巴戟天多糖通过上调沉默信息调节因子1抑制炎性牙周膜细胞NOD样受体热蛋白结构域相关蛋白3的表达及活性

doi:10.7518/hxkq.2023.2023114

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (6): 662-670.doi: 10.7518/hxkq.2023.2023114

巴戟天多糖通过上调沉默信息调节因子1抑制炎性牙周膜细胞NOD样受体热蛋白结构域相关蛋白3的表达及活性

蔡红宣¹(), 王正安², 张赞¹, 戴晶怡¹, 司为幸¹, 符起亚¹, 杨静文³(), 田亚光⁴^,⁵()

^1.海南医学院口腔医学院，海口 570100
^2.海南医学院第一附属医院口腔科，海口 570100
^3.北京大学口腔医院修复科，北京 100080
^4.海南医学院附属海南医院口腔科，海口 570100
^5.海南省人民医院口腔科，海口 570100

收稿日期:2023-04-14 修回日期:2023-09-27 出版日期:2023-12-01 发布日期:2023-11-27
通讯作者: 杨静文,田亚光 E-mail:Cai_hongxuan@163.com;jingwen.yang@foxmail.com;yaguangtian@163.com
作者简介:蔡红宣，医师，硕士，E-mail：Cai_hongxuan@163.com
基金资助:
海南省重点研发计划项目(ZDYF2021SHFZ229);海南省自然科学基金(822CXTD534)

Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1

Cai Hongxuan¹(), Wang Zheng’an², Zhang Zan¹, Dai Jingyi¹, Si Weixing¹, Fu Qiya¹, Yang Jingwen³(), Tian Yaguang⁴^,⁵()

^1.School of Stomatology, Hainan Medical University, Haikou 570100, China
^2.Dept. of Stomatology, The First Affiliated Hospital of Hainan Medical College, Haikou 570100, China
^3.Dept. of Prosthodontics, Peking University School and Hospital of Stomatology, Beijing 100080, China
^4.Dept. of Stomatology, Hainan Affiliated Hospital of Hainan Medical University, Haikou 570100, China
^5.Dept. of Stomatology, Hainan General Hospital, Haikou 570100, China

Received:2023-04-14 Revised:2023-09-27 Online:2023-12-01 Published:2023-11-27
Contact: Yang Jingwen,Tian Yaguang E-mail:Cai_hongxuan@163.com;jingwen.yang@foxmail.com;yaguangtian@163.com
Supported by:
Key Research and Development Project of Hainan(ZDYF2021SHFZ229);Hainan Provincial Natural Science Foundation of China(822CXTD534);Correspondence: Tian Yaguang, E-mail: yaguangtian@163.com;Yang Jing-wen, E-mail: jingwen.yang@foxmail.com

摘要/Abstract

摘要：

目的探讨炎性微环境下巴戟天多糖（MOP）对牙周膜细胞沉默信息调节因子1（SIRT1）及NOD样受体热蛋白结构域相关蛋白3（NLRP3）表达的影响。方法将30只大鼠随机分为对照组（n=6）和模型组（n=24），模型组采用正畸丝结扎法建立牙周炎模型，3周后每组各处死6只大鼠，Micro-CT检测确认建模成功。剩余模型组大鼠随机分为牙周炎自然恢复组、生理盐水（NS）组和MOP组。MOP组于大鼠左上颌第一磨牙腭侧注射MOP［200 mg/（kg·3d），50 µL，持续4周］，NS组注射等体积NS，牙周炎自然恢复组不做任何处理。取大鼠左上颌骨组织，采用苏木精-伊红（HE）染色观察牙周膜细胞病理改变，免疫组织化学检测SIRT1、NLRP3表达量。体外培养人牙周膜成纤维细胞（hPDLFs），CCK-8检测MOP对细胞活性的影响。将第4代细胞分为对照组、炎症组（10 µg/mL脂多糖）及实验组（5 µmol/L MOP，5 µmol/L MOP+10 µg/mL脂多糖）。利用实时定量聚合酶链反应（qRT-PCR）及Western blot检测SIRT1和NLRP3表达变化，免疫沉淀及酶联免疫法（ELISA）分别检测NLRP3乙酰化及白细胞介素（IL）-1β、IL-18含量。采用Prism 9.0软件对数据进行统计学分析。结果在体内实验中，MOP组较牙周炎自然恢复组和NS组NLRP3表达下降，SIRT1表达升高（P<0.05），炎细胞浸润减少。在体外实验中，与对照组相比，炎症组NLRP3 mRNA和蛋白表达量均增高（P<0.05），SIRT1表达降低（P<0.01）；MOP能上调炎症细胞SIRT1表达（P<0.05），降低NLRP3表达及其乙酰化水平（P<0.05），减少上清液中IL-1β、IL-18含量（P<0.01）。结论炎性牙周膜细胞SIRT1表达降低，NLRP3表达升高；MOP干预能促进SIRT1表达，导致NLRP3表达受抑制，同时通过去乙酰化作用使NLRP3乙酰化水平下降，从而NLRP3活性降低，由此发挥抑制炎症作用。

