葡萄籽原花青素治疗大鼠复发性溃疡性结肠炎的作用及其机理研究
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摘要
目的：用三硝基苯磺酸(TNBS)-50%乙醇溶液二次致炎法建立大鼠复发结肠炎模型,观察葡萄籽原花青素(GSPE)对其治疗作用,并初步探讨其作用机理。
方法：雄性Wistar大鼠分为6组：正常对照组、急性结肠炎组、结肠炎自发性愈合组、复发性结肠炎模型对照组、复发性结肠炎模型加柳氮磺吡啶(SASP)治疗组(SASP治疗组)、复发性结肠炎模型加GSPE治疗组(GSPE治疗组)。大鼠直肠给予80mg/kg TNBS-50%乙醇溶液建立UC模型,观察14d后,用30mg/kg TNBS-50%乙醇溶液诱导复发性结肠炎。用200mg/kgGSPE灌胃对其进行治疗,并以500mg/kgSASP作为阳性对照,给药7d后处死大鼠,取结肠标本评价炎症程度并检测各项指标,结肠炎症的评价指标包括大体形态与组织病理学变化、组织与血清中髓过氧化物酶(MPO)活力；生化法检测组织与血清中丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)含量、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)及诱导型一氧化氮合酶(iNOS)活力。
结果：①与正常对照组、急性结肠炎组及结肠炎自发性愈合组比较,复发性结肠炎模型对照组大鼠结肠湿重指数明显升高(P＜0.05)；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠结肠湿重指数明显降低(P＜0.05)。
②大体观察显示,复发性结肠炎模型对照组大鼠结肠粘膜溃疡面较大、部分结肠粘膜充血、水肿；GSPE治疗组大鼠结肠粘膜溃疡面积缩小,肠壁内表面光滑,大体形态学损伤评分下降(P＜0.01)；组织学检查结果显示,与复发性结肠模型对照组比较,GSPE治疗组大鼠结肠组织病理学损伤评分明显下降(P＜0.05)。
③与正常对照组比较,复发性结肠炎模型对照组大鼠血清与结肠组织中MPO和iNOS活力、MDA和NO含量明显升高；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠血清与结肠组织中MPO活力、MDA和NO含量明显降低(P＜0.05或P＜0.01)；GSPE治疗组大鼠血清中iNOS活力与复发性结肠炎模型对照组比较也明显降低(P＜0.05)。
④与正常对照组比较,复发性结肠炎模型对照组大鼠血清与结肠组织中SOD和GSH-Px活力及GSH含量明显降低(P＜0.05或P＜0.01)；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠血清与结肠组织中GSH-Px、SOD活力及GSH含量明显升高(P＜0.05或P＜0.01)。
⑤GSPE治疗组与SASP治疗组比较,除大鼠血清中MPO活力在GSPE治疗组中降低更明显外(P＜0.05),其余指标均无明显差异。
结论：(1)采用TNBS/乙醇在首次致炎两周后进行第2次致炎,可以模拟UC缓解-复发交替出现的特点,是较理想的人类UC模型,可以应用于UC缓解期复发的研究。(2)GSPE对大鼠溃疡性结肠炎复发阶段有明显的治疗作用,其作用机制与抗氧化、减轻脂质过氧化损伤、清除自由基、抑制iNOS活力减少NO过量生成有关。
目的：考察葡萄籽原花青素(GSPE)治疗三硝基苯磺酸(TNBS)-50%乙醇溶液诱导大鼠复发性溃疡性结肠炎(UC)的量效关系,并观察GSPE对TNBS诱导的复发性UC大鼠结肠组织中MAPK与NF-κB信号转导系统的调节作用,以深入探讨其治疗作用机理。
方法：用TNBS-50%乙醇二次致炎法建立大鼠UC复发的模型,将雄性Wistar大鼠随机分为正常对照组、模型对照组、柳氮磺吡啶(SASP)组和GSPE低(100mg/kg)、中(200mg/kg)、高(400mg/kg)剂量组,于第二次致炎24h后灌胃对其进行治疗,连续给药7天后处死大鼠,取结肠标本评价炎症程度并检测各项指标,结肠炎症的评价指标包括结肠湿重指数(结肠湿重/结肠长,mg/cm)、大体形态与组织病理学变化、组织与血清中髓过氧化物酶(MPO)活力；生化法检测组织与血清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和一氧化氮合酶(iNOS)活力、及丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)含量。取结肠标本,制备组织匀浆后离心取上清液用酶联免疫ELISA法检测结肠组织中细胞外信号调节蛋白激酶(ERK), c-Jun氨基端激酶(JNK),p38丝裂原活化蛋白激酶(p38MAPK)、c-Jun蛋白(c-Jun)、c-Fos蛋白(c-Fos)、激活蛋白1(AP-1)及核因子-κB(NF-κB)的表达水平。
