生物钟蛋白Bmal1对实验性牙周炎相关肾损伤的影响

doi:10.7518/hxkq.2024.2023245

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (2): 163-171.doi: 10.7518/hxkq.2024.2023245

生物钟蛋白Bmal1对实验性牙周炎相关肾损伤的影响

马浩楠¹(), 李琼¹, 商雅琦¹, 辛禧瑞¹, 刘歆婵², 武洲³, 于维先⁴^,⁵()

^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.日本九州大学齿学研究院，日本福冈 812-8582
^4.吉林大学口腔医院老年口腔科，长春 130021
^5.吉林省口腔生物医学国际联合研究中心，长春 130021

收稿日期:2023-08-07 修回日期:2023-09-25 出版日期:2024-04-01 发布日期:2024-03-26
通讯作者: 于维先 E-mail:mahn21@mails.jlu.edu.cn;ywx@jlu.edu.cn
作者简介:马浩楠，医师，硕士，E-mail：mahn21@mails.jlu.edu.cn
基金资助:
吉林省科技计划项目(20220402069GH)

Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury

Ma Haonan¹(), Li Qiong¹, Shang Yaqi¹, Xin Xirui¹, Liu Xinchan², Wu Zhou³, Yu Weixian⁴^,⁵()

^1.Dept. of Periodontology, Hospital of Stomatology, Jilin University, Changchun 130021, China
^2.Dept. of Implantology, Hospital of Stomatology, Jilin University, Changchun 130021, China
^3.Institute of Dentistry, Kyushu University, Fukuoka 812-8582, Japan
^4.Dept. of Geriatric Dentistry, Hospital of Stomatology, Jilin University, Changchun 130021, China
^5.Jilin Province International Joint Research Center for Oral Biomedicine, Changchun 130021, China

Received:2023-08-07 Revised:2023-09-25 Online:2024-04-01 Published:2024-03-26
Contact: Yu Weixian E-mail:mahn21@mails.jlu.edu.cn;ywx@jlu.edu.cn
Supported by:
Jilin Provincial Science and Technology Plan Project(20220402069GH)

摘要/Abstract

摘要：

目的通过建立大鼠牙周炎模型，探讨生物钟蛋白Bmal1对慢性牙周炎相关肾损伤的影响。方法将12只雄性Wistar大鼠随机分为对照组和牙周炎组，每组6只。采用正畸结扎丝对牙周炎组大鼠双侧上颌第一磨牙进行结扎处理，对照组大鼠不进行任何干预措施。8周后，检测2组大鼠的牙周临床指标，包括牙周探诊深度、牙龈出血指数和牙齿松动度。采用Micro-CT对大鼠上颌骨进行扫描和三维图像重建，评估牙槽骨吸收情况。采用苏木精-伊红（HE）和过碘酸雪夫（PAS）染色对牙周组织和肾组织进行病理学观察。采用生化试剂盒检测肾脏功能指标肌酐、白蛋白、血尿素氮的水平，以及氧化应激指标超氧化物歧化酶、谷胱甘肽和丙二醛的水平。MitoSOX red染色检测肾组织内活性氧（ROS）含量。实时荧光定量聚合酶链反应（RT-qPCR）和免疫组织化学染色检测大鼠肾组织中Bmal1、核因子-E2相关因子2（Nrf2）和血红素加氧酶1（HO-1）的基因及蛋白表达水平。结果与对照组相比，牙周组织Micro⁃CT及HE染色结果显示，牙周炎组上颌第一磨牙区出现明显的骨吸收和附着丧失；肾组织HE及PAS染色结果表明，牙周炎组大鼠肾组织有显著的组织病理损伤；肾功能和氧化应激指标结果显示，牙周炎组的氧化应激水平出现异常而肾功能指标无明显异常；MitoSOX red结果显示，牙周炎组肾组织内ROS含量升高；RT-qPCR和免疫组织化学结果显示，牙周炎组肾组织内Bmal1、Nrf2和HO-1表达水平降低。结论生物钟蛋白Bmal1在牙周炎大鼠肾脏的氧化损伤过程中发挥重要作用。

关键词: 牙周炎, 肾损伤, 昼夜节律, 氧化应激, 生物钟蛋白Bmal1

Abstract:

