芍药苷通过抑制NLRP3炎症小体治疗溃疡性结肠炎小鼠的研究
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摘要
目的研究芍药苷(Paeoniflorin,PF)在溃疡性结肠炎(Ulcerative colitis,UC)小鼠结肠组织中的动态分布,并探讨其治疗UC的途径及其作用机制。方法 C57BL/6小鼠在实验第1~7天自由饮用3%葡聚糖硫酸钠(Dextran sulfate sodium,DSS)水溶液制备UC模型,第8天,UC小鼠灌服黄芩汤(含芍药苷),采用超高效液相色谱串联四极杆飞行时间质谱仪(UPLC-Q-TOF-MS)检测芍药苷在结肠中的含量变化,检测时间点为给药0.5,1,3,12 h。另取C57BL/6小鼠随机分为正常组、模型组、柳氮磺吡啶组(Sulfasalazine,SASP)和芍药苷组,每组8只。同时,SASP组(50 mg·kg~(-1))和芍药苷组(25 mg·kg~(-1))分别灌胃给药;正常组和模型组小鼠灌胃给予生理盐水,连续10 d,并记录体质量变化。实验第11天,取小鼠脾脏称质量;采用动物血细胞计数仪检测单核细胞数量;苏木素—伊红染色(HE)观察结肠形态变化;免疫组化法检测结肠和肠系膜中巨噬细胞的分布;超高分辨率激光共聚焦扫描显微镜(Confocal)检测结肠组织巨噬细胞和核苷酸结合寡聚化结构域样受体3(Nucleotide-binding oligomerization domain-like receptor family,pyrin domain-containing 3,NLRP3)蛋白共定位。ELISA试剂盒检测结肠匀浆上清白细胞介素-1β(IL-1β)的含量。结果 UPLC-Q-TOF-MS检测结果表明,芍药苷在结肠中可以检测到,并且芍药苷可显著减轻小鼠结肠溃疡症状并显著促进UC小鼠体质量(P<0.05)和脾指数恢复(P<0.001),降低UC小鼠外周血单核细胞比例(P<0.01)。减少结肠和肠系膜中巨噬细胞浸润的数量,并通过抑制结肠巨噬细胞中NLRP3炎症小体活化进而降低结肠组织上清中IL-1β含量(P<0.01)。结论芍药苷可以在结肠中检测到,并可改善UC小鼠病理症状,抑制肠系膜和结肠组织中巨噬细胞的浸润,抑制结肠巨噬细胞中NLRP3蛋白并抑制细胞因子IL-1β的释放。
Objective To investigate the therapeutic effect of PF on UC and its possible pathways. Methods Theconcentrations of PF in DSS-induced UC mice after orally fed with HQT(0.5,1,3,12 h) were detected byUPLC-Q-TOF-MS. In vivo, animals were divided into 4 groups randomly: Control group, DSS group, SASPgroup,PF group(25 mg·kg~(-1));apart from the normal group,the remaining groups were administrated with 3%DSS,there after provided with regular water for 3 days. PF and SASP were given intragastrically from day 1 to day10 and the mice were sacrificed on day 11. The basal body weight changes of each group, spleen index andmacroscopic appearances of colon were observed. The concentration of monocytes in peripheral blood was detected byPeripheral blood automatic analyzer and the distribution of macrophages(F4/80 +) in colon was detected byimmunohistochemistry. Protein co-localization of NLRP3 and colon macrophagocytes was observed by confocalmicroscope. In vitro experiment,RAW264.7 was stimulated with LPS(1 μg·m L-1) and treated with PF(12.5,25, 50 μmol · L-1). Results PF was detect in the colon. PF could improve the disease symptom of mice(P <0.05),reduce the proportion of monocytes in peripheral blood(P < 0.01),and reduce the number of macrophageinfiltration in the colon,inhibit protein expression of NLRP3 in the macrophage. In vitro experiments showed that PFpossessed anti-inflammatory effect by inhibit the secretion of NO(P < 0.001). Conclusion PF can be absorbedinto colon and maybe a potential effective drug for ulcerative colitis.
