Mitochondrial Superoxide is the Key for Promoting Cell Migration in Wound Healing

Research Article

Thromb Haemost Res. 2022; 6(1): 1072.

Mitochondrial Superoxide is the Key for Promoting Cell Migration in Wound Healing

Ping Weidong, Wang Xin, Wang Xiaowei, Li Fei, Huang Chunyan, Zhao Qiming*

Department of Plastic Surgery, Zhejiang Hospital, Hangzhou, 310013, Zhejiang, China

*Corresponding author: Zhao Qiming, Department of Plastic Surgery, Zhejiang Hospital, No. 12 Lingyin Road, Xihu District, Hangzhou, 310013, Zhejiang, China; E-mail: [email protected]

Received: December 30, 2021; Accepted: January 27, 2022; Published: February 03, 2022


Introduction: The focus of current study was to investigate the role of mitochondrial superoxide for promoting cell migration in healing responses.

Methods: To simulate the skin wound healing process, scratch wound assay in A549 cells was utilized. Subsequently, mitochondrial superoxide and intracellular calcium flux (Ca2+) were measured by MitoSox red reagent and Fluo-4 staining. Paraquat (PQ), N-acetyl cysteine (NAC), superoxide dismutase (SOD) mimics (EUK-134), free radical scavenge (MCI-186), and diphenyliodonium (DPI) were added into medium to evaluate the influences of reactive oxygen species (ROS) and superoxide on cell migration.

Results: Compared with before wound (BW), mitochondrial superoxide increased time-dependent manner. Correspondingly, Ca2+ transients was rapidly elevated after scratch wound and gradually declined toward an steady level, as evidenced by Fluo-4 fluorescence intensity. Scratch wound assay displayed that cell migration in PQ 0.1 mM group was stronger than that of Control group, whilst it was restrained after administration of 10 mM NAC. Additionally, cell migration in EUK-134 and MCI-186 group was conspicuously lower than that of Control group and similar with the degree of inhibiting role of DPI, indicating that mitochondrial superoxide served the crux influence on promoting cell migration after scratch.

Conclusion: ROS and superoxide production during scratch wound facilitated cell migration. Importantly, mitochondrial superoxide played the positive effects on healing response.

Keywords: Reactive oxygen species; Mitochondrial superoxide; Wound healing; Cell migration


ROS: Reactive Oxygen Species; mtROS: Mitochondrial ROS; Ca2+: Calcium Flux; PQ: Paraquat; NAC: N-acetyl Cysteine; SOD: Superoxide Dismutase; DPI: Diphenyliodonium


Wound healing is a complicated regenerative process, which is indispensable for mammals to survive [1]. Currently, human wounds become a leading threat to public health, influencing millions of people worldwide [2]. In general, nonhealing wound is a main cause of morbidity and mortality. To our knowledge, cell migration exerts the crux roles on biological events including wound closure, repair and regeneration [3]. Flat membrane protrusion in the front of cells, known as lamellipodia, the first procedure of cell migration, is driven by actin polymerization modulated by Rac1 [4]. Thus, exploring the factor that influence cell migration is crucial.

It’s well known that reactive oxygen species (ROS) are produced in wound sites and serve as signaling during wound healing [5,6]. Except for being generated by membrane and cytoplasm, mitochondrion is also a principal source of ROS. Correspondingly, the role of mitochondrial ROS (mtROS) for wound closure has been verified in previous studies [7,8]. Besides, previous study has verified that endothelial cell migration is partly dependent on mitochondriagenerated ROS [9]. A similar study has also demonstrated that mtROS is closely associated with cell migration [10], supporting its promoting roles in wound closure.

Superoxide, as the member of ROS family, serves the function for cell migration, which have been pointed out. For instance, Mu et al. [11] have revealed that Chlorotyrosine boosts smooth muscle cell migration via increasing superoxide generation. Another study has uncovered that superoxide production supports epithelial cell migration, thereby promoting wound closure [12]. In terms of relation between mitochondrial superoxide and cell migration, Dias Amoedo et al. [13] have reported that inhibition of mitochondrial superoxide generation is helpful to suppress the migration of A549 cell, and then target lung adenocarcinoma. However, how mitochondrial superoxide influences migration, thereby wound healing remains poorly understood. In this study, we investigated whether mitochondrial ROS involved cell migration or not. Our results demonstrated that mitochondrial superoxide exerted the crux roles on wound healing.

Materials and Methods

Cell culture and treatment

A549 cells (non-small cell lung cancer cell line) were purchased from cell resource center of shanghai institute for life sciences (Shanghai, China), and cultured in DMEM medium (Gibco, Carlsbad, MD, USA) supplemented with 10% FBS and 1% penicillin/ streptomycin (Meilunbio, Dalian, China).

Wound healing assay

To simulate the skin wound healing process, scratch wound assay was utilized referring to previous study [14]. Briefly, A549 cells were grown to form monolayers in 6-well plates, and then scratch was performed with 200 μl pipette tip (Figure 1). After washing with DMEM medium, the scratched cells were returned to incubator for further culture up to 12 h. To detect the influences of ROS and superoxide, paraquat (PQ), N-acetyl cysteine (NAC), SOD mimics (EUK-134), free radical scavenge (MCI-186), and diphenyliodonium (DPI) were added into medium, respectively.