缝隙连接蛋白43通过调控凋亡参与大鼠牙周炎相关肾损伤

doi:10.7518/hxkq.2024.2023352

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (3): 296-303.doi: 10.7518/hxkq.2024.2023352

缝隙连接蛋白43通过调控凋亡参与大鼠牙周炎相关肾损伤

辛雨¹(), 傅若冰¹, 辛禧瑞¹, 商雅琦¹, 刘歆婵², 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:2023-10-21 修回日期:2024-02-26 出版日期:2024-06-01 发布日期:2024-05-24
通讯作者: 于维先 E-mail:xin_yu21@mail.jlu.edu.cn;ywx@jlu.edu.cn
作者简介:辛雨，医师，硕士，E-mail：xin_yu21@mail.jlu.edu.cn
基金资助:
吉林省科技厅国际合作项目(20220402069GH)

Role of connexin 43 in a rat model of periodontitis-induced renal injury

Xin Yu¹(), Fu Ruobing¹, Xin Xirui¹, Shang Yaqi¹, Liu Xinchan², 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 Geriatric Stomatology, Hospital of Stomatology, Jilin University, Changchun 130021, China
^4.Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Changchun 130021, China

Received:2023-10-21 Revised:2024-02-26 Online:2024-06-01 Published:2024-05-24
Contact: Yu Weixian E-mail:xin_yu21@mail.jlu.edu.cn;ywx@jlu.edu.cn
Supported by:
International Cooperation Project of Jilin Provincial Department of Science and Technology(20220402-069GH)

摘要/Abstract

摘要：

目的本研究旨在探讨缝隙连接蛋白43（Cx43）在大鼠牙周炎诱导慢性肾损伤模型中的作用。方法按照完全随机数字表法将12只SPF级Wistar雄性大鼠分为对照组和牙周炎组，每组6只。对照组大鼠不做处理，牙周炎组大鼠采用钢丝结扎大鼠双侧上颌第一磨牙的颈部，构建牙周炎模型。建模8周后检查大鼠牙周临床指标。显微CT（micro‑CT）扫描大鼠上颌骨重建其三维结构并分析牙槽骨吸收情况；组织病理学检测牙周及肾组织的病理改变；MitoSOX red试剂检测肾组织中活性氧（ROS）含量；生化试剂盒检测血清氧化应激生物标志物；实时荧光定量聚合酶链反应（qRT-PCR）检测肾组织中Cx43、核因子-κB（NF-κB）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、BCL2-Associated X的蛋白质（Bax）、B淋巴细胞瘤-2基因（Bcl-2）和半胱氨酸天冬氨酸蛋白酶3（Caspase-3）mRNA表达水平，蛋白免疫印迹（Western blot）法检测肾组织中Cx43、NF-κB、IL-1β、Bax、Bcl-2和Caspase-3蛋白表达水平。结果 micro-CT三维重建结果显示，牙周炎组大鼠第一磨牙牙槽骨骨质吸收明显，牙槽嵴高度降低，且釉牙骨质界到牙槽嵴顶的距离显著大于对照组。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；牙周炎组大鼠肾组织中肾小球基底膜轻度增厚，鲍曼氏囊腔扩张，肾小管刷状缘破坏。MitoSOX red染色结果显示牙周炎组肾组织中ROS含量明显升高。生化检测结果显示，牙周炎组大鼠血清中超氧化物歧化酶和谷胱甘肽水平降低，丙二醛水平升高。qRT-PCR和Western blot结果显示，牙周炎组肾组织中Cx43、IL-1β、IL-6、Bax和Caspase-3 mRNA及Cx43、IL-1β、NF-κB、Bax和Caspase-3蛋白表达水平较对照组上升，而Bcl-2 mRNA及蛋白表达水平下降。结论牙周炎可能通过上调大鼠肾组织中Cx43的表达激活NF-κB信号分子，引起大鼠肾脏组织中炎症水平和凋亡水平升高，最终诱导肾脏损伤的发生。

关键词: 牙周炎, 肾损伤, 缝隙连接蛋白43, 大鼠, 凋亡

Abstract:

