Therapeutic Effect And Mechanism Of The Exosomes Secreted From Human Umbilical Cord Mesenchymal Stem Cells Cultured With Hypoxia On Allergic Rhinitis In Mice

Background and Objectives:Allergic rhinitis is a common chronic inflammatory disease of the upper respiratory tract,characterized by continuous nasal congestion,rhinorrhea,sneezing and nasal pruritus.About 500 million people around the world are suffering from it.Allergic rhinitis not only seriously affects the quality of life and work efficiency of patients,but also brings many adverse reactions and economic burdens from long-term drug treatment to patients.At present,there is no clinical effective drug for the treatment of the disease.In addition,the emergence of patient resistance and tolerance to drugs also seriously affect its therapeutic effect.Therefore,it is important to find a safer and more effective treatment scheme for allergic rhinitis.Recent studies have confirmed that mesenchymal stem cells(MSCs)and their secreted exosomes have immunosuppressive effects and are a promising treatment for allergic rhinitis.Umbilical mesenchymal stem cells(UCMSCs),with potent proliferation,no ethical barriers and no tumorigenesis and no immunological rejection,are an ideal source of stem cells for the treatment of various diseases.However,long-term in vitro expansion of UCMSCs reduces its differentiation potential and immunomodulatory function.A growing number of studies have shown that simulating the in vivo environment(i.e.hypoxia)of stem cell therapy can improve their viability,promote cell proliferation,and maintain their stem cell characteristics.This project aims to investigate the effects of long-term hypoxic treatment on UCMSCs characteristics and to elucidate the mechanism of exosomes secreted by UCMSCs in the treatment of allergic rhinitis.Methods1.Isolation,culture and identification of human UCMSCs: UCMSCs from human umbilical cord were separated and cultured by tissue adhesion method,and the following characteristics of UCMSCs under normoxic or low oxygen conditions were compared: 1)cell count and telomerase activity analysis was used to detect UCMSCs amplification;2)flow cytometry was used to examine surface molecular markers of UCMSCs;3)lipogenic,osteogenic and chondrogenic differentiation experiment to detect multiple differentiation potential of UCMSCs in vitro;4)soft agar colony formation experiment was used to determine the tumorigenicity of UCMSCs;5)G-band analysis was used to detect chromosome karyotype of UCMSCs.2.Preparation,identification and composition analysis of UCMSCs-derived conditioned media(CM)and exosomes: UCMSCs were cultured under normoxic or hypoxic conditions,and CM and exosomes were prepared by conventional methods.The following characteristics of CM or exosomes were compared under normoxic or hypoxic conditions: 1)The contents of soluble cytokines such as TGF,IL-10,TNF,IL-6 and IL-1 in CM were detected by Elisa assay;2)Exosomes were separated from UCMSC-CM by super high speed centrifugation;3)The mi RNA contents and differential expressions were analyzed by small molecule RNA sequencing;4)The main biological functions of differential mi RMA expression were analyzed using GO and KEGG pathway enrichment methods.3.The therapeutic effect and mechanism of the CM or exosomes derived from UCMSCs cultured with long-term hypoxia on allergic rhinitis in mice: 1)A mouse model for allergic rhinitis was established by OVA;2)Behavioral observation was conducted by calculating the numbers of nose scratching and sneezing;3)the inflammatory cell infiltration and tissue remodeling of mouse nasal mucosa tissue were detected by HE staining;4)the m RNA expressions of Th1,Th2,Treg,IL-4 and IL-10 were detected by Q-PCR in mouse nasal mucosa tissue.4.Effect and mechanism analysis of exosomes secreted from UCMSCs cultured with long-term hypoxia on dendritic cell(DC)functions: 1)Immature DC cells were isolated from human peripheral blood monocytes by continuous adherent method;2)The immature DC cells were differentiated into mature DC(m DC)by stimulation with OVA plus LPS,and the effects of exosomes on DC maturation were also examined,in the control,immature DC cells were differentiated into tolerant DC cells with IL-10 and TGF induction;3)The proportion of the CD11 c and HLA-DR double positive m DC,and the expression levels of the costimulatory molecules CD80,CD40,CD86,and CD83 were analyzed by flow cytometry.Results1.Long-term hypoxia treatment promotes UCMSCs proliferation and differentiation,and inhibits cell senescence: 1)UCMSCs isolated by adherent method was a