Activin B Regulates BMSCs-induced Cell Migration In Wounding Healing Through JNK-ERK Signaling Pathway

Cutaneous wound healing is a biological process involving a complex and coordinated series of events that include inflammation, new tissue formation, and tissue remodeling.At present, many approaches that can accelerate wound healing process have been developed; however, faster healing velocity and better healing quality remain challenging tasks faced by clinicians. Skin grafting is currently the most effective method for the management of large skin defects. In recent years, the discovery that bone marrow-derived mesenchymal stem cells (BMSCs) are able to differentiate into skin cell types and participate in the regeneration of skin has provided a promising alterative strategy for the treatment of severe skin wounds. Transplantation of BMSCs into full-thickness burn wounds has bee found to be able to significantly induce epidermal thickening, increase the number of dermal nerve fibers, accelerate would healing, and greatly improve healing quality, Moreover, BM-MSCs participate in wound re-epithelialization and regeneration of sweat glands and blood vessels [4]. Therefore, BMSCs represent ideal seeder cells for skin tissue engineering.A wide variety of growth factors and cytokines are involved in the regulation of all phases of wound healing. Activins are members of the transforming growth factor-β (TGF-β) superfamily that exert their biological effects through binding to specific receptors. In adult tissue, activin signaling can regulate wound healing and re-epithelialization and plays a key role in cutaneous wound healing. Furthermore, activins and their antagonist follistatin are involved in the control of hair follicle development and hair growth cycles. In our previous studies, we found that activin B promoted keratinocyte migration through the JNK/MAPK pathway and thereby accelerated wound healing. Furthermore, our recent results indicated that activin B not only promoted epithelial wound closure in vivo through the RhoA-Rock-cJun signaling pathway but also stimulated the proliferation of hair follicle cells and regeneration of hair follicles at the wound areaStem cell-mediated regeneration and repair involve migration of stem cells from the sites where they colonize to wound sites. Such migration process is controlled by chemokines and their receptors. Among these chemokines, activin/TGF-P is known to regulate cell migration, proliferation, differentiation, and extracellular matrix synthesis and secretion during the wound healing process.BMSCs highly express activin receptors on their surface, and deletion of Smad3, a downstream signal molecule of the activin/TGF-β pathway, resulted in decreased migration of BMSCs, suggesting that the activin/TGF-β pathway might play an important role in the regulation of biological functions of BMSCs.Considering that BMSCs can accelerate would healing and promote regeneration of skin appendages, and that activins can promote the migration of BMSCs, we hypothesized that activin B might improve BMSC-mediated wound healing. To test this hypothesis, we evaluated the impact of combined administration of BMSCs and activin B on wound healing in rats. In addition, we explored the mechanisms behind the impact of activin B treatment on BMSC-mediated wound healing in vitro. Our study will provide a theoretical basis for the development of new strategies for the management of wounds.(1) PurposeExplore the impact of Activin B regulates BMSCs treated rat skin wounds healing. (2) MethodsRats were anesthetized by an intraperitoneal injection of10%chloral hydrate (w/v;0.003mL/g body weight). After dorsal hair was depilated using a hair removal wax, full-thickness skin wounds of1×1cm2were created on the back of rats. The rats were given free access to food and water after wound creation. After wounding, four groups of rats were administered with0.4ml PBS,10ng/mL activin B,6-8X106/mL BMSCs, and10ng/mL activin B+6-8×106/mL BMSCs, respectively. Wound areas were measured photographically every day after wounding, and the rate of wound closure was calculated using the following formula:Wound closure rate (%)=[(original wound area-open area on final day)/original wound area] X100%. On postoperative days3,7,14and21, the complete wound with a0.5cm margin was carefully removed, rinsed in PBS, fixed in4%PFA, rinsed again in PBS, dehydrated in a graded ethanol series, cleared in dimethylbenzene and embedded in paraffin. Five-micrimeter sections were prepared, deparaffinized in dimethylbenzene and rehydrated. H&E staining was then performed according to standard procedures.