| [Background] Type 2 diabetes(T2D)accounts for more than 90% of diabetes patients.In recent years,the incidence of T2 D has dramatically increased,which has become a chronic metabolic disease with high incidence around the world,causing serious damage to human health and a huge economic burden to the society.T2 D is a systemic disease that predisposes patients to a variety of oral diseases,including dental caries,gingivitis,periodontitis,and maxillofacial infections.In addition,T2 D increases the difficulty of the treatment for oral diseases and leads to poor prognosis.Current treatments for T2 D mainly include oral antidiabetic agents and exogenous insulin infusion,which can ameliorate hyperglycemia temporarily but fail to achieve long-term efficacy and reverse the course of the disease.Therefore,it is of great significance to clarify the regulatory mechanism of T2 D and establish effective treatment strategies for maintaining oral and systemic health.As a kind of widely studied adult stem cells,mesenchymal stem cells(MSCs)are capable of multi-differentiation,immunomodulation,and microenvironment improvement,and have the advantages of low immunogenicity and in vitro amplification,which has shown great promises in the treatment of autoimmune diseases,diabetes,cardiovascular diseases,neurodegenerative diseases and so on.The mechanism underlying the therapeutic effects of MSCs has always been a hot topic in the field of cell therapy.Previous studies have shown that MSCs exert their efficacy through differentiating into a variety of tissue cells after transplantation into the body.In recent years,a growing number of studies have found that,instead of directly differentiating into adult cells,the therapeutic effects of MSCs are produced via regulating tissue cells and microenvironment,which mainly depends on the paracrine function of MSCs,including a variety of cytokines and other bioactive molecules.Although previous studies have found that MSCs show promising therapeutic effects on T2 D,and the therapeutic mechanism has been partially clarified,there are still many problems that remain unclear.In particular,MSCs undergo apoptosis and are cleared by the host within a short time after systemic infusion,while still producing systemic and sustained therapeutic effects.The paradox suggests that apoptosis may be involved in the treatment of MSCs,and the specific mechanism remains to be further explored.Extracellular vesicles(EVs)are a kind of tiny vesicles secreted by cells,containing proteins,lipids,nucleic acids and other biological active ingredients.EVs act as an important player in the communication between cells,which participates in the development,homeostasis maintenance,injury repair and other biological processes.As an important component of MSCs’ secretome,EVs have been shown to play a key role in the therapeutic application of MSCs.In the process of apoptosis,cells also secrete a large number of EVs,which are known as apoptotic extracellular vesicles(Apo EVs).Current studies mainly focus on EVs derived from living cells,while few studies have been conducted on Apo EVs.Therefore,their biological characteristics and roles in the treatment of MSCs are still unclear.Macrophages are a key component of the innate immune system,and their dysfunction causes chronic systemic inflammation,which plays an important role in the occurrence and development of T2 D.In particular,the pro-inflammatory activation of macrophages and the progressive infiltration of bone marrow monocyte-derived macrophages(Mo MFs)are important features and pathological mechanisms of T2 D.In addition,macrophages are the main phagocytes in the body,responsible for the removal of a large number of apoptotic cells and their products generated on a daily basis.The process of apoptotic cell clearance by macrophages is called efferocytosis,which can not only eliminate the damaged cells in time,but also enhance the secretion of anti-inflammatory factors.Efferocytosis can reduce the inflammatory response,which plays an important role in maintaining the homeostasis of the body.However,it remains unknown whether MSC-derived Apo EVs can be engulfed by macrophages and regulate their function after systemic infusion,and what effects Apo EVs exert on metabolic diseases.In conclusion,the following hypothesis are proposed in this study: MSCs undergo apoptosis after systemic transplantation and releases specific Apo EVs which are engulfed by macrophages and regulate their functions so as to reduce chronic inflammation and alleviate T2 D.