LincRNA-EPS对牙龈卟啉单胞菌LPS诱导下小胶质细胞炎症反应的影响研究

doi:10.12439/kqhm.1005-4979.2025.04.004

摘要/Abstract

摘要：

目的：探究基因间区长链非编码RNA-EPS（long intergenic non-coding RNA-EPS，lincRNA-EPS）对牙龈卟啉单胞菌脂多糖（Porphyromonas gingivalis lipopolysaccharide，P.g-LPS）诱导下小胶质细胞炎症反应的影响。方法：体外用P.g-LPS刺激小鼠小胶质细胞系（BV-2细胞），实时定量聚合酶链反应（real‐time quantitative polymerase chain reaction，RT‐qPCR）检测lincRNA-EPS的表达变化。通过质粒转染构建lincRNA-EPS过表达的BV-2细胞，划痕实验观察lincRNA-EPS过表达对P.g-LPS诱导下BV-2细胞迁移能力的影响，RT‐qPCR、酶联免疫吸附实验（enzyme linked immunosorbent assay，ELISA）及蛋白质印迹法检测其炎症相关基因白细胞介素1β（interleukin 1β，IL-1β）、IL-6、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、精氨酸酶-1（arginase-1，Arg1）、核因子κB蛋白家族成员p65（nuclear factor kappa B protein family member p65，NF-κB p65）及磷酸化p65（phosphorylated p65，pp65）的mRNA和蛋白表达水平变化。免疫荧光染色法观察lincRNA-EPS过表达后BV-2细胞中p65的亚细胞定位。结果：对BV-2细胞进行体外P.g-LPS诱导后，lincRNA-EPS的表达下调（P<0.05）。过表达lincRNA-EPS可显著抑制P.g-LPS刺激后的BV-2细胞迁移及IL-1β、IL-6、TNF-α、iNOS的表达（P<0.05），上调静息状态时的Arg1表达（P<0.05）。过表达lincRNA-EPS后，BV-2细胞中总p65表达未见明显改变，p65入核减少，pp65表达水平降低（P<0.05）。结论：LincRNA-EPS可通过抑制p65磷酸化和核转位，减轻BV-2细胞在体外P.g-LPS刺激后的炎症反应。结论：康复新液联合甲硝唑可有效促进IMTM拔除术后患者的恢复，预防干槽症等并发症的发生，对牙槽骨吸收也具有一定的改善作用。

Abstract:

Objective: To investigate whether long intergenic non-coding RNA-EPS (lincRNA-EPS) affects microglial inflammatory response induced by Porphyromonas gingivalis lipopolysaccharides (P.g-LPS). Methods: Mouse microglia cell lines (BV-2 cells) were stimulated with P.g-LPS in vitro and the expression changes of lincRNA-EPS were detected by real‐time quantitative polymerase chain reaction (RT‐qPCR). BV-2 cells were transfected with plasmids to construct lincRNA-EPS overexpressed BV-2 cells. The effects of lincRNA-EPS overexpression on P.g-LPS-induced migration ability of BV-2 cells were observed by cell scratch assay, and the expression changes of inflammation-related genes interleukin 1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), arginase-1 (Arg1), nuclear factor kappa B protein family member p65 (NF-κB p65) and phosphorylated p65 (pp65) were detected by RT‐ qPCR, enzyme-linked immunosorbent assay (ELISA) and Western blotting. The subcellular localization of p65 in BV-2 cells was detected by immunofluorescence staining. Results: The expression of lincRNA-EPS was down-regulated after in vitro P.g-LPS induction of BV-2 cells (P<0.05). Overexpression of lincRNA-EPS significantly inhibited the P.g-LPS-induced migration of BV-2 cells and the expressions of IL-1β, IL-6, TNF-α, and iNOS (P<0.05). In contrast, the expression of Arg1 was up-regulated at resting state (P<0.05). After overexpression of lincRNA-EPS, the expression of total p65 in BV-2 cells was not significantly changed, while the p65 in the nucleus and pp65 expression were decreased (P<0.05). Conclusion: This study demonstrated that lincRNA-EPS can attenuate the inflammatory response of BV-2 cells stimulated by P.g-LPS in vitro by inhibiting the phosphorylation and nuclear translocation of p65.

