芍药汤对溃疡性结肠炎大鼠TLR4/NF-κB通路的影响
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摘要
探讨芍药汤对溃疡性结肠炎(UC)大鼠TLR4/NF-κB信号通路的影响。将56只大鼠随机分成正常组(双蒸水)、模型组(双蒸水)、阳性组(美沙拉嗪,10 mL·kg~(-1))和芍药汤高、中、低剂量组(2.4,1.2,0.6 g·mL~(-1))。采用2,4-二硝基氯苯加(DNCB)醋酸/乙醇复合法建立UC大鼠模型后,连续灌胃给药7 d,每日2次。治疗周期结束后,次日观察各组大鼠一般表现及进行疾病活动指数评分;处死,取结肠,测长度及大体形态评分(DAI);HE染色观察结肠组织病理形态学变化;紫外分光光度法检测血液和结肠组织髓过氧化物酶(MPO)的含量;ELISA法测定血液和结肠组织P-选择素、巨噬细胞移动抑制因子(MIF)和血栓素B_2(TXB_2)的含量;免疫组化法和Western blot法检测结肠组织中TLR4和NF-κB蛋白表达。结果显示,与模型组比较,不同剂量的芍药汤能改善UC大鼠的一般表现,其中芍药汤高剂量组DAI评分显著降低(P<0.001);芍药汤高、中剂量组能显著抑制UC大鼠结肠长度缩短和病理损伤,且能显著降低血清和结肠组织中MPO,P-选择素,MIF,TXB_2的含量(P<0.001);免疫组化及Western blot法结果均显示芍药汤高剂量组结肠组织中TLR4和NF-κB蛋白表达量较模型组显著降低(P<0.001)。上述结果表明,芍药汤对溃疡性结肠炎有保护和修复作用,其机制可能与阻断TLR4/NF-κB通路,抑制MPO,P-选择素,MIF和TXB_2的表达有关。
The aim of this paper was to investigate the effect of Shaoyao Tang on ulcerative colitis(UC) in rats via regulation of TLR4/NF-κB signal pathway. A total of 56 Wistar rats were randomly divided into 6 groups: normal control group(double distilled water), model group(double distilled water), mesalazine group(10 mL·kg~(-1)), high dose, middle dose and low dose Shaoyao Tang groups(2.4, 1.2, and 0.6 g·mL~(-1)). After UC rat models were established by 2, 4-dinitrochlorobenzene(DNCB)/ethanol enema, the rats received double distilled water or corresponding drugs twice a day for 7 days. After the treatment cycle, the general performance and disease activity index(DAI) of rats were observed on the next day. Then the rats were sacrificed. The length of colon was measured. Macroscopic and histological score of colon were evaluated. Histopathological changes of colon were observed by HE staining. Ultraviolet spectrophotometry detection was used to detect the content of myeloperoxidase(MPO) in blood and colon tissues. The levels of P-selectin, macrophage migration inhibitory factor(MIF) and thromboxane B_2(TXB_2) in blood and colon tissues were determined by ELISA. Immunohistochemistry and Western blot analysis were performed to detect the protein expressions of TLR4 and NF-κB in colon tissues. The results showed that as compared with the model group, Shaoyao Tang of different doses improved the general performance of UC rats. Moreover, high-dose Shaoyao Tang group showed the most obvious effect in scoring of disease activity index(P<0.001); both medium and high doses of Shaoyao Tang significantly inhibited the colon shortening and pathological injury, with significantly decreased expression levels of MPO, P-selectin, MIF and TXB_(2 )in serum and colon tissues of UC rats(P<0.001). Immunohistochemistry and Western blot assay showed that the levels of TLR4 and NF-κB protein expression in the colon tissues of Shaoyao Tang high-dose group were remarkably lower than that in the model group(P<0.001). This study shows that Shaoyao Tang has protective and repairing effects on UC, and its possible mechanism is achieved probably by regulating the TLR4/NF-κB pathway and inhibiting the expressions of MPO, P-selectin, MIF and TXB_2.