结果：①大体观察显示,模型对照组大鼠结肠粘膜溃疡面积较大、部分结肠粘膜充血,与模型对照组比较,GSPE各剂量组大鼠结肠粘膜溃疡面积缩小,肠壁内表面光滑,大体形态学损伤评分明显下降(P＜0.05或P＜0.01)；组织学检查结果显示,与模型对照组比较,GSPE各剂量组大鼠结肠组织病理学损伤评分明显下降(P＜0.05)。
②与正常对照组比较,模型对照组大鼠体重明显下降,结肠湿重指数及血清与结肠组织中的MPO活性均明显增加(P＜0.05或P＜0.001)；与模型对照组比较,GSPE各剂量组大鼠体重下降程度较轻(P＜0.05),结肠湿重指数及血清与结肠组织中的MPO活性均明显降低(P＜0.05,P＜0.01或P＜0.001),随GSPE剂量的增加,大鼠血清中MPO活力逐渐降低。
③与正常对照组比较,模型对照组大鼠血清与结肠组织中的MDA含量明显升高而SOD活力明显降低(P＜0.05,P＜0.01或P＜0.001)；与模型对照组比较,GSPE低、中、高剂量组大鼠结肠组织和血清中MDA含量均明显降低而SOD活力均明显升高(P＜0.05,P＜0.01或P＜0.001)。
④与正常对照组比较,模型对照组大鼠血清与结肠组织中的GSH含量和GSH-Px活力均明显降低(P＜0.01或P＜0.001)；与模型对照组比较,GSPE各剂量组大鼠结肠组织和血清中GSH含量和GSH-Px活力(除血清中GSH-Px活力)均明显升高(P＜0.05,P＜0.01或P＜0.001)。
⑤与正常对照组比较,模型对照组大鼠血清与结肠组织中的NO含量和iNOS活力均明显升高(P＜0.05或P＜0.01)；与模型对照组比较,GSPE各剂量组大鼠结肠组织和血清中NO含量和iNOS活力均明显降低(P＜0.05,P＜0.01或P＜0.001)。
⑥与正常对照组比较,模型对照组大鼠结肠组织中ERK, JNK和p38MAPK含量显著升高(P＜0.05或P＜0.01)；GSPE低、中、高剂量组大鼠结肠组织中ERK,JNK和p38MAPK含量与模型对照组比较明显降低(P＜0.01或P＜0.001),其中,GSPE各剂量组大鼠结肠组织的p38MAPK含量随着GSPE治疗剂量的增加而逐渐降低(呈剂量依赖性)。
⑦与正常对照组比较,模型对照组大鼠结肠组织中c-Jun与c-Fos含量显著升高(P＜0.05或P＜0.01)；GSPE低、中剂量组大鼠结肠组织c-Jun与c-Fos含量与模型对照组比较明显降低(P＜0.05或P＜0.01),而柳氮磺吡啶组和GSPE高剂量组大鼠肠组织中c-Jun与c-Fos含量与模型对照组比较无明显差异(P＞0.05)。
⑧与正常对照组比较,模型对照组大鼠结肠组织中AP-1与NF-κB含量显著升高(P＜0.05或P＜0.01)；GSPE低、中、高剂量组大鼠结肠组织中AP-1及NF-κB含量与模型对照组比较均明显降低(P＜0.05,P＜0.01或P＜0.001),其中,GSPE各治疗组大鼠结肠组织的AP-1含量随着治疗剂量的增加而逐渐降低(呈剂量依赖性),与柳氮磺吡啶组比较,GSPE高剂量组大鼠结肠组织中AP-1含量降低更明显(P＜0.05)。
结论：(1)GSPE在治疗TNBS/乙醇二次致炎诱导的大鼠复发性结肠炎时,除大体损伤评分、结肠组织与血清MPO活力、组织GSH-Px活力和NO含量及血清GSH含量变化呈剂量依赖性,其它指标：如体重变化、结肠湿重指数、结肠组织与血清MDA含量及SOD活力等均未表现出明显的剂量依赖性,这可能是由于低剂量或中剂量的GSPE已经表现了明显的治疗作用,其治疗效果没有因剂量的增加而明显的增强。
(2) MAPK信号传导通路系统与NF-κB信号转导通路均与UC发病关系密切,在UC的发生、发展中占有重要地位,GSPE可能是通过抑制MAPK与NF-κB信号转导通路对大鼠复发性UC起治疗作用。
Aim:The aim of the present study was to investigate the therapeutic effect and mechanism of proanthocyanidins from grape seeds (GSPE) in the treatment of recurrent ulcerative colitis (UC) in rats.
