Studies On The Effects And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomal MiR-199a-5p In Sulfur Mustard-induced Acute Lung Injury

Background and Objective:Sulfur mustard(SM)is a typical representative of chemical warfare agent that is difficult to prevent and cure after exposure.Since its introduction on the battle field in the World War I,it has caused a large number of casualties and a series of medical problems.Due to its special physical and chemical properties,such as high volatility and easiness of manufacture,storage and deployment,the prevention and treatment solutions to solve SM exposure is one of the foci in the field of global public security.It is also one of the most possible used tools in terrorism and therefore a global concern of counter-terrorism.So,it is of great significance to explore the toxicological mechanism of SM and develop efficient preventive and therapeutic drugs.As a powerful bifunctional alkylation blistering agent,SM alkylates intracellular and extracellular components of whole-body tissues.It will lead to a cascade of systemic damage,especially severe acute lung injury(ALI),and eventually death.Oxidative stress and apoptosis are considered to be vital processes for the SM toxicity mechanism.Our group has previously confirmed the therapeutic effect of exosomes derived from bone marrow stromal cells(BMSCs-Ex)in promoting the repair of alveolar epithelial barrier and alleviating lung injury.However,the key functional components and the underlying mechanisms have not been fully elaborated.Considering that human umbilical cord mesenchymal stem cells(HMSCs)have the advantages of abundant sources,no influence on the donor,easy collection,no allogeneic rejection,and avoidance of ethical controversy,they are regarded as an ideal cell-based therapy for various diseases.Due to the curative effect of BMSCs-Ex and HMSCs,this paper further explores the role and mechanism of HMSCs-derived exosomes(HMSCs-Ex)in treating ALI induced by SM.Methods:Part Ⅰ: The studies on effects of HMSCs-Ex in SM-induced acute lung injury1.Extraction and characterization of HMSCs-Ex.The primary cultured HMSCs were identified by flow cytometry and differentiation experiment according to the phenotypic characteristics and multipotent differentiation capacity of stem cells.Further,we separated the exosomes from the supernatant of HMSCs by ultracentrifugation.To confirm whether the isolated HMSCs-Ex could be used for subsequent experiments,we used transmission electron microscope to observe the morphology,nanoparticle tracking analyzer to measure the diameter distribution and Western blot to detect the external and internal biomarkers of the samples.2.After establishing the SM-exposed mouse model and injecting of HMSCs-Ex by tail vein for 14 days,the survival rate and body weight were observed and recorded to evaluate the therapeutic effect of HMSCs-Ex.3.We further investigated the function of HMSCs-Ex on in SM-induced acute lung injury.We examined the pathological damage of hematoxylin-eosin(H&E)-stained lung tissue,wet/dry weight ratio(W/D)of lung sections and protein concentration(TP)in alveolar lavage fluid(BALF).4.Considering the key role of oxidative stress and apoptosis in SM toxicity,we explored the effect of HMSCs-Ex in suppressing oxidative stress and apoptosis in vivo and in vitro.The intracellular reactive oxygen species(ROS)production in the lung was determined using dihydroethidium(DHE)staining.The oxidative DNA injury and lipid peroxidation injury was determined by detecting the level of 8-hydroxydeoxyguanosine(8-OHd G)and malondialdehyde(MDA).The ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG)and the level of superoxide dismutase(SOD)activity were measured to determine the production of endogenous antioxidant enzymes in lung tissues and BEAS-2B cells.Part Ⅱ: The studies on mechanism of miR-199a-5p derived from HMSCs-Ex in SM-induced acute lung injury1.We further explored the molecular mechanism of HMSCs-Ex in alleviating SMinduced oxidative injury and apoptosis in vitro.RT-q PCR and Western blot were applied to detect the m RNA and protein level of NRF2,HO1 and NQO1 in SM-exposed BEAS-2B cells after different treatments.2.10 most abundant micro RNA(miRNA)inhibitors were collected based on the data information of miRNA in HMSCs-Ex.Cell Counting Kit-8(CCK-8)method were applied to explore the potential key components of HMSCs-Ex in mediating the inhibition of SMinduced oxidative stress and apoptosis.The expression of relative miRNA in each group was verified using RT-q PCR method.3.To verify the function of exosomal miR-199a-5p,miR-199a-HMSCs-Ex overexpressed model was established by transfecting HMSCs-Ex with miR-199a-5p.RTq PCR and Western Blot were used to detect the oxidative stress and apoptosis changes as well as the m RNA and protein level of NRF2,HO1 and NQO1 expressions in BEAS-2B cells after the treatment of miR-199a-HMSCs-Ex.4.Since miRNA mainly down-regulates gene expression at the transcriptional or post transcriptional level by binding with messenger RNA(m RNA)and plays important roles in biological processes,we investigated the potential target of miR-199a-5p derived from