spindle-shaped and the view of entire cells was a vortex shape;2)Cell count and telomerase activity analysis showed that hypoxic pretreatment promoted UCMSCs proliferation;3)Galactosidase staining indicated that hypoxic pretreatment inhibited UCMSCs senescence;4)Flow cytometry showed that there were a high expression of CD105,CD44,CD73 and CD90,and no expression of CD34,CD45 and HLA-DR in UCMSCs;5)UCMSCs were directionally differentiated into osteocytes,Chondrocytes and adipocytes in vitro;6)Q-PCR results showed that hypoxia pretreatment was beneficial to maintain the expressions of the stemness genes OCT4,Sox2 and Nanog of UCMSCs;7)Soft-agar cloning experiments showed that under the low-oxygen treatment conditions,UCMSCs were cultured to P20 generation without tumorigenesis;8)G band analysis showed that UCMSCs cultured under low oxygen conditions until the P20 generation had a normal karyotype.2.CM secreted from UCMSCs pretreated with hypoxia had a strong therapeutic effect on allergic rhinitis in OVA-induced mice,and the mechanism might be related to decreasing the infiltration of Th1,Th2 and Treg cells in nasal mucosa tissues: 1)The behavioral observation of nose scratching and sneezing showed that compared to the normal oxygen species,CM secreted from UCMSCs pretreated with hypoxia significantly improved the symptoms of allergic rhinitis in mice compared to the group with normal oxygen supplied;2)HE staining indicated that CM secreted from UCMSCs pretreated with hypoxia significantly inhibited inflammatory cell infiltration and tissue remodeling in nasal mucosa tissue;3)Elisa analysis indicated that CM secreted from UCMSCs pretreated with hypoxia remarkably reduced the total serum Ig E content;4)Q-PCR analysis showed that CM secreted from UCMSCs pretreated with normal oxygen inhibited the expressions of Gata3 and IL-4,promoted the expressions of T-bet,Foxp3,and IL-10,whereas the CM secreted from UCMSCs pretreated with hypoxia inhibited the expressions of Gata3,T-bet,Foxp3,IL-4,and IL-10.3.UCMSCs pretreated with hypoxia had no significant effect on soluble cytokine content of CM,but affected the mi RNA content and expression profile of exosomes: 1)Elisa analysis showed that UCMSCs pretreated with hypoxia or normal oxygen did not affect the soluble cytokine content of CM;2)mi RNA sequencing results showed that hypoxia pretreatment affected mi RNA expression profile of UCMSCs-derived exosomes;3)GO and KEGG signaling pathway enrichment analysis showed that the differentially expressed mi RNAs mainly affected the common stimulation signaling pathway in T lymphocytes.4.The exosomes secreted from UCMSCs pretreated with hypoxia enhanced the therapeutic effect on allergic rhinitis in mice: 1)Exosomes secreted from UCMSCs pretreated with hypoxia significantly improved the symptoms of allergic rhinitis in mice;2)HE staining indicated that exosomes secreted from UCMSCs after hypoxia pretreatment remarkably inhibited inflammatory cell infiltration and tissue remodeling in nasal mucosa tissues of mice;3)Q-PCR analysis showed that the exosomes from UCMSCs with normal oxygen inhibited IL-4 expression,promoted the expression of IL-10,whereas the exosomes secreted from UCMSCs with hypoxia pretreatment inhibited both IL-4 and IL-10 expressions.5.Exosomes secreted from UCMSCs pretreated with hypoxia inhibited the differentiation maturation of DC cells and the expressions of the costimulatory molecules in vitro: Flow cytometry showed that exosomes secreted from UCMSC pretreated with hypoxia significantly reduced the proportion of CD11 c + HLA-DR + positive cells and inhibited the expressions of costimulatory molecules CD40 and CD80.Conclusions1.Hypoxic pretreatment enhanced the proliferation and differentiation ability of UCMSCs,promoted the expressions of mesenchymal stem cell marker genes,and had no effect on its absence of hematopoietic stem cell-related genes,low immunogenicity,and non-tumorigenic characteristics.2.Hypoxic pretreatment promoted the therapeutic effects of UCMSCs-derived secretions including CM and exosomes on OVA-induced allergic rhinitis in mice.3.Exosomes derived from UCMSCs pretreated with hypoxia enhanced the therapeutic effect of OVA-induced allergic rhinitis in mice by changing the mi RNA content and expression profile in UCMSCs-derived exosomes.4.The exosomes secreted from UCMSCs pretreated with hypoxia inhibited the differentiation and maturation of DC cells,and attenuated the expressions of the co-stimulatory molecules in DC cells,subsequently enhancing their therapeutic effect on OVA-induced allergic rhinitis in mice.