Objective To investigate the therapeutic effect of PF on UC and its possible pathways. Methods Theconcentrations of PF in DSS-induced UC mice after orally fed with HQT(0.5,1,3,12 h) were detected byUPLC-Q-TOF-MS. In vivo, animals were divided into 4 groups randomly: Control group, DSS group, SASPgroup,PF group(25 mg·kg~(-1));apart from the normal group,the remaining groups were administrated with 3%DSS,there after provided with regular water for 3 days. PF and SASP were given intragastrically from day 1 to day10 and the mice were sacrificed on day 11. The basal body weight changes of each group, spleen index andmacroscopic appearances of colon were observed. The concentration of monocytes in peripheral blood was detected byPeripheral blood automatic analyzer and the distribution of macrophages(F4/80 +) in colon was detected byimmunohistochemistry. Protein co-localization of NLRP3 and colon macrophagocytes was observed by confocalmicroscope. In vitro experiment,RAW264.7 was stimulated with LPS(1 μg·m L-1) and treated with PF(12.5,25, 50 μmol · L-1). Results PF was detect in the colon. PF could improve the disease symptom of mice(P <0.05),reduce the proportion of monocytes in peripheral blood(P < 0.01),and reduce the number of macrophageinfiltration in the colon,inhibit protein expression of NLRP3 in the macrophage. In vitro experiments showed that PFpossessed anti-inflammatory effect by inhibit the secretion of NO(P < 0.001). Conclusion PF can be absorbedinto colon and maybe a potential effective drug for ulcerative colitis.
引文
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[13]张保国,梁晓夏,刘庆芳.黄芩汤现代药效学研究与临床运用[J].中国中药杂志,2008,33(22):2587-2590.
[14]农辉,黄雪.葡聚糖硫酸钠诱导结肠炎模型的研究进展[J].世界华人消化杂志,2014,22(22):3245-3250.
[15]王玮,周艳宏,李夏雨,等.巨噬细胞在溃疡性结肠炎癌变中的作用[J].中南大学学报(医学版),2012,37(6):637-641.
[16]Zaki MH,Lamkanfi M,Kanneganti TD.The Nlrp3 inflammasome:contributions to intestinal homeostasis[J].Trends in immunology,2011,32(4):171-179.
[17]Hirota SA,Ng J,Lueng A,et al.NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis[J].Inflammatory bowel diseases,2011,17(6):1359-1372.
[2]MC GRIMM,WE PULLMAN,GM BENNETT,et al.Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa[J].Journal of gastroenterology and hepatology,1995,10(4):387-395.
[3]Martinez FO,Helming L,Gordon S.Alternative activation of macrophages:an immunologic functional perspective[J].Annual Review of Immunology,2009,27:451-483.
[4]Stow JL,Ching LP,Offenh?user C,et al.Cytokine secretion in macrophages and other cells:pathways and mediators[J].Immunobiology,2009,214(7):601-612.
[5]于小凤,吕行政,董委波.黄芩汤治疗溃疡性结肠炎临床观察[J].中国中医急症,2010,19(9):1510-1529.
[6]丁晓刚.黄芩汤有效部位组方抗大鼠溃疡性结肠炎的实验研究[D].北京中医药大学,2003.
[7]Wang C,Tang X,Zhang L.Huangqin-Tang and ingredients in modulating the pathogenesis of ulcerative colitis[J].Evidence-based Complementary and Alternative Medicine,2017,2017:1-7.
[8]Jia Z,He J.Paeoniflorin ameliorates rheumatoid arthritis in rat models through oxidative stress,inflammation and cyclooxygenase 2[J].Experimental and Therapeutic Medicine,2016,11(2):655-659.
[9]Zhang J,Li H,Huo R,et al.Paeoniflorin selectively inhibits LPSprovoked B-cell function[J].Journal of pharmacological science,2015,128(1):8-16.
[10]汪斌,朱向东,段永强,等.芍药苷对溃疡性结肠炎大鼠结肠组织病理形态及血清抗炎和促炎因子平衡的影响[J].中国中西医结合消化杂志,2013,21(1):1-4.
[11]李涛,王怡薇,王彦礼,等.黄芩汤血浆中多成分LC-MS法测定及其药代动力学特征研究[J].药学学报,2013,(6):917-924.
[12]刘芳兰,蒋莹.芍药苷对小鼠胶原诱导性关节炎抑制作用[J].亚太传统医药,2015,(17):10-11.
[13]张保国,梁晓夏,刘庆芳.黄芩汤现代药效学研究与临床运用[J].中国中药杂志,2008,33(22):2587-2590.
[14]农辉,黄雪.葡聚糖硫酸钠诱导结肠炎模型的研究进展[J].世界华人消化杂志,2014,22(22):3245-3250.
[15]王玮,周艳宏,李夏雨,等.巨噬细胞在溃疡性结肠炎癌变中的作用[J].中南大学学报(医学版),2012,37(6):637-641.
[16]Zaki MH,Lamkanfi M,Kanneganti TD.The Nlrp3 inflammasome:contributions to intestinal homeostasis[J].Trends in immunology,2011,32(4):171-179.
[17]Hirota SA,Ng J,Lueng A,et al.NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis[J].Inflammatory bowel diseases,2011,17(6):1359-1372.