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DING Huamin, GUO Yuchen, QIN Chunxia, SONG Zhibing, SUN Lili. Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 211-216. doi: 10.12206/j.issn.2097-2024.202306031
Citation: DING Huamin, GUO Yuchen, QIN Chunxia, SONG Zhibing, SUN Lili. Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 211-216. doi: 10.12206/j.issn.2097-2024.202306031

Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene

doi: 10.12206/j.issn.2097-2024.202306031
  • Received Date: 2023-06-19
  • Rev Recd Date: 2023-10-26
  • Available Online: 2024-05-22
  • Publish Date: 2024-05-25
  •   Objective  To establish a mice model of atopic dermatitis with acute itching and investigate the antipruritic effect and its mechanism of Xiaofeng Zhiyang granules(XFZYG).   Methods  A mice model of atopic dermatitis was prepared by induction method. Mice were sensitized by calcipotriol and ovalbumin (OVA) applying to the right ear daily for 10 days, and then stimulated by OVA injected intradermally into the right cheek to resulting in acute itching. These mice were divided into 5 groups: blank control group, model group, low dose (7.2 g/kg) and high dose (14.4 g/kg) of XFZYG, and positive control group (montelukast 5 mg/kg). Drugs were administered by gavage at 12 h and 30 min before stimulation. The leukotriene levels in the serum of the mice were measured by Elisa and the basophil ratio and activation status in the blood were measured by flow cytometry.   Results  The mean number of scratches in the model group was 56 between 30 min and 60 min after stimulation, while the mean number of scratches in the low and high dose of XFZYG groups were 42 and 23 respectively, which were significantly lower than those in the model group (P<0.05). The serum leukotriene levels and the proportion of basophils in the low and high dose of XFZYG groups were significantly lower than those in the model group (P<0.05).   Conclusion  XFZYG had certain therapeutic effect on acute itching of atopic dermatitis in mice, and the mechanism of its action was related to the reduction of leukotriene level and basophil ratio in serum of mice with atopic dermatitis .
  • [1] LAUGHTER M R, MAYMONE M B C, MASHAYEKHI S, et al. The global burden of atopic dermatitis: lessons from the Global Burden of Disease Study 1990-2017[J]. Br J Dermatol, 2021, 184(2):304-309. doi:  10.1111/bjd.19580
    [2] TSAI T F, RAJAGOPALAN M, CHU C Y, et al. Burden of atopic dermatitis in Asia[J]. J Dermatol, 2019, 46(10):825-834. doi:  10.1111/1346-8138.15048
    [3] 樊文龙, 陈东宇, 王红心, 等. 亚洲5国居民1990—2019年特应性皮炎发病趋势年龄-时期-队列分析[J]. 中国公共卫生, 2023, 39(5):650-655. doi:  10.11847/zgggws1138656
    [4] 庞威, 樊杰, 孙继泽, 等. 消风止痒颗粒中防风薄层鉴别优化[J]. 人参研究, 2022, 34(6):29-31.
    [5] 庞博, 徐英莉, 刘晓峰, 等. 氯雷他定与消风止痒颗粒对3种荨麻疹动物模型的药效评价研究[J]. 世界中医药, 2022, 17(21):3026-3032. doi:  10.3969/j.issn.1673-7202.2022.21.008
    [6] 葛杰. 消风止痒颗粒、氯雷他定片内服联合血液透析治疗尿毒症皮肤瘙痒症的临床研究[J]. 中华中医药学刊, 2018, 36(6):1497-1499.
    [7] WANG F, TRIER A M, LI F X, et al. A basophil-neuronal axis promotes itch[J]. Cell, 2021, 184(2):422-440,e17.
    [8] PUAR N, CHOVATIYA R, PALLER A S. New treatments in atopic dermatitis[J]. Ann Allergy Asthma Immunol, 2021, 126(1):21-31. doi:  10.1016/j.anai.2020.08.016
    [9] 邱彩雄, 李康良, 何爽, 等. 皮肤源性急慢性瘙痒动物模型研究进展[J]. 中国医药导报, 2022, 19(33):45-48.
    [10] SIRACUSA M C, KIM B S, SPERGEL J M, et al. Basophils and allergic inflammation[J]. J Allergy Clin Immunol, 2013, 132(4):789-801;quiz788. doi:  10.1016/j.jaci.2013.07.046
    [11] OBATA K, MUKAI K R, TSUJIMURA Y, et al. Basophils are essential initiators of a novel type of chronic allergic inflammation[J]. Blood, 2007, 110(3):913-920. doi:  10.1182/blood-2007-01-068718
    [12] NOTI M, WOJNO E D, KIM B S, et al. Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis[J]. Nat Med, 2013, 19(8):1005-1013. doi:  10.1038/nm.3281
    [13] POTO R, QUINTI I, MARONE G, et al. IgG autoantibodies against IgE from atopic dermatitis can induce the release of cytokines and proinflammatory mediators from basophils and mast cells[J]. Front Immunol, 2022, 13:880412. doi:  10.3389/fimmu.2022.880412
    [14] FOGH K, HERLIN T, KRAGBALLE K. Eicosanoids in skin of patients with atopic dermatitis: Prostaglandin E2 and leukotriene B4 are present in biologically active concentrations[J]. J Allergy Clin Immunol, 1989, 83(2):450-455. doi:  10.1016/0091-6749(89)90132-2
    [15] CAPRA V, THOMPSON M D, SALA A, et al. Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends[J]. Med Res Rev, 2007, 27(4):469-527. doi:  10.1002/med.20071
    [16] OYOSHI M K, HE R, LI Y T, et al. Leukotriene B4-driven neutrophil recruitment to the skin is essential for allergic skin inflammation[J]. Immunity, 2012, 37(4):747-758. doi:  10.1016/j.immuni.2012.06.018
    [17] 孟聪聪, 王晓琴, 韩秀萍. 孟鲁司特治疗特应性皮炎研究进展[J]. 中国麻风皮肤病杂志, 2017, 33(12):759-762.
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Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene

