黄芩汤调控Nrf2通路对溃疡性结肠炎大鼠氧化应激作用的影响
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摘要
研究黄芩汤对溃疡性结肠炎大鼠的氧化应激作用的影响,探讨其抗氧化作用机制。经中国中医科学院中药研究所伦理委员会审议同意后,运用2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic, TNBS)联合乙醇造模法建立大鼠溃疡性结肠炎(ulcerative colitis, UC)模型,将SD大鼠按体重随机分为正常组、模型组、柳氮磺砒啶组(salazosulfapyridine, SASP, 0.5 g·kg~(-1))、黄芩汤高、中、低剂量组(20、10、5 g·kg~(-1))。连续给药5天后,取血清以及结肠组织,生化法检测大鼠血清中过氧化氢酶(catalase, CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-px)、髓过氧化物酶(myeloperoxidase, MPO)、超氧化物歧化酶(superoxide dismutase, SOD)的活性, ELISA法测定大鼠血清中过氧化脂(lipid peroxide, LPO)的浓度,免疫组织化学法观察核因子E_2相关因子Nrf2 (nuclear factor erythroid-2-related factor 2)的阳性表达,利用Western blot技术检测Nrf2下游抗氧化酶血红素氧化酶1 (heme oxygenase, HO-1)、醌氧化还原酶1 (NAD[P]H:quinone oxidoreductase 1, NQO-1)的蛋白表达。与正常组相比,模型组血清SOD、CAT、GSH-px活性均有显著性下降,而模型组血清MPO活性有显著性增加(P<0.05或P<0.01),与模型组相比,黄芩汤高、中剂量组中CAT的活性增加有显著性差异(P<0.05或P<0.01),黄芩汤高、中、低剂量组中GSH-px活性均显著增加(P<0.05);与正常组相比,模型组血清中LPO的含量显著升高(P<0.01),而与模型组相比,黄芩汤高、中剂量组均能显著降低LPO的含量(P<0.05);免疫组化检测发现,阳性药柳氮磺胺吡啶组与黄芩汤高剂量组Nrf2表达有统计学意义(P<0.05); Western blot结果显示,与模型组相比,黄芩汤各给药组与柳氮磺胺吡啶组均能够增加组织内HO-1和NQO1的表达,并且呈现一定的剂量依赖关系。黄芩汤具有显著的抗氧化应激作用,能明显改善UC大鼠的体征、精神状况及排便情况,其对于UC的治疗作用机制可能与激活Nrf2通路,增加HO-1、NQO-1等Ⅱ相代谢酶的表达,降低血清中LPO、MPO的含量,增强SOD、CAT、GSH-px的活性有关。
This study aimed to investigate the effect of Huangqin Tang(HQT) on oxidative stress associated with ulcerative colitis in rats, and to explore its antioxidant mechanism. After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, the rats were given 2,4,6-trini‐trobenzenesulfonic(TNBS)/ethanol mixture to induce the ulcerative colitis(UC), and were randomly divided into normal group, model group, the salazosulfapyridine(SASP) group, and high, middle or low dose(20, 10, 5 g·kg~(-1))of HQT groups. After 5 days of treatment, the activity of catalase(CAT) from micrococcus lysodeikticus, glutathione peroxidase(GSH-px), myeloperoxidase(MPO), superoxide dismutase(SOD) were detected by biochemical assays. The levels of lipid peroxide(LPO) were detected by ELISA. The positive protein expression of nuclear factor erythroid-2-related factor 2(Nrf2) were detected by immunohistochemistry method and the downstream antioxidant enzymes of Nrf2 were determined by Western blot analyses. The levels of SOD, CAT and GSH-px activities in the normal group were significantly higher than the model group, while the serum MPO activity in the model group was obviously increased(P<0.05 or P<0.01). Compared with the model group, there was a significant difference in the activity of CAT in the high and middle dose groups of HQT(P<0.05 or P<0.01), the activity of GSH-px in the high, middle and low dose groups of HQT were apparently higher than the model group(P<0.05); The serum levels of LPO in the model group were significantly higher than those in the normal group(P<0.05), while the up-regulating effects on LPO were reversed by the high and middle dose groups of HQT(P<0.05). The expression of Nrf2 in the high-dose group of HQT and SASP group was statistically significant(P<0.05). The results of Western blot showed that compared with the model group, each of the HQT and SASP group could increase the heme oxygenase(HO-1) and NAD[P]H: quinone oxidoreductase 1(NQO-1) expression in a dose-dependence manner. HQT has significant anti-oxidative stress and obviously improves the signs, mental status and defecation of UC rats. The mechanism of action for HQT maybe related to activate the Nrf2 pathway and increase the expression of Ⅱ phase metabolic enzymes such as HO-1 and NQO-1, reduce the content of LPO and MPO in serum and enhance the activity of SOD, CAT and GSH-px.
