The Regulation Of JNK/ERK Signal Pathway On Activin B-mediated Fibroblasts In Skin Wound Healing

Research Backgroud and ObjectiveAs the largest organ of the human body,the skin is the first barrier to maintain homeostasis,and to defense the foreign matter from pathogens.Wound often accompanies with skin injury,and if do not treat as soon as possible,it will not only affect the skin function,but also become account for infection,thus it is important to treat the wound in the early stage in order to prevent body from internal environment disorders and body consumption.As a result,skin wound healing is an urgent problem to be addressed in clinical medicine.Also,skin wound healing is a complex biological process,mainly experiences four interdependent phases,which are local hemostatic stage,inflammatory response stage,cell proliferation and granulation tissue formation stage and tissue remodeling stage.Fibroblast,which is differentiated from embryonic mesenchymal cells is the most important cell in the loose connective tissue and major cell of dermis in human body.It acts as the main repair cells and runs through the whole process of skin wound healing.In the early stage of wound healing,fibroblasts secrete extracellular matrix which is composed of procollagen,collagenase and fibronectin,and form granulation tissue with new capillaries and inflammatory cells together.After cell proliferation and external matrix deposition,fibroblasts change the proportion of collagen of the wound under the action of collagenase and growth factors to participate in the late transformation of the organization.In the process of wound healing,cell proliferation,migration and differentiation is regulated by cell growth factors.These cell growth factors mainly include EGF,bFGF,TGF-?,PDGF etc.Activin belongs to the TGF-? superfamily because of its typical TGF-? superfamily structure.It has a wide range of biological activities.Studies have shown that the expression of Activin is found in mammalian reproductive,embryonic development,erythrocyte differentiation,and post-injury repair.Our team previous studies have found that Activin B can promote the migration of keratinocytes by MAPK signaling pathway to promote skin wound healing in mice.Mitogen-activated protein kinase(MAPK)is an important signaling system composed of a cascade of activated silk/threonine protein kinases that mediates cell biologic responses and is widely found in mammals.In cell migration,differentiation,proliferation and other biological functions have played an important role.Owing to the fact that fibroblasts are one of the major cells in wound healing,we speculate that Activin B may be involved in fibroblasts repairing the wound and is accomplished through the MAPK signaling pathway.In order to explore the MAPK signaling pathway whether participate and how to regulate fibroblasts in wound healing,Activin was designed to stimulate the fibroblast to observe changes in its agonists first,and then to block or activate the signaling pathway specifically to investigate the role of signal pathway.Activin-mediated changes in fibroblast can tell how it regulates fibroblasts.This experiment was designed to study how MAPK signal pathway mediate Activin B regulating fibroblasts in vitro in skin wound healing.And we targeted to provide a new theoretical basis for the treatment of skin wounds and scars.Research MethodsIn Vivo1.Animal Model:Cutting an around 8mm wound on the back of C57BL/6(SPF Class)female mice which need to be anesthetized by 2%pentobarbital sodium and denuded by honey wax depilatory paste.The particular cutting wound should contain the whole layer of the skin and reach the subcutaneous.Would need to be exposed after operation,and be observed every day.2.Division:Mice should be divided into PBS Group and Activin B Group randomly and fed with water and food.3.Record and calculate the recovery rate of the wound tissue:recorded the healing rate in the same condition every day and calculated the healing rate by using IPP image analyze system.