| Background and purpose:Idiopathic pulmonary fibrosis is a chronic fibrotic interstitial pneumonia of unknown etiology,characterized by diffuse collagen deposition in the lungs,structural destruction of alveolar tissue,and formation of fibrous scarring and honeycomb capsules.Clinical manifestations include irreversible pulmonary ventilation and hyperventilation,severely reduced quality of survival,and an extremely poor prognosis.Currently,the treatment options for idiopathic pulmonary fibrosis are extremely limited.Mesenchymal stem cells(MSC)are considered as a novel biological therapy for repairing damage to various organs and tissues in the human body due to their unique ability to induce proliferation and differentiation.In recent years,basic research on human umbilical cord-mesenchymal stem cells(hUC-MSCs)for the treatment of various lung diseases has gradually increased,and their efficacy has been confirmed to some extent.However,the direct clinical application of hUC-MSCs has many problems that cannot be ignored,such as lack of cell sources,decreased proliferation and differentiation potential after passaging,limited cell residence time in damaged areas,insufficient homing ability and risk of tumorigenesis,which seriously hinder their translation from the laboratory to the clinic.Exosomes(exos)are tiny vesicles between 50 and 150 nm in size that are secreted by cells and contain lipids,nucleic acids,proteins and other components that can be received by recipient cells to facilitate intercellular communication.Previous studies have shown that MSCs perform their biological functions mainly in a paracrine manner and that exosomes,as the main mediators,have a significant therapeutic potential.The use of MSC-exos not only avoids many of the risks associated with direct MSC transplantation,but also facilitates storage and transport.Among the bioactive substances encapsulated in exosomes,microRNA(miRNA)has attracted much attention because of its ability to target and regulate genes.It has been demonstrated that miRNAs from donor cells can be transported to recipient cells via exosomes and"reprogram" the genes within them.At the same time,exosomes are able to protect the miRNAs within them from high temperature,acid-base and repeated freeze-thaw environments.Previous experiments of our group found that hypoxic pretreatment could inhibit the activation of mitochondrial apoptotic pathway in hUC-MSCs and thus enhance their anti-apoptotic ability,and significantly strengthen the paracrine function of hUC-MSCs.It was found that hypoxic pretreatment significantly enhanced the expression of miRNAs in MSCs and their exosomes,and strengthened their repair ability to damaged tissues.Therefore,an in-depth study of the effects of hypoxia-pretreated hUC-MSCsexos on lung fibrosis and exploring the regulatory mechanisms of miRNAs within them will provide a possibility for their clinical application.Contents:Part Ⅰ Culture of hypoxic hUC-MSCs and extraction and identification of their exosomesMethods:The hUC-MSCs were cultured and a model of hypoxic hUC-MSCs was constructed,and their exosomes were extracted using ultracentrifugation technique,and the extracted exosomes were identified by electron microscopic observation,particle size analysis and protein immunoblotting(Western blot)for signature proteins.western blot was used to detect the HIF-1α and Rab27a in hypoxic hUC-MSCs expression levels to clarify the effect of hypoxia on the secretion of exosomes from hUC-MSCs.Results:The ultracentrifugation technique can extract exosomes successfully and efficiently.Hypoxia can activate the HIF-1α/Rab27a pathway in hUC-MSCs and promote their exosome secretion.Part Ⅱ Interventional effects of hypoxic hUC-MSCs-exos on pulmonary fibrosisMethods:This part includes animal and cellular experiments.A mouse model of pulmonary fibrosis was constructed using intraperitoneal injection of bleomycin induction,and the intervention was performed by tail vein injection of exosomes at the end of modeling.The survival status and weight changes of mice were recorded,and lung tissues were retained for pathological HE,Masson and immunohistochemical staining,and the expression levels of pulmonary fibrosis marker proteins were detected by Western blot to judge the intervention effect.The EMT model was constructed by TGF-β1 induction of human alveolar epithelial A549 cells and co-cultured with exosomes,and the entry of exosomes