Effect Of Simulated Microgravity On Differentiation Of Mesenchymal Stem Cells Into Dermal Fibroblasts And The Related Molecular Mechanisms

With the development of the aerospace enginggring,astronauts nowadays spend more time and take more tasks in the space.As the body’s first defense barrier,the skin is the most vulnerable tissue during human spaceflight missions.Studies have shown that the microgravity environment in space has an impact on the skin of the astronauts,such as thinning of the dermis,decreased elasticity,increased sensitivity,etc,which indicates that the microgravity environment has damaged the physiological properties of the skin.If the damaged skin is not repaired in time,serious consequences will result.However,the effect of the microgravity environment on the skin repair process remains to study.From the perspective of space exploration missions,how to ensure the skin health of astronauts in spaceflight is also a critical and urgent scientific problem.Therefore,it is necessary to understand whether and how the microgravity environment affects the wound healing process and the underlying mechanisms.Dermal fibroblasts are not only the main structural components of the dermis but also the most important tissue repair cells during the skin damage repair.The multi-directional differentiation potential of bone marrow mesenchymal stem cells(BMSCs)can play an important role in the repair of damaged tissue.Studies have confirmed that BMSCs can differentiate into dermal fibroblasts and participate in the repair process of damaged skin.But in space microgravity conditions,the changed characteristics of this differentiation process and the biological mechanisms involved are still unknown.Given this,this study mainly explores the effect of simulated microgravity(SMG)on the differentiation of BMSCs into dermal fibroblasts and its related molecular mechanisms and provides experimental evidence and potential targets of intervention and reconstruction for skin damage repair under gravity unloading.The main contents and results of this study are as follows:(1)The isolation and culture of BMSCs and the construction of a system for the differentiation into dermal fibroblastsBMSCs were isolated and extracted from the hindlimb of SD rats.Cell surface markers were detected by flow cytometry,and the results showed that CD29 and CD54 were positive,while CD45 and CD11b/c were negative,which was consistent with the characteristics of BMSCs surface markers.L-DMEM containing ascorbic acid and connective tissue growth factor(CTGF)was used as the induction medium to induce the differentiation of BMSCs into dermal fibroblasts.The expression of dermal fibroblast-related markers Collagen Ⅰ(COL Ⅰ),Collagen Ⅲ(COL Ⅲ),Desmin and Fibroblast Specific Protein-1(fibroblast specific protein-1,FSP-1)was detected by q RT-PCR,Sirius red staining and Western blot after induction.The results showed that the expression levels of these m RNAs and proteins were significantly increased.(2)Effects of simulated microgravity on the differentiation of BMSCs into dermal fibroblastsThe two-dimensional rotary incubator developed by the Institute of Mechanics of the Chinese Academy of Sciences was used to produce the SMG effect on the cultured cells,and the BMSCs were inoculated into the rotary incubator to induce differentiation.The results showed that compared with the normal gravity(NG)group,the m RNA expressions of dermal fibroblasts markers COL I,COL III,Desmin and FSP-1 were significantly decreased in the simulated microgravity group.The secretion of collagen I and collagen III was strikingly decreased,and the protein expressions of Desmin and FSP-1 were also obviously decreased.The results showed that the simulated microgravity effect inhibited the differentiation of BMSCs into dermal fibroblasts.(3)The molecular mechanisms of simulated microgravity effect in inhibiting the differentiation of BMSCs dermal fibroblastsThe results showed that during the differentiation process under NG,the expression of β-catenin at both m RNA and protein levels was significantly increased.Meanwhile,during the differentiation process under the SMG effect,the expression ofβ-catenin at both levels was significantly decreased.After activating the Wnt/β-catenin signaling pathway with Li Cl under the effect of SMG,the expression of dermal fibroblasts markers COL I,COL III,Desmin and FSP-1 at both m RNA and protein levels was significantly increased after induction.This indicated that the induced differentiation of BMSCs into dermal fibroblasts was restored by up-regulating the Wnt/β-catenin signaling pathway under the effect of SMG.The results showed that there was no change in total ERK1/2 detected by Western blot during the differentiation process under NG,but the expression of phosphorylated ERK1/2 was significantly increased.By contrast,no change in total ERK1/2 during differentiation under SMG effect,but the expression of phosphorylated ERK1/2 was significantly decreased.After activation of ERK1/2 with tert-Butylhydroquinone(t BHQ)under SMG effect,the expression of β-catenin was significantly increased,and the expression of dermal fibroblasts markers COL I,COL III,Desmin and FSP-1 was significantly increased after induction.These findings suggest that the ERK/β-catenin signaling pathway plays an critical role in force signal transduction during microgravity inhibition of BMSCs differentiation into dermal fibroblasts.In conclusion,this study confirmed that the SMG effect inhibited the Wnt/β-catenin signaling pathway by inhibiting ERK1/2-mediated force signal transduction,and further inhibited the differentiation of BMSCs into dermal fibroblasts,suggesting that the ERK/β-catenin signaling pathway may be a potential target that intervenes the differentiation of BMSCs into dermal fibroblasts under microgravity.The results of this study provide an experimental and theoretical reference for the therapeutic strategies of wound healing and skin reconstruction in aerospace medicine.