痤疮丙酸杆菌生物膜诱导角质形成细胞发生炎症反应的分子机制研究

Molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes biofilms in keratinocytes

目的探讨痤疮丙酸杆菌生物膜诱导人原代角质形成细胞发生炎症反应的分子机制。方法体外构建痤疮丙酸杆菌生物膜模型,激光共聚焦显微镜观察其三维立体结构。将培养的人原代角质形成细胞分成3组:二甲基亚砜(DMSO)对照组(仅加入0.01%DMSO)、痤疮丙酸杆菌悬浮菌组(加入痤疮丙酸杆菌悬浮菌,简称悬浮菌组)、痤疮丙酸杆菌生物膜悬液组(加入痤疮丙酸杆菌生物膜悬液,简称生物膜悬液组)。实时荧光定量PCR(RT-qPCR)检测孵育6 h后各组白细胞介素6(IL-6)、IL-8以及肿瘤坏死因子α(TNF-α)mRNA相对表达水平,酶联免疫吸附试验(ELISA)检测孵育24 h后各组IL-6、IL-8及TNF-α游离蛋白水平,Western印迹法检测角质形成细胞Toll样受体2(TLR2)蛋白表达水平。进一步用TLR2/丝裂原激活蛋白激酶(MAPK)/核因子κB(NF-κB)信号通路关键分子的阻断剂(C29、ST2825、BAY11-7082、SB203580、U0126-EtOH)联合痤疮丙酸杆菌生物膜悬液作用于人原代角质形成细胞,同时设DMSO对照组和痤疮丙酸杆菌生物膜悬液阳性对照组,然后检测IL-6、IL-8及TNF-αmRNA和游离蛋白表达水平。多组间比较采用单因素方差分析,两两多重比较采用Bonferroni法。结果激光共聚焦显微镜下,痤疮丙酸杆菌生物膜形成草坪般的三维立体结构,生物膜生长状态良好。RT-qPCR及ELISA显示,生物膜悬液组、悬浮菌组、DMSO对照组炎症因子IL-6、IL-8、TNF-αmRNA及游离蛋白表达水平差异均有统计学意义(mRNA:F=89.70、312.17、46.09,均P<0.001;游离蛋白:F=886.12、634.25、307.01,均P<0.001);生物膜悬液组IL-6、IL-8、TNF-αmRNA及游离蛋白表达均显著高于悬浮菌组和DMSO对照组(均P<0.001);悬浮菌组IL-6 mRNA表达及TNF-α游离蛋白表达均显著高于DMSO对照组(P<0.001、=0.003),但IL-6游离蛋白、TNF-αmRNA、IL-8 mRNA及游离蛋白表达与DMSO对照组相比差异无统计学意义(均P>0.05)。Western印迹结果显示,生物膜悬液组和悬浮菌组TLR2蛋白表达均明显高于DMSO对照组。经不同MAPK/NF-κB通路抑制剂预处理及痤疮丙酸杆菌生物膜悬液共孵育,C29组、ST2825组、BAY11-7082组、SB203580组、U0126-EtOH组及DMSO对照组IL-6、IL-8、TNF-αmRNA及游离蛋白表达水平均显著低于生物膜悬液组(均P<0.05)。结论痤疮丙酸杆菌生物膜诱导人角质形成细胞发生炎症反应能力较强,可能通过激活TLR2/MAPK/NF-κB信号通路实现。

Objective To investigate molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes(C.acnes)biofilms in human primary keratinocytes.Methods A C.acnes biofilm model was established in vitro,and confocal fluorescence microscopy was performed to examine its three-dimensional structure.The cultured human primary keratinocytes were divided into 3 groups:a dimethyl sulfoxide(DMSO)control group(treated with 0.01%DMSO alone),a C.acnes suspension group(co-incubated with C.acnes suspensions),and a C.acnes biofilm group(co-incubated with C.acnes biofilms).Real-time fluorescence-based quantitative PCR(RT-qPCR)was performed to determine the relative mRNA expression of interleukin(IL)-6,IL-8,and tumor necrosis factor(TNF)-αin the groups after 6-hour culture,enzyme-linked immunosorbent assay to detect the free protein levels of IL-6,IL-8,and TNF-αin the groups after 24-hour culture,and Western blot analysis to determine the protein expression of Toll-like receptor 2(TLR2)in keratinocytes.In addition,some human primary keratinocytes were pretreated with key molecular blockers targeting the TLR2/mitogen-activated protein kinase(MAPK)/nuclear factor(NF)-κB signaling pathway(C29,ST2825,BAY11-7082,SB203580,U0126-EtOH),and then co-incubated with C.acnes biofilms;the DMSO control group and the C.acnes biofilm group receiving no pretreatment were simultaneously set as negative and positive controls,respectively.The mRNA and free protein expression levels of IL-6,IL-8,and TNF-αwere then detected in the above groups.One-way analysis of variance was used for comparisons among multiple groups,and the Bonferroni method was used for multiple comparisons.Results Confocal fluorescence microscopy demonstrated a three-dimensional C.acnes biofilm structure resembling a lawn,and the biofilm grew well.RT-qPCR and ELISA showed significant differences in the mRNA and free protein expression levels of IL-6,IL-8,and TNF-αamong the C.acnes biofilm group,C.acnes suspension group and DMSO control group(mRNA:F=89.70,312.17,46.09,respectively,all P<0.001;free protein:F=886.12,634.25,307.01,respectively,all P<0.001);in detail,the mRNA and free protein expression levels of IL-6,IL-8,and TNF-αwere significantly higher in the C.acnes biofilm group than in the C.acnes suspension group and DMSO control group(all P<0.001);the C.acnes suspension group showed significantly increased expression levels of IL-6 mRNA and TNF-αfree protein compared with the DMSO control group(P<0.001,=0.003,respectively),while there were no significant differences in the expression of IL-6 free protein,TNF-αmRNA,or IL-8 mRNA and free protein between the 2 groups(all P>0.05).Western blot analysis showed that the TLR2 protein expression was significantly higher in the C.acnes suspension group and C.acnes biofilm group than in the DMSO control group.After the pretreatment with molecular blockers targeting the MAPK/NF-κB signaling pathway and co-incubation with C.acnes biofilms,the mRNA and free protein expression levels of IL-6,IL-8 and TNF-αwere all significantly lower in the C29 group,ST2825 group,BAY11-7082 group,SB203580 group,U0126-EtOH group,as well as in the DMSO control group compared with the C.acnes biofilm group(all P<0.05).Conclusion The C.acnes biofilms exhibited a strong ability to induce inflammatory responses in human keratinocytes,possibly through the activation of the TLR2/MAPK/NF-κB signaling pathway.