doi: 10.12206/j.issn.2097-2024.202306031

Abstract:   Objective  To establish a mice model of atopic dermatitis with acute itching and investigate the antipruritic effect and its mechanism of Xiaofeng Zhiyang granules(XFZYG).   Methods  A mice model of atopic dermatitis was prepared by induction method. Mice were sensitized by calcipotriol and ovalbumin (OVA) applying to the right ear daily for 10 days, and then stimulated by OVA injected intradermally into the right cheek to resulting in acute itching. These mice were divided into 5 groups: blank control group, model group, low dose (7.2 g/kg) and high dose (14.4 g/kg) of XFZYG, and positive control group (montelukast 5 mg/kg). Drugs were administered by gavage at 12 h and 30 min before stimulation. The leukotriene levels in the serum of the mice were measured by Elisa and the basophil ratio and activation status in the blood were measured by flow cytometry.   Results  The mean number of scratches in the model group was 56 between 30 min and 60 min after stimulation, while the mean number of scratches in the low and high dose of XFZYG groups were 42 and 23 respectively, which were significantly lower than those in the model group (P<0.05). The serum leukotriene levels and the proportion of basophils in the low and high dose of XFZYG groups were significantly lower than those in the model group (P<0.05).   Conclusion  XFZYG had certain therapeutic effect on acute itching of atopic dermatitis in mice, and the mechanism of its action was related to the reduction of leukotriene level and basophil ratio in serum of mice with atopic dermatitis .