[Aim] Based on the T2 D disease model and bone marrow mesenchymal stem cells(BMMSCs),this study was designed to explore: 1.Whether apoptosis contributes to the therapeutic effects of systemically infused BMMSCs on T2D;2.Whether BMMSC-derived Apo EVs exert therapeutic effects on T2 D after systemic infusion;3.Whether the systemically transplanted Apo EVs can be engulfed by macrophages and regulate their functions;4.What are the specific molecular mechanisms mediating the uptake of Apo EVs by macrophages.The aim of this study is to deeply reveal the molecular mechanism underlying MSCmediated cytotherapy and to further clarify the in vivo fate,biological function and therapeutic potential of Apo EVs,so as to promote the establishment of a novel EV-mediated T2D treatment strategy.[Methods] 1.Analyses of in vivo apoptosis and therapeutic effects of systemically infused BMMSCs.Human BMMSCs were isolated and cultured by density gradient centrifugation,and were identified by flow cytometric analysis of surface markers,colony formation assay as well as osteogenic,adipogenics and chondrogenic differentiation assay;Fluorescent dye labeled BMMSCs were injected through systemic infusion and the distribution of BMMSCs was detected by in vivo imaging and tissue section analyses;The survival of BMMSCs in vivo was observed by laser confocal microscopy imaging at different time points and the apoptosis of BMMSCs were detected by flow cytometry;A T2 D mouse model induced by high fat diet(HFD)was established and BMMSCs were therapeutically transplanted following a sequential multiple infusion strategy;The efficacy of BMMSCs was observed through glucose tolerance test,insulin tolerance test,insulin signaling pathway test,histological staining as well as organ and systemic lipid test;BMMSCs were pretreated with the apoptosis inhibitor,and the role of apoptosis in MSC-mediated treatment of T2 D was explored by comparing their therapeutic effects with normal BMMSCs.2.Analyses of the therapeutic effects of BMMSC-derived apoptotic cells(Apo MSCs)and Apo EVs after systemic infusion in vivo.Apoptosis of BMMSCs was induced in vitro,and Apo MSCs without secretory components were prepared and identified by TUNEL staining and flow cytometry;Apo MSCs were injected systemically and their therapeutic effects on T2 D mice were analyzed;BMMSCs-Apo EVs were isolated by gradient centrifugation and identified by morphologic observation through transmission electron microscopy,particle size distribution analysis through nanoparticle tracking analyzer,and apoptosis marker detection through western blot,immunofluorescence and flow cytometry;Apo EVs were injected systemically and their therapeutic effects on HFDinduced T2 D mice were analyzed;The therapeutic efficacy of Apo EVs was further verified on a gene defect-induced T2 D mouse model through glucose tolerance test,insulin tolerance test and blood lipid test. 3.Analyses of ApoEVs’ proteomic features,in vivo fate and biological effects.Proteomic technique were used to analyze the differentially expressed proteins in BMMSCs and Apo EVs,and functional analysis was performed on the proteins enriched in Apo EVs;Fluorescent dye labeled Apo EVs were systemically infused and the distribution of Apo EVs was detected by in vivo imaging and tissue section analyses;Immunofluorescence staining and flow cytometry were used to observe whether Apo EVs could be engulfed by macrophages;The number of macrophages was detected by immunofluorescence staining and flow cytometry;The expressions of chemokines involved in the recruitment of macrophages and related receptors were detected by RT-q PCR,immunofluorescence staining and ELISA;The migration of monocytes were detected via flow cytometric analysis of peripheral blood monocytes and in vivo tracking of monocyte migration;The polarization phenotype of macrophages was observed by immunofluorescence staining and RT-q PCR;The levels of inflammatory cytokines in peripheral blood were detected by ELISA.4.Analyses of the expression of calreticulin(CRT)on Apo EV surface and its role in Apo EV uptake.Bone marrow macrophages were isolated,cultured and identified;Fluorescent dye labeled Apo EVs was added to bone marrow macrophages in vitro and the uptake of Apo EVs was detected by immunofluorescence and flow cytometry at different time and concentration gradients;The phenotype of bone