Effect Of Simulated Microgravity On The Migration Of Human Skin Fibroblasts And The Related Mechanism

As an opening of a new era of space exploration,people start paying more and more attention to the safety and health of astronauts in addition to space equipment.The skin is the largest organ of the human body and plays an important role in protecting the body.Multiple studies have shown that the microgravity environment in space has a variety of effects on skin and even wound healing.However,the specific effect and related pathways of microgravity acting on wound healing are still a mystery.Skin fibroblasts are main structural components of dermis and the main source of extracellular matrix.In the repair of skin damage,fibroblasts will migrate to the injury site and participate in the reconstruction of the dermis,and their migration directly affects the efficiency and outcome of wound healing.However,the effect of the microgravity environment in space on the migration of skin fibroblasts and the related mechanisms are still unclear.Therefore,an increased understanding of the interaction of microgravity with wound healing and skin fibroblasts migration may indicate novel insights into the mechanisms of wound healing during space flight.We utilized a two-dimensional clinostat to model Simulated microgravity(SMG)and detected the impact of SMG on skin fibroblasts migration and the related mechanisms at the in vitro.The effect of SMG on skin wound healing was investigated at the in vivo by hindlimb unloading.The main experimental results are as follows:(1)Constructed methods to simulate microgravity effects at the in vivo and in vitro levelsConstructed methods to simulate microgravity effects by two-dimensional clinostat at the in vitro level;Constructed methods to simulate microgravity effects by hindlimb unloading model at the in vivo levels.(2)SMG inhibits the migration of skin fibroblasts HFF-1 by depolymerizing F-actin and down-regulating YAPThe cell wound healing results showed that the wound healing rate of the SMG group was significantly lower than that of the normal gravity(NG)group at 24 h and 36 h of migration.After SMG treatment for 36 h,western bolt(WB)experiments results showed that SMG treatment reduced the expression of YAP in HFF-1 and increased YAP phosphorylation ratio.The results of immunofluorescence(IF)showed that SMG limited YAP translocation into the nucleus;After adding LPA to SMG treatment for 36 h,the WB results showed that the expression of inhibited YAP increased significantly and the phosphorylation ratio decreased.Cell wound healing assay showed that LPA significantly promoted the migration of cells inhibited by SMG at 24 h and 36 h.The above results indicated that SMG inhibited skin fibroblasts migration by reducing YAP expression and increasing YAP phosphorylation ratio.The effect of SMG treatment on the cytoskeleton was further detected by immunofluorescence,and it was found that SMG promoted F-actin depolymerization.Adding Jasp to treating with SMG promoted F-actin polymerization to a similar level as the NG group.And,cell wound healing assay showed that Jasp could significantly enhance cells migration inhibited by SMG.In addition,it was found by WB experiment that after adding Jasp to SMG treatment for 36 h,the expression of inhibited YAP was significantly increased,and YAP phosphorylation ratio was significantly decreased.The results shows that SMG inhibits the migration of skin fibroblasts HFF-1 by depolymerizing F-actin and down-regulating YAP.(3)SMG inhibits skin wound healing by downregulating YAPThe injured rats were treated under hindlimb unloading,and pictures were taken on the 0,2,4,6,and 8th days under unloading to calculate the wound healing rate.The results showed that the wound healing rate of the SMG group was significantly lower than that of the NG group on the 4th and 6th day.After LPA gel were applied to the wound(the SMG+LPA group),it was found that the wound healing rate down-regulated by SMG increased significantly;The wound tissue was taken to make tissue sections and stained on the 8th day under hindlimb unloading.The SMG group had the fewest fibroblasts in the wound,which was significantly lower than the NG group and the SMG+LPA group.The results showed that SMG may inhibit skin damage repair by affecting the migration of skin fibroblasts and reducing the number of fibroblasts in the wound;LPA treatment may promote skin injury repair under SMG conditions by affecting YAP to restore the migration ability of skin fibroblasts to the injury site.This study revealed the effect of SMG on skin fibroblasts migration,clarified the mechanism of F-actin/YAP pathway regulating HFF-1 cell migration and skin wound healing under SMG,and indicated a theoretical reference for exploring how microgravity environment in space affects skin wound healing.