HMSCs-Ex.Bioinformatics prediction and double luciferase reporter gene experiment were used to find the possible target gene of miR-199a-5p.To verify the relationship between miR-199a-5p and the target gene,RT-q PCR and Western Blot experiments were applied to detect the changes of the target gene in m RNA and protein levels in SM-exposed BEAS-2B cells after different administration.Next,we studied the biological function of Caveolin1(CAV1),which was the target gene of miR-199a-5p.To explore the role of miR-199a-5p in HMSCs-Ex in inhibiting the expression of CAV1 and activating NRF2 signaling pathway,RT-q PCR,Western blot and immunofluorescence staining were applied to detect the changes in m RNA and protein levels of CAV1,NRF2,HO1 and NQO1 expressions after the treatment of miR-199a-HMSCs-Ex.To further clarify the relationship between CAV1,miR-199a-5p and the NRF2 signaling pathway,we constructed overexpression of CAV1-in BEAS-2B cell lines.The effects of miR-199a-HMSCs-Ex treatment on the protein expression of CAV1 and NRF2 signaling pathways were compared.5.We further verified the protective effects of miR-199a-5p in HMSCs-Ex on SMinduced lung injury by RT-q PCR,H&E staining,immunohistochemical staining and Western blot experiment in vivo.Results:Part Ⅰ: The studies on effects of HMSCs-Ex in SM-induced acute lung injury1.The cultured cells had the potential of adipogenesis,osteogenesis and chondrogenesis and they were identified as undifferentiated mesenchymal stem cells and the isolated HMSCs-Ex were qualified for subsequent experiments due to their high purity.2.HMSCs-Ex treatment ameliorated the decrease in the survival rate induced by SM and alleviated the consequences of diarrhea and weight loss in mice.It showed better effect than that of the NAC group,which is accepted as a potential antidote against SM-induced toxicity(P< 0.05).HFLs-Ex,the negative control of HMSCs-Ex,has no significant effect on improving survival rate(P> 0.05).3.HMSCs-Ex treatment reduced alveolar capillary permeability,inhibited inflammatory cell migration and significantly alleviated SM-induced acute lung injury(P?0.001).4.HMSCs-Ex treatment alleviated the SM-induced oxidative stress and apoptosis by reducing reactive oxygen species(ROS),lipid peroxidation products(MDA)and increasing the activities of antioxidant enzymes(GSH and SOD),thus promoting the recovery of lung injury in vivo.A consistent result was obtained at the cellular level.(P values were all less than 0.05 and some were less than 0.01.)Part Ⅱ: The studies on mechanism of miR-199a-5p derived from HMSCs-Ex in SM-induced acute lung injury1.RT-q PCR and Western blot showed that HMSCs-Ex inhibited SM-induced oxidative stress and apoptosis by regulating the translocating of NRF2 protein from the cytoplasm to the nucleus and increasing the expression of downstream antioxidant enzymes HO1 and NQO1.2.Cell viability assay and RT-q PCR suggested that miR-199a-5p,one of the main components in HMSCs-Ex,might play a key role in inhibiting SM-induced oxidative stress and apoptosis.3.The miR-199a-HMSCs-Ex cell model was constructed.RT-q PCR and Western blot results showed that exosomal miR-199a-5p inhibited oxidative stress and apoptosis by activating NRF2 signal pathway and thus alleviating SM-induced lung injury.4.The mutual targeting relationship between CAV1 and miR-199a-5p was detected by bioinformatics prediction and double luciferase reporter gene experiment.RT-q PCR and Western blot indicated that miR-199a-5p could down-regulate the expression of CAV1 at m RNA and protein levels,thus verifying the targeting relationship between CAV1 and miR-199a-5p.The results of RT-q PCR,Western blot and immunofluorescence staining experiments indicated that the overexpression of miR-199a-5p in HMSCs-Ex induced a further decrease of CAV1 and the activation of the NRF2 signaling pathway.It was suggested that CAV1 was a key player in the negative regulation of the NRF2 pathway in miR-199aHMSCs-Ex-mediated lung protection.5.Animal experiments showed that miR-199a-HMSCs-Ex treatement exerted a more obvious improvement on SM-induced lung injury compared with the HMSCs-Ex group(P?0.05),while miR-199a-5p further promoted the activation of NRF2 signaling pathway through targeting CAV1 so as to reduce the SM-induced oxidative stress and apoptosis.Conclusion:In this study,HMSCs-Ex was used to treat ALI induced by SM for the first time.It was found that HMSCs-Ex can effectively improve the survival rate of SM-exposed mice,and restored SM-induced lung injuries by inhibiting oxidative stress and apoptosis in vitro and in vivo.We also found that miR-199a-5p,which was abundant in HMSCs-Ex,played an important role in reducing oxidative stress and inhibiting apoptosis through the activation of NRF2 signaling pathway.In addition,target gene analysis confirmed that CAV1 was a target gene regulated by miR-199a-5p,and its overexpression inhibited the upregulation of miR-199a-HMSCs-Ex on the activation of the NRF2 signaling pathway.Collectively,our data indicated that miR-199a-5p was one of the key molecules in HMSCs-Ex that attenuated SMassociated oxidative stress and apoptosis via mediating the CAV1/NRF2 signaling pathway.