饥饿条件下活性氧通过PINK1/Parkin通路调控人牙周膜细胞的线粒体自噬

doi:10.7518/hxkq.2022.06.004

华西口腔医学杂志 ›› 2022, Vol. 40 ›› Issue (6): 645-653.doi: 10.7518/hxkq.2022.06.004

饥饿条件下活性氧通过PINK1/Parkin通路调控人牙周膜细胞的线粒体自噬

范智博¹(), 金珂², 李胜鸿¹, 徐洁¹, 徐晓梅¹()

^1.西南医科大学附属口腔医院正畸科，泸州 646099
^2.绵阳市中心医院口腔科，绵阳 621099

收稿日期:2022-05-24 修回日期:2022-10-05 出版日期:2022-12-01 发布日期:2022-11-23
通讯作者: 徐晓梅 E-mail:505603632@qq.com;xuxiaomei@hotmail.com
作者简介:范智博，医师，硕士，E-mail：505603632@qq.com
基金资助:
四川省科技厅应用基础项目(2021YJ0151);西南医科大学校级科研项目(2021LZMS019);西南医科大学校级科研项目(2022Z02)

Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation

Fan Zhibo¹(), Jin Ke², Li Shenghong¹, Xu Jie¹, Xu Xiaomei¹()

^1.Dept. of Orthodontics, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646099, China
^2.Dept. of Stomatology, Mianyang Central Hospital, Mianyang 621099, China

Received:2022-05-24 Revised:2022-10-05 Online:2022-12-01 Published:2022-11-23
Contact: Xu Xiaomei E-mail:505603632@qq.com;xuxiaomei@hotmail.com
Supported by:
Application Foundation Project of Sichuan Provincial Science and Technology Department(2021YJ-0151);School Scientific Research Project of Southwest Medical University(2021LZMS019);School Scientific Research Project of Southwest Medical University(2022Z02);Correspondence: Xu Xiaomei, E-mail: xuxiaomei@hotmail.com

摘要/Abstract

摘要：

目的探究人牙周膜细胞（hPDLC）在饥饿条件下活性氧（ROS）与PINK1/Parkin通路介导hPDLC线粒体自噬的具体机制。方法分离培养正常牙周组织的hPDLSC，利用Earle’s平衡盐溶液（EBSS）模拟饥饿环境诱导hPDLC线粒体自噬，利用N-乙酰-L-半胱氨酸（NAC）抑制ROS生成以探讨ROS在hPDLC线粒体自噬的作用，利用环孢素A（CsA）抑制PINK1/Parkin通路以研究ROS与PINK1/Parkin通路在饥饿条件下激活hPDLC中的作用。采用流式细胞术及JC-1线粒体膜电位检测试剂盒，检测线粒体膜电位；采用透射电镜观察线粒体自噬体的生成及线粒体形态变化；采用线粒体红色荧光探针定位线粒体，溶酶体绿色荧光探针定位溶酶体；采用DCFH-DA ROS荧光探针检测ROS生成强度；采用实时荧光定量聚合酶链反应（RT-qPCR）检测细胞中线粒体自噬基因（Tomm20、Timm23）及PINK1/Parkin通路的表达水平，采用蛋白质印迹（Western blot）检测细胞中线粒体自噬蛋白（Tomm20、Timm23）及PINK1/Parkin通路蛋白表达水平。结果 EBSS饥饿作用30 min后，诱导激活hPDLC线粒体自噬的作用最强，ROS表达增加，且线粒体自噬相关基因（Tomm20、Timm23）下调（P<0.001），PINK1/Parkin通路表达上调（P<0.001）。NAC抑制ROS的产生后，自噬被抑制，同时Tomm20、Timm23表达上调（P<0.001，P<0.05），PINK1/Parkin通路表达下调（P<0.001，P<0.05）。而当CsA抑制PINK1/Parkin通路表达时（P<0.05，P<0.05），自噬被逆转，同时Tomm20、Timm23表达上调（P<0.001，P<0.01）。结论 ROS在饥饿条件下主要通过PINK1/Parkin通路增强hPDLC线粒体自噬。

关键词: Earle’s平衡盐溶液, 活性氧, 线粒体自噬, 人牙周膜细胞, PINK1/Parkin通路

Abstract:

