HU W S,LIN C L.Hemorrhoid is associated with increased risk of peripheral artery occlusive disease:a nationwide cohort study[J].J Epidemiol,2017,27(12):574-577.
[22]姬培震,张怡,李雪萍,等.附子理中汤灌肠对脾肾阳虚型溃疡性结肠炎大鼠NF-κB,TNF-α,IL-1β表达的影响[J].中国实验方剂学杂志,2015,21(14):124-128.