The aim of this paper was to investigate the effect of Shaoyao Tang on ulcerative colitis(UC) in rats via regulation of TLR4/NF-κB signal pathway. A total of 56 Wistar rats were randomly divided into 6 groups: normal control group(double distilled water), model group(double distilled water), mesalazine group(10 mL·kg~(-1)), high dose, middle dose and low dose Shaoyao Tang groups(2.4, 1.2, and 0.6 g·mL~(-1)). After UC rat models were established by 2, 4-dinitrochlorobenzene(DNCB)/ethanol enema, the rats received double distilled water or corresponding drugs twice a day for 7 days. After the treatment cycle, the general performance and disease activity index(DAI) of rats were observed on the next day. Then the rats were sacrificed. The length of colon was measured. Macroscopic and histological score of colon were evaluated. Histopathological changes of colon were observed by HE staining. Ultraviolet spectrophotometry detection was used to detect the content of myeloperoxidase(MPO) in blood and colon tissues. The levels of P-selectin, macrophage migration inhibitory factor(MIF) and thromboxane B_2(TXB_2) in blood and colon tissues were determined by ELISA. Immunohistochemistry and Western blot analysis were performed to detect the protein expressions of TLR4 and NF-κB in colon tissues. The results showed that as compared with the model group, Shaoyao Tang of different doses improved the general performance of UC rats. Moreover, high-dose Shaoyao Tang group showed the most obvious effect in scoring of disease activity index(P<0.001); both medium and high doses of Shaoyao Tang significantly inhibited the colon shortening and pathological injury, with significantly decreased expression levels of MPO, P-selectin, MIF and TXB_(2 )in serum and colon tissues of UC rats(P<0.001). Immunohistochemistry and Western blot assay showed that the levels of TLR4 and NF-κB protein expression in the colon tissues of Shaoyao Tang high-dose group were remarkably lower than that in the model group(P<0.001). This study shows that Shaoyao Tang has protective and repairing effects on UC, and its possible mechanism is achieved probably by regulating the TLR4/NF-κB pathway and inhibiting the expressions of MPO, P-selectin, MIF and TXB_2.
引文
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[2] Lee S H, Kwon J E, Cho M L. Immunological pathogenesis of inflammatory bowel disease [J]. Intest Res, 2018, 16(1): 26.
[3] Shouval D S, Rufo P A. The role of environmental factors in the pathogenesis of inflammatory bowel diseases: a review[J]. JAMA Pediatr, 2017, 171(10): 999.
[4] 王移飞,王凤仪,徐兰萍,等.芍药汤经HMGB1调节湿热型溃疡性结肠炎大鼠MyD88和NF-κB的分子机制[J].中国实验方剂学杂志,2018,24(12):86.
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[6] Cortez M, Carmo L S, Rogero M M, et al. A high-fat diet increases IL-1, IL-6, and TNF-α production by increasing NF-κB and attenuating PPAR-γ expression in bone marrow mesenchymal stem cells [J]. Inflammation, 2013, 36: 379.
[7] 宁丽琴, 叶柏, 沈洪, 等. 国医大师徐景藩治疗溃疡性结肠炎的用药规律分析和分子靶点预测[J].中国中药杂志, 2018,43(5):1042.
[8] 莫新民, 唐利文. 三黄丸治疗溃疡性结肠炎临床观察[J]. 中国中医基础医学杂志, 2003, 9(7):547.
[9] Ajdukovic J, Salamunic I, Hozo I, et al.Soluble P-selectin glycoprotein ligand-a possible new target in ulcerative colitis[J]. Bratisl Lek Listy, 2015, 116(3):147.
[10] Ali A A, Abd Al Haleem E N, Khaleel S A, et al. Protective effect of cardamonin against acetic acid-induced ulcerative colitis in rats[J]. Pharmacol Rep, 2017, 69(2):268.
[11] Liu G, Yu L, Fang J, et al.Methionine restriction on oxidative stress and immune response in dss-induced colitis mice[J]. Oncotarget, 2017, 8(27):44511.
[12] 杨权, 陈守国, 许宁. 溃疡性结肠炎患者血浆P选择素的测定及其意义[J]. 海南医学, 2010, 21(2):13.
[13] Di Sabatino A, Santilli F, Guerci M, et al.Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF-α treatment[J]. J Thromb Haemost, 2016, 116(3):486.
[14] Horikawa N, Suzuki T, Uchiumi T, et al. Cyclic AMP-dependent Cl-secretion induced by thromboxane A2 in isolated human colon[J]. J Psychophysiol, 2005, 562(Pt 3):885.
[15] Yukitake H, Takizawa M, Kimura H. Macrophage migration inhibitory factor as an emerging drug target to regulate antioxidant response element system[J]. Oxid Med Cell Longev, 2017, 2017:8584930.
[16] Mitsuyama K, Suzuki A, Tomiyasu N, et al. Transciption factor-targeted therapies in inflammatory bowel disease[J]. Disgestion, 2001, 63(suppl1):68.
[17] Shusterman T, Sela S, Cohen H, et al. Effect of the antioxidant Mesna (2-mercaptoethanesulfonate) on experimental colitis[J]. Dig Dis Sci, 2003, 48(6): 1177.