This study aimed to investigate the effect of Huangqin Tang(HQT) on oxidative stress associated with ulcerative colitis in rats, and to explore its antioxidant mechanism. After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, the rats were given 2,4,6-trini‐trobenzenesulfonic(TNBS)/ethanol mixture to induce the ulcerative colitis(UC), and were randomly divided into normal group, model group, the salazosulfapyridine(SASP) group, and high, middle or low dose(20, 10, 5 g·kg~(-1))of HQT groups. After 5 days of treatment, the activity of catalase(CAT) from micrococcus lysodeikticus, glutathione peroxidase(GSH-px), myeloperoxidase(MPO), superoxide dismutase(SOD) were detected by biochemical assays. The levels of lipid peroxide(LPO) were detected by ELISA. The positive protein expression of nuclear factor erythroid-2-related factor 2(Nrf2) were detected by immunohistochemistry method and the downstream antioxidant enzymes of Nrf2 were determined by Western blot analyses. The levels of SOD, CAT and GSH-px activities in the normal group were significantly higher than the model group, while the serum MPO activity in the model group was obviously increased(P<0.05 or P<0.01). Compared with the model group, there was a significant difference in the activity of CAT in the high and middle dose groups of HQT(P<0.05 or P<0.01), the activity of GSH-px in the high, middle and low dose groups of HQT were apparently higher than the model group(P<0.05); The serum levels of LPO in the model group were significantly higher than those in the normal group(P<0.05), while the up-regulating effects on LPO were reversed by the high and middle dose groups of HQT(P<0.05). The expression of Nrf2 in the high-dose group of HQT and SASP group was statistically significant(P<0.05). The results of Western blot showed that compared with the model group, each of the HQT and SASP group could increase the heme oxygenase(HO-1) and NAD[P]H: quinone oxidoreductase 1(NQO-1) expression in a dose-dependence manner. HQT has significant anti-oxidative stress and obviously improves the signs, mental status and defecation of UC rats. The mechanism of action for HQT maybe related to activate the Nrf2 pathway and increase the expression of Ⅱ phase metabolic enzymes such as HO-1 and NQO-1, reduce the content of LPO and MPO in serum and enhance the activity of SOD, CAT and GSH-px.
引文
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[14]Ayman M,Mousa O,Khalid M,et al.Commiphora molmol protects against methotrexate-induced nephrotoxicity by upregu lating Nrf2/ARE/HO-1 signaling[J].Biomed Pharmacother,2018,106:499-509.
[15]Yu J,Wu XX,Wang CX,et al.NQO1 dependent non-canonical necroptosis mediated by ROS and RIP1/RIP3 in parallel in glioma cancer cells[J].Chin J Pharmacol Toxicol(中国药理学与毒理学杂志),2018,32:326-327.
[16]Yang X,Guo T,Wang Y,et al.Therapeutic effect of ginsenoside Rd in rats with TNBS-induced recurrent ulcerative colitis[J].Arch Pharm Res,2012,35:1231-1239.
[17]Krajka-ku?niak V,Paluszczak J,Baer-Dubowska W.The Nrf2-ARE signaling pathway:an update on its regulation and possible role in cancer prevention and treatment[J].Pharmacol Rep,2017,69:393-402.
[18]Wang GW,Zhang XL,Wu QH,et al.The hepatoprotective effects of Sedum sarmentosum extract and its isolated major constituent through Nrf2 activation and NF-κB inhibition[J].Phytomedicine,2018,53:263-273.
[19]Sarlos P,Kovesdi E,Magyari L,et al.Genetic update on inflam matory factors in ulcerative colitis:review of the current litera ture[J].World J Gastrointest Pathophysiol,2014,5:304-321.
