木瓜总三萜和奥美拉唑联用对吲哚美辛诱导大鼠胃溃疡的治疗作用研究
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摘要
观察木瓜总三萜和奥美拉唑联用对吲哚美辛诱导胃溃疡大鼠治疗作用,并探寻其可能作用机制。将大鼠随机分为正常组、模型组、奥美拉唑(3.6 mg·kg~(-1))组、木瓜总三萜(100 mg·kg~(-1))组、木瓜总三萜和奥美拉唑联用(100 mg·kg~(-1)+3.6 mg·kg~(-1))组。除正常组外,各组按照20 mg·kg~(-1)灌胃给予吲哚美辛,每天1次,连续7 d,然后灌胃给予相应药物,每天1次,连续14 d。末次给药次日,每组随机选取一半大鼠进行胃黏膜血流量、胃液量测定和血液中TNF-α,IL-1β,IL-6,IL-4,IL-10含量检测;每组余下大鼠在末次给药后4 h行幽门结扎术,8 h后取胃,收集胃内容物,检测胃内分泌物体积、pH和分泌物总酸度,然后进行胃损伤组织学分析、MPO活性、cAMP含量检测和形态学分析;实时定量PCR法检测TNF-α,IL-1β,IL-6,IL-4,IL-10,VEGFA,A_(2A)R mRNA表达,Western blot法检测胃组织中VEGFA,A_(2A)R,PKA,p-PKA,CREB,p-CREB,EGF,EGFR,p-EGFR,MUC6,TFF2蛋白表达。结果显示,木瓜总三萜和奥美拉唑联用可显著增加胃黏膜血流量、胃黏液量,降低胃内分泌物体积、分泌物总酸度和黏膜损伤,降低血液和胃组织中TNF-α,IL-1β,IL-6含量和表达,升高IL-4,IL-10含量和表达,抑制胃组织中MPO活性,增加胃组织中cAMP含量,上调胃组织中VEGFA和A_(2A)R mRNA和p-PKA,p-CREB,VEGFA,A_(2A)R,EGF,p-EGFR,MUC6和TFF2蛋白表达,升高p-PKA/PKA,p-CREB/CREB和p-EFGR/EFGR比值,其中木瓜总三萜和奥美拉唑联用治疗效果比两者单用效果更加明显。结果表明木瓜总三萜和奥美拉唑联用对吲哚美辛诱导大鼠胃溃疡有显著治疗作用,其作用机制可能与其调节A_(2A)R/AKT/CREB,A_(2A)R/VEGFA,EGF/EGFR和MUC6/TFF2信号通路,抑制促炎因子生成、增加胃黏膜血流量、上调促进黏膜细胞增殖因子和保护因子蛋白表达有关。
The aim of this paper was to observe the combination therapy with total triterpenoids of Chaenomeles speciosa and omeprazole on indomethacin-induced gastric ulcer in rats, and explore its possible mechanism. Rats were randomly divided into normal group, model group, omeprazole monotherapy(3.6 mg·kg~(-1)) group, total triterpenoids of C. speciosa monotherapy(100 mg·kg~(-1)) group, total triterpenoids of C. speciosa and omeprazole combination therapy(100 mg·kg~(-1)+3.6 mg·kg~(-1)) group. Except for the normal group, the other groups were given indomethacin(20 mg·kg~(-1)) by oral once a day for 7 consecutive days. Then the treated groups were given corresponding drugs by gavage, once a day for 14 consecutive days. The next day after the last administration, half of the rats in each group were measured the gastric mucosal blood flow, gastric juice volume and serum TNF-α, IL-1β, IL-6, IL-4 and IL-10. After the remaining rats in each group were underwent pyloric ligation 4 hours after the last administration, the gastric endocrine volume, pH value and total acidity of gastric secretion were measured, then histological analysis was performed, MPO activity, cAMP content and histomorphological analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of gastric tissue TNF-α,IL-1β, IL-6, IL-4, IL-10, VEGFA, A_(2A)R; the protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue were detected by Western blot. The results indicated that total triterpenoids of C. speciosa and omeprazole combination therapy might significantly increase gastric mucosal blood flow, gastric mucus volume, reduce gastric endocrine volume, secretion acidity and mucosal damage, decrease the levels of TNF-α,IL-1β and IL-6, increase the levels of IL-4 and IL-10 in blood and gastric tissue, inhibit the activity of MPO, increase the content of cAMP in gastric tissue, up-regulate the mRNA expressions of VEGFA, A_(2A)R and protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue, elevate p-PKA/PKA, p-CREB/CREB and p-EFGR/EFGR. Moreover, the combination therapy with total triterpenoids of C. speciosa and omeprazole was more obvious than those of two monotherapies. These aforementioned findings suggested that the combination therapy with total triterpenoids of C. speciosa and omeprazole on indomethacin-induced gastric ulcer have significant therapeutic effect on indomethacin induced gastric ulcer in rats, its mechanism might be related to regulating A_(2A)R/AKT/CREB, A_(2A)R/VEGFA, EGF/EGFR and MUC6/TFF2 signaling pathways, inhibiting pro-inflammatory factors, increasing gastric mucosal blood flow, up-regulating mucosal cell proliferation factors and promoting mucosal protective factors.
