自噬在延期牙再植术后炎症性牙根吸收中的作用研究

口腔生物医学 ›› 2021, Vol. 12 ›› Issue (3): 149-154.

自噬在延期牙再植术后炎症性牙根吸收中的作用研究

雷心怡¹,张旗²

1. 同济大学附属口腔医院
2. 同济大学口腔医学院·同济大学附属口腔医院牙体牙髓科

收稿日期:2021-03-01 修回日期:2021-07-20 出版日期:2021-09-25 发布日期:2021-09-27
通讯作者: 张旗 E-mail:qizhang@tongji.edu.cn
基金资助:
牙周膜成纤维细胞外泌体递送的骨保护素对再植牙根吸收的保护作用及机制研究;骨保护素在牙根吸收中的多重保护作用和机制的研究

Effects of autophagy in inflammatory root resorption after delayed tooth replantation

Received:2021-03-01 Revised:2021-07-20 Online:2021-09-25 Published:2021-09-27

摘要/Abstract

摘要： 目的：观察大鼠延期牙再植术后自噬水平变化，探讨自噬在延期牙再植术后炎症性牙根吸收中的作用。方法：选取24只4周龄SD大鼠，拔除双侧上颌第一磨牙，干燥30 min后植回牙槽窝，术后1、3、7、28 d取材，染色观察炎症浸润情况、牙根表面吸收情况、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)等炎症因子表达及LC3B和Beclin-1等自噬相关蛋白的表达情况。使用1μg/mL脂多糖(LPS)处理人牙周膜成纤维细胞(hPDLFs)，细胞免疫荧光染色观察不同时间点LC3B和Beclin-1的表达，LPS中分别添加自噬促进剂雷帕霉素和自噬抑制剂氯喹，实时定量RT-PCR检测炎症和自噬相关蛋白的基因转录水平。结果：牙再植术后第1、3、7天牙周组织中炎症细胞浸润、炎症因子分泌逐渐增加，第7天时炎症性牙根吸收明显，第28天炎症明显好转。自噬相关蛋白表达在术后第1天明显上升，第3、7天表达下降，第28天再次增多。体外细胞免疫荧光染色结果显示自噬相关蛋白在LPS刺激24 h时被激活，72 h时被抑制，体外细胞与体内组织自噬相关蛋白表达趋势一致。实时定量RT-PCR结果显示，在LPS处理的hPDLFs中加入自噬促进剂后，炎症因子表达明显减少。结论：自噬参与延期牙再植术后炎症性牙根吸收的发生发展，并可能起保护作用。

关键词: [关键词] 自噬, 延期牙再植, 炎症性牙根吸收

Abstract: Objective：To observe the changes of autophagy levels after delayed tooth replantation in rats and investigate the effect of autophagy in inflammatory root resorption after delayed tooth replantation. Methods： Twenty-four 4-week-old SD rats were divided into different groups. Maxillary first molars were extracted, dried for 30 minutes and then replanted into the alveolar sockets. Rats were sacrificed on day 1, 3, 7 and 28 after surgery, the inflammation infiltration situation, root resorption situation, expression of inflammatory cytokines, TNF-α and IL-1β, and expression of autophagy proteins LC3B and Beclin-1 were detected by tissue staining. Immunofluorescence staining was performed on human periodontal ligament fibroblasts (hPDLFs) treated with LPS to detect the expression of LC3B and Beclin1 at different time points. Meanwhile, the inflammation and autophagy-related gene expression in hPDLFs treated with LPS and autophagy promoter (Rapamycin) or autophagy inhibitor (chloroquine) was examined through real-time RT-PCR. Results：Massive inflammatory cell infiltration and inflammatory cytokines TNF-α and IL-1β around the root were observed on the 1st and 3rd day after surgery. On the 7th day, inflammatory infiltration level increased gradually and increased secretion of TNF-α and IL-1β was detected through immunohistochemical staining. The TRAP staining result showed obvious inflammatory resorption on the 7th day. The inflammation alleviated on the 28th day. During this process, autophagy proteins LC3 and Beclin-1 increased significantly on the 1st day after surgery, then declined on the 3rd and 7th day and increased again on the 28th days. In vitro experiments, autophagy proteins LC3B and Beclin-1 were activated in hPDLFs after LPS 24h stimulation, while inhibited after 72h LPS stimulation. The trends of autophagy proteins in vitro were consistent with the in vivo results. Expression of inflammation genes were significantly reduced in hPDLFs treated with autophagy promoter and LPS. Conclusions：Autophagy participates in the development of root inflammatory resorption after delayed tooth replantation, and may play a protective role.