[2]Hindryckx P,Baert F,Hart A,et al.Clinical trials in ulcerative colitis:a historical perspective[J].J Crohns Colitis,2015,9:580.
[3]Ng WK,Wong SH,Ng SC.Changing epidemiological trends of inflammatory bowel disease in Asia[J].Int Res,2016,14:111-119.
[4]Jin Y,Yang J,Lin L,et al.The attenuation of scutellariae radix extract on oxidative stress for colon injury in lipopolysaccharide induced RAW264.7 cell and 2,4,6-trinitrobrnzene sulfonic acid induced ulcerative colitis rats[J].Phramacogn Mag,2016,12:153-159.
[5]Wang YW,Zhang HH,Wang YL,et al.Effect of Huangqin Tang on the regulatory NF-κB p65 signal pathway in rats with ulcerative colitis[J].Acta Pharm Sin(药学学报),2015,50:21-27.
[6]Nariaki F,Jun K.Increase in prostate stem cell antigen expression in prostatic hyperplasia induced by testosterone and 17β-estradiol in C57BL mice[J].J Steroid Biochem Mol Biol,2016,158:56-62.
[7]Morris GP,Beck PL,Herridge MS,et al.Hapten-induced model of chronic inflammation and ulceration in the rat colon[J].Gastroenterology,1989,96:795-803.
[8]Zou Y,Lin J,Li W,et al.Huangqin-tang ameliorates dextran sodium sulphate-induced colitis by regulating intestinal epithelial cell homeostasis,inflammation and immune response[J].Sci Rep,2016,6:39299.
[9]Xi JS.The Clinical Study of Huangqin Tang on Ulcerative Colitis(黄芩汤治疗溃疡性结肠炎的临床研究)[D].Beijing:Beijing University of Chinese Medicine,2006.
[10]Wang DF,Wang YL,Wang YW,et al.Effect of Huangqin Tang on the function of regulatory TLR4/MyD88 signal pathway in rats with ulcerative colitis[J].Acta Pharm Sin(药学学报),2016,51:1558-1563.
[11]Rani V,Deep U,Singh RK,et al.Oxidative stress and metabolic disorder;pathogenesis and therapeutic strategies[J].Life Sci,2016,148:183-193.
[12]Eve DJ,Steele MR,Sanberg PR,et al.Hyperbaric oxygen therapy as a potential treatment for post-traumatic stress disorder with traumatic brain injury[J].Neuropsychiatr Dis Treat,2016,12:2689-2705.
[13]Zhang X,Wang X,Sun X,et al.Differences in cognitive function of rats with traumatic brain injuries following hyperbaric oxygen therapy[J].Med Sci Monit,2016,22:2608-2615.
[14]Ayman M,Mousa O,Khalid M,et al.Commiphora molmol protects against methotrexate-induced nephrotoxicity by upregu lating Nrf2/ARE/HO-1 signaling[J].Biomed Pharmacother,2018,106:499-509.
[15]Yu J,Wu XX,Wang CX,et al.NQO1 dependent non-canonical necroptosis mediated by ROS and RIP1/RIP3 in parallel in glioma cancer cells[J].Chin J Pharmacol Toxicol(中国药理学与毒理学杂志),2018,32:326-327.
[16]Yang X,Guo T,Wang Y,et al.Therapeutic effect of ginsenoside Rd in rats with TNBS-induced recurrent ulcerative colitis[J].Arch Pharm Res,2012,35:1231-1239.
[17]Krajka-ku?niak V,Paluszczak J,Baer-Dubowska W.The Nrf2-ARE signaling pathway:an update on its regulation and possible role in cancer prevention and treatment[J].Pharmacol Rep,2017,69:393-402.
[18]Wang GW,Zhang XL,Wu QH,et al.The hepatoprotective effects of Sedum sarmentosum extract and its isolated major constituent through Nrf2 activation and NF-κB inhibition[J].Phytomedicine,2018,53:263-273.
[19]Sarlos P,Kovesdi E,Magyari L,et al.Genetic update on inflam matory factors in ulcerative colitis:review of the current litera ture[J].World J Gastrointest Pathophysiol,2014,5:304-321.