活性氧/c-Jun氨基末端激酶/核因子-κB信号分子通过调控凋亡参与牙周炎诱导肝损伤

doi:10.7518/hxkq.2022.05.005

华西口腔医学杂志 ›› 2022, Vol. 40 ›› Issue (5): 532-540.doi: 10.7518/hxkq.2022.05.005

活性氧/c-Jun氨基末端激酶/核因子-κB信号分子通过调控凋亡参与牙周炎诱导肝损伤

曹牛奔¹(), 刘笑梦¹, 邓愉¹, 刘歆婵², 辛雨¹, 于维先¹^,³^,⁴()

^1.吉林大学口腔医院牙周病科，长春 130021
^2.吉林大学口腔医院种植科，长春 130021
^3.吉林大学口腔医院老年口腔科，长春 130021
^4.吉林省牙发育及颌骨重塑与再生重点实验室，长春 130021

收稿日期:2022-03-24 修回日期:2022-07-20 出版日期:2022-10-01 发布日期:2022-10-17
通讯作者: 于维先 E-mail:caonb20@mail.jlu.edu.cn;ywx@jlu.edu.cn
作者简介:曹牛奔，医师，硕士，E-mail：caonb20@mail.jlu.edu.cn
基金资助:
吉林省财政厅科技项目(JCSZ2021893-22);吉林省科技发展计划项目(20190201058JC)

Reactive oxygen species/c-Jun N-terminal kinase/nuclear factor kappa-B signaling molecules are involved in pe-riodontitis-induced liver injury by regulating apoptosis

Cao Niuben¹(), Liu Xiaomeng¹, Deng Yu¹, Liu Xinchan², Xin Yu¹, Yu Weixian¹^,³^,⁴()

^1.Dept. of Periodontics, Hospital of Stomatology, Jilin University, Changchun 130021, China
^2.Dept. of Dental Implantology, Hospital of Stomatology, Jilin University, Changchun 130021, China
^3.Dept. of Geria-tric Stomatology, Hospital of Stomatology, Jilin University, Changchun 130021, China
^4.Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Chang-chun 130021, China

Received:2022-03-24 Revised:2022-07-20 Online:2022-10-01 Published:2022-10-17
Contact: Yu Weixian E-mail:caonb20@mail.jlu.edu.cn;ywx@jlu.edu.cn
Supported by:
Science and Technology Project of Jilin Provincial Department of Finance(JCSZ2021893-22);Science and Technology Development Plan Project of Jilin Province(20190201058JC);Correspondence: Yu Weixian, E-mail: ywx@jlu.edu.cn

摘要/Abstract

摘要：

目的牙周炎和非酒精性脂肪性肝病（NAFLD）的发生发展与活性氧（ROS）的大量积累密切相关。ROS参与调控c-Jun氨基末端激酶（JNK）/核因子-κB（NF-κB）信号分子的激活，当该信号分子被ROS过度激活后可引发机体内环境紊乱。因此，本研究旨在探究ROS/JNK/NF-κB信号分子在牙周炎诱导肝损伤中的作用机制。方法将12只SPF级雄性Wistar大鼠随机分为对照组和牙周炎组，在牙周炎组大鼠双侧上颌第一磨牙颈部进行钢丝结扎构建牙周炎模型，8周后检查大鼠牙周临床指标并处死，Micro-CT重建牙槽骨三维结构并分析牙槽骨吸收情况，组织病理学分析牙周及肝组织的病理改变，MitoSOX red试剂检测肝组织中ROS含量，生化试剂盒检测肝功指标和氧化应激生物标志物，实时荧光定量聚合酶链反应（qRT-PCR）检测肝组织中NF-κB、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、BCL2-Associated X的蛋白质（Bax）和B淋巴细胞瘤-2基因（Bcl-2）mRNA表达水平，蛋白免疫印迹（Western blot）法检测肝组织中磷酸化c-Jun氨基末端激酶（P-JNK）、JNK、NF-κB、Bax、Bcl-2和半胱氨酸天冬氨酸蛋白酶3（Caspase-3）蛋白表达水平，末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色法检测肝细胞凋亡。结果 Micro-CT结果显示，牙周炎组大鼠牙槽骨骨质吸收明显，且釉牙骨质界到牙槽嵴顶的距离明显大于对照组。组织病理学结果显示，牙周炎组大鼠牙周组织内可见大量炎症细胞浸润和牙槽骨明显吸收；肝组织结构破坏，可见大量气球样变和红色脂滴形成。MitoSOX red染色结果显示牙周炎组肝组织中ROS含量明显升高。生化检测结果显示，牙周炎组血清中谷草转氨酶（AST）和谷丙转氨酶（ALT）含量升高；肝组织中超氧化物歧化酶（SOD）和谷胱甘肽（GSH）含量降低，丙二醛（MDA）含量升高。qRT-PCR和Western blot结果显示，牙周炎组肝组织中IL-6、TNF-α、Bax和NF-κB mRNA及P-JNK/JNK、NF-κB、Caspase-3和Bax蛋白表达水平较对照组显著上升，而Bcl-2 mRNA和蛋白表达水平下降。TUNEL染色结果显示牙周炎组肝组织中凋亡细胞数量明显增多。结论 ROS/JNK/ NF-κB信号分子通过调控细胞凋亡参与牙周炎诱导肝损伤。

关键词: 牙周炎, 肝损伤, 氧化应激, c-Jun氨基末端激酶, 核因子-κB, 凋亡

Abstract:

