钙池操纵性钙内流对特应性皮炎上皮屏障功能的影响
Impacts of store-operated calcium entry on the function of epithelial barrier in atopic dermatitis
曹灿 1刘光辉 2王畅畅 1马如雪 1贾欣睿 3钟剑 1高亚东1
作者信息
- 1. 武汉大学中南医院过敏反应科 湖北 武汉 430071
- 2. 华中科技大学协和深圳医院过敏防治中心 广东 深圳 518052
- 3. 武汉市第三医院皮肤科 湖北 武汉 430060
- 折叠
摘要
目的:探讨钙池操纵性钙内流(SOCE)及其介导的钙信号在特应性皮炎(AD)上皮屏障破坏中的作用.方法:6周龄BALB/c小鼠,采取胶带粘贴及 1%DNCB致敏法建立AD模型,分为对照组、AD组、SOCE抑制剂BTP2处理的AD组(AD-BTP2)和外用糖皮质激素处理的AD组(AD-TCS).采用组织学检查表皮厚度,通过皮水分丢失(TEWL)测定小鼠皮肤屏障功能.体外培养角质形成细胞HaCaT,分为对照组、尘螨(HDM)提取物刺激组和HDM刺激并BTP2干预组(HDM-BTP2),采用MTT法进行细胞增殖试验,RT-PCR、Western Blot法分别检测SOCE及上皮屏障相关基因的表达.FITC-右旋糖酐通透性实验评估HaCaT细胞屏障功能.结果:AD组小鼠较对照组小鼠的经皮水分丢失和表皮增厚均更为显著(P<0.000 1);而BTP2处理后AD小鼠的经皮水分丢失(P<0.05)和表皮增厚(P<0.000 1)进一步加重.HDM刺激可以促进HaCaT细胞的增殖,而BTP对细胞增殖没有影响.HDM刺激后HaCaT细胞SOCE相关蛋白Orai1～3,STIM1～2和SARAF的mRNA表达与对照组细胞无差异,但Orai1蛋白表达显著高于对照组(P<0.05).HDM刺激HaCaT细胞后屏障相关闭合蛋白Occludin和兜甲蛋白 Loricrin表达下调;而 BTP2 处理后可以恢复 Occludin和增加 E-cadherin的表达.HDM组 HaCaT细胞的FITC-右旋糖酐通透性高于对照组细胞,而1 μmol/L BTP2可以显著降低HDM诱导的FITC-右旋糖酐通透性改变.结论:在AD动物模型和细胞水平,SOCE抑制剂BTP2对上皮屏障具有相反的效应,提示SOCE在2型炎症中作用的复杂性,相关机制还需要进一步研究.
Abstract
Objective:To explore the impacts of store-operated calcium entry(SOCE)on the epithelial barrier of atopic dermatitis(AD).Methods:AD model in 6-week BALB/c mouse was built by tape stripping and 1%DNCB challenging and mice were divided into 4 groups:control group,AD group,AD-BTP2 group,and AD-TCS group.The effects of inhibiting SOCE in vivo were evaluated by trans-epidermal water loss(TEWL)and histology of the epidermis.HaCaT cells were cultured in vi-tro and cell proliferation was tested by MTT assay after HDM and BTP2 stimulation.RT-PCR and Western-Blot of SOCE and epithelial barrier-related genes in HaCaT cells proceeded for expression changes and carried out FITC-dextran assay for cell barrier function evaluation.Results:TEWL and epidermis thickness significantly increased in AD group mice than in the control group(P<0.000 1).However,the BTP2-treated group showed a more significant increase in TEWL(P<0.05)and epi-dermis thickness(P<0.000 1)as compared with the AD group.HDM stimulation promotes HaCaT cell proliferation,but BTP2 showed no effects.The mRNA expression levels of SOCE-related genes had no significant change after HDM stimulation in HaCaT cells.The mRNA expression of SOCE-related proteins Orai1-Orai3,STIM1,STIM1,and SARAF in HaCaT cells after HDM stimulation did not differ from those in control cells,but the protein expression of Orai1 was significantly higher(P<0.05).Occludin and Loricrin,two barrier-related proteins,decreased after HDM stimulation,and BTP2 treatment could restore the expression of Occludin and elevate E-cadherin expression.HaCaT cells in the HDM group showed higher permeability of FITC-dextran than the control group,and BTP2(1 μmol/L)treatment significantly decreased the HDM-induced permeability changes.Conclu-sion:BTP2,the inhibitor of SOCE,shows opposite influences in the AD mouse model and HaCaT cell,which suggests the complexity of SOCE in type 2 inflammation,and further study of the underly-ing mechanisms needs to be conducted.
关键词
钙池操纵性钙内流/特应性皮炎/上皮屏障Key words
Store-Operated Calcium Entry/Atopic Dermatitis/Epithelial Barrier引用本文复制引用
出版年
2024
NETL
NSTL
万方数据