Objective This study aimed to explore the specific mechanism, mediated by the reactive oxygen species (ROS) and PINK1/Parkin pathway, of the mitochondrial autophagy of human periodontal ligament cells (hPDLCs) under starvation conditions. Methods hPDLCs were isolated and cultured from normal periodontal tissues. Earle’s balanced salt solution (EBSS) was used to simulated a starvation environment and thus stimulate hPDLCs mitochondrial autophagy. N-Acetyl-L-cysteine (NAC) was used to inhibit ROS production to explore the role of ROS in hPDLC mitochondrial autophagy. Cyclosporin A was used to inhibit the PINK1/Parkin pathway to study the role of ROS and the PINK1/Parkin pathway in hPDLCs activation under starvation. The mitochondrial membrane potential was detected by flow cytometry with a JC-1 mitochondrial membrane potential detection kit. The morphological structure of mitochondria and the formation of mitochondrial autophagosome were observed by transmission electron microscopy. Mito tracker red cmxros and lyso tracker green staining were used to observe the localization of mitochondria and lysosomes. The formation intensity of ROS was detected with a DCFH-DA ROS fluorescent probe. The expression levels of mitochondrial autophagy genes (Tomm20 and Timm23) and the PINK1/Parkin pathway were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of mitochondrial autophagy proteins (Tomm20 and Timm23) and PINK1/Parkin protein were detected by Western blot. Results EBSS starvation for 30 min induced the strongest activation of hPDLCs mitochondrial autophagy, increased the expression of ROS, downregulated the expression of mitochondrial autophagy-related genes (Tomm20 and Timm23) (P<0.001), and upregulated the PINK1/Parkin pathway (P<0.001). After NACinhibited ROS production, mitochondrial autophagy was also inhibited. Meanwhile, the expression of Tomm20 and Timm23 was upregulated (P<0.001 and P<0.05), and the expression of the PINK1/parkin pathway (P<0.001 and P<0.05) was down regulated. When cyclosporin A inhibited the expression of the PINK1/Parkin pathway (P<0.05 and P<0.05), it reversed the mitochondrial autophagy of hPDLCs (P<0.001 and P<0.01) and also upregulated the expression of Tomm20 and Timm23 (P<0.001 and P<0.01). Conclusion ROS enhanced the mitochondrial autophagy of hPDLCs primarily through the PINK1/Parkin pathway under starvation conditions.

Key words: Earle’s balanced salt solution, reactive oxygen species, mitophagy, human periodontal ligament cell, PINK1/Parkin pathway

中图分类号:

Q?256

范智博, 金珂, 李胜鸿, 徐洁, 徐晓梅. 饥饿条件下活性氧通过PINK1/Parkin通路调控人牙周膜细胞的线粒体自噬[J]. 华西口腔医学杂志, 2022, 40(6): 645-653.

Fan Zhibo, Jin Ke, Li Shenghong, Xu Jie, Xu Xiaomei. Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation[J]. West China Journal of Stomatology, 2022, 40(6): 645-653.