The aim of this paper was to observe the combination therapy with total triterpenoids of Chaenomeles speciosa and omeprazole on indomethacin-induced gastric ulcer in rats, and explore its possible mechanism. Rats were randomly divided into normal group, model group, omeprazole monotherapy(3.6 mg·kg~(-1)) group, total triterpenoids of C. speciosa monotherapy(100 mg·kg~(-1)) group, total triterpenoids of C. speciosa and omeprazole combination therapy(100 mg·kg~(-1)+3.6 mg·kg~(-1)) group. Except for the normal group, the other groups were given indomethacin(20 mg·kg~(-1)) by oral once a day for 7 consecutive days. Then the treated groups were given corresponding drugs by gavage, once a day for 14 consecutive days. The next day after the last administration, half of the rats in each group were measured the gastric mucosal blood flow, gastric juice volume and serum TNF-α, IL-1β, IL-6, IL-4 and IL-10. After the remaining rats in each group were underwent pyloric ligation 4 hours after the last administration, the gastric endocrine volume, pH value and total acidity of gastric secretion were measured, then histological analysis was performed, MPO activity, cAMP content and histomorphological analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of gastric tissue TNF-α,IL-1β, IL-6, IL-4, IL-10, VEGFA, A_(2A)R; the protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue were detected by Western blot. The results indicated that total triterpenoids of C. speciosa and omeprazole combination therapy might significantly increase gastric mucosal blood flow, gastric mucus volume, reduce gastric endocrine volume, secretion acidity and mucosal damage, decrease the levels of TNF-α,IL-1β and IL-6, increase the levels of IL-4 and IL-10 in blood and gastric tissue, inhibit the activity of MPO, increase the content of cAMP in gastric tissue, up-regulate the mRNA expressions of VEGFA, A_(2A)R and protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue, elevate p-PKA/PKA, p-CREB/CREB and p-EFGR/EFGR. Moreover, the combination therapy with total triterpenoids of C. speciosa and omeprazole was more obvious than those of two monotherapies. These aforementioned findings suggested that the combination therapy with total triterpenoids of C. speciosa and omeprazole on indomethacin-induced gastric ulcer have significant therapeutic effect on indomethacin induced gastric ulcer in rats, its mechanism might be related to regulating A_(2A)R/AKT/CREB, A_(2A)R/VEGFA, EGF/EGFR and MUC6/TFF2 signaling pathways, inhibiting pro-inflammatory factors, increasing gastric mucosal blood flow, up-regulating mucosal cell proliferation factors and promoting mucosal protective factors.
引文
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[9] 覃慧林,张永峰,贺海波,等.木瓜总三萜抗吲哚美辛诱导RGM-1细胞凋亡的作用[J].中国临床药理学与治疗学,2016,21(12):1347.
[10] 贺海波,张永峰,李小妹,等.木瓜三萜片与奥美拉唑胶囊联用治疗非甾体抗炎药致上消化道出血临床研究[J].中药药理与临床,2017,33(2):209.