(3) ResultsWe then evaluated whether activin B promotes BMSCs mediated wound healing by applying PBS, activin B, BMSCs, or activin B plus BMSCs to wound sites and monitoring wound closure. Compared to administration with PBS, activin B or BMSCs, and activin B plus BMSCs significantly promoted wound healing. On days7and14after administration, the rates of wound closure were significantly higher in the activin B plus BMSCs group than in the PBS, BMSCs, and activin B groups. On day12, the wounds have healed completely in all rats administered with BMSCs plus activin B, whereas the other three groups still showed incomplete wound closure. Complete wound closure occurred on day14for the activin B group and BMSCs group, and on day16for the PBS group. Histological analysis confirmed that wound re-epithelialization has completed on day14in the activin B, BMSCs, and BMSCs plus activin B groups. Compared to the PBS and BMSCs groups, regeneration of hair follicles was observed in the activin B group and combination group. (4) ConclusionTaken together, these data suggest that combined activin B and BMSCs could not only promote wound re-epithelialization and healing but also stimulate the regeneration of hair follicles.(1) PurposeIn vitro and in vivo to investigate whether Activin B regulate the contractile function of BMSCs motility and wound healing.(2) MethodsIn vitro the application of immunohistochemistry to detect the expression of K15, K19;using cell wound healing assay.cell chemotaxis experiments,cells Actin structure observation of cell migration in experimental records;In vivo using Brdu to label and observe changes of cell proliferation during wound healing.(3) ResultsThe expression of K15, K19:BMSCs at passage4were seeded in24-well plates with one coverslip in each well at a density of4×105cells/well. Divided two groups, which were incubated in5%FBS plus DMEM medium containing PBS,5%FBS plus10ng/mL activin DMEM medium containing.Until21days from the train the first day of every other day, remove the coverglass,cell immunochemistry. The results show:Activin B group was not significantly higher than PBS group.We further used Brdu to detect cell proliferation around the wound found that the number of Brdu-positive cells in epidermal basal layer of BMSCs, Activin B group and activin B plus BMSCs group was significantly higher than PBS group,but BMSCs.Activin B group was not significantly higher than activin B plus BMSCs group.Accordingly, we further believe that Activin B through someone signaling pathway regulate the biological activity of BMSCs, thus promote proliferation and migration of BMSCs.To investigate whether activin B regulates actin stress fiber formation in BMSCs, we examined the distribution and formation of actin stress fibers in BMSCs treated with activin B by phallotoxin staining. actin stress fibers were mainly distributed along the membrane at30min after treatment. At2and4hours, numerous actin stress fibers were arranged across the cell bodies. At6hours, only few actin stress fibers were seen at the periphery of cells. In contrast, the distribution and number of actin stress fibers showed no significant changes during the whole process in PBS-treated cells. Apparently, activin B could induce actin stress fiber formation in BMSC cells.Since actin polymerization plays a key role in cell migration, we next examined the impact of activin B on the migration of BMSCs using scratch wound healing assay and transwell migration assay. Many BMSCs cells treated with10ng/mL activin B have migrated to the scratch wound72h after the scratch, while few PBS-treated BMSCs were found in the scratch wound. In the transwell migration assay, the number of cells that migrated to the lower chamber was significantly higher in BMSCs cells treated with10ng/mL activin B than in PBS-treated BM-MSCs. Taken together, these data suggest that activin B induces actin stress fiber formation and BMSC migration.(4) Conclusionthese data suggest that activin B induces actin stress fiber formation and BM-MSC migration.so as to promote wound healing.