West China Journal of Stomatology ›› 2022, Vol. 40 ›› Issue (6): 645-653.doi: 10.7518/hxkq.2022.06.004

Previous Articles     Next Articles

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. 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


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

CLC Number: