| [Background]Skin is one of the most important organs of the human body,and skin injuries are common complaints among people of all ages.The skin injuries in the oral and maxillofacial region not only seriously affect the homeostasis of the body’s multiple systems,but also cause a huge psychological burden,thus bringing a series of social and economic problems.Currently,to clarify the key regulatory mechanism and explore more effective treatment strategies is a hot and difficult point in tissue repair and regenerative medicine.In recent years,mesenchymal stem cells(MSCs)have been widely used in the treatment of various diseases.MSCs transplantation can effectively treat various diseasessuch as rheumatoid arthritis,myocardial infarction,liver fibrosis,multiple sclerosis and systemic lupus erythematosus.Our previous research has also found that MSCs treatment alleviated scar formation during cutaneous wound healing and improved the healing rate and quality efficaciously.Notably,the mechanism underlying MSCs therapy has always been the focus in this field.Previous studies have shown that MSCs can differentiate into a variety of adult cells and integrate with host tissues to exert their therapeutic effects after in vivo transplantation.Meanwhile,some studies have shown that in most cases,transplanted MSCs participate in the repair and regeneration of tissues via paracrine function such as secretion of PGE2,VEGF,IL-4 and other immune regulatory factors,instead of direct differentiation into tissue cells.At present,despite the findings in elucidating the mechanism underlying MSCs therapy,some phenomena observed in MSCs treatment remain to be explored.In specific,it has been found that a large number of MSCs undergo apoptosis within a short time after transplantation,while MSCs exert sustainable therapeutic effects.The evidence indicates that apoptotic MSCs may also contribute to the therapeutic effects.For now,whether apoptosis plays a role in promoting regeneration remains largely unknown.MSCs release extracellular vesicles(EVs)after transplantation into recipient tissues,which are secreted by cells and mediate signal communication or regulation between cells.At present,the role of MSC-derived EVs in maintaining homeostasis,disease diagnosis and treatment has attracted more and more attention,with the underlying mechanism being gradually revealed.It is worth noting that the transplanted MSCs undergo apoptosis shortly after entering the host tissue,and during the process of apoptosis,MSCs release specific apoptotic extracellular vesicles(Apo EVs).Therefore,it is of great potential to clarify the possible regulatory role of Apo EVs in tissue repair and regeneration.Among the many hypotheses about the therapeutic mechanisms of MSCs,one of the most critical strategies is to promote the angiogenesis during tissue repair and regeneration,while enhancing the function of endothelial cells(ECs)is a pivotal cellular basis for promoting angiogenesis.In specific,autophagy,which maintains the metabolic balance and functional homeostasis of cells by degrading misfolded proteins and damagedorganelles after the formation of autophagy lysosomes,is essential for regulating and promoting ECs-mediated vascular formation.In summary,this study proposes the following hypothesis: during MSCs treatment,the transplanted MSCs underwent apoptosis and then released Apo EVs,which were taken up by ECs and activated autophagy to enhance their functions,leading to promotion of angiogenesis during tissue healing.At present,there are lack of studies on the biological role of Apo EVs,so it is of great theoretical significance and practical value to explore the enhancement of angiogenesis by Apo EVs derived from apoptotic MSCs through autophagy pathway.[Aim]This study aims to study the new mechanism of MSCs treatment based on skin defect model,elucidating that transplanted MSCs undergo apoptosis and release Apo EVs to enhance the bioactivity and quantity of ECs lysosome,investigating that Apo EVs promote TFEB protein expression and intra-nuclear transfer to activate ECs autophagy,and clarifying that Apo EVs enhance ECs vascularization function and the angiogenesis of regenerated tissues through ECs autophagy.This study will provide a strong experimental basis and theoretical support for the in-depth understanding of the mechanism of MSCs treatment,the clarification of EVs released by MSCs mediating intercellular signal communication as well as tissue repair and regeneration,and the establishment of new strategies based on EVs treatment.[Methods]1.Isolation,culture,identification and therapeutic effect verification of BMMSCs.Firstly,rat BMMSCs were cultured by whole bone marrow irrigation.Secondly,the surface markers of BMMSCs were identified by flow cytometry.Further,the clonal formation and proliferation ability of BMMSCs were detected by crystal violet and thiazole blue staining.Subsequently,the multidirectional differentiation ability of BMMSCs were examined by ALP staining,alizarin red staining,oil red O staining and western blot.Finally,we successfully established a wound healing model for skin defects and evaluated the therapeutic effects of BMMSCs via taking photos of the wound healing region on the 7th and 14 th days after BMMSCs injection,and examining the histologicalchanges by H&E and Masson staining.In addition,we observed the survival and apoptosis of BMMSCs after injection in vivo by using immunofluorescence staining.2.Separation,extraction and identification of ApoEVs and evaluation of their effect on ECs’ function.Firstly,we collected Apo EVs during the apoptosis process of BMMSCs by gradient centrifugation and characterized Apo EVs by scanning electron microscopy,particle size analysis,immunofluorescence staining and western blot.Then,we collected Apo EVs in vitro and injected them into the skin defect model.We observed the healing condition of the wound at 7 days and 14 days after the treatment,examined the histological changes by H&E and Masson staining,and detected the expression of angiogenesis related factor CD31 in the healing area by immunofluorescent staining.Finally,we explored the role of Apo EVs in ECs’ proliferation,migration,tube formation and expression of vascular-related genes by using in vitro culture model.3.In vitro evaluation of the effects of Apo EVs on ECs’ lysosomes and the biological effects of TFEB on ECs.In order to further clarify the underlying mechanism,we used immunofluorescence and RT-q PCR to detect the impacts of Apo EVs on lysosomes in ECs and analyzed the changes of lysosomes’ quantity and bioactivity.In the meanwhile,the intra-nuclear transfer of key regulatory protein TFEB were observed by western blot and immunofluorescence staining.Finally,we down-regulated the protein expression level of TFEB in ECs by transfection with si-TFEB to detect the role of TFEB in Apo EVs-mediated promotion of ECs proliferation,migration,and tube formation.4.Assessment of the impact of autophagy on ApoEVs-mediated promotion of ECs angiogenesis.Firstly,we evaluated the influence of Apo EVs on the expression of autophagy-related genes and proteins in ECs by western blot and RT-q PCR.Furthermore,we used western blot and immunofluorescence staining to detect the impact of Apo EVs on the change of ECs autophagy flow and the role of TFEB in the Apo EVs-mediated increase of ECs’ autophagy.Meanwhile,we used in vitro models to observe the role of autophagy in the promotion of the proliferation,migration and lumen formation of ECs by Apo EVs.Finally,we verified the involvement of autophagy in Apo EVs-mediated enhancement of skin vascularization in vivo via H&E and Masson histological staining andimmunofluorescence staining of CD31.[Results]1.Rat BMMSCs were successfully isolated and showed spindle-shaped appearance in culture.The BMMSCs,which are positive for mesenchymal stem cells surface markers and negative for hematopoietic stem cells surface markers,possess the ability of self-renewal and multi-directional differentiation.Compared with the control group injected with PBS,subcutaneous injection of BMMSCs suspension effectively promoted the healing of cutaneous wounds.Nevertheless,it is worth noting that the number of BMMSCs decreased sharply within a short period of time after injection,along with occurrence of apoptosis.2.Apo EVs released from apoptotic BMMSCs were collected,and subcutaneously injected into skin defect model which effectively promoted cutaneous wound healing and increased the number of newly formed blood vessels in the tissue.As to the direct effect of Apo EVs on angiogenesis,in vitro experiments found that Apo EVs promote the angiogenesis function of ECs via enhancing proliferation,migration,lumen formation and vascular-related genes expression.3.Tracing of Apo EVs by fluorescence labeling showed that Apo EVs could co-locate with the ECs’ lysosome and increase its bioactivity and quantity.Further exploration displayed that Apo EVs upregulated the expression of the key protein TFEB in ECs and promote its transfer from cytoplasm to nucleus.In order to prove that the function of Apo EVs was mediated by TFEB protein,the expression of TFEB protein was inhibited by si RNA targeting,which was found to block the promotion effect of Apo EVs on ECs’ angiogenesis.4.It was found by RT-q PCR and western blot that Apo EVs could up-regulate the expression of ECs autophagy-related genes.In the meanwhile,this result was also verified by immunofluorescence detection of ECs autophagic flow.Furthermore,si RNA targeted inhibition experiments revealed that TFEB protein played an important role in the expression of ECs autophagy-related proteins,and the promotion effect of Apo EVs on ECs autophagy level was mediated by TFEB protein.Finally,through in vivo model,autophagy was uncovered to mediate the impact of Apo EVs on promoting angiogenesis in skin wound healing,thus accelerating the healing of cutaneous wound.[Conclusion]1.BMMSCs therapy can effectively promote the healing of cutaneous wound,while a large number of BMMSCs undergo apoptosis after transplantation.2.When BMMSCs undergo apoptosis,a large number of Apo EVs are released,which can directly act on ECs and enhance their vascularization function,thus promoting angiogenesis during cutaneous wound healing and improving the therapeutic effect on skin defect.3.ApoEVs released in the process of apoptosis can be ingested by ECs and activate the cell lysosome,increasing its bioactivity and quantity.Meanwhile,Apo EVs can increase the expression of TFEB protein in ECs and promote the transfer of TFEB protein from cytoplasm to nucleus.TFEB protein can mediate the effect of Apo EVs on angiogenesis by ECs.4.TFEB protein can directly regulate the expression of genes and proteins related to ECs autophagy that plays a key role in strengthening the angiogenic function of ECs.Therefore,Apo EVs can promote ECs autophagy through TFEB protein and further enhance their angiogenic function,thus promoting angiogenesis of regenerated tissues. |
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