Objective This study aims to investigate the role of gap junction mediated by connexin 43 (Cx43) in renal injury induced by periodontitis in rats. Methods Twelve SPF-grade Wistar male rats were divided into a control group and a periodontitis group by using a completely random number table method, with six rats in each group. The control group rats were not treated, while the periodontitis group rats were subjected to wire ligation of the neck of their bilateral maxillary first molars to construct a periodontitis model. After 8 weeks of modeling, the rats were examined for clinical indicators of the periodontium. micro-CT scanning of the maxilla reconstructed its 3D structure and analyzed the absorption of alveolar bone. Histopathological changes in periodontal and renal tissues were detected. MitoSOX red reagent was used to determine reactive oxygen species (ROS) content in renal tissues. A biochemical reagent kit was used to detect serum oxidative stress biomarkers. Real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR) was employed to determine Cx43, nuclear factor kappa-B (NF-κB) , interleukin (IL)-1β, IL-6, BCL2-Associated X (Bax), B-lymphomatoma-2 gene (Bcl-2), and Caspase-3 mRNA were determined. Western blot analysis was used to detect Cx43, NF-κB, IL-1β, Bax, Bcl-2 and Caspase-3 protein. Results micro-CT 3D reconstruction showed significant bone resorption of the first molar alveolar bone in the periodontitis group rats and decreased height of the alveolar ridge. The distance from the enamel cementum boundary to the top of the alveolar ridge in the periodontitis group was significantly higher than that inthe control group. The histopathological results showed a large number of inflammatory cells that infiltrated the periodontal tissue of the periodontitis group, and the alveolar bone was significantly absorbed. Rats in the periodontitis group also exhibited mild thickening of the glomerular basement membrane, dilation of the Bowman’s capsule, and destruction of the brush-like edge of the renal tubules in the renal tissue. The MitoSOX red staining results showed a significant increase in ROS content in the renal tissue of the periodontitis group. The biochemical test results showed that the levels of superoxide dismutase and glutathione in the serum of rats with periodontitis decreased, while that of malondialdehyde increased. The results of qRT-PCR and Western blot showed that the expression levels of Cx43, IL-1β, IL-6, Bax, Caspase-3 mRNA and Cx43, IL-1β, NF-κB, Bax, Caspase-3 proteins in the periodontitis group significantly increased compared with those in the control group, while the expression levels of Bcl-2 mRNA and protein decreased. Conclusion Periodontitis may activate NF-κB signaling molecules by upregulating the expression of Cx43 in rat kidney tissues, leading to increased levels of inflammation and apoptosis and ultimately inducing kidney injury.

Key words: periodontitis, renal injury, connexin 43, rat, apoptosis

中图分类号:

R781.4

辛雨, 傅若冰, 辛禧瑞, 商雅琦, 刘歆婵, 于维先. 缝隙连接蛋白43通过调控凋亡参与大鼠牙周炎相关肾损伤[J]. 华西口腔医学杂志, 2024, 42(3): 296-303.

Xin Yu, Fu Ruobing, Xin Xirui, Shang Yaqi, Liu Xinchan, Yu Weixian. Role of connexin 43 in a rat model of periodontitis-induced renal injury[J]. West China Journal of Stomatology, 2024, 42(3): 296-303.

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基因	引物序列（5’-3’）	碱基数/bp	基因号
Cx43	F：ACTGAGCCCATCCAAAGACT	20	NM_010288.3
	R：GCTGTAATTCGCCCAGTTTT	20
IL-1β	F：GCAACTGTTCCTGAACTCAAC	21	NM_008361.4
	R：ATCTTTTGGGGTCCGTCAACT	21
IL-6	F：ATTGTATGAACAGCGATGATGCAC	20	NM_012489.2
	R：CCAGGTAGAAACGGAACTCCAGA	20
Bax	F：CCACCAAGAAGCTGAGCGA	19	NM_017059.2
	R：GCTGCCACACGGAAGAAGA	19
Bcl-2	F：CTCTGTGGATGACTGAGTACCTG	23	NM_016993.2
	R：GAGCAGCGTCTTCAGAGACAG	21
Caspase-3	F：GGGGAGCTTGGAACGCTAAG	20	NM_009810.3
	R：CACACACACAAAGCTGCTCC	20
β-actin	F：GGAGATTACTGCCCTGGCTCCTA	23	NM_031144.3
	R：GACTCATCGTACTCCTGCTTGCTG	24

牙周临床指标	对照组	牙周炎组
PD/mm	0.20±0.02	2.45±0.08***
BI	0.19±0.20	2.54±0.24***
TM	0.00±0.00	1.00±0.27*

生化指标	对照组	牙周炎组
SOD/（U/mL）	188.67±8.65	145.3±5.27**
GSH/（U/L）	10.14±0.98	4.99±0.42**
MDA/（nmol/mL）	6.19±0.87	8.83±0.72*

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缝隙连接蛋白43通过调控凋亡参与大鼠牙周炎相关肾损伤

Role of connexin 43 in a rat model of periodontitis-induced renal injury

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