溃结康对溃疡性结肠炎小鼠结肠抗氧化作用及 Nrf2/ARE信号通路的影响
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摘要
目的:探讨溃结康对溃疡性结肠炎小鼠肠粘膜抗氧化应激作用及Nrf2/ARE信号通路的影响。方法:建立葡聚糖硫酸钠诱导小鼠实验性溃疡性结肠炎模型,实验分为正常对照组、模型组、柳氮磺胺吡啶0.45g/kg组、溃结康12.8g/kg、6.4g/kg、3.2g/kg组。造模同时灌胃给药,连续7天,第8天实验结束时,采集结肠组织,生化法检测小鼠肠粘膜中T-SOD、MDA、GSH-PX、CAT的活性;实时荧光定量PCR检测结肠NOX1、NOX2、Nrf2、Keap-1、HO-1、NQO-1mRNA表达,Western blot法检测结肠NOX1、NOX2、p-Nrf2、Keap-1、HO-1、NQO-1蛋白表达。结果:模型组小鼠结肠SOD、GSH-PX及CAT抗氧化酶活性显著降低,MDA生成显著增加,Keap1、NOX1、NOX2mRNA及蛋白表达均显著上调,Nrf2mRNA表达显著增加,II相解毒酶NQO-1、HO-1mRNA表达无明显变化。溃结康12.8g/kg、6.4g/kg组可提高SOD、GSH-PX活性,上调Nrf2mRNA、p-Nrf2蛋白、NQO-1基因及蛋白,下调NOX1、NOX2mRNA表达;溃结康12.8g/kg组还可显著提高CAT活性,降低MDA含量,上调HO-1mRNA及蛋白,下调Keap-1mRNA,NOX1、NOX2蛋白表达;溃结康6.4g/kg组NOX2蛋白表达明显降低,Keap-1mRNA及蛋白表达明显降低;溃结康3.2g/kg组NOX1 mRNA表达明显降低,而其它指标无明显变化。结论:溃结康具有一定的抗氧化应激损伤作用,可能为其治疗溃疡性结肠炎的作用机制之一。
Objective: To investigate the antioxidant effect of Kuijiekang Decoction(KJKD) and its influence on Nrf2/ARE signaling pathway of mice with ulcerative colitis. Methods:Animals were randomly divided into the control group,the model group,SASP group(0.45 g/kg)and KJKD groups(12.8 g/kg、6.4 g/kg、3.2 g/kg). The ulcerative colitis mice model was induced by dextran sodium sulfate(DSS) and all mice were administered with corresponding drugs for 7 days. After the last administration, mice were sacrificed and to collect colonic mucosa,the activities of T-SOD、MDA、GSH-PX、CAT were measured by chemical methods, mRNA and protein expressions of NOX1、NOX2、Nrf2、keap-1、HO-1、NQO-1 were detected by realtime polymerase chain reaction(RT-PCR)and western blottingResults:Compared with the control group,antioxidant enzyme activities of SOD、GSH-PX、CAT were decreased while MDA content was increased, mRNA and protein expressions of NOX1、NOX2 were increased,the level of Nrf2 mRNA was increased while Keap1 mRNA expression was decreased,the protein level of Keap1 was significantly increased. There was no obvious changes of NQO-1、HO-1 mRNA expressions,.Compared with the model group,antioxidant enzyme activities of SOD、GSH-PX were improved,the mRNA expressions of NOX1、NOX2 were decreased in KJKD groups(12.8 g/kg、6.4 g/kg). Meanwhile KJKD(12.8 g/kg) also enhanced the activity of CAT,markedly reduced the content of MDA,and up regulated the expressions of Nrf2、NQO-1 and HO-1 mRNA,down-regulated the mRNA expression of Keap-1 and protein levels of NOX1、NOX2,and enhanced the protein levels of p-Nrf2、NQO-1、HO-1.The protein level of NOX2 was significantly reduced,the gene expressions and protein levels of Nrf2 and NQO-1 were increased and Keap-1 mRNA and protein expressions were lower in KJKD group(6.4 g/kg) than those in the model group. KJKD(3.2 g/kg) down regulated the expression of NOX1 mRNA, but had no significantly effect on other index.Conclusion:KJKD can ameliorate oxidative stress injury on ulcerative colitis mice,which may be one of mechanisms in treating ulcerative colitis.
