Metformin-induced Autophagy Regulates Skin-derived Stem Cell Activity And Promotes The Regeneration Of Mechanically Stretched Skin

BackgroundSoft tissue expansion is one of the most common techniques used in plastic surgery to repair skin and soft tissue defects and to reconstruct organs.The technique involves placing a silicone expander through an injection port under the skin and regularly filling it with liquid.The mechanical stretch stimulates the growth of new skin on the surface of the expander;this skin flap is further used for reconstruction.The advantages of skin and soft tissue expansion are evident.After the operation,new skin tissue similar in texture,color,and sensory function to the healthy skin surrounding the recipient area is observed,and the occurrence of scars and deformities in the donor area is significantly reduced.However,the expansion process is long,and its efficiency is low.The process usually requires several months of repeated injections with sterile saline,during which complications may occur,increasing patients’ physical pain and decreasing their quality of life.The ability of skin to regenerate on applying mechanical traction is limited,and when the regeneration is insufficient,complications may occur and affect the efficacy of follow-up treatment.Therefore,there is an urgent need to explore new tissue expansion methods to promote expanded skin regeneration and to provide better clinical outcomes.During skin and soft tissue expansion,mechanical traction causes cell proliferation and consequently a continuous growth of the skin flap.The mechanism of mechanical traction-induced skin regeneration has always been a research hotspot in the field of skin expansion.Currently,phenomena,such as physiological hyperplasia,elastic extension,mechanical creep,and displacement of adjacent skin tissue,are known to play a role in skin expansion.Hyperplastic tissue is an important new component of expanded skin: the higher its proportion in the skin flap,the better the outcome of postoperative repair.Previous studies have shown that the use of stem cells(such as fat and bone marrow stem cells)can effectively promote expanded skin regeneration.,Skin-derived stem cell such as hair follicle stem cells(FSCs)and epidermal stratum basale cells,may play an even more critical role in this process.FSCs seem to be located in the protuberance of the outer root sheath of hair follicles,also known as the bulge region,which participates in the periodic growth of hair follicles and repair and regeneration of damaged skin.FSCs’ proliferation,differentiation,and apoptosis are closely regulated by various signaling pathways,such as the bone morphological protein/transforming growth factor β(BMP/TGF-β),Wnt/β-catenin(Wnt/β-catenin),Notch,Sonic-Hedgehog pathways.We demonstrated previously that a local injection of HFSCs amplified in vitro into the expanded skin can participate in skin regeneration through direct differentiation.However,the clinical application of stem cells expanded in vitro is limited by ethical,safety,and other issues;therefore,it is necessary to find alternative methods.The pharmacological activation of local skin-derived stem cells may be a possible way to promote the growth of expanded skin.Recent studies reported that a topical application of metformin(MET)activates the mouse skin-derived stem cells and promotes mouse hair growth by inducing autophagy.However,the specific effect of MET on FSCs is not completely understood and requires further experimental verification.MET is a commonly used autophagy inducer that mainly acts through activating the AMP-dependent protein kinase(AMPK)signaling pathway and inhibiting mammalian rapamycin target protein(mTOR).Autophagy is a highly conservative evolutionary process in eukaryotes.Under adverse conditions,lysosomes degrade intracellular agingand degeneration-related macromolecular substances and damaged organelles,decompose them into smaller substances,and recycle them to support cellular metabolism and organelle renewal.Previous studies have shown that drug-induced autophagy accelerates skin wound healing by promoting tissue vascularization.When the local autophagy of the wound is inhibited,wound healing time is prolonged.Therefore,pharmacies that induce autophagy may be beneficial in promoting tissue repair and regeneration.Autophagy can be observed within expanded skin,as shown in our previous studies,but its effect on the growth of expanded skin is not completely understood.From the above studies,we hypothesize that topical application of MET may regulate the proliferative activity of skin-derived stem cells by inducing autophagy,thus promoting the regeneration of expanded skin.AimThe purpose of this study was to investigate the effect of topically applied MET on the growth of expanded skin and explore the underlying molecular mechanism using a rat scalp expansion model.The regulatory effect