首页|加味荆芩汤对玫瑰痤疮模型小鼠皮损组织炎症及免疫反应的影响

加味荆芩汤对玫瑰痤疮模型小鼠皮损组织炎症及免疫反应的影响

Effects of Jiawei Jingqin Decoction(加味荆芩汤)on Inflammatory and Immune Responses in Rosacea Model Mice

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目的 探讨加味荆芩汤治疗玫瑰痤疮的可能作用机制.方法 50只8周龄雌性BALB/c小鼠随机分为正常对照组、模型组和加味荆芩汤低、中、高剂量组,每组10只.除正常对照组外,其余各组小鼠予背部注射40µl抗菌肽LL-37(浓度为320 μmol/L),每12h重复注射1次,共注射4次,以制备玫瑰痤疮小鼠模型,正常对照组小鼠同时间点背部皮内注射40 µl PBS溶液.造模成功后加味荆芩汤低、中、高剂量组小鼠分别给予加味荆芩汤4.35、8.71、17.42 g/(kg·d)灌胃,正常对照组和模型组小鼠给予20 ml/(kg·d)生理盐水灌胃.各组均每日1次,持续5天.观察各组小鼠皮损变化,并进行红斑面积测定及红斑程度评分;取注射部位皮肤组织HE染色观察病理形态学变化,甲苯胺蓝染色检测肥大细胞数量;ELISA法检测皮损组织中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平;RT-qPCR法检测玫瑰痤疮发病机制中固有免疫关键因子[Toll样受体2(TLR2)及下游分子丝氨酸蛋白酶激肽释放酶5(KLK5)、基质金属蛋白酶9(MMP-9)]与相关炎症因子[IL-1β、IL-6、TNF-α]mRNA,适应性免疫CD4+T细胞相关因子干扰素γ(INF-γ)mRNA表达;免疫组化染色法检测KLK5、MMP-9蛋白表达及CD4+T阳性细胞数;Western blot法检测CD4+T细胞相关因子[IFN-γ、CC趋化因子受体5(CCR5)、信号转导转录激活因子1(STAT1)、信号转导转录激活因子3(STAT3)]蛋白表达水平.结果 正常对照组小鼠背部注射部位皮肤正常,未见皮损及红斑,病理显示未见炎性细胞浸润.模型组小鼠红斑程度评分及红斑面积较正常对照组显著升高(P<0.05),病理显示大量炎细胞浸润,部分可见深层结缔组织损伤.与模型组比较,加味荆芩汤中、高剂量组小鼠红斑程度评分及红斑面积均显著降低(P<0.05),炎性渗出及炎性细胞浸润有所减少;加味荆芩汤低剂量组差异无统计学意义(P>0.05),炎细胞浸润较模型组略有减轻.与正常对照组比较,模型组小鼠皮损组织肥大细胞数,IL-1β、IL-6、TNF-α水平,IL-1β、IL-6、TNF-α、TLR2、INF-γ、KLK5、MMP-9 mRNA表达,KLK5、MMP-9蛋白表达,CD4+T阳性细胞数,IFN-γ、STAT1蛋白表达均升高(P<0.05).与模型组比较,加味荆芩汤中、高剂量组肥大细胞数、IL-1β水平,IL-1β、TNF-αmRNA表达及CD4+T阳性细胞数降低;加味荆芩汤高剂量组IL-6、TNF-α水平,IL-6、TLR2、INF-γ、MMP-9 mRNA表达,MMP-9、KLK5蛋白表达,CD4+T阳性细胞数,IFN-γ、STAT1蛋白表达均降低(P<0.05).结论 加味荆芩汤对玫瑰痤疮小鼠模型皮肤症状具有显著改善作用,其作用机制可能与调控皮肤免疫炎症反应、进而减轻致病因子及炎症因子的释放相关.
Objective To investigate the potential mechanisms of Jiawei Jingqin Decoction(加味荆芩汤)in the treatment of rosacea.Methods Fifty 8-week-old female BALB/c mice were randomly divided into normal control group,model group,and Jiawei Jingqin Decoction low,medium,and high-dose groups,with 10 mice in each group.Except for the normal control group,mice in the other groups were injected with 40μl of antimicrobial peptide LL-37 at a concentration of 320 μmol/L on the back,repeated once every 12 hours for a total of 4 injections,to induce the ro-sacea mouse model.The mice in the normal control group were injected with 40 μl of PBS solution at the same time.After successful modeling,the mice in the Jiawei Jingqin Decoction low,medium,and high-dose groups were orally administered Jiawei Jingqin Decoction at doses of 4.35,8.71,and 17.42 g/(kg·d)respectively,while the mice in the normal control group and the model group were orally administered 20 ml/(kg·d)of normal saline.All groups were treated once daily for 5 days.The changes in skin lesions were observed,and the erythema area was measured and scored.Skin tissue at the injection site was collected for histopathological examination using HE staining,and the number of mast cells was detected using toluidine blue staining.The levels of interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-alpha(TNF-α)in the skin lesion tissues were measured using the ELISA method.The mRNA expression levels of key factors in the innate immune response pathway of rosacea disease mechanism,including Toll-like receptor 2(TLR2),downstream molecules serine protease kallikrein 