DING Huamin, GUO Yuchen, QIN Chunxia, SONG Zhibing, SUN Lili. Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 211-216. doi: 10.12206/j.issn.2097-2024.202306031
Citation: DING Huamin, GUO Yuchen, QIN Chunxia, SONG Zhibing, SUN Lili. Therapeutic effect of Xiaofeng Zhiyang granules on acute itching in mice with atopic dermatitis by decreasing leukotriene[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 211-216. doi: 10.12206/j.issn.2097-2024.202306031
  • 特应性皮炎(AD)是一种慢性、复发性、炎症性皮肤病,在全球疾病负担研究(GBD)中以伤残调整寿命年的率衡量时,其造成的疾病负担在非致命性疾病中居于第15位,在所有疾病和损伤中居于59位[1],同时AD造成的负担还与一个国家的国内生产总值呈正相关[2]。在过去的几十年里,亚洲国家居民的AD患病率持续上升,AD亦成为中国当前严重的公共卫生问题之一。中国居民AD的发病率逐年增加,儿童和老年人在亚洲五国均具有较高的AD发病风险,且中国居民的AD发病风险随时期的推进而加重[3]

    消风止痒颗粒现行质量标准收载于《部颁标准》中药成方制剂第十五册,标准编号:WS3-B-2975-98,处方中包括防风、蝉蜕、地骨皮、苍术(炒)、亚麻子、当归、地黄、木通、荆芥、石膏和甘草十一味药材[4]。消风止痒颗粒不仅对接触性荨麻疹作用明显,有缓解皮肤红肿,抑制风团之效[5],还能改善尿毒症患者皮肤瘙痒症状[6]。本研究旨在通过消风止痒颗粒对AD引起的急性瘙痒的缓解作用,探究其可能的作用机制,为中药在止痒方面的研究提供一定的基础和参考依据。

    • 健康C57BL/6小鼠60只,雄性,体重18~20 g,SPF级,购自上海西普尔-必凯实验动物有限公司,实验动物生产许可证号:SCXK(沪)2018-0006。饲养条件:室温20~25 ℃,湿度40 %~70 %,标准饲料,动物自由饮食和饮水。

    • 消风止痒颗粒由烟台东诚大洋制药有限公司提供,批号:Z20113054,规格:每袋3 g,临用前用纯净水配制成所需浓度。卡泊三醇(aladdin,批号:C126438);卵清蛋白(OVA,SAITONC,批号:A80003);无水乙醇(aladdin,批号:E111991);小鼠白三烯C4(LTC4)Elisa试剂盒(江苏晶美生物科技有限公司,批号:JM-02678M1);红细胞裂解液(BD,Cat:555899);FITC 抗小鼠 FcεRIα 抗体(BioLegend,Cat:134305);PE 抗小鼠 IgE 抗体(BioLegend,Cat:406907);PerCP/Cyanine5.5 抗小鼠 CD49b 抗体(Bio Legend,Cat:103519);APC 抗小鼠 CD200R (OX2R)抗体(BioLegend,Cat:123915)。

    • JA2003A型电子天平(上海精天电子仪器有限公司);A51119500C型全自动酶标仪(美国Thermo科技公司);5420型高速离心机(美国Eppendorf公司);CytoFLEX型流式细胞仪(美国贝克曼公司)。

    • 参照Wang等的小鼠AD急性瘙痒模型制备方法[7]。8周龄的C57小鼠,每天先用卡泊三醇(0.5 nmol,溶在15 μl的100 %乙醇中)涂抹右侧耳皮肤(右耳内外侧各7.5 μl),然后用卵清蛋白(OVA,5 mg/ml,溶在20 μl磷酸盐缓冲盐水PBS中)进行涂抹(右耳内外侧各10 μl),连续处理10 d(从第0天到第9天)以使小鼠敏感。对照组小鼠先用15 μl乙醇处理,然后用20 μl PBS处理。在第10天,在小鼠右颊皮内注射OVA(2.5 mg/ml,溶在生理盐水中)20 μl进行激发。