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参考文献 28

1	Dutra EH, Nanda R, Yadav S. Bone response of loaded periodontal ligament[J]. Curr Osteoporos Rep, 2016, 14(6): 280-283.
2	Huang H, Williams RC, Kyrkanides S. Accelerated orthodontic tooth movement: molecular mechanisms[J]. Am J Orthod Dentofacial Orthop, 2014, 146(5): 620-632.
3	Wang M, Zhang L, Lin F, et al. Dynamic study into autophagy and apoptosis during orthodontic tooth movement[J]. Exp Ther Med, 2021, 21(5): 430.
4	Yang J, Zhou R, Ma Z. Autophagy and energy metabolism[J]. Adv Exp Med Biol, 2019, 1206: 329-357.
5	Wong SQ, Kumar AV, Mills J, et al. Autophagy in aging and longevity[J]. Hum Genet, 2020, 139(3): 277-290.
6	Deretic V. Autophagy in inflammation, infection, and immunometabolism[J]. Immunity, 2021, 54(3): 437-453.
7	Dikic I, Elazar Z. Mechanism and medical implications of mammalian autophagy[J]. Nat Rev Mol Cell Biol, 2018, 19(6): 349-364.
8	Wang M, Zhang L, Lin F, et al. Dynamic study into autophagy and apoptosis during orthodontic tooth movement[J]. Exp Ther Med, 2021, 21(5): 430.
9	Xu J, Zhao X, Zeng J, et al. Role of autophagy in the periodontal ligament reconstruction during orthodontic tooth movement in rats[J]. J Dent Sci, 2020, 15(3): 351-363.
10	Wan H, Tang B, Liao X, et al. Analysis of neuronal phosphoproteome reveals PINK₁ regulation of BAD function and cell death[J]. Cell Death Differ, 2018, 25(5): 904-917.
11	Gu X, Qi Y, Feng Z, et al. Lead (Pb) induced ATM-dependent mitophagy via PINK₁/Parkin pathway[J]. Toxicol Lett, 2018, 291: 92-100.
12	周晓芸, 宋雨鸿, 薛丹, 等. 基于线粒体自噬探讨参苓白术散对COPD骨骼肌损伤细胞的保护作用机制[J]. 广东药科大学学报, 2020, 36(3): 369-374.
	Zhou XY, Song YH, Xue D, et al. Protective mechanism of Shenling Baizhu powder on the injured cells of CO-PD skeletal muscle based on mitochondrial autophagy[J]. J Guangdong Pharm Univ, 2020, 36(3): 369-374.
13	Ren Y, Shen HM. Critical role of AMPK in redox regulation under glucose starvation[J]. Redox Biol, 2019, 25: 101154.
14	Parzych KR, Klionsky DJ. An overview of autophagy: morphology, mechanism, and regulation[J]. Antioxid Redox Signal, 2014, 20(3): 460-473.
15	Kim KH, Lee MS. Autophagy—a key player in cellular and body metabolism[J]. Nat Rev Endocrinol, 2014, 10(6): 322-337.
16	Li Y, Jacox LA, Coats S, et al. Roles of autophagy in orthodontic tooth movement[J]. Am J Orthod Dentofacial Orthop, 2021, 159(5): 582-593.
17	Huang H, Williams RC, Kyrkanides S. Accelerated orthodontic tooth movement: molecular mechanisms[J]. Am J Orthod Dentofacial Orthop, 2014, 146(5): 620-632.
18	Niklas A, Proff P, Gosau M, et al. The role of hypoxia in orthodontic tooth movement[J]. Int J Dent, 2013, 2013: 841840.
19	Li L, Tan J, Miao Y, et al. ROS and autophagy: interactions and molecular regulatory mechanisms[J]. Cell Mol Neurobiol, 2015, 35(5): 615-621.
20	蒋玉坤, 胡芝爱, 关禹哲, 等. 应力诱导自噬的机械转导过程研究进展[J]. 四川大学学报(医学版), 2021, 52(6): 929-935.
	Jiang YK, Hu ZA, Guan YZ, et al. Research progress in mechanotransduction process of mechanical-stress-indu-ced autophagy[J]. J Sichuan Univ (Med Sci), 2021, 52(6): 929-935.
21	Morel E, Dupont N, Codogno P. Primary cilium-dependent autophagy in the response to shear stress[J]. Biochem Soc Trans, 2021, 49(6): 2831-2839.
22	Zou R, Wu S, Wang Y, et al. Role of integrin‑linked kinase in static compressive stress‑induced autophagy via phosphatidylinositol 3 kinase in human periodontal ligament cells[J]. Int J Mol Med, 2021, 48(3): 167.
23	Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: the clash between damage and metabolic needs[J]. Cell Death Differ, 2015, 22(3): 377-388.
24	Onishi M, Yamano K, Sato M, et al. Molecular mechanisms and physiological functions of mitophagy[J]. EMBO J, 2021, 40(3): e104705.
25	Battaglia AM, Chirillo R, Aversa I, et al. Ferroptosis and cancer: mitochondria meet the “Iron Maiden” cell death[J]. Cells, 2020, 9(6): 1505.
26	Tanaka K. The PINK1-Parkin axis: an overview[J]. Neurosci Res, 2020, 159: 9-15.
27	Dagda RK, Cherra SJ 3rd, Kulich SM, et al. Loss of PI-NK-1 function promotes mitophagy through effects on o-xidative stress and mitochondrial fission[J]. J Biol Chem, 2009, 284(20): 13843-13855.
28	Wang C, Yang Q, Han Y, et al. A reduced level of the long non-coding RNA SNHG8 activates the NF-kappaB pathway by releasing functional HIF-1alpha in a hypo-xic inflammatory microenvironment[J]. Stem Cell Res Ther, 2022, 13(1): 229.

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饥饿条件下活性氧通过PINK1/Parkin通路调控人牙周膜细胞的线粒体自噬

Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation

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