中图分类号:

高建芳, 苏俭生. LincRNA-EPS对牙龈卟啉单胞菌LPS诱导下小胶质细胞炎症反应的影响研究[J]. 《口腔颌面外科杂志》, 2025, 35(4): 268-277.

GAO Jianfang, SU Jiansheng. Effect of lincRNA-EPS on the microglial inflammatory response induced by P.g-LPS: An experimental study[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(4): 268-277.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I4/268

图/表 9

表1 RT‐qPCR引物序列

Table 1 Primer sequences of RT‐qPCR

基因名	正向引物序列(5'-3')	反向引物序列(5'-3')
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
TNF-α	AGCCGATGGGTTGTACCTTG	ATAGCAAATCGGCTGACGGT
lincRNA-EPS	ATGTGTATTAGAGTTTTGCCT	TCTTTTCAAGCCCATATGTGA
p65	AGGCTTCTGGGCCTTATGTG	TGCTTCTCTCGCCAGGAATAC
Arg1	CTCCAAGCCAAAGTCCTTAGAG	AGGAGCTGTCATTAGGGACATC
iNOS	GTTCTCAGCCCAACAATACAAGA	GTGGACGGGTCGATGTCAC

图1 不同浓度P.g-LPS刺激后BV-2细胞活力及炎症基因的表达水平随时间的变化 A. CCK8检测细胞活力；B—D. P.g-LPS刺激后BV-2细胞中炎症相关基因mRNA表达水平变化。***表示P<0.001，与对照组（0 μg·mL^-1）比较。

Figure 1 Changes in BV-2 cell viability and expression level of inflammatory genes over time after stimulation with different concentrations of P.g-LPS

图2 P.g-LPS诱导后lincRNA-EPS表达水平的变化 **表示P<0.01，***表示P<0.001，与对照组（0 μg·mL^-1）比较。

Figure 2 Changes in lincRNA-EPS expression after P.g-LPS stimulation

图3 通过质粒转染在BV-2细胞中过表达lincRNA-EPS A.质粒转染后的免疫荧光染色图；B. lincRNA-EPS mRNA相对表达量，***表示P<0.001，与NC组比较。

Figure 3 Overexpression of lincRNA-EPS in BV-2 cells by plasmid transfection

图4 LincRNA-EPS过表达对BV-2细胞迁移能力的影响 A.划痕实验（×40）；B、C.依次为12 h和24 h的细胞迁移率，*表示P<0.05，与N组比较；#表示P<0.05，##表示P<0.01，与NL组比较。

Figure 4 Effect of lincRNA-EPS overexpression on the migration ability of BV-2 cells

图5 LincRNA-EPS过表达后BV-2细胞炎症基因的表达水平变化 A—C.各组细胞中炎症相关基因的mRNA相对表达水平；D—F.各组细胞培养上清液中炎症因子的浓度。**表示P<0.01，与N组比较；#表示P<0.05，##表示P<0.01，与NL组比较。

Figure 5 Changes in the expression levels of inflammatory genes in BV-2 cells after lincRNA-EPS overexpression

图6 LincRNA-EPS过表达后BV-2细胞极化相关基因及蛋白的表达水平变化 A—C.各组细胞中iNOS的表达水平；D—F.各组细胞中Arg1的表达水平。*表示P<0.05，**表示P<0.01，与N组比较；##表示P<0.01，与NL组比较。

Figure 6 Changes in the expression levels of polarization-related genes and proteins in BV-2 cells after lincRNA-EPS overexpression

图7 LincRNA-EPS的亚细胞定位（×400）

Figure 7 Subcellular localization of lincRNA-EPS（×400）

图8 LincRNA-EPS过表达后p65、pp65的表达变化 A.各组细胞中p65的免疫荧光染色图像（×400）；B. p65和DAPI共定位的Pearson相关系数统计结果；C.各组细胞中p65的mRNA表达水平；D. p65、pp65的蛋白条带；E、F.依次为各组细胞中p65、pp65的蛋白表达水平统计图。*表示P<0.05，与N组比较，#表示P<0.05，与NL组比较。

Figure 8 Changes in the expression levels of p65 and pp65 after lincRNA-EPS overexpression

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