5(KLK5)and matrix metal-loproteinase 9(MMP-9),as well as related inflammatory factors(IL-1β,IL-6,TNF-α),and adaptive immune CD4+T cell-related factors interferon-gamma(INF-γ)mRNA were detected using RT-qPCR.Immunohistochemical stain-ing was performed to detect the protein expression of KLK5,MMP-9,and the number of CD4+T-positive cells.Western blot analysis was used to determine the protein expression levels of CD4+T cell-related factors including IFN-γ,CC chemokine receptor 5(CCR5),signal transducer and activator of transcription 1(STAT1),signal transducer and activator of transcription 3(STAT3).Results The skin at the injection site on the back of mice in the normal control group appeared normal without any skin lesions or erythema,and histopathological examination showed no in-filtration of inflammatory cells.The model group had significantly higher erythema scores and larger erythema areas compared to the normal control group(P<0.05),and histopathological examination revealed a significant infiltration of inflammatory cells,with some signs of deep connective tissue damage.Compared to the model group,the Jiawei Jingqin Decoction medium and high-dose groups showed a significant decrease in erythema scores and erythema areas(P<0.05),as well as a reduction in inflammatory exudates and inflammatory cell infiltration.However,the Jiawei Jingqin Decoction low-dose group did not show significant difference(P>0.05),but there was a slight reduction in inflammatory cell infiltration compared to the model group.When compared to the normal control group,the rosacea skin tissues of the model group showed an increase in the number of mast cells,levels of IL-1β,IL-6,TNF-α,mRNA expression of IL-1β,IL-6,TNF-α,TLR2,INF-γ,KLK5,MMP-9,protein expression of KLK5,MMP-9,and the number of CD4+T-positive cells(P<0.05).Compared to the model group,the Jiawei Jingqin Decoction medium and high-dose groups showed a decrease in the number of mast cells,levels of IL-1 β,mRNA expression of IL-1β,TNF-α,and the number of CD4+T-positive cells.The Jiawei Jingqin Decoction high-dose group showed a decrease in IL-6,TNF-α levels,mRNA expression of IL-6,TLR2,INF-γ,MMP-9,protein expression of MMP-9,KLK5,and the number of CD4+T-positive cells,as well as a decrease in IFN-y and STAT1 protein expression(P<0.05)compared to the Model group.Conclusion Jiawei Jingqin Decoction has significant improvement effect on the skin symptoms of roseacne in a mouse model.Its mechanism might be related to regulating skin immune inflamma-tory reactions,thereby reducing the release of pathogenic factors and inflammatory factors.

rosaceaCD4+T positive cellimmune responsesimmune responsesJiawei Jingqin Decoction(加味荆芩汤)

杨瑾、赵丽娟、伍迪、张旭、叶建州

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云南省中医医院,云南省昆明市五华区光华街120号,650021

玫瑰痤疮 CD4+T细胞 炎症因子 免疫反应 加味荆芩汤

国家中医药管理局第七批全国老中医药专家学术经验继承工作项目国家中医药管理局全国名老中医药专家传承工作室建设项目云南省高层次中医药后备人才培养对象云南省科学技术厅中医联合专项

国中医药人教函[2022]76号云卫中医发展发[2021]1号202101AZ070001-216

2024

中医杂志
中华中医药学会 中国中医科学院

中医杂志

CSTPCD北大核心
影响因子:1.464
ISSN:1001-1668
年,卷(期):2024.65(3)
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