Methods:Rats were randomized into six groups:normal control group, acute colitis group, spontaneous healing colitis group, recurrent colitis model control group, SASP group and GSPE group. To induce recurrent colitis, rats were instilled with 2,4, 6-trinitrobenzenesulfonic acid (TNBS) (80 mg/kg) into the colon through the cannula in the first induced phase, and then the rats were second instilled with TNBS (30 mg/kg) into the colon on the 16th day after the first induction UC. Rats were intragastrically administered GSPE (200 mg/kg) per day for 7 days after twice-induction of colitis by TNBS. Sulfasalazine (SASP) at 500 mg/kg was used as a positive control drug. Rats were killed 7 days after GSPE treatment. The colonic injury and inflammation were assessed by macroscopic and macroscopic damage scores, colon weight/length ratio (mg/cm) and myeloperoxidase (MPO) activity. Then, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) activities, and the levels of malonyldialdehyde (MDA), glutathione (GSH) and nitric oxide (NO) in serum and colonic tissues were measured.
Results:Compared with recurrent UC group, GSPE treatment facilitated recovery of pathologic changes in the colon after induction of recurrent colitis, as demonstrated by reduced colonic weight/length ratio and macroscopic and microscopic damage scores (P<0.05 or P<0.01). The MPO and iNOS activities with MDA and NO levels in serum and colon tissues of colitis rats were significantly decreased in GSPE group compared with the recurrent UC group (P<0.05 or P<0.01). In addition, GSPE treatment was associated with notably increased SOD, GSH-Px activities and GSH levels of colon tissues and serum of rats. No statistical difference was observed in any biochemical analysis between the SASP and GSPE treatment group, except for the MPO activity in serum of the GSPE group that were significantly lower than the SASP group.
Conlusions:GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response, inhibiting inflammatory cell infiltration and antioxidation damage, promoting damaged tissue repair to improve colonic oxidative stress, and inhibiting of colonic iNOS activity to reduce the production of NO.
Aim:To elucidate the molecular mechanisms involved in the therapeutic effects of proanthocyanidins from grape seeds (GSPE), we explore whether GSPE regulates the inflammatory response of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced recurrent colitis in rats at the levels of MAPK and NF-κB signal transduction pathways.
Method:Rats were intragastrically administered different doses of GSPE (100,200, and 400 mg/kg) per day for 7 days after recurrent colitis was twice-induced by intracolonic injection of TNBS dissolved in 50% ethanol. Sulfasalazine (SASP) at 500 mg/kg was used as a positive control drug. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities, malonyldialdehyde (MDA) and nitric oxide (NO) levels and a histological study of the lesions. We determined GSH production as well as the GSH-Px and SOD activities by biochemical methods. The expression levels of extracellular signal regulated kinase (ERK), c-Jun NH2-terminal terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun and c-Fos, nuclear transcription factor activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) in the colon tissues were all measured by enzyme-linked immunosorbent assay (ELISA) methods.
Result:GSPE treatment was associated with a remarkable amelioration in macroscopic and microscopic colitis scores, decreased colonic and serum MPO and iNOS activity as well as MDA and NO levels in TNBS-induced recurrent colitis. The GSH-Px, SOD activity and GSH levels in colonic and serum of rats treated with GSPE were significantly increased as compared to recurrent control group. GSPE reduced the expression levels of ERK, JNK, p38MAPK, c-Jun, c-Fos, AP-1, and NF-κB in the colon mucosa.
Conclusion:(1) GSPE showed significantly dose-dependent effects on some indices of TNBS-induced recurrent colitis in rats, such as MPO and GSH-Px activity, NO and GSH levels in colonic tissues. GSPE did not show dose-dependent effects, however, on colonic MDA levels, weight/length ratio, macroscopic damage scores, macroscopic damage scores, and serum GSH-Px activity et al. It may by that the low or medium dose of GSPE already shows maximal therapeutic effect. (2) GSPE exerted a protective effect on recurrent colitis in rats by inhibition of MAPKs and NF-κB signal transduction pathways, modificating the inflammatory response and promoting damaged tissue repair to improve colonic oxidative stress.
