摘要
目的革兰氏阴性菌能够诱发并加重慢性鼻窦炎(CRS)炎症反应,但病理机制有待进一步证实,而细胞焦亡被证实与细菌性炎症反应密切相关,因此本研究重点探讨来源于革兰氏阴性菌的关键成分脂多糖(LPS)是否能够通过诱导并加剧CRS患者鼻黏膜上皮细胞焦亡来促进CRS的炎症进展。方法利用临床上对照组和CRS组患者的鼻黏膜组织,采用IHC检测焦亡相关蛋白的表达。提取并培养正常原代人鼻黏膜上皮细胞(N-HNEpCs)和CRS原代人鼻黏膜上皮细胞(CRS-HNEpCs)。随后将2种细胞分为4组(对照组、LPS 1 mg/L组、LPS 5 mg/L组和LPS 25 mg/L组);CCK-8检测细胞存活率,EthD-I荧光染色结合GSDMD检测细胞焦亡,免疫荧光技术、Western blot、RT-qPCR检测炎性介质IL-18、IL-1β和NLRP3炎症小体(NLRP3、ASC、cleaved Caspase-1)的表达。结果IHC显示CRS患者鼻黏膜组织中NLRP3、cleaved Caspase-1、IL-18和IL-1β蛋白表达相较对照组显著增加(P<0.01)。体外实验结果表明,不同浓度的LPS刺激均可降低N-HNEpCs和CRS-HNEpCs的存活率(P<0.05),并可增加EthD-I荧光染色阳性细胞数和焦亡关键蛋白GSDMD的表达(P<0.05),且呈现出剂量依赖关系;免疫荧光技术、Western blot和RT-qPCR结果均显示5 mg/L LPS刺激后,N-HNEpCs和CRS-HNEpCs内炎性介质IL-18、IL-1β和NLRP3炎症小体的表达显著上调(P<0.05)。结论LPS可通过激活NLRP3炎症小体从而触发正常原代人鼻黏膜上皮细胞焦亡,并可进一步促进CRS原代人鼻黏膜上皮细胞焦亡进展,提示革兰氏阴性菌可能通过诱发鼻黏膜上皮细胞焦亡从而引起并加剧CRS病变。
It is well-recognized that gram-negative bacteria can induce and aggravate inflammatory process in chronic rhinosinusitis(CRS).However,the pathological mechanism needs to be further confirmed.Pyroptosis has been found to be closely related to inflammatory process.This study aimed at exploring whether LPS,a key component derived from Gram-negative bacteria,could induce and aggravate pyroptosis in primary human nasal epithelial cells(HNEpCs)in CRS.Nasal mucosa tissues were collected from CRS patients and control individuals.The expression levels of pyroptosis-related proteins were detected by IHC.Normal HNEpCs(N-HNEpCs)and CRS HNEpCs(CRS-HNEpCs)were extracted from nasal mucosa tissues and cultured.Then N-HNEpCs and CRSHNEpCs were randomly divided into four groups(Control group,LPS 1 mg/L group,LPS 5 mg/L group and LPS 25 mg/L group).Cell survival rate was detected by CCK-8;EthD-I staining combined with GSDMD was used to detect pyroptosis;the expression of inflammatory mediators IL-18,IL-1βand NLRP3 inflammasome(NLRP3,ASC,cleaved Caspase-1)were detected by immunofluorescence,Western blotting and RT-qPCR.IHC showed that the expressions of NLRP3,cleaved Caspase-1,IL-18 and IL-1βwere significantly increased(P<0.01)in CRS patients.In vitro,LPS stimulation of different concentrations could decrease the survival rate of N-HNEpCs and CRS-HNEpCs(P<0.05),while elevate the number of EthD-I staining positive cells and the expression of GSDMD(P<0.05)in a dose-dependent manner.Immunofluorescence,Western blot and RT-qPCR results showed that 5 mg/L LPS stimulation could significantly up-regulate the expression of inflammatory mediators IL-18,IL-1βand NLRP3 inflammasome in N-HNEpCs and CRS-HNEpCs(P<0.05).In conclusion,LPS can induce pyroptosis in N-HNEpCs by activating NLRP3 inflammasome,and can further promote the progression of pyroptosis in CRS-HNEpCs,suggesting that Gramnegative bacteria may cause and aggravate CRS lesions by inducing nasal epithelial cells pyroptosis.
作者
王睿智
朱锦祥
方彩珊
李丹
刘沁东
徐玮祯
阮岩
周敏
WANG Ruizhi;ZHU Jinxiang;FANG Caishan;LI Dan;LIU Qindong;XU Weizhen;RUAN Yan;ZHOU Min(First Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Department of Otolaryngology,First Affiliated Hospital,Guangzhou University of Chinese Medicine,Guangzhou 510405,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2023年第5期409-419,共11页
Immunological Journal
基金
国家自然科学基金(81974581,82274590)
广州市科技局基础与应用基础研究项目(2022-01-01-11-3016-0012)。
