JNK/NF-κB/Caspase-3在小鼠实验性牙周炎诱导肾细胞凋亡中的作用研究

摘要/Abstract

摘要： 目的：构建小鼠实验性牙周炎模型，探究c-Jun氨基末端激酶（JNK）/核因子-κB（NF-κB）/含半胱氨酸的天冬氨酸蛋白水解酶-3（Caspase-3）在牙周炎诱导的肾功能异常中的作用。方法：选取12只SPF级雄性C57小鼠随机分成对照组、牙周炎组，后者上颌第二磨牙颈部结扎丝线构建牙周炎模型，4周后处死，显微CT（Micro-CT）评估牙槽骨吸收情况；苏木精-伊红（HE）和过碘酸雪夫（PAS）染色观察小鼠牙周及肾脏的病理变化；测定肾脏丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）及血清中肾功能指标肌酐（Cre）和白蛋白（Alb）的含量。线粒体超氧化物红色荧光探针染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）染色评估肾脏氧化应激及凋亡情况；实时荧光定量PCR分析小鼠肾组织中NF-κB、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、Bcl2-相关X蛋白（Bax）及B细胞淋巴瘤-2基因（Bcl-2）的表达水平。免疫组织化学染色法测定肾脏JNK、磷酸化c-Jun氨基末端激酶（p-JNK）、NF-κB和Caspase-3的表达水平。结果：牙周炎组小鼠上颌第二磨牙牙龈红肿，Micro-CT见牙槽骨吸收，釉牙骨质界至牙槽嵴顶距离增加；牙周炎小鼠牙龈上皮钉突不规则伸长，伴牙槽嵴顶吸收，肾小球结构损伤及包曼氏囊腔扩张；牙周炎小鼠的肾小球基底膜基质增厚，肾小管刷状缘减少。牙周炎组的肾MDA、血清Cre和Alb升高（P＜0.05），肾SOD和GSH下降（P＜0.05）；牙周炎小鼠肾脏活性氧（ROS）（P＜0.05）及细胞凋亡数量均增多（P＜0.05）；牙周炎小鼠肾组织中IL-6、TNF-α、Bax、NF-κB的表达上升（P＜0.05），Bcl-2表达下降（P＜0.05），JNK、p-JNK、NF-κB和Caspase-3的表达显著增加（P＜0.05）。结论：JNK/NF-κB/Caspase-3可能通过影响肾细胞凋亡，在牙周炎诱导的肾功能异常中发挥重要作用。

Abstract: Objective:?To investigate the role of c-Jun N-terminal kinase (JNK)/nuclear factor-κB (NF-κB)/Caspase-3 in kidney dysfunction related to periodontitis by establishing a mouse model of experimental periodontitis. Methods:?Twelve SPF-grade male C57 mice were randomly divided into a control group and a periodontitis group. Periodontitis was induced by ligating silk around the necks of the bilateral maxillary second molars. Four weeks later, the mice were sacrificed, and the three-dimensional structure of the alveolar bone was reconstructed using Micro-CT to analyze bone resorption. Histological changes in periodontal and kidney tissues were observed using hematoxylin-eosin (HE) and periodic acid-schiff (PAS) staining. Biochemical kits were used to detect kidney oxidative stress biomarkers malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and kidney function indicators creatinine (Cre) and albumin (Alb). Mitochondrial superoxide red fluorescent probe staining was employed to assess ROS levels in kidney tissues, while terminal dexynucleotidyltransferase-mediated dUTPnick end labeling (TUNEL) staining evaluated renal cell apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure expression levels of NF-κB, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), B-cell lymphoma 2 (Bcl-2), and in B-cell lymphoma-2-associated X protein (Bax) kidney tissues. Immunohistochemistry was performed to detect phosphorylated JNK (p-JNK), JNK, NF-κB, and Caspase-3 expressions in kidney tissues. Results: Compared to the control group, mice in the periodontitis group exhibited gingival hyperemia and swelling around the maxillary second molars. Micro-CT analysis revealed significant alveolar bone resorption in the periodontitis group, with an increased distance from the enamel-dentin junction to the alveolar ridge crest. Iirregular elongation of the gingival epithelial rete pegs, accompanied by resorption at the alveolar ridge crest, damage to the glomerular structure, and expansion of Bowman's space were showed. The thickening of the glomerular basement membrane and a reduction in the brush border of renal tubules in the periodontitis group were indicated. Biochemical assays demonstrated elevated MDA levels, decreased SOD and GSH levels in the kidney tissue of periodontitis mice (P＜0.05), along with increased serum levels of Cre and Alb (P＜0.05). Elevated ROS levels in the renal tissue of periodontitis mice was shown(P＜0.05). TUNEL staining results indicated an increase in the number of apoptotic renal cells in the periodontitis group (P＜0.05). qRT-PCR and immunohistochemistry showed that the expressions of IL-6, TNF-α, Bax, and NF-κB in the kidney tissue of periodontitis mice were increased (P＜0.05), while the expression of Bcl-2 was decreased (P＜0.05). The expression of JNK, p-JNK, NF-κB, and Caspase-3 was significantly elevated (P＜0.05). Conclusions: The JNK/NF-κB/Caspase-3 may play a crucial role in affecting renal cell apoptosis, contributing to kidney dysfunction induced by periodontitis.

傅若冰邓愉刘歆婵商雅琦胡梦婷车贞贞于维先. JNK/NF-κB/Caspase-3在小鼠实验性牙周炎诱导肾细胞凋亡中的作用研究[J]. 口腔生物医学, 2025, 16(1): 26-33.

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