Objective To investigate the mechanism of circadian clock protein Bmal1 (Bmal1) on renal injury with chronic periodontitis, we established an experimental rat periodontitis model. Methods Twelve male Wistar rats were randomly divided into control and periodontitis groups (n=6, each group). The first maxillary molars on both sides of the upper jaw of rats with periodontitis were ligated by using orthodontic ligature wires, whereas the control group received no intervention measures. After 8 weeks, clinical periodontal parameters, including probing depth, bleeding index, and tooth mobility, were evaluated in both groups. Micro-CT scanning and three-dimensional image reconstruction were performed on the maxillary bones of the rats for the assessment of alveolar bone resorption. Histopatholo-gical observations of periodontal and renal tissues were conducted using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Renal function indicators, such as creatinine, albumin, and blood urea nitrogen levels, and oxidative stress markers, including superoxide dismutase, glutathione, and malondialdehyde levels, were measured using biochemical assay kits. MitoSOX red staining was used to detect reactive oxygen species (ROS) content in the kidneys. The gene and protein expression levels of Bmal1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in rat renal tissues were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Results Micro-CT and HE staining results showed significant bone resorption and attachment loss in the maxillary first molar region of the periodontitis group. Histological examination through HE and PAS staining revealed substantial histopathological damage to the renal tissues of the rats in the periodontitis group. The findings of the assessment of renal function and oxidative stress markers indicated that the periodontitis group exhibited abnormal levels of oxidative stress, whereas the renal function levels showed abnormalities without statistical significance. MitoSOX Red staining results showed that the content of ROS in the renal tissue of the periodontitis group was significantly higher than that of the control group, and RT-qPCR and immunohistochemistry results showed that the expression levels of Bmal1, Nrf2, and HO-1 in the renal tissues of the rats in the periodontitis group showed a decreasing trend. Conclusion Circadian clock protein Bmal1 plays an important role in the oxidative damage process involved in the renal of rats with periodontitis.

Key words: periodontitis, renal injury, circadian rhythm, oxidative stress, circadian clock protein Bmal1

中图分类号:

R781.4

马浩楠, 李琼, 商雅琦, 辛禧瑞, 刘歆婵, 武洲, 于维先. 生物钟蛋白Bmal1对实验性牙周炎相关肾损伤的影响[J]. 华西口腔医学杂志, 2024, 42(2): 163-171.

Ma Haonan, Li Qiong, Shang Yaqi, Xin Xirui, Liu Xinchan, Wu Zhou, Yu Weixian. Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury[J]. West China Journal of Stomatology, 2024, 42(2): 163-171.

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基因	引物序列（5’-3’）	基因号
β-actin	F：GGAGATTACTGCCCTGGCTCCTA	NM_024362.2
	R：AAGGGCTCCAAGGTCCACAG
Bmal1	F：GCTTTGAGGTGACCAGCAAGTACA	NM_031144.3
	R：AAGGGCTCCAAGGTCCACAG
Nrf2	F：TTGGCAGAGACATTCCCATTTGTA	NM_001399173.1
	R：GAGCTATCGAGTGACTGAGCCTG
HO-1	F：GAGACGGCTTCAAGCTGGTGATG	NM_012580.2
	R：GTTGAGCAGGAACGCAGTCTTGG

指标	对照组	牙周炎组
PD/mm	0.22±0.02	2.03±0.12***
BI	0.17±0.41	2.33±0.52***
TM	0.00±0.00	1.17±0.41***

项目	对照组	牙周炎组
BMD/mm³	974.67±13.80	841±21.28***
BV/TV	0.79±0.05	0.30±0.04***
Tb.Th/mm	0.26±0.04	0.12±0.02**
CEJ?ABC/mm	0.82±0.02	1.12±0.04***

指标	对照组	牙周炎组
SOD	80.65±2.05	68.54±4.08*
GSH	59.37±1.44	38.65±4.38***
MDA	1.54±0.02	3.55±0.21***
白蛋白	29.55±1.38	33.71±3.10
尿素氮	6.40±1.35	7.20±0.97
肌酐	34.54±4.45	41.54±2.16

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生物钟蛋白Bmal1对实验性牙周炎相关肾损伤的影响

Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury

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