of MET on the proliferation of skin-derived stem cells in expanded skin was the focal point of this research.MethodsIn the first part of the experiment,a rat scalp expansion model was developed using a custom-made 1 mL circular expander.The Sprague-Dawley male rats were divided into two groups: MET and double distilled water(control).A 1 mL expansion capsule was implanted at the top of the skull,and normal saline was injected regularly(1 mL every three days)into the expander one week after the operation.MET(300 mM)or water was topically applied on the expanded skin of rats in the MET and control group respectively,every day.After injecting the volume to 10 mL,the skin flap was sampled and tested.The tissue samples were stained with hematoxylin-eosin(H&E)and Masson’s trichrome for histological observation;blood vessel density was analyzed by CD31 immunohistochemical staining;Ki67,proliferating cell nuclear antigen(PCNA),aurora l kinase B(Aurora B),and phosphorylated histone H3(pH3)immunohistochemical staining were used to evaluate cell proliferation.Western blotting was employed to detect specific proteins and their relative levels,and Sirius red staining was performed for visualizing collagen fibers.Skin cells and collagen fibers ultrastructures were examined by transmission electron microscopy,and the blood perfusion of the skin flap was visualized using a laser perfusion imager.ResultsThe results showed that the topical application of MET results in a significant thickening of the expanded skin,an increase in the epidermal cell layers,and a more compact arrangement of the cells compared with those in the control group.The collagen volume fraction and amount of type I and III collagen in the dermis of the skin flap were significantly higher in the MET group than the control group.Sirius red staining revealed more abundant mature collagen in the MET group than the control group.More regular and uniform collagen arrangement was observed in the MET group,whereas the control group exhibited loose collagen organization and collagen fracture.Blood perfusion in the expanded skin and the CD31 signal were significantly higher in the MET group than the control group.These results confirmed that a topical application of MET promotes the regeneration of expanded skin and enhances the proliferation of hair follicle bulge cells.Furthermore,double immunofluorescence staining allowed the precise localization of the proliferative cells in the expanded skin samples.PCNA and CK14 co-staining positive cells were labeled as value-added keratinocytes in the basal layer of the epidermis,and the PCNA and CK15 co-staining positive cells were labeled as value-added FSCs.The number of CK14+/PCNA+ cells in the basal layer of the epidermis and CK15+/PCNA+ cells in the outer root sheath and bulge area was significantly higher in the MET group than the control group.The relative levels of CK14 and CK15 determined by western blotting and the intensity of the signal of PCNA,Aurora B,and pH3 for the rats in the MET group confirmed that a topical application of MET increases the proliferation of keratinocytes and HFSCs and promotes skin expansion.Besides,we found the many autophagy structures in the MET group.Autophagy in expanded skin was assessed using the following methods: transmission electron microscopy scanning and by evaluating the levels of autophagy-related proteins(LC3,Becline-1,and P62).Next,we established a head skin expansion model in SD rats using the autophagy inhibitor 3-methyladenine(3-MA)to observe whether MET promotes dilated skin regeneration by inducing autophagy.The results showed that MET induces autophagy in the epidermis and dermis of the skin flap and significantly up-regulates the expression of LC3b immunofluorescence staining.3-MA administration inhibited MET-induced autophagy,consequently decreasing the effect of MET on expanded skin regeneration and on HFSCs proliferation.In the final part of the experiment,we explored the potential mechanism underlying the effect of MET on autophagy in expanded skin.Using the rat scalp expansion model,we administered Compound C,an inhibitor of the AMPK pathway,to investigate if the AMPK-mTOR signaling pathway mediates MET effects on autophagy.The results showed that the administration of Compound C counteracted the effect of MET on promoting expanded skin regeneration and skin-derived stem cells’ proliferation.ConclusionThis study confirms that MET promotes the growth and regeneration of expanded skin by increasing the proliferation of skin-derived stem cells and enhancing vascularization.Our research highlights that autophagy enhancement is essential for MET activity,and found that the AMPK-mTOR signaling pathway may as a major regulator of MET-induced autophagy in expanded skin.These results indicated that MET could be useful in improving skin expansion procedures and may also be benefit for its clinical applications.