下调G蛋白偶联受体C家族5A表达抑制脂多糖诱导的人牙龈成纤维细胞炎症反应  

Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts

作　　者：胡芋含 商玲玲 葛少华 Hu Yuhan;Shang Lingling;Ge Shaohua(Department of Periodontology,School and Hospital of Stomatology,Cheeloo College of Medicine,Shandong University&Shandong Key Laboratory of Oral Tissue Regeneration&Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration&Shandong Provincial Clinical Research Center for Oral Diseases,Jinan 250012,China)

机构地区：[1]山东大学齐鲁医学院口腔医学院·口腔医院牙周科、山东省口腔组织再生重点实验室口腔生物材料与组织再生山东省工程研究中心、山东省口腔疾病临床医学研究中心,济南250012

出　　处：《中华口腔医学杂志》2024年第4期344-353,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82170964,82201064)。

摘　　要：目的探究下调G蛋白偶联受体C家族5A(GPRC5A)表达对脂多糖诱导的人牙龈成纤维细胞(GFs)炎症反应的影响并探索其机制,为深入探讨G蛋白偶联受体(GPCR)在牙周炎中的调控作用及其机制奠定基础。方法于2022年12月至2023年2月在山东大学口腔医学院·口腔医院口腔颌面外科、牙周科收集3名牙周健康者(牙周健康组)和3例牙周炎患者(牙周炎组)的牙龈组织,采用免疫组化染色检测两组牙龈组织中GPRC5A的表达。收集健康牙龈组织提取GFs,健康牙龈组织来自2022年12月至2023年2月在山东大学齐鲁医学院口腔医学院·口腔医院口腔颌面外科招募的6例16~20岁需拔除埋伏阻生齿的患者。应用胶原酶消化法提取并培养GFs,设置30、50和80μmol/L浓度转染GPRC5A小干扰RNA(siGPRC5A)的实验组和阴性对照小干扰RNA(siNC),利用实时荧光定量PCR(RT-qPCR)检测siGPRC5A的沉默效率;设置阴性对照、脂多糖、siGPRC5A+脂多糖和siGPRC5A 4个组,通过RT-qPCR和蛋白质印迹法分别验证应用siGPRC5A对1 mg/L脂多糖诱导的GFs炎症状态下GPRC5A在基因和蛋白水平的表达;下调GPRC5A表达后,RT-qPCR检测脂多糖诱导的GFs白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子-α(TNF-α)、前列腺素内过氧化物合酶2(PTGS2)基因的表达;通过蛋白质印迹法、免疫荧光染色检测核因子-κB(NF-κB)信号通路的激活情况。结果免疫组化结果显示,牙周炎组牙龈组织中GPRC5A表达(0.132±0.006)显著高于牙周健康组(0.036±0.019)(t=8.24,P=0.001)。RT-qPCR结果显示,在脂多糖作用2、6、12和24 h时,脂多糖组GPRC5A基因水平表达量(分别为0.026±0.002、0.042±0.005、0.004±0.000、0.016±0.000)均分别显著高于阴性对照组(分别为0.004±0.000、0.004±0.000、0.002±0.000、0.007±0.000)(均P<0.001),且6 h时达峰值。50μmol/L siGPRC5A(31.16±3.29)与siNC组(100.00±4.88)相比能有效沉默GFs中GPRC5A的表达(F=297.98,P<0.001)。RT-qPCR和蛋白质印ObjectiveTo clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A(GPRC5A)on lipopolysaccharide(LPS)-induced inflammatory response in human gingival fibroblasts(GFs),thus to provide a foundation for delving into the role of G protein coupled receptor(GPCR)in periodontitis.MethodsGingival tissue samples were collected from 3 individuals periodontally healthy(health group)and 3 patients with periodontitis(periodontitis group)in Shandong Stomatological Hospital from December 2022 to February 2023.The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining.GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology,Cheeloo College of Medicine,Shandong University from December 2022 to February 2023.GFs were isolated with enzymic digestion and transfected with 30,50 and 80μmol/L small interfering RNA-GPRC5A(siGPRC5A)or small interfering RNA-negative control(siNC),regarded as the experimental group and the negative control one,respectively.The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR(RT-qPCR).Experiments were then conducted using these cells which were divided into four groups of negative control(NC),LPS,siGPRC5A+LPS and siGPRC5A.The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown.RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS,namely,interleukin(IL)-1β,IL-6,IL-8,tumor necrosis factor(TNF)-α,prostaglandin endoperoxide synthase 2(PTGS2)after GPRC5A knockdown.Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B(NF-κB)signaling pathway.ResultsImmunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group(0.132±0.006)increased co

关 键 词：牙周炎 G蛋白偶联受体 G蛋白偶联受体C家族5A 核因子ΚB 炎症因子 牙龈成纤维细胞 

分 类 号：R781.42[医药卫生—口腔医学]