查看更多>>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80 μmol/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。50 μmol/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和蛋白质印迹法结果显示，脂多糖组GPRC5A在基因和蛋白水平的表达（分别为1.30±0.10、1.43±0.03）均显著高于阴性对照组（分别为1.00±0.01、1.00±0.00）（均P<0.001），siGPRC5A组（分别为0.27±0.03、0.71±0.00）均显著低于阴性对照组（分别为1.00±0.01、1.00±0.00）（均P<0.001），siGPRC5A+脂多糖组（分别为0.39±0.03、1.06±0.16）均显著低于脂多糖组（分别为1.30±0.10、1.43±0.03）（均P<0.001）。RT-qPCR结果显示，脂多糖组IL-8、IL-1β、IL-6、TNF-α、PTGS2基因表达水平均显著高于阴性对照组，siGPRC5A+脂多糖组均显著低于脂多糖组（均P<0.001）。蛋白质印迹法结果表明，脂多糖组p65和IκBα蛋白磷酸化水平均显著高于阴性对照组，而siGPRC5A+脂多糖组均显著低于脂多糖组（均P<0.01）。免疫荧光染色结果显示，对照组NF-κB p65集中表达于胞质，在脂多糖刺激下p65部分向核内转位，应用siGPRC5A后能一定程度上抑制脂多糖诱导的p65核内转位。 结论 GPRC5A在GFs炎症状态下表达增加，下调GPRC5A的表达能抑制脂多糖诱导的炎症反应，且GPRC5A可通过NF-κB信号通路发挥炎症调控的作用。 Objective To 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. Methods Gingival 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 Shandong Stomatological Hospital from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/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. Results Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in GPRC5A expression (31.16±3.29) compared with that of the NC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001 F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.
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