[18] Liu X, Wang J M. Anti-inflammatory effects of iridoid glycosides fraction of Folium syringae leaves on TNBS-induced colitis in rats[J]. J Ethnopharmacol, 2011, 133:780.
[19] CesarF O, Shunji I, Mohammad A K R, et al. Epithelial Toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis[J]. Clin Vaccine Immunol, 2006, 3(1):132.
[20] Ukil A, Maity S, Das P K. Protection from experimental colitis by theaflavin-3,3′-digallate correlates with inhibition of IKK and NF-kappaB activation[J]. Br J Pharmacol, 2006, 149:121.
[2] Lee S H, Kwon J E, Cho M L. Immunological pathogenesis of inflammatory bowel disease [J]. Intest Res, 2018, 16(1): 26.
[3] Shouval D S, Rufo P A. The role of environmental factors in the pathogenesis of inflammatory bowel diseases: a review[J]. JAMA Pediatr, 2017, 171(10): 999.
[4] 王移飞,王凤仪,徐兰萍,等.芍药汤经HMGB1调节湿热型溃疡性结肠炎大鼠MyD88和NF-κB的分子机制[J].中国实验方剂学杂志,2018,24(12):86.
[5] Sohn K H, Jo J M, Cho W J, et al. Bojesodok-eum, a herbal prescription, ameliorates acute inflammation in association with the inhibition of NF-κB-mediated nitric oxide and proinflammatory cytokine production [J]. Evid Based Complement Alternat Med, 2012, doi: 10.1155/2012/457370.
[6] Cortez M, Carmo L S, Rogero M M, et al. A high-fat diet increases IL-1, IL-6, and TNF-α production by increasing NF-κB and attenuating PPAR-γ expression in bone marrow mesenchymal stem cells [J]. Inflammation, 2013, 36: 379.
[7] 宁丽琴, 叶柏, 沈洪, 等. 国医大师徐景藩治疗溃疡性结肠炎的用药规律分析和分子靶点预测[J].中国中药杂志, 2018,43(5):1042.
[8] 莫新民, 唐利文. 三黄丸治疗溃疡性结肠炎临床观察[J]. 中国中医基础医学杂志, 2003, 9(7):547.
[9] Ajdukovic J, Salamunic I, Hozo I, et al.Soluble P-selectin glycoprotein ligand-a possible new target in ulcerative colitis[J]. Bratisl Lek Listy, 2015, 116(3):147.
[10] Ali A A, Abd Al Haleem E N, Khaleel S A, et al. Protective effect of cardamonin against acetic acid-induced ulcerative colitis in rats[J]. Pharmacol Rep, 2017, 69(2):268.
[11] Liu G, Yu L, Fang J, et al.Methionine restriction on oxidative stress and immune response in dss-induced colitis mice[J]. Oncotarget, 2017, 8(27):44511.
[12] 杨权, 陈守国, 许宁. 溃疡性结肠炎患者血浆P选择素的测定及其意义[J]. 海南医学, 2010, 21(2):13.
[13] Di Sabatino A, Santilli F, Guerci M, et al.Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF-α treatment[J]. J Thromb Haemost, 2016, 116(3):486.
[14] Horikawa N, Suzuki T, Uchiumi T, et al. Cyclic AMP-dependent Cl-secretion induced by thromboxane A2 in isolated human colon[J]. J Psychophysiol, 2005, 562(Pt 3):885.
[15] Yukitake H, Takizawa M, Kimura H. Macrophage migration inhibitory factor as an emerging drug target to regulate antioxidant response element system[J]. Oxid Med Cell Longev, 2017, 2017:8584930.
[16] Mitsuyama K, Suzuki A, Tomiyasu N, et al. Transciption factor-targeted therapies in inflammatory bowel disease[J]. Disgestion, 2001, 63(suppl1):68.
[17] Shusterman T, Sela S, Cohen H, et al. Effect of the antioxidant Mesna (2-mercaptoethanesulfonate) on experimental colitis[J]. Dig Dis Sci, 2003, 48(6): 1177.
[18] Liu X, Wang J M. Anti-inflammatory effects of iridoid glycosides fraction of Folium syringae leaves on TNBS-induced colitis in rats[J]. J Ethnopharmacol, 2011, 133:780.
[19] CesarF O, Shunji I, Mohammad A K R, et al. Epithelial Toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis[J]. Clin Vaccine Immunol, 2006, 3(1):132.
[20] Ukil A, Maity S, Das P K. Protection from experimental colitis by theaflavin-3,3′-digallate correlates with inhibition of IKK and NF-kappaB activation[J]. Br J Pharmacol, 2006, 149:121.