[11] Ganguly K,Swarnakar S.Chronic gastric ulceration causes matrix metalloproteinases-9 and -3 augmentation:alleviation by melatonin[J].Biochimie,2012,94(12):2687.
[12] Carvalho N S,Silva M M,Silva R O,et al.Protective effects of simvastatin against alendronate-induced gastric mucosal injury in rats[J].Dig Dis Sci,2016,61(2):400.
[13] Laine L,Weinstein W M.Histology of alcoholic hemorrhagic "gastritis":a prospective evaluation[J].Gastroenterology,1988,94(6):1254.
[14] Sostres C,Gargallo C J,Lanas A.Nonsteroidal anti-inflammatory drugs and upper and lower gastrointestinal mucosal damage[J].Arthritis Res Ther,2013,doi:10.1186/ar4175.
[15] Takeuchi K.Pathogenesis of NSAID-induced gastric damage:importance of cyclooxygenase inhibition and gastric hypermotility[J].World J Gastroenterol,2012,18(18):2147.
[16] Cheng Y T,Lu C C,Yen G C.Phytochemicals enhance antioxidant enzyme expression to protect against NSAID-induced oxidative damage of the gastrointestinal mucosa[J].Mol Nutr Food Res,2017,doi:10.1002/mnfr.201600659.
[17] Rivollier A,He J P,Kole A,et al.Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon[J].J Exp Med,2012,209(1):139.
[18] 于艳,贾天柱,才谦.茅苍术及其麸炒品对胃溃疡大鼠抗炎作用的比较研究[J].中国中药杂志,2016,41(4):705.
[19] Odashima M,Otaka M,Jin M,et al.Attenuation of gastric mucosal inflammation induced by aspirin through activation of A2A adenosine receptor in rats[J].World J Gastroenterol,2006,12(4):568.
[20] Milne G R,Palmer T M.Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor[J].Scientific World J,2011,11:320.
[21] Jones M K,Kawanaka H,Baatar D,et al.Gene therapy for gastric ulcers with single local injection of naked DNA encoding VEGF and angiopoietin-1[J].Gastroenterology,2001,121(5):1040.
[22] Chatterjee A,Chatterjee S,Biswas A,et al.Gallic acid enriched fraction of Phyllanthus emblica potentiates indomethacin-induced gastric ulcer healing via e-NOS-dependent pathway[J].Evid Based Complement Alternat Med,2012,doi:10.1155/2012/487380.
[23] Magierowski M,Magierowska K,Hubalewska-Mazgaj M,et al.Carbon monoxide released from its pharmacological donor,tricarbonyldichlororuthenium (Ⅱ) dimer,accelerates the healing of pre-existing gastric ulcers[J].Br J Pharmacol,2017,174(20):3654.
[24] Wang B,Zhao H Y,Zhou L,et al.Effect of Kuiyangping on expressions of EGF and EGFR mRNA in gastric mucosa in rats with experimental gastric ulcer[J].J Beijing Univ Med,2008,31(11):759.
[25] Hoffmann W.TFF2,a MUC6-binding lectin stabilizing the gastric mucus barrier and more (review)[J].Int J Oncol,2015,47(3):806.
[2] Cappell M S,Schein J R.Diagnosis and treatment of nonsteroidal anti-inflammatory drug-associated upper gastrointestinal toxicity[J].Gastroenterol Clin North Am,2000,29(1):97.
[3] Yuan J Q,Tsoi K K,Yang M,et al.Systematic review with network Meta-analysis:comparative effectiveness and safety of strategies for preventing NSAID-associated gastrointestinal toxicity[J].Aliment Pharmacol Ther,2016,43(12):1262.
[4] Ko I G,Kim S E,Jin J J,et al.Combination therapy with polydeoxyribonucleotide and proton pump inhibitor enhances therapeutic effectiveness for gastric ulcer in rats[J].Life Sci,2018,203:12.
[5] 王海燕,覃慧林,张永峰,等.木瓜总三萜对佐剂性关节炎大鼠关节滑膜组织中Akt,NF-κB和促炎因子的表达影响[J].中国实验方剂学杂志,2017,23(5):141.
[6] 石孟琼,覃慧林,张永峰,等.木瓜总三萜对脂多糖诱导下RAW264.7细胞炎症模型细胞因子的影响[J].中药药理与临床,2016,32(3):76.