Objective The occurrence and development of periodontitis and nonalcoholic fatty liver disease (NAFLD) are closely related to the accumulation of reactive oxygen species (ROS). ROS are involved in regulating the activation of c-Jun N-terminal kinase (JNK)/nuclear factor kappa-B (NF-κB) signaling molecules. When the signaling molecules are overactivated by ROS, the internal environment of the body can be disturbed. Therefore, this study aimed to explore the mechanism by which ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury. Methods Twelve SPF male Wistar rats were randomly divided into control and periodontitis groups. The perio-dontitis model of rats was established by wire ligation in the neck of bilateral maxillary first molars. After 8 weeks, the periodontal clinical indexes of the rats were examined, and the rats were sacrificed. Micro-CT reconstruction of a three-dimensional alveolar bone structure and analysis of alveolar bone absorption were conducted. Pathological changes in the periodontal and liver tissues were analyzed by histopathology. MitoSOX red reagent was used to detect the ROS content in liver tissue. Biochemical kits were used to detect liver function and oxidative stress biomarkers. The mRNA expression levels ofinterleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), NF-κB, BCL2-associated X (Bax), and B-cell lymphoma-2 (Bcl-2) in liver tissue were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of phosphorylated c-Jun N-terminal kinase (P-JNK), JNK, NF-κB, Caspase-3, Bax, and Bcl-2 in liver tissue were detected by Western blot. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results Micro-CT results showed that the mice in the periodontitis group had obvious alveolar bone resorption and significantly greater distance from the cemento-enamel junction to the alveolar bone crest than those in the control group. Histopathological results showed that a large number of inflammatory cells were infiltrated in the periodontal tissue of the periodontitis group. In addition, the resorption of alveolar ridge bone was obvious and liver tissue structure was destroyed, with balloon-like changes and red lipid droplets. MitoSOX red staining results showed that the ROS level was significantly higher in the liver tissue of the periodontitis group than in that of the control group. Biochemical test results showed that the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the serum of the periodontitis group were higher than those in the serum of the control group. The levels of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue decreased, whereas the that of malondialdehyde (MDA) increased. Western blot and qRT-PCR results revealed that the mRNA levels of IL-6, TNF-α, Bax, and NF-κB and the protein levels of P-JNK/JNK, NF-κB, Caspase-3, and Bax were significantly higher in the liver tissue of the perio-dontitis group than in that of the control group. Meanwhile, the mRNA and protein levels of Bcl-2 were lower in the periodontitis group than in the control group. TUNEL staining showed that the number of apoptotic cells was significantly higher in the periodontitis group than in the control group. Conclusion ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury by regulating apoptosis.

Key words: periodontitis, liver injury, oxidative stress, c-Jun N-terminal kinases, nuclear factor kappa-B, apoptosis

中图分类号:

Q 257

曹牛奔, 刘笑梦, 邓愉, 刘歆婵, 辛雨, 于维先. 活性氧/c-Jun氨基末端激酶/核因子-κB信号分子通过调控凋亡参与牙周炎诱导肝损伤[J]. 华西口腔医学杂志, 2022, 40(5): 532-540.

Cao Niuben, Liu Xiaomeng, Deng Yu, Liu Xinchan, Xin Yu, Yu Weixian. Reactive oxygen species/c-Jun N-terminal kinase/nuclear factor kappa-B signaling molecules are involved in pe-riodontitis-induced liver injury by regulating apoptosis[J]. West China Journal of Stomatology, 2022, 40(5): 532-540.

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基因	引物序列（5’—3’）	碱基数/bp	基因号
IL-6^[14]	F：ATTGTATGAACAGCGATGATGCAC	20	NM_012489.2
	R：CCAGGTAGAAACGGAACTCCAGA	20
TNF-α^[14]	F：GGCGTGTTCATCCGTTCTC	24	NM_012675.3
	R：CTTCAGCGTCTCGTGTGTTTCT	23
NF-κB^[14]	F：CGACGTATTGCTGTGCCTTC	19	NM_199267.2
	R：TTGAGATCTGCCCAGGTGGTA	22
Bax^[15]	F：CCACCAAGAAGCTGAGCGA	19	NM_017059.2
	R：GCTGCCACACGGAAGAAGA	19
Bcl-2^[15]	F：CTCTGTGGATGACTGAGTACCTG	23	NM_016993.2
	R：GAGCAGCGTCTTCAGAGACAG	21
β-actin^[14]	F：GGAGATTACTGCCCTGGCTCCTA	23	NM_031144.3
	R：GACTCATCGTACTCCTGCTTGCTG	24

牙周临床指标	对照组	牙周炎组
PD/mm	0.21±0.03	2.26±0.08***
BI	0.20±0.20	2.60±0.25***
TM	0.00±0.00	1.00±0.32*

生化指标	对照组	牙周炎组
SOD/（U·mgprot^-1）	83.27±3.72	67.10±2.22*
GSH/（μmol·gprot^-1）	63.78±3.22	31.26±5.34**
MDA/（nmol·mgprot^-1）	1.56±0.08	2.14±0.13*
AST/（U·L^-1）	36.00±1.29	63.57±1.94***
ALT/（U·L^-1）	34.16±0.51	50.79±1.38***

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活性氧/c-Jun氨基末端激酶/核因子-κB信号分子通过调控凋亡参与牙周炎诱导肝损伤

Reactive oxygen species/c-Jun N-terminal kinase/nuclear factor kappa-B signaling molecules are involved in pe-riodontitis-induced liver injury by regulating apoptosis

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