(1) PurposeIn vitro to examine whether the MAPK signaling pathway is involved in activin B-induced BMSC migration(2) MethodsBMSCs at passage4were treated with the JNK specific inhibitor of SP600125(5μmol) of p38inhibitor of SB202190(5μmol) and ERK inhibitor of SL327(5μmol),treated cells30min after use phallotoxin staining to investigate activin B regulates actin stress fiber formation in BMSCs and cells migration. The expression of activin B-induced p-JNK, p-ERK, and p38protein were detected using Western Blotting. (3) ResultsThe levels of JNK and ERK phosphorylation were significantly increased10min after activin B treatment and reached the peak at30min. Two hours later, JNK phosphorylation returned to the baseline level and ERK phosphorylation slightly decreased. In contrast, p38phosphorylation showed no significant changes after activin B treatment (data not shown).To further determine whether JNK and ERK signaling mediates activin B-induced actin stress fiber formation and BM-MSC migration, specific inhibitors were used to treat BMSCs two hours after activin B treatment. Both SP600125, a specific inhibitor of JNK, and SL327, a specific inhibitor of ERK significantly inhibited activin B-induced actin stress fiber formation. In contrast, the p38-specific inhibitor SB202190showed no significant impact on activin B-induced actin stress fiber formation.Similar results were also obtained in scratch wound healing assays. Many BMSCs cells treated with combined activin B and SB202190have migrated to the scratch wound72h after the scratch, but there very few PBS-treated BM-MSCs, BMSCs treated with combined activin B and SP600125, and those treated with combined activin B and SL327in the scratch wound.(4) ConclusionCollectively, these data suggest that activation of JNK and ERK, but not p38, is required for activin B-induced actin stress fiber formation and BM-MSC migration.(1) PurposeThe purpose of the present study is to discuss the optimum transplantation approaches of bone marrow mesenchymal stem cells (BMSCs) combination with Activin B on skin wound healing.(2) Methods18SD rats were randomly divided into the epidermal transplantation group, the tail vein transplantation group and control group. After full-thickness skin wounds were created on each mouse on the back of rats, the two experimental group rats were administered with phosphate buffer solution (PBS) containing1X106BMSCs marked with Green Fluorescent Dye CFSE and0.4ml10ng/ml activin B for three days respectively, and control group was administered with0.4ml PBS. In order to evaluate the therapeutic effects of different transplantation approaches, we detected residual situation of the CFSE-labeled BMSCs in the wounded skin and different visceral organs by both the In Vivo imaging system and frozen section3and7days respectively after transplantation. Detection of CFSE-labeled BMSCs was lasted for14days until to full-thick skin healing. Wounded areas were recorded and wound healing rates were calculated the3,7and14days respectively after skin wounding, and then wounded skin was harvested, paraffin-embedded and Hematoxylin and eosin (H&E) stained.(3) ResultsWe examined wound healing in rats transplanted with CFSE-labeled BMSCs using an in vivo imager. On days3and7after cell transplantation, CFSE green fluorescence was detected in rats administered with BMSCs plus activin B epidermal transplantation groups and BMSCs plus activin B tail vein transplantation group, but not in those administered with PBS. In frozen sections, CFSE+BMSCs with BMSCs plus activin B epidermal transplantation groups have incorporated into wound sites on day3,the trend of migration to the wound BMSCs plus activin B tail vein transplantation group. On day7, CFSE+BMSCs have disappeared at wound sites and migrated to the wound margin in rats administered with BMSCs plus activin B epidermal transplantation groups and BMSCs plus activin B tail vein transplantation group. Organs frozen section results:expression of BMSCs plus activin B epidermal transplantation groups and BMSCs plus activin B tail vein transplantation group lungs green fluorescence of the strongest,the PBS group were not see the green fluorescence.On days7and14after administration, the rates of wound closure were significantly higher in the activin B plus BM-MSCs group than in the PBS, but wound healing rate in the epidermal transplantation group and the tail vein transplantation group had no significant difference. Histological analysis confirmed that wound re-epithelialization has completed on day14in the BMSCs plus activin B epidermal transplantation groups and BMSCs plus activin B tail vein transplantation group.Compared to the PBS groups, regeneration of hair follicles was observed.(4) ConclusionBMSCs transplantation via the epidermal and the tail vein can both migrate to the wounded sites and promote the wound healing. Compared with the tail vein transplantation group, wound repair in the epidermal transplantation group was more simple and feasible.