炎性微环境牙周膜成纤维细胞整合素α5对NLRP3表达的影响  

Effect of integrinα5 on NLRP3 expression in periodontal ligament fibroblasts within an inflammatory microenvironment

作　　者：戴晶怡 蔡红宣 司为幸 张赞 王珠瑞 李孟森 田亚光 DAI Jingyi;CAI Hongxuan;SI Weixing;ZHANG Zan;WANG Zhurui;LI Mengsen;TIAN Yaguang(School of Stomatology,Hainan Medical University,Haikou 570100,China;Key Laboratory of Tumorigenesis and Intervention in Hainan Province,Hainan Medical University,Haikou 571199,China;Department of Stomatology,Hainan Affiliated Hospital of Hainan Medical University,Haikou 570100,China;Departmentof Stomatology,Hainan General Hospital,Haikou 570100,China)

机构地区：[1]海南医科大学口腔医学院,海南海口570100 [2]海南医科大学海南省肿瘤发生与干预重点实验室,海南海口571199 [3]海南医科大学附属海南医院口腔科,海南海口570100 [4]海南省人民医院口腔科,海南海口570100

出　　处：《口腔疾病防治》2025年第1期24-32,共9页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：海南省自然科学基金(822CXTD534);海南省重点研发计划项目(ZDYF2021SHFZ229)。

摘　　要：目的探讨炎性微环境牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)整合素α5(integrinα5)对NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)表达的影响。方法获得单位实验动物伦理委员会的批准,以脂多糖(lipopolysaccharide,LPS)(0.5、5、50μg/mL)预处理大鼠PDLFs 24 h后更换为无血清DMEM培养基,24 h后收集上清液分别与含10%无外泌体血清的DMEM培养基按体积比1∶1混合获得条件培养基(conditioned medium,CM),记作0.5-CM、5-CM、50-CM,另将含10%无外泌体血清的DMEM培养基记作0-CM。CM组以上述各浓度的CM培养PDLFs;抑制剂组以含有不同浓度整合素α5抑制剂ATN-161(0、0.025、0.25、2.5、25、250μg/mL)的0-CM培养PDLFs;CCK-8法检测上述各组CM和整合素α5抑制剂ATN-161对细胞活性的影响。根据CCK-8结果,在进一步的抑制剂干预实验中,将PDLFs培养于0-CM、不含/含25μg/mL ATN-161的5-CM及含25μg/mL ATN-161的0-CM中,依次为0-CM组、5-CM组、ATN-161+5-CM组及ATN-161组。利用Western blot及qRT-PCR检测整合素α5和NLRP3表达变化。体内实验将24只大鼠随机分为4组(n=6),对照组为健康大鼠,不做任何处理,其余3组大鼠每3天于大鼠左上颌第一磨牙腭侧分别注射40μL的含25μg/mL ATN-161的0-CM或5-CM(不含/含25μg/mL ATN-161),记作ATN-161组、5-CM组和ATN-161+5-CM组。第30天取大鼠左上颌骨组织行Micro-CT、HE染色及免疫组化检测。结果CCK-8检测显示,12 h、24 h时各组CM和25μg/mL及以下浓度的ATN-161对细胞活性抑制无显著差异(P>0.05);48 h时50-CM和25、250μg/mL ATN-161均显著抑制细胞活性(P<0.05)。在体外实验,相较于0-CM组,5-CM组大鼠PDLFs中整合素α5和NLRP3在蛋白和mRNA水平表达均显著升高(P<0.05);25μg/mL ATN-161干预显著抑制5-CM培养条件下整合素α5和NLRP3的表达(P<0.05)。在体内实验,与对照组相比,5-CM组和ATN-161+5-CM组牙槽骨吸收明显,牙周膜炎�Objective To investigate the effect of integrinα5 on the expression of NOD-like receptor thermal protein domain associated protein 3(NLRP3)in periodontal ligament fibroblasts(PDLFs)within an inflammatory microenvironment.Methods This study was approved by the Ethics Committee of Laboratory animals.After rat PDLFs were treated with LPS(0.5,5,and 50μg/mL)for 24 h,the primary medium was discarded and replaced with serum-free culture medium.After 24 h,the supernatant was collected and mixed with DMEM medium containing 10%exosome-free serum at a volume ratio of 1:1 to obtain conditioned medium(CM).The groups were labeled as the 0.5-CM,5-CM,and 50-CM groups.In addition,PDLFs cultured in DMEM medium containing 10%exosome-free serum were considered the 0-CM group.PDLFs were cultured with the above CM.In the inhibitor group,PDLFs were cultured in 0-CM containing different concentrations of integrinα5 inhibitor ATN-161(0,0.025,0.25,2.5,25,and 250μg/mL).The effect of CM and integrinα5 inhibitor ATN-161 on cell viability was assessed using the CCK-8 assay.According to the CCK-8 results,in further inhibitor intervention experiments,PDLFs were cultured in 0-CM,5-CM(without/with 25μg/mL ATN-161),and 0-CM containing 25μg/mL ATN-161,which were labeled as the 0-CM,5-CM,ATN-161+5-CM,and ATN-161 groups,respectively.The expression changes of integrinα5 and NLRP3 were detected using Western blot and qRT-PCR techniques.For in vivo experiments,24 rats were randomly divided into four groups(n=6).The control group contained healthy rats that received no treatment.The rats in the other three groups were injected with 40μL of 0-CM containing 25μg/mL ATN-161 or 5-CM(without or with 25μg/mL ATN-161)on the palatal side of the left maxillary first molar every three days;these groups were classified as the ATN-161,5-CM,and ATN-161+5-CM groups,respectively.On the 30th day,the left maxillary tissue of rats was used for Micro-CT,HE staining,and immunohistochemical detection.Results The CCK-8 assay showed that CM,25μg/mL ATN-161,and ATN-

关 键 词：牙周炎 整合素Α5 整合素α5抑制剂 牙周膜成纤维细胞 旁分泌 免疫反应 炎性微环境 NOD样受体热蛋白结构域相关蛋白3 条件培养基 

分 类 号：R78[医药卫生—口腔医学]