摘要
目的:探讨牙龈卟啉单胞菌(P.gingivalis)脂多糖(LPS)对人牙龈成纤维细胞(HGFs)焦亡的影响及可能的调控机制。方法:收集14例牙周炎患者和14例牙周健康者的牙龈组织样本,免疫组化染色检测焦亡相关分子消皮素D(GSDMD)、信号分子环鸟苷酸-腺苷酸合成酶(cGAS)及干扰素基因刺激蛋白(STING)的蛋白表达水平,并分析其与牙周临床指标的相关性。伴或不伴线粒体DNA(mtDNA)抑制剂溴化乙锭(ETBR)/STING抑制剂H151处理HGFs时,采用P.gingivalis LPS和腺苷三磷酸(ATP)共同刺激HGFs,流式细胞术检测线粒体活性氧(mtROS)总量变化,实时荧光定量PCR检测胞质中mtDNA水平,Western blot检测cGAS/STING和炎症小体NOD样受体蛋白3(NLRP3)及焦亡相关蛋白表达水平;免疫荧光染色观察胞内NLRP3和含CARD结构域的凋亡相关斑点样蛋白(ASC)共定位水平,扫描电镜观察细胞焦亡形态,并评估乳酸脱氢酶(LDH)释放水平。结果:与牙周健康者相比,牙周炎患者的牙龈组织中GSDMD、cGAS和STING表达增高(P<0.01),且三者表达水平与牙龈指数(GI)、探诊深度(PD)、临床附着丧失(CAL)均呈正相关(P<0.001)。P.gingivalis LPS+ATP刺激后,HGFs的mtROS水平升高,胞质mtDNA释放增加(P<0.001),cGAS、STING、NLRP3及焦亡相关蛋白表达水平升高(P<0.01),NLRP3和ASC共定位水平升高(P<0.001),同时导致HGFs细胞肿胀与变形,胞膜出现孔隙,LDH释放水平升高(P<0.001);而H151或ETBR预处理均能抑制上述改变(P<0.01)。结论:P.gingivalis LPS可能通过促进mtDNA释放激活cGAS/STING信号通路,进而活化NLRP3炎性小体,最终诱导HGFs焦亡,参与调控牙周免疫炎症反应。
Objective:To investigate the effects and potential mechanism of Porphyromonas gingivalis(P.gingivalis)and its lipopolysaccharide(LPS)on pyroptosis of human gingival fibroblasts(HGFs).Methods:Gingivae were collected from 14 periodontitis patients and 14 periodontally healthy individuals.The levels of gasdermin D(GSDMD),cyclic GMP-AMP synthase(cGAS)and stimulator of interferon gene(STING)were detected by immunohistochemistry,and their correlations with periodontal clinical parameters were analyzed.HGFs were stimulated with P.gingivalis LPS and adenosine triphosphate(ATP),with or without the treatment of STING inhibitor H151 or mitochondrial DNA(mtDNA)inhibitor ethidium bromide(ETBR).Total mitochondrial reactive oxygen species(mtROS)were detected by flow cytometry,and levels of mtDNA in cytoplasm were detected by real-time PCR.Levels of GSDMD,cGAS,STING,inflammasome NOD-like receptor protein 3(NLRP3)and pyroptosis-related proteins were detected by Western blot.The co-localization of intracellular NLRP3 and apoptosis-associated speck-like protein containing a CARD(ASC)was observed by immunofluorescence staining.The morphology of pyroptotic cells was observed by scanning electron microscopy.Meanwhile,levels of lactate dehydrogenase(LDH)were evaluated.Results:Compared with periodontally healthy individuals,the expression levels of GSDMD,cGAS and STING in gingivae of periodontitis patients were increased(P<0.01),and were positively correlated with gingival index(GI),probing depth(PD)and clinical attachment loss(CAL)(P<0.001).After stimulation with P.gingivalis LPS and ATP,the levels of mtROS and cytoplasmic mtDNA increased(P<0.001).P.gingivalis LPS significantly elevated the expression of cGAS,STING,the NLRP3 inflammasome and pyroptosis-related proteins(P<0.01),while also enhancing the co-localization of NLRP3 and ASC(P<0.001).These cells also displayed marked swelling and deformation,with pore formation in the plasma membrane,and the levels of LDH were significantly increased(P<0.001).Furthermore,these changes were effectively inhibited by H151 or ETBR(P<0.01).Conclusions:P.gingivalis and its LPS can promote the release of mtDNA,activate the NLRP3 inflammasome and promote pyroptosis in HGFs via cGAS/STING pathway,thereby exacerbating periodontal immune-inflammatory responses.
作者
李哲青
姚世怡
孙颖
仝悦
LI Zheqing;YAO Shiyi;SUN Ying;TONG Yue(Department of Periodontology,The Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China;State Key Laboratory Cultivation Base of Research,Prevention and Treatment for Oral Diseases(Nanjing Medical University),Nanjing 210029,China;Jiangsu Province Engineering Research Center of Stomatological Translational Medicine(Nanjing Medical University),Nanjing 210029,China;Department of Endodontics,The Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《口腔生物医学》
2026年第1期10-18,共9页
Oral Biomedicine
基金
江苏省自然科学基金(BK20231268)
江苏省卫健委重点项目(K2024035)
江苏省科教能力提升工程——江苏省研究型医院(YJXYYJSDW4),江苏省医学创新中心(CXZX202227)。