Objective: To investigate the antioxidant effect of Kuijiekang Decoction(KJKD) and its influence on Nrf2/ARE signaling pathway of mice with ulcerative colitis. Methods:Animals were randomly divided into the control group,the model group,SASP group(0.45 g/kg)and KJKD groups(12.8 g/kg、6.4 g/kg、3.2 g/kg). The ulcerative colitis mice model was induced by dextran sodium sulfate(DSS) and all mice were administered with corresponding drugs for 7 days. After the last administration, mice were sacrificed and to collect colonic mucosa,the activities of T-SOD、MDA、GSH-PX、CAT were measured by chemical methods, mRNA and protein expressions of NOX1、NOX2、Nrf2、keap-1、HO-1、NQO-1 were detected by realtime polymerase chain reaction(RT-PCR)and western blottingResults:Compared with the control group,antioxidant enzyme activities of SOD、GSH-PX、CAT were decreased while MDA content was increased, mRNA and protein expressions of NOX1、NOX2 were increased,the level of Nrf2 mRNA was increased while Keap1 mRNA expression was decreased,the protein level of Keap1 was significantly increased. There was no obvious changes of NQO-1、HO-1 mRNA expressions,.Compared with the model group,antioxidant enzyme activities of SOD、GSH-PX were improved,the mRNA expressions of NOX1、NOX2 were decreased in KJKD groups(12.8 g/kg、6.4 g/kg). Meanwhile KJKD(12.8 g/kg) also enhanced the activity of CAT,markedly reduced the content of MDA,and up regulated the expressions of Nrf2、NQO-1 and HO-1 mRNA,down-regulated the mRNA expression of Keap-1 and protein levels of NOX1、NOX2,and enhanced the protein levels of p-Nrf2、NQO-1、HO-1.The protein level of NOX2 was significantly reduced,the gene expressions and protein levels of Nrf2 and NQO-1 were increased and Keap-1 mRNA and protein expressions were lower in KJKD group(6.4 g/kg) than those in the model group. KJKD(3.2 g/kg) down regulated the expression of NOX1 mRNA, but had no significantly effect on other index.Conclusion:KJKD can ameliorate oxidative stress injury on ulcerative colitis mice,which may be one of mechanisms in treating ulcerative colitis.
引文
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[2]Tomasello G,Mazzola M,Leone A,et al.Nutrition,oxidative stress and intestinal dysbiosis:influence of diet on gut microbi-ota in inflammatory bowel disease.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2016,160(4)∶461～466.
[3]祁燕,万春平,李小丝,等.溃结康对溃疡性结肠炎小鼠肠粘膜炎症因子及肠屏障功能相关蛋白的影响.中药药理与临床,2017,33(6)∶120～124.
[4]魏丹丹,林旭红,王慧超,等.香草乙酮改善葡聚糖硫酸钠诱发溃疡性结肠炎小鼠的炎症反应与NOXs-ROS-p38MAPK信号通路的关系.生理学报,2015,67(1)∶74～82.
[5]Zhu H,Li Y R.Oxidative stress and redox signaling mecha-nisms of inflammatory bowel disease∶updated experimental and clinical evidence.Exp Biol Med,2012,237(5)∶474～480.
[6]马涛.溃疡性结肠炎组织中NOX1、NOX2表达量与肠黏膜氧化应激反应屏障功能损伤的相关性.海南医学院学报,2017,23(19)∶2638～2640.
[7]Deng W,Abliz A,Xu S,et al.Severity of pancreatitis associationed intestinal mucosal barrier injury is reduced following treatment with the NADPH oxidase inhibitor apocynin.Mol Med Rep,2016,14(4)∶3525～3534.
[8]Hayes P,Dhillon S,O Neill K,et al.Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease.Cell Moe Gastroenterol Hepatol,2015,1(5)∶489～502.
[9]Reuter S,Gupta S C,Chaturvedi M,et al.Oxidative stress inflammation and cancer∶ how are they linked.Free Radic Biol Med,2010,49(11)∶1603～1616.
[10]Jones RM,Luo L,Ardita C S,et al.Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated gener-ation of reactive oxygen species.EMBO J,2013,32(23)∶3017～3028.
[11]Dringen R.Metabolism and functions of glutathione in brain.Pro Neurobio,2000,62∶649～671.
[12]Zhou C,Li H,Yao Y,et al.Delayed remote ischemic precondi-tioning produces an additive cardioprotection to sevoflurane post-conditioning through an enhanced heme oxygenase 1 level partlyvia nuclear factor erythroid 2-related factor 2 nuclear transloca-tion.Journal of Cardiovascular Pharmacology & Therapeu-tics,2014,19(6)∶558～566.
[13]Menegon S,Columbano A,Giordano S.The Dual Roles of NRF2 in Cancer.Trends Mol Med,2016,22(7)∶578～593.
[14]Loboda A,Damulewicz M,Pyza E,et al.Role of Nrf2 /HO-1system in developmen,oxidative stress response and diseasesan evolutionarily conserved mechanis.Cell Mol Life Sci,2016,73(17)∶3221～3324.
[15]Wu KC,Cui J Y,Klaassen CD.Effect of graded Nrf2 activa-tion on phase- I and-II drug metabolizing enzymes and trans-portersin mouse liver.PLoS One,2012,7(7)∶e39006.