    • 60只雄性C57BL/6小鼠随机分为空白对照组、模型组、消风止痒颗粒低(7.2 g/kg)和高剂量组(14.4 g/kg)、阳性对照组(孟鲁司特,5 mg/kg)5组,每组12只,适应性饲养1周后开始进行造模。消风止痒颗组低剂量是按体表面积换算方法由人临床使用剂量(15岁以上患者1日分2~3次口服36 g,按60 kg体重计算,即0.6 g/(kg·d))换算成小鼠剂量(7.2 g/kg),高剂量设为低剂量的2倍,即14.4 g/kg。各组小鼠给药途径均为灌胃给药,各组药物均在给药前现配。消风止痒颗粒低剂量组药物加饮用水后配成1 g/ml浓度, 消风止痒颗粒高剂量组药物加饮用水后配成2 g/ml浓度;阳性对照组孟鲁司特加饮用水后配成0.695 mg/ml浓度,空白对照组用饮用水灌胃,各组小鼠均按7.2 ml/kg体积灌胃给药。各组于激发前12 h及激发前30 min各灌胃给药一次。

    • 瘙痒行为在第10天上午8点至11点之间进行。测试前2 d将右侧脸颊备皮,测试前1 d(即第9天)将动物在测试室中适应60 min。测试当天给药30 min后在小鼠右颊皮内注射OVA(2.5 mg/ml,溶在0.9 %生理盐水中)20 μl进行激发,录像60 min,记数30~60 min小鼠抓挠次数。抓挠的定义是前脚或后脚直接对背部、脸颊或耳朵进行连续抓挠的情况,当这种情况在鼠将前爪或后爪离开或放在嘴巴为计数1次。

    • 在小鼠瘙痒行为学实验结束后,每组随机取6只小鼠进行眼眶取血,在离心管中4 ℃静置2 h后,3 000 r/min离心10 min,取上清液到新的离心管中备用。血清样本按Elisa试剂盒操作说明书进行实验。

    • 外周血淋巴细胞样本制备:每组各取3只小鼠,腹主动脉取血于抗凝管中(500 μl左右),每个样品加入1 ml红细胞裂解液,室温孵育15 min,1 000 r/min离心5 min,弃去上清液,再次加入1 ml红细胞裂解液,室温孵育15 min,1 000 r/min离心5 min,弃去上清液后加入1 ml PBS重悬离心,得到外周血淋巴细胞。抗体孵育:各样品管外周血淋巴细胞用300 μl的PBS重悬,每管加入1 μl抗体,4 ℃孵育30 min,流式细胞仪进样检测。

    • 实验数据以均值±标准差($ \bar{x} $±s)表示,采用GraphPad Prism软件进行统计处理,采用单因素方差分析(one-way ANOVA)进行差异的显著性检验。P<0.05为差异具有统计学意义。

    • 空白对照组小鼠无明显的急性抓挠行为,模型组小鼠激发后出现明显的抓挠行为,模型组抓挠次数均值为56次,与模型组相比,各给药组均不同程度显著减少小鼠抓挠次数(P<0.05),阳性对照组为34次、消风止痒颗粒低剂量组为42次、消风止痒颗粒高剂量组为23次,结果见图1。消风止痒颗粒能明显减少小鼠抓挠次数,改善小鼠AD急性瘙痒症状。

    • 与空白对照组相比,模型组小鼠血清中白三烯含量明显升高(P<0.001);各给药组相较于模型组小鼠血清中白三烯含量均显著下降(P<0.05),结果见图2。结果表明,消风止痒颗粒能显著降低AD小鼠血清中白三烯水平,降低炎症水平。

    • 模型组小鼠血液中CD49b+细胞和FceRla+细胞以及CD200R+细胞的比例显著高于空白对照组(P<0.05),消风止痒颗粒高剂量组与模型组相比较,能明显降低CD49b+细胞和FceRla+细胞以及CD200R+细胞的比例(P<0.05),结果如图3图5所示。CD49b和FceRla以及CD200R为嗜碱性粒细胞表面抗原[7],两者升高表明小鼠血液中嗜碱性粒细胞比例升高,而消风止痒颗粒能降低由特异性皮炎引起增多的嗜碱性粒细胞,减少炎症反应,进而减轻瘙痒症状。