marrow macrophages was detected by RT-q PCR and ELISA;The expression of CRT on Apo EV surface was detected by western blot,ELISA and fluorescence staining;Apo EVs were pretreated with CRT neutralizing antibody to observe the effects on the uptake of Apo EVs by macrophages in vitro and in vivo,and the effects on the regulation of macrophage function by Apo EVs;The expression of CRT was down-regulated by si RNA to observe the effects on the uptake of Apo EVs by macrophages in vitro and in vivo,and the effects on the regulation of macrophages function by Apo EVs;The expression of CRT was down-regulated by si RNA and the therapeutic effects of Apo EVs on T2 D was observed.[Results] 1.BMMSCs undergo apoptosis after systemic infusion and apoptosis mediates its therapeutic effects on T2 D.The isolated and cultured BMMSCs displayed typically spindle shape,with the ability of clone formation,osteogenesis,adipogenesis and chondrogenesis differentiation,and the surface markers of BMMSCs met the identification criteria of MSCs.In vivo tracing experiments showed that the systemically infused BMMSCs was mainly distributed in the liver,followed by the lung and spleen.Further observation showed that the number of BMMSCs was significantly reduced within a short time after transplantation in vivo,and most of the cells underwent apoptosis.Systemic infusion of BMMSCs in the T2 D model effectively improved glucose intolerance,insulin resistance,insulin signaling,hepatic steatosis and lipid metabolism,while the therapeutic effects of BMMSCs pretreated with apoptosis inhibitor were significantly inhibited.2.BMMSC-derived Apo EVs effectively treated T2 D.Firstly,Apo MSCs without secretory components were prepared and identified,which showed no significant therapeutic effects on a T2 D model after systemic infusion.Then,BMMSC-derived Apo EVs were isolated,characterized and identified.Systemic infusion of Apo EVs in the HFD-induced T2 D model was found to be effective in alleviating T2D-related symptoms.By introducing the gene defect-induced T2 D model,it was further verified that Apo EVs could effectively improve glucose intolerance,insulin resistance and abnormal lipid metabolism.3.Systemically infused Apo EVs target macrophages to regulate their function and thus improve chronic inflammation in T2 D.Proteomic analysis showed that Apo EVs are enriched with many functional proteins,which are involved in a variety of biological processes including immune and metabolic regulation.Through in vivo tracing experiment,it was found that systemically infused Apo EVs were mainly distributed in the liver,which were mainly engulfed by macrophages.Further analyses showed that,after uptake by macrophages,Apo EVs inhibited the recruitment of Mo MFs and regulated the transformation of macrophage polarization phenotype from pro-inflammatory to antiinflammatory,thus alleviating the systemic chronic inflammation.4.CRT mediates the uptake of Apo EVs so as to contribute to Apo EV-mediated regulation of macrophages and treatment of T2 D.Apo EVs can be engulfed by macrophages in vitro at a time-and concentration-dependent manner.The "Eat-me" key signal molecule CRT was found to be present on the surface of Apo EVs.The use of CRT neutralizing antibody or the down-regulation of CRT expression by si RNA can inhibit the uptake of Apo EVs by macrophages and suppress the regulatory effects of Apo EVs on macrophages.Furthermore,down-regulation of CRT expression was found to inhibit the therapeutic effects of Apo EVs on the T2 D model.[Conclusion] 1.Apoptosis mediates the therapeutic effects of BMMSCs on T2D;2.Systemic infusion of apoptotic BMMSCs exerts no significant therapeutic effects on T2D;3.Systemic infusion of BMMSC-derived Apo EVS can effectively improve T2D;4.Apo EVs are enriched with functional proteins that are involved in immune and metabolism modulation;5.Apo EVs are engulfed by macrophages after systemic infusion and regulate their functions to reduce chronic inflammation;6.CRT is a key molecule that mediates the uptake of Apo EVs by macrophages.This study revealed the therapeutic mechanism that MSCs undergo apoptosis and release Apo EVs to treat T2 D,clarified Apo EVs’ in vivo fate of targeting macrophages and the underlying molecular mechanism,and verified the modulatory effects and therapeutic potential of Apo EVs,thus providing theoretical support for the establishment of novel T2 D treatment strategies based on EVs. |
PDF Full Text Request