牙龈卟啉单胞菌对载脂蛋白E基因敲除小鼠Th17/Treg失衡及血管内皮功能的影响

doi:10.13591/j.cnki.kqyx.2025.10.001

摘要/Abstract

摘要：

目的探究牙龈卟啉单胞菌(Porphyromonas gingivalis,P. gingivalis)对载脂蛋白E基因敲除(apolipoprotein E knockout,ApoE^-/-)小鼠Th17/Treg失衡及血管内皮功能的影响。方法高脂饮食下对8周龄ApoE^-/-小鼠给予P. gingivalis牙周涂菌,隔天一次,持续8周。通过Micro-CT检测小鼠牙槽骨吸收量,利用16S rRNA测序技术对小鼠口腔菌群进行分析,通过HE染色、油红O染色检测ApoE^-/-小鼠动脉粥样硬化斑块形成情况,采用生化检测技术分析小鼠血清中甘油三酯、总胆固醇、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇水平;利用实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)、免疫组织化学技术(immunohistochemistry,IHC)等检测模型主动脉斑块中的血管细胞黏附分子(vascular cell adhesion molecule,VCAM)、细胞间黏附分子(intercellular adhesion molecule,ICAM)、基质金属蛋白酶(matrix metallopeptidase,MMP)-2和MMP-9等内皮损伤指标;通过IHC、qRT-PCR、流式细胞术等技术检测主动脉斑块Th17和Treg相关蛋白白介素-17(interleukin-17,IL-17)、叉头/翼螺旋转录因子(forkhead box P3,Foxp3)及相关基因特征性转录视黄酸相关孤儿受体γt(retinoic acid related orphan receptor γt,RORγt)的表达水平和脾脏中的Th17和Treg比例。结果 P. gingivalis感染的ApoE^-/-小鼠牙槽骨丧失量增加(P<0.01),口腔菌群多样性下降(P<0.05),菌群组成发生改变,动脉粥样硬化斑块面积比例增加(P<0.01),甘油三酯、总胆固醇、低密度脂蛋白胆固醇浓度升高(P<0.05),高密度脂蛋白胆固醇浓度降低(P<0.01);主动脉斑块中ICAM、VCAM蛋白表达升高(P<0.05),ICAM、VCAM、MMP-2和MMP-9基因水平升高(P<0.05);主动脉斑块中的IL-17相关蛋白及RORγt基因水平升高(P<0.01),Foxp3相关蛋白及基因水平下降(P<0.01),脾脏中的Th17/Treg细胞比例失衡(P<0.05)。结论 P. gingivalis感染引起ApoE^-/-小鼠口腔的菌群失调,促进Th17/Treg平衡失调,加剧动脉粥样硬化过程中内皮功能障碍。

关键词: 牙龈卟啉单胞菌, 动脉粥样硬化, Th17/Treg失衡, 内皮功能障碍, 口腔菌群失调

Abstract:

Objective To investigate the effect of Porphyromonas gingivalis (P. gingivalis) on Th17/Treg imbalance and vascular endothelial function in ApoE^-/- mice. Methods Eight-week-old ApoE^-/- mice were inoculated orally with P. gingivalis every other day and received high-fat diet for 8 weeks. The alveolar bone loss was assessed by Micro-CT and the oral microflora of mice was analyzed by 16S rRNA sequencing technology. HE staining and oil red O staining were used to detect plaque formation in ApoE^-/- mice. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels of mice were detected by biochemical detection technology. Real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC) were used to detect the indicators of endothelial injury in the model aortic plaques, including vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), matrix metallopeptidase (MMP)-2 and MMP-9. The relative proportions of Th17 and Treg in aortic plaque and spleen were detected by IHC, qRT-PCR and flow cytometry, including interleukin-17 (IL-17), retinoic acid related orphan receptor γt (RORγt) and forkhead box P3 (Foxp3). Results In ApoE^-/- mice infected with P. gingivalis, the severity of alveolar bone loss was increased (P<0.01); the diversity of oral microbiota was decreased (P<0.05), and the composition of oral microbiota was changed. The proportion of atherosclerotic plaque area was increased (P<0.01), paralleled by elevated serum levels of TG, TC and LDL-C (P<0.05), alongside a significant decrease in HDL-C (P<0.01). Aortic plaques exhibited upregulated protein expression of VCAM and ICAM as well as heightened mRNA levels of ICAM, VCAM, MMP-2 and MMP-9 (P<0.05). The levels of IL-17 related protein and RORγt transcriptional activity in aortic plaque were increased (P<0.01), while the levels of Foxp3 related protein and gene were decreased (P<0.01). The ratio of Th17/Treg cells in spleen was imbalanced (P<0.05). Conclusion Periodontitis caused by the imbalance of the oral flora of ApoE^-/- mice induced by P. gingivalis can cause Th17/Treg imbalance in aortic plaque and exacerbate the endothelial dysfunction during atherosclerosis.

Key words: Porphyromonas gingivalis, atherosclerosis, imbalance of Th17/Treg, endothelial dysfunction, oral bacterial dysbiosis

中图分类号:

R781.4

王新越, 杨洁, 郝婷, 刘玉, 吴文蕾. 牙龈卟啉单胞菌对载脂蛋白E基因敲除小鼠Th17/Treg失衡及血管内皮功能的影响[J]. 口腔医学, 2025, 45(10): 721-730.

WANG Xinyue, YANG Jie, HAO Ting, LIU Yu, WU Wenlei. Effect of Porphyromonas gingivalis on Th17/Treg imbalance and vascular endothelial function in ApoE^-/- mice[J]. Stomatology, 2025, 45(10): 721-730.

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