Key words: [Key words] Autophagy, Delayed tooth replantation, Inflammatory root resorption

雷心怡张旗. 自噬在延期牙再植术后炎症性牙根吸收中的作用研究[J]. 口腔生物医学, 2021, 12(3): 149-154.

参考文献 18

[1]	Souza BDM, Dutra KL, Kuntze MM, et al.Incidence of root resorption after the replantation of avulsed teeth: A meta-analysis[J].J Endod, 2018, 44(8):1216-1227
[2]	Panzarini SR, Okamoto R, Poi WR, et al.Histological and immunohistochemical analyses of the chronology of healing process after immediate tooth replantation in incisor rat teeth[J].Dent Traumatol, 2013, 29(1):15-22
[3]	Roskamp L, Westphalen VD, Carneiro E, et al.Relationship between extra-alveolar time and atopy in the prognosis of the replantation of avulsed teeth[J].J Trauma, 2010, 69(6):E79-E81
[4]	Hecova H, Tzigkounakis V, Merglova V, et al.A retrospective study of 889 injured permanent teeth[J].Dent Traumatol, 2010, 26(6):466-475
[5]	Bastos JV, Silva TA, Colosimo EA, et al.Expression of inflammatory cytokines and chemokines in replanted permanent teeth with external root resorption[J].J Endod, 2017, 43(2):203-209
[6]	Ahn HJ, Nam OH, Lee HS, et al.Expression of inflammatory cytokines and MMPs on replanted teeth at different extra-alveolar time: An ex vivo and in vivo study[J].Int J Paediatr Dent, 2016, 26(4):301-309
[7]	Lapaquette P, Guzzo J, Bretillon L, et al.Cellular and molecular connections between autophagy and inflammation[J].Mediators Inflamm, 2015, 2015:398483-
[8]	Harris J, Lang T, Thomas JPW, et al.Autophagy and inflammasomes[J].Mol Immunol, 2017, 86:10-15
[9]	Ge Y, Huang M, Yao YM.Autophagy and proinflammatory cytokines: Interactions and clinical implications[J].Cytokine Growth Factor Rev, 2018, 43:38-46
[10]	Chen LY, Mo SZ, Hua YM.Compressive force-induced autophagy in periodontal ligament cells downregulates osteoclastogenesis during tooth movement[J].J Periodontol, 2019, 90(10):1170-1181
[11]	Sato K, Muramatsu T, Tsuchiya Y, et al.Proliferation,migration and apoptosis of periodontal ligament cells after tooth replantation[J].Oral Dis, 2010, 16(3):263-268
[12]	Deretic V, Saitoh T, Akira S.Autophagy in infection,inflammation and immunity[J].Nat Rev Immunol, 2013, 13(10):722-737
[13]	Deretic V, Levine B.Autophagy balances inflammation in innate immunity[J].Autophagy, 2018, 14(2):243-251
[14]	Jones SA, Mills KH, Harris J.Autophagy and inflammatory diseases[J].Immunol Cell Biol, 2013, 91(3):250-258
[15]	Nakahira K, Haspel JA, Rathinam VA, et al.Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome[J].Nat Immunol, 2011, 12(3):222-230
[16]	Ilyas G, Cingolani F, Zhao EP, et al.Decreased macrophage autophagy promotes liver injury and inflammation from alcohol[J].Alcohol: Clin Exp Res, 2019, 43(7):1403-1413
[17]	Sokos D, Everts V, de Vries TJ.Role of periodontal ligament fibroblasts in osteoclastogenesis: A review[J].J Periodontal Res, 2015, 50(2):152-159
[18]	Yucel-Lindberg T, B?ge T.Inflammatory mediators in the pathogenesis of periodontitis[J].Expert Rev Mol Med, 2013, 15:7-