Mechanism Of Reactive Oxygen Species Promoting Regeneration Of Skeletal Muscles Following Gecko Tail Amputation

Objective Reactive oxygen species(ROS) are involved in multiple physiological functions including facilitation of cell apoptosis,promoting inflammatory responses and contributing to aging.Recent evidences have shown that ROS are able to promote appendage regeneration by regulation of several pathways. Gekko japonicas can regenerate its tail after amputation, whether ROS are implicated in the regulation of the tail regeneration and the underlying mechanism remains unclear. In the present study, we aim to understand the sources of the ROS originated following gecko tail amputation, and to unveil the mechanism of ROS regulating tail regeneration.Methods The compound 2',7'-dichlorofluorescin diacetate(H2DCFDA,Calbiochem) was used to monitor the accumulation of ROS in the amputated gecko tail. Fluorescent DCF was formed through ROS oxidation.Diphenyleneiodonium or apocynin(APO, Sigma-Aldrich)were injected intraperitoneally to establish ROS inhibition model.Masson staining of skeletal muscles were used to observe the morphology following treatment of the regenerate with APO at0 d,3d and 7d. The ultrastructural organelles of it were visualized using transmission electron microscopy. transcriptome analysis on skeletal muscles either at 0d, 3d and 7d following tail amputation or the regenerate treatment with APO. Quantitative real-time PCR was further performed to validate the expression of PERE and NADPH oxidases. The regulatory molecules of LC3B-II/LC3B-I,MP2K7 and phosphorylation of its downstream kinases JNK, ERK1/2, p38 were also examined by Western Blot.Results1. ROS sustained high expression in skeletal muscle following gecko tail amputation. The high rates of ROS production were found at 7d regenerates,while ROS production decreased at 14 d.2. APO treatment can effectively inhibit the production of ROS,and accordingly inhibits the regeneration of amputated tails.3. APO treatment can decrease the cross sectional area of skeletal muscles by inhibition of ROS production, as shown by masson staining.4. The ultrastructural organelles visualized using transmission electron microscopy have shown that inhibition of ROS production results in the misaligned Z-discs of skeletal muscle following APO treatment. Also, it affects the autophagy of the muscles.5. Transcriptome analysis has shown that both PERE and NADPH are involved in the production of ROS in the skeletal muscle following gecko tail amputation.6. APO treatment reduced the expression of ULK1 and MP2K7,as well as their phosphorylation, which were associated with the formation of autophage.Conclusions1. Reactive oxygen species generated from skeletal muscles are required for gecko tail regeneration, and its inhibition reduced the length of regenerating tail.2. Both PERE and NADPH oxidases(including p40 phox,gp91 phox, p47 phox) are mainly involved in the ROS production in the skeletal muscle following gecko tail amputation.3. ROS are involved in the autophage of injured skeletal muscle through regulation of MAP2K7 and LC3 B.