方法：雄性Wistar大鼠分为6组：正常对照组、急性结肠炎组、结肠炎自发性愈合组、复发性结肠炎模型对照组、复发性结肠炎模型加柳氮磺吡啶(SASP)治疗组(SASP治疗组)、复发性结肠炎模型加GSPE治疗组(GSPE治疗组)。大鼠直肠给予80mg/kg TNBS-50%乙醇溶液建立UC模型,观察14d后,用30mg/kg TNBS-50%乙醇溶液诱导复发性结肠炎。用200mg/kgGSPE灌胃对其进行治疗,并以500mg/kgSASP作为阳性对照,给药7d后处死大鼠,取结肠标本评价炎症程度并检测各项指标,结肠炎症的评价指标包括大体形态与组织病理学变化、组织与血清中髓过氧化物酶(MPO)活力；生化法检测组织与血清中丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)含量、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)及诱导型一氧化氮合酶(iNOS)活力。
结果：①与正常对照组、急性结肠炎组及结肠炎自发性愈合组比较,复发性结肠炎模型对照组大鼠结肠湿重指数明显升高(P＜0.05)；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠结肠湿重指数明显降低(P＜0.05)。
②大体观察显示,复发性结肠炎模型对照组大鼠结肠粘膜溃疡面较大、部分结肠粘膜充血、水肿；GSPE治疗组大鼠结肠粘膜溃疡面积缩小,肠壁内表面光滑,大体形态学损伤评分下降(P＜0.01)；组织学检查结果显示,与复发性结肠模型对照组比较,GSPE治疗组大鼠结肠组织病理学损伤评分明显下降(P＜0.05)。
③与正常对照组比较,复发性结肠炎模型对照组大鼠血清与结肠组织中MPO和iNOS活力、MDA和NO含量明显升高；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠血清与结肠组织中MPO活力、MDA和NO含量明显降低(P＜0.05或P＜0.01)；GSPE治疗组大鼠血清中iNOS活力与复发性结肠炎模型对照组比较也明显降低(P＜0.05)。
④与正常对照组比较,复发性结肠炎模型对照组大鼠血清与结肠组织中SOD和GSH-Px活力及GSH含量明显降低(P＜0.05或P＜0.01)；与复发性结肠炎模型对照组比较,GSPE治疗组大鼠血清与结肠组织中GSH-Px、SOD活力及GSH含量明显升高(P＜0.05或P＜0.01)。
⑤GSPE治疗组与SASP治疗组比较,除大鼠血清中MPO活力在GSPE治疗组中降低更明显外(P＜0.05),其余指标均无明显差异。
结论：(1)采用TNBS/乙醇在首次致炎两周后进行第2次致炎,可以模拟UC缓解-复发交替出现的特点,是较理想的人类UC模型,可以应用于UC缓解期复发的研究。(2)GSPE对大鼠溃疡性结肠炎复发阶段有明显的治疗作用,其作用机制与抗氧化、减轻脂质过氧化损伤、清除自由基、抑制iNOS活力减少NO过量生成有关。
目的：考察葡萄籽原花青素(GSPE)治疗三硝基苯磺酸(TNBS)-50%乙醇溶液诱导大鼠复发性溃疡性结肠炎(UC)的量效关系,并观察GSPE对TNBS诱导的复发性UC大鼠结肠组织中MAPK与NF-κB信号转导系统的调节作用,以深入探讨其治疗作用机理。
方法：用TNBS-50%乙醇二次致炎法建立大鼠UC复发的模型,将雄性Wistar大鼠随机分为正常对照组、模型对照组、柳氮磺吡啶(SASP)组和GSPE低(100mg/kg)、中(200mg/kg)、高(400mg/kg)剂量组,于第二次致炎24h后灌胃对其进行治疗,连续给药7天后处死大鼠,取结肠标本评价炎症程度并检测各项指标,结肠炎症的评价指标包括结肠湿重指数(结肠湿重/结肠长,mg/cm)、大体形态与组织病理学变化、组织与血清中髓过氧化物酶(MPO)活力；生化法检测组织与血清中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和一氧化氮合酶(iNOS)活力、及丙二醛(MDA)、谷胱甘肽(GSH)和一氧化氮(NO)含量。取结肠标本,制备组织匀浆后离心取上清液用酶联免疫ELISA法检测结肠组织中细胞外信号调节蛋白激酶(ERK), c-Jun氨基端激酶(JNK),p38丝裂原活化蛋白激酶(p38MAPK)、c-Jun蛋白(c-Jun)、c-Fos蛋白(c-Fos)、激活蛋白1(AP-1)及核因子-κB(NF-κB)的表达水平。