into A549 cells was observed using confocal fluorescence microscopy,and its effect on EMT was detected by Elisa and Western blot.Results:In the animal experiments,mice showed dry hair,reduced activity,shortness of breath and weight loss after the administration of bleomycin induction,and the successful construction of the mouse model of pulmonary fibrosis could be judged by combining with the histopathological manifestations of lung.After the intervention of hUC-MSCs-exos or hypoxic hUC-MSCs-exos,the mice showed reduced symptoms and weight recovery.Combining with the results of HE and Masson staining of lung tissues and the expression levels of collagen deposition,Collagen-I,PAI-1 and αSMA,it could be judged that the exosome treatment was effective and that hypoxic hUCMSCs-exos was effective in reduced interstitial Collagen-I deposition in mice lungs was more effective.In cellular experiments,both hUC-MSCs-exos and hypoxic hUC-MSCs-exos were able to enter the cytoplasm of A549 cells,inhibit the level of secretion of Collagen-Ⅰ and Fibronectin in A549 cells after TGF-β1 stimulation,and down-regulate the expression of αSMA,Vimentin and SMAD4 in A549 cells,effectively reducing EMT.Notably,hypoxic hUC-MSCs-exos significantly inhibited the secretion of Collagen-I in A549 cells,demonstrating an excellent ability to attenuate EMT.Part Ⅲ Mechanistic study of hypoxic hUC-MSCs-exos to reduce pulmonary fibrosisMethods:A comprehensive search was conducted in the GEO database using the search terms "umbilical","mesenchymal stem cell" and "exosome".RT-qPCR was performed to detect the changes in the expression levels of these miRNAs after co-culture of TGFβ1-induced A549 cells with hypoxic hUC-MSCs-exos,and the key miRNAs,miRNA146a-5p(miR-146a-5p),in hypoxic hUC-MSCs-exos were finally screened out.The binding site of miR-146a-5p to SMAD4 was predicted by bioinformatics,and the targeting relationship between the two was verified using a dual luciferase reporter gene assay.A549 cell models with high and low expression of miR-146a-5p were constructed using miR-146a-5p mimics,miR-146a-5p inhibitor,and the effects of miR-146a-5p on SMAD4 expression levels and EMT of A549 cells were examined.A549 cell model with low expression of SMAD4 was constructed and cotransfected with miR-146a-5p inhibitor,and the effect of miR-146a-5p/SMAD4 on EMT of A549 cells was examined.Further,RT-qPCR was performed to detect the effect of hypoxia on miR-146a-5p expression in hUC-MSCs and their exosomes and the expression level of miR-146a-5p within A549 cells after co-culture with hypoxic hUC-MSCs-exos.Finally,miR-146a5p expression in hypoxic hUC-MSCs-exos was inhibited and its effects on SMAD4 expression levels and EMT in A549 cells were examined.Results:SMAD4 is regulated by miR-146a-5p target gene.The expression of miR-146a-5p in A549 cells gradually decreased with the prolongation of TGF-β1 stimulation.Overexpression of miR-146a-5p attenuated TGF-β1-induced EMT and significantly inhibited SMAD4 expression in A549 cells,knockdown of miR-146a-5p expression promoted EMT and significantly upregulated SMAD4 expression,and silencing of SMAD4 antagonized the promotion of EMT caused by knockdown of miR-146a-5p expression.The miR-146a-5p expression level of hUC-MSCs and their exosomes increased time-dependently after hypoxia treatment,and miR-146a-5p could translocate to A549 cells via hUC-MSCs-exos,exerting the effects of attenuating EMT and suppressing SMAD4 expression.miR-146a-5p expression in hUC-MSCs-exos was inhibited after suppressing hypoxic hUC-MSCs-exos-5p expression,this effect was significantly attenuated.Conclusions:1.The ultracentrifugation technique can successfully isolate and extract the exosomes of hUC-MSCs,and the hypoxic pretreatment has a facilitating effect on the secretion of hUC-MSCs exosomes.2.hUC-MSCs-exos and hypoxic hUC-MSCs-exos can reduce the number of myofibroblasts in the lung tissue of mice with pulmonary fibrosis,inhibit the deposition of extracellular matrix such as collagen,and reduce pulmonary fibrosis.3.hUC-MSCs-exos and hypoxic hUC-MSCs-exos were able to enter the cytoplasm of alveolar epithelial cells to play a role in reducing EMT.4.Hypoxia pretreatment could upregulate the expression of miR-146a-5p in hUCMSCs-exos,and miR-146a-5p targeted to inhibit SMAD4 and thus reduce EMT in alveolar epithelial cells,exerting an anti-fibrotic effect. |
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