    • AD是一种以皮肤持续瘙痒为特征的复发性、慢性、非感染性炎症性皮肤病。其发病机制是多种因素共同作用的结果,涉及表皮屏障功能受损、免疫异常、宿主遗传因素和环境因素的相互作用。表皮的最外层,角质层和颗粒层的紧密连接形成蛋白质和脂质屏障,防止皮肤种水分的流失、微生物病原体的入侵,以及过敏原、毒素和刺激物引起的炎症反应。AD发生后,皮肤屏障受损,表皮分化蛋白(特别是聚丝蛋白)和紧密连接蛋白表达减少,脂质,特别是长链脂肪酸和神经酰胺缺乏。由于抗菌肽(AMP)对环境病原体(包括金黄色葡萄球菌)的反应不足,使皮肤对微生物的屏障作用也被削弱[8]。国内外报道中,皮肤源性瘙痒动物模型是药物止痒研究实验最常使用的类型,由于不同药物作用机制、疾病生理病理特征不同,使用的模型也不尽相同。近年来,越来越多的新品系、新技术、新途径应用于皮肤源性急慢性瘙痒动物模型的建立,为机制研究提供基础,同时也出现了更加经济、造模周期短和更符合疾病生理病理特征等优势动物模型,如同时使用4-氨基吡啶(4-AP)和组胺能更好地诱发动物瘙痒行为,AD的卡泊三醇模型操作更简便,瘙痒更明显,并且具备更多 AD 临床特征[9]。本研究造模时参考Wang等的方法[7],在小鼠皮内注射OVA激发后的30~60 min内,小鼠出现明显的抓挠行为,说明该方法能很好的诱发小鼠瘙痒行为。通过对各组小鼠30 min内抓挠次数的统计,发现消风止痒颗粒给药后能显著减少小鼠急性瘙痒后的抓挠次数,说明口服消风止痒颗粒能减轻AD后急性瘙痒的症状。

      尽管嗜碱性粒细胞是哺乳动物体内最不常见的粒细胞群体,但在许多人类疾病中,如过敏性疾病、器官排斥反应、自身免疫疾病及癌症,都有发现其积累。例如,嗜碱性粒细胞被认为与过敏性接触性皮炎、AD、药物过敏反应、超敏反应、哮喘、大疱性类天疱疮、狼疮肾炎、克罗恩病、皮肤和肾脏同种异体移植反应以及急性和慢性骨髓性白血病的发病机制有关[10]。还有许多研究表明,在急性AD、慢性IgE介导的皮炎、气道炎症和嗜酸性粒细胞性食管炎疾病中,嗜碱性粒细胞在诱导TH2细胞因子介导的炎症中发挥重要作用[11-13]。本研究通过流式细胞术检测了小鼠血清中CD49b+和FceRla+细胞的比例,发现口服消风止痒颗粒能明显减少由AD诱导增加的嗜碱性粒细胞,说明消风止痒颗粒是通过降低小鼠血清中嗜碱性粒细胞数量来达成缓解瘙痒的效果。此外,有研究报道,AD患者的病变皮肤或尿液中检测到多种前列腺素和白三烯,如PGE2、LTB4和CysLTE4[14, 15],提示前列腺素和白三烯可能参与AD 症状的发展。在OVA诱导的小鼠AD模型中,抓挠行为诱导中性粒细胞向皮肤浸润,浸润的中性粒细胞产生的LTB4作用于中性粒细胞和Th 2细胞上表达的BLT1,促进其向皮肤浸润[16]。有文献报道了作为白三烯拮抗剂的孟鲁司特可治疗AD,本实验选用了孟鲁司特作为特异性阳性对照药[17]。在分离小鼠血清后,检测了血清中白三烯含量,结果发现,消风止痒颗粒能明显降低AD小鼠血清中白三烯水平,说明消风止痒颗粒口服后改善AD小鼠瘙痒症状的机制可能与降低血清中白三烯水平有关。

      综上所述,本研究证实了消风止痒颗粒能够改善AD小鼠的急性瘙痒症状,其机制可能与减少小鼠血液中嗜碱性粒细胞数量及血清中白三烯水平相关。本研究为将来进一步进行消风止痒颗粒在AD急性瘙痒方面的研究提供了深入的实验与理论依据。

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