结果：①大体观察显示,模型对照组大鼠结肠粘膜溃疡面积较大、部分结肠粘膜充血,与模型对照组比较,GSPE各剂量组大鼠结肠粘膜溃疡面积缩小,肠壁内表面光滑,大体形态学损伤评分明显下降(P＜0.05或P＜0.01)；组织学检查结果显示,与模型对照组比较,GSPE各剂量组大鼠结肠组织病理学损伤评分明显下降(P＜0.05)。
②与正常对照组比较,模型对照组大鼠体重明显下降,结肠湿重指数及血清与结肠组织中的MPO活性均明显增加(P＜0.05或P＜0.001)；与模型对照组比较,GSPE各剂量组大鼠体重下降程度较轻(P＜0.05),结肠湿重指数及血清与结肠组织中的MPO活性均明显降低(P＜0.05,P＜0.01或P＜0.001),随GSPE剂量的增加,大鼠血清中MPO活力逐渐降低。
③与正常对照组比较,模型对照组大鼠血清与结肠组织中的MDA含量明显升高而SOD活力明显降低(P＜0.05,P＜0.01或P＜0.001)；与模型对照组比较,GSPE低、中、高剂量组大鼠结肠组织和血清中MDA含量均明显降低而SOD活力均明显升高(P＜0.05,P＜0.01或P＜0.001)。
④与正常对照组比较,模型对照组大鼠血清与结肠组织中的GSH含量和GSH-Px活力均明显降低(P＜0.01或P＜0.001)；与模型对照组比较,GSPE各剂量组大鼠结肠组织和血清中GSH含量和GSH-Px活力(除血清中GSH-Px活力)均明显升高(P＜0.05,P＜0.01或P＜0.001)。
⑤与正常对照组比较,模型对照组大鼠血清与结肠组织中的NO含量和iNOS活力均明显升高(P＜0.05或P＜0.01)；与模型对照组比较,GSPE各剂量组大鼠结肠组织和血清中NO含量和iNOS活力均明显降低(P＜0.05,P＜0.01或P＜0.001)。
⑥与正常对照组比较,模型对照组大鼠结肠组织中ERK, JNK和p38MAPK含量显著升高(P＜0.05或P＜0.01)；GSPE低、中、高剂量组大鼠结肠组织中ERK,JNK和p38MAPK含量与模型对照组比较明显降低(P＜0.01或P＜0.001),其中,GSPE各剂量组大鼠结肠组织的p38MAPK含量随着GSPE治疗剂量的增加而逐渐降低(呈剂量依赖性)。
⑦与正常对照组比较,模型对照组大鼠结肠组织中c-Jun与c-Fos含量显著升高(P＜0.05或P＜0.01)；GSPE低、中剂量组大鼠结肠组织c-Jun与c-Fos含量与模型对照组比较明显降低(P＜0.05或P＜0.01),而柳氮磺吡啶组和GSPE高剂量组大鼠肠组织中c-Jun与c-Fos含量与模型对照组比较无明显差异(P＞0.05)。
⑧与正常对照组比较,模型对照组大鼠结肠组织中AP-1与NF-κB含量显著升高(P＜0.05或P＜0.01)；GSPE低、中、高剂量组大鼠结肠组织中AP-1及NF-κB含量与模型对照组比较均明显降低(P＜0.05,P＜0.01或P＜0.001),其中,GSPE各治疗组大鼠结肠组织的AP-1含量随着治疗剂量的增加而逐渐降低(呈剂量依赖性),与柳氮磺吡啶组比较,GSPE高剂量组大鼠结肠组织中AP-1含量降低更明显(P＜0.05)。
结论：(1)GSPE在治疗TNBS/乙醇二次致炎诱导的大鼠复发性结肠炎时,除大体损伤评分、结肠组织与血清MPO活力、组织GSH-Px活力和NO含量及血清GSH含量变化呈剂量依赖性,其它指标：如体重变化、结肠湿重指数、结肠组织与血清MDA含量及SOD活力等均未表现出明显的剂量依赖性,这可能是由于低剂量或中剂量的GSPE已经表现了明显的治疗作用,其治疗效果没有因剂量的增加而明显的增强。
(2) MAPK信号传导通路系统与NF-κB信号转导通路均与UC发病关系密切,在UC的发生、发展中占有重要地位,GSPE可能是通过抑制MAPK与NF-κB信号转导通路对大鼠复发性UC起治疗作用。
Aim:The aim of the present study was to investigate the therapeutic effect and mechanism of proanthocyanidins from grape seeds (GSPE) in the treatment of recurrent ulcerative colitis (UC) in rats.