(Healing rate=(Original wound size-unrecovered wound size)/original wound size*100%)4.Wound area re-epithelialization and granulation tissue re-formation:took the whole layer of the skin within 0.5cm of the wound after 3,5 and 7 days of the operation.Paraffin embedding,section,H&E staining and observing the re-epithelialization process,the formation of the granulation tissue and the change of the internal structure of the granulation tissue.5.Expression of type I collagen in granulation tissue within wound area:took the whole layer of the skin within 0.5cm of the wound after 3,5 and 7 days of the operation.Paraffin embedding,section and observed the expression of the type I collagen in granulation tissue by using Immunoassay and observed the collagenous fiber by using Masson Staining.In Vitro1.Cultured Fibroblasts:peeling the skin from C57BL/6 neonatal rat and placed the dermal r horizontally in a Petri dish with 0.25%trypsin and digested at 4? over night.Then removing the epithelium and then discard dermis by using the curved tweezers until the dermis is completely broken.Oscillating the broken dermis violently with complete medium.Filtering the dermis by using 100?m filter,centrifuging.Suspending the cell with new complete medium.And finally the cells were cultured in the culture medium after suspension.2.Elisa,RT-PCR:Dividing the group into PBS and Activin B by passing 4 to 6 proliferation of the fibroblast.Extracting the supernatant fluid,mRNA and using Elisa and RT-PCR to figure out the influence of Activin B on the secretion of the collagen of the fibroblasts.3.CCK8:Dividing the group into PBS,Activin B,Activin B+SP600125,Activin B+SL-327 and Activin B+p38 by passing 4 to 6 proliferation of the fibroblast.Figuring out the influence of the Activin B on the proliferation of the fibroblasts by using CCK8 reagent kits.4.Transwell Assay:Dividing the group into PBS,Activin B,Activin B+SP600125,Activin B+SL-327 and Activin B+p38 by passing 4 to 6 proliferation of the fibroblast.To figure out the influence of the Activin B on the chemotaxis of the fibroblasts.5.Wound Healing:Dividing the group into PBS,Activin B,Activin B+SP600125,Activin B+SL-327 and Activin B+p38 by passing 4 to 6 proliferation of the fibroblast.To figure out the influence of the Activin B on the wound healing of the fibroblasts.6.Western Blot:Dividing the group into PBS,Activin B,Activin B+SP600125,Activin B+SL-327 and Activin B+p38 by passing 4 to 6 proliferation of the fibroblast.Extracting the protein and figuring out the influence of the Activin B on the signal pathway of mediating fibroblasts.Research Result1.In Vivo:the recovery rate of the Activin B group was faster than that of PBS group after 3 and 5 days of the operation.At the meantime,the generation of granulation tissue of Activin B group was also faster than that of PBS group postoperative 3,5 and 7 days.Besides,in Activin B group,a large number of fibroblasts can be observed under microscope in day 3 while new capillaries were mostly changing to mature capillaries in day 5.Thus,Activin B can accelerate the wound healing by stimulating the formulation of the granulation tissue.In the results of immunoassay and Masson Staining,no significant difference was found in the type I collagen and collagenous fiber between Actvin B and PBS groups.And therefore Activin B has no influence on the secretion of the collagen of fibroblasts in the animal experiment.2.In Vitro:After the stimulation of the Activin B,the proliferation of fibroblasts at 12h,24h and 72h was significantly higher than that of the PBS group.The result of Transwell Assay also showed that the chemotaxis amount of the fibroblast in Activin B group is larger than that in PBS group.Besides,the result of wound healing suggested that the wound clousare rate of Activin B group was faster than that of PBS group.Thus,Activin B can advance the proliferation,chemotaxis and migration of fibroblasts.3.The result of Western Blot suggested that the Activin B group had a higher expression of p-JNK and p-ERK than that in PBS group after the stimulation of Activin B.And p-JNK had the highest expression at the time of 10 min while p-ERK was at 30min and both showed decrease afterward.Besides,we found that proliferation,chemotaxis and migration of fibroblasts was inhibited by JNK inhibitor SP600125;the proliferation and chemotaxis of fibroblasts was inhibited by ERK inhibitor SL-327,whereas there was no obvious inhibition of migration,so we can be inferred that Activin B regulated fibroblast proliferation,chemotaxis and migration through JNK and ERK/MAPK signaling pathway.4.The expression of p-p38 in Activin B group showed no significant change comparing to that in PBS group.There was no change in proliferation and migration of fibroblasts by using the inhibitor SB202190 of the p38 pathway.However,the chemotaxis of fibroblasts was inhibited significantly by using SB202190.Therefore,although p38 signaling did not involved in Activin B regulating fibroblast proliferation and migration,it still is an important regulator of chemotaxis in fibroblastsResearch Conclusion:Activin B regulates fibroblasts in proliferation,chemotaxis and migration through JNK and ERK/MAPK signaling pathways in wound healing.P38/MAPK signaling pathway is an important regulator of fibroblast chemotaxis.