[7] 贺海波,张永峰,李小妹,等.木瓜三萜对吲哚美辛致胃黏膜损伤小鼠胃酸分泌及胃黏膜屏障的影响[J].生物资源,2017,39(3):211.
[8] 熊兴军,李小妹,何毓敏,等.木瓜总三萜对DSS诱导溃疡性结肠炎小鼠PPARγ/SIRT1/NF-κB p65信号通路及黏膜屏障的影响[J].中国中药杂志,2018,43(21):179.
[9] 覃慧林,张永峰,贺海波,等.木瓜总三萜抗吲哚美辛诱导RGM-1细胞凋亡的作用[J].中国临床药理学与治疗学,2016,21(12):1347.
[10] 贺海波,张永峰,李小妹,等.木瓜三萜片与奥美拉唑胶囊联用治疗非甾体抗炎药致上消化道出血临床研究[J].中药药理与临床,2017,33(2):209.
[11] Ganguly K,Swarnakar S.Chronic gastric ulceration causes matrix metalloproteinases-9 and -3 augmentation:alleviation by melatonin[J].Biochimie,2012,94(12):2687.
[12] Carvalho N S,Silva M M,Silva R O,et al.Protective effects of simvastatin against alendronate-induced gastric mucosal injury in rats[J].Dig Dis Sci,2016,61(2):400.
[13] Laine L,Weinstein W M.Histology of alcoholic hemorrhagic "gastritis":a prospective evaluation[J].Gastroenterology,1988,94(6):1254.
[14] Sostres C,Gargallo C J,Lanas A.Nonsteroidal anti-inflammatory drugs and upper and lower gastrointestinal mucosal damage[J].Arthritis Res Ther,2013,doi:10.1186/ar4175.
[15] Takeuchi K.Pathogenesis of NSAID-induced gastric damage:importance of cyclooxygenase inhibition and gastric hypermotility[J].World J Gastroenterol,2012,18(18):2147.
[16] Cheng Y T,Lu C C,Yen G C.Phytochemicals enhance antioxidant enzyme expression to protect against NSAID-induced oxidative damage of the gastrointestinal mucosa[J].Mol Nutr Food Res,2017,doi:10.1002/mnfr.201600659.
[17] Rivollier A,He J P,Kole A,et al.Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon[J].J Exp Med,2012,209(1):139.
[18] 于艳,贾天柱,才谦.茅苍术及其麸炒品对胃溃疡大鼠抗炎作用的比较研究[J].中国中药杂志,2016,41(4):705.
[19] Odashima M,Otaka M,Jin M,et al.Attenuation of gastric mucosal inflammation induced by aspirin through activation of A2A adenosine receptor in rats[J].World J Gastroenterol,2006,12(4):568.
[20] Milne G R,Palmer T M.Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor[J].Scientific World J,2011,11:320.
[21] Jones M K,Kawanaka H,Baatar D,et al.Gene therapy for gastric ulcers with single local injection of naked DNA encoding VEGF and angiopoietin-1[J].Gastroenterology,2001,121(5):1040.
[22] Chatterjee A,Chatterjee S,Biswas A,et al.Gallic acid enriched fraction of Phyllanthus emblica potentiates indomethacin-induced gastric ulcer healing via e-NOS-dependent pathway[J].Evid Based Complement Alternat Med,2012,doi:10.1155/2012/487380.
[23] Magierowski M,Magierowska K,Hubalewska-Mazgaj M,et al.Carbon monoxide released from its pharmacological donor,tricarbonyldichlororuthenium (Ⅱ) dimer,accelerates the healing of pre-existing gastric ulcers[J].Br J Pharmacol,2017,174(20):3654.
[24] Wang B,Zhao H Y,Zhou L,et al.Effect of Kuiyangping on expressions of EGF and EGFR mRNA in gastric mucosa in rats with experimental gastric ulcer[J].J Beijing Univ Med,2008,31(11):759.
[25] Hoffmann W.TFF2,a MUC6-binding lectin stabilizing the gastric mucus barrier and more (review)[J].Int J Oncol,2015,47(3):806.