Methods:Rats were randomized into six groups:normal control group, acute colitis group, spontaneous healing colitis group, recurrent colitis model control group, SASP group and GSPE group. To induce recurrent colitis, rats were instilled with 2,4, 6-trinitrobenzenesulfonic acid (TNBS) (80 mg/kg) into the colon through the cannula in the first induced phase, and then the rats were second instilled with TNBS (30 mg/kg) into the colon on the 16th day after the first induction UC. Rats were intragastrically administered GSPE (200 mg/kg) per day for 7 days after twice-induction of colitis by TNBS. Sulfasalazine (SASP) at 500 mg/kg was used as a positive control drug. Rats were killed 7 days after GSPE treatment. The colonic injury and inflammation were assessed by macroscopic and macroscopic damage scores, colon weight/length ratio (mg/cm) and myeloperoxidase (MPO) activity. Then, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) activities, and the levels of malonyldialdehyde (MDA), glutathione (GSH) and nitric oxide (NO) in serum and colonic tissues were measured.
Results:Compared with recurrent UC group, GSPE treatment facilitated recovery of pathologic changes in the colon after induction of recurrent colitis, as demonstrated by reduced colonic weight/length ratio and macroscopic and microscopic damage scores (P<0.05 or P<0.01). The MPO and iNOS activities with MDA and NO levels in serum and colon tissues of colitis rats were significantly decreased in GSPE group compared with the recurrent UC group (P<0.05 or P<0.01). In addition, GSPE treatment was associated with notably increased SOD, GSH-Px activities and GSH levels of colon tissues and serum of rats. No statistical difference was observed in any biochemical analysis between the SASP and GSPE treatment group, except for the MPO activity in serum of the GSPE group that were significantly lower than the SASP group.
Conlusions:GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response, inhibiting inflammatory cell infiltration and antioxidation damage, promoting damaged tissue repair to improve colonic oxidative stress, and inhibiting of colonic iNOS activity to reduce the production of NO.
Aim:To elucidate the molecular mechanisms involved in the therapeutic effects of proanthocyanidins from grape seeds (GSPE), we explore whether GSPE regulates the inflammatory response of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced recurrent colitis in rats at the levels of MAPK and NF-κB signal transduction pathways.
Method:Rats were intragastrically administered different doses of GSPE (100,200, and 400 mg/kg) per day for 7 days after recurrent colitis was twice-induced by intracolonic injection of TNBS dissolved in 50% ethanol. Sulfasalazine (SASP) at 500 mg/kg was used as a positive control drug. The inflammatory response was assessed by gross appearance, myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities, malonyldialdehyde (MDA) and nitric oxide (NO) levels and a histological study of the lesions. We determined GSH production as well as the GSH-Px and SOD activities by biochemical methods. The expression levels of extracellular signal regulated kinase (ERK), c-Jun NH2-terminal terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun and c-Fos, nuclear transcription factor activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) in the colon tissues were all measured by enzyme-linked immunosorbent assay (ELISA) methods.
Result:GSPE treatment was associated with a remarkable amelioration in macroscopic and microscopic colitis scores, decreased colonic and serum MPO and iNOS activity as well as MDA and NO levels in TNBS-induced recurrent colitis. The GSH-Px, SOD activity and GSH levels in colonic and serum of rats treated with GSPE were significantly increased as compared to recurrent control group. GSPE reduced the expression levels of ERK, JNK, p38MAPK, c-Jun, c-Fos, AP-1, and NF-κB in the colon mucosa.
Conclusion:(1) GSPE showed significantly dose-dependent effects on some indices of TNBS-induced recurrent colitis in rats, such as MPO and GSH-Px activity, NO and GSH levels in colonic tissues. GSPE did not show dose-dependent effects, however, on colonic MDA levels, weight/length ratio, macroscopic damage scores, macroscopic damage scores, and serum GSH-Px activity et al. It may by that the low or medium dose of GSPE already shows maximal therapeutic effect. (2) GSPE exerted a protective effect on recurrent colitis in rats by inhibition of MAPKs and NF-κB signal transduction pathways, modificating the inflammatory response and promoting damaged tissue repair to improve colonic oxidative stress.
引文
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