关键词: 牙周炎, 巴戟天多糖, 沉默信息调节因子1, NOD样受体热蛋白结构域相关蛋白3, 去乙酰化

Abstract:

Objective This study aims to investigate the effect of morinda officinalis polysaccharides (MOP) in inflammatory microenvironment on the expression of silent information regulator sirtuin 1 (SIRT1) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in periodontal ligament cells. Methods Thirty rats were randomly divided into control group (n=6) and model group (n=24). The model group used orthodontic wire ligation to establish periodontitis, and six rats from each group were killed after 3 weeks. The successful modeling was confirmed by Micro-CT. The remaining rats in the model group were randomly divided into natural recovery group, normal saline (NS) group, and MOP group. In the MOP group, MOP [200 mg/(kg·3d), 50 µL for 4 weeks] was injected into the palatal side of the left maxillary first molar of the rats, while the NS group was injected with equal volume of NS. The natural recovery group did not undergo any treatment. The left maxilla tissues of the rats were collected, and pathological changes in perio-dontal ligament cells were observed by hematoxylin-eosin (HE) staining. The expression of SIRT1 and NLRP3 was detected by immunohistochemistry. Cultivate periodontal ligament fibroblasts in vitro and detect the effect of MOP on cell activity using CCK-8. The 4th generation cells were divided into control group, inflammation group (10 µg/mL lipopolysaccharide), and experimental group (5 µmol/L MOP, 5 µmol/L MOP+10 µg/mL lipopolysaccharide). The expression of SIRT1 and NLRP3 was detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot analyses. The acetylation of NLRP3 and the contents of interleukin (IL)-1β and IL-18 were detected by immunoprecipitation and enzyme-linked immunosorbent assay, respectively. Statistical analysis of data was conducted using Prism 9.0 software. Results In the vivo experiments, the expression of NLRP3 and SIRT1 in the MOP group decreased significantly compared with that in the natural recovery group and NS group, while the expression of SIRT1 increased (P<0.05) and inflammatory cell infiltration decreased. In the in vitro experiments, the expression of NLRP3 mRNA and protein in the inflammation group increased (P<0.05), while the expression of SIRT1 significantly decreased (P<0.01); MOP upregulated the expression of SIRT1 in inflammatory cells (P<0.05), reduced the expression of NLRP3 and its acetylation level significantly (P<0.05), suppressed the content of IL-1β and IL-18 in the supernatant (P<0.01). Conclusion The SIRT1 expression decreased, and that of NLRP3 expression increased in inflammatory periodontal ligament cells. MOP intervention promoted SIRT1 expression, resulting in the inhibition of NLRP3. Meanwhile, the acetylation level of NLRP3 reduced through deacetylation, leading to the decreased activity of NLRP3. Thus, MOP acted as inflammatory suppressor.

Key words: periodontitis, morinda officinalis polysaccharides, silent information regulator transcript 1, NOD-like receptor thermal protein domain associated protein 3, deacetylation

中图分类号:

Q 257

蔡红宣, 王正安, 张赞, 戴晶怡, 司为幸, 符起亚, 杨静文, 田亚光. 巴戟天多糖通过上调沉默信息调节因子1抑制炎性牙周膜细胞NOD样受体热蛋白结构域相关蛋白3的表达及活性[J]. 华西口腔医学杂志, 2023, 41(6): 662-670.

Cai Hongxuan, Wang Zheng’an, Zhang Zan, Dai Jingyi, Si Weixing, Fu Qiya, Yang Jingwen, Tian Yaguang. Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1[J]. West China Journal of Stomatology, 2023, 41(6): 662-670.

图/表 7

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参考文献 29

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基因	引物序列（5’-3’）
NLRP3	F：AAGCACCTGTTGTGCAATCTGAAG
	R：GGGAATGGCTGGTGCTCAATAC
SIRT1	F：AATATATCCCGGACAGTTCCAGCC
	R：ATCCTTTGGATTCCTGCAACCTGC
β-actin	F：ATGCTCTCCCTCACGCCATCCT
	R：ATCGGAACCGCTCGTTGCCAAT

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巴戟天多糖通过上调沉默信息调节因子1抑制炎性牙周膜细胞NOD样受体热蛋白结构域相关蛋白3的表达及活性

Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1

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