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Linolamide in Apoptotic Bodies: A Key Factor in Mesenchymal Stem Cell Immunotherapy for Two Abortion Models

ABSTRACT: Background: Unexplained recurrent spontaneous abortion (URSA) is a distressing pregnancy disorder with no effective medical intervention. As a potential treatment for URSA, human umbilical cord mesenchymal stem cells (hucMSCs) undergo apoptosis and release apoptotic bodies (ABs) shortly after transplantation. However, the medical effects and the exact doses of ABs in the therapy for URSA remain unclear. Methods: Staurosporine (STS) and ultraviolet (UV) induced apoptosis in hucMSCs. Differential centrifugation, transmission electron microscopy, immunofluorescent staining, and flow cytometry were used to isolate and characterize hucMSC-derived ABs (hucMSC-ABs), respectively. Two mouse abortion models (LPS-induced and CBA/J×DBA/2 immune-mediated) mimicked URSA. Flow cytometry, real-time quantitative PCR, immunofluorescence staining, and western blotting were used to analyze macrophage polarization and inflammatory pathways. Untargeted metabolomic analysis was used to detect the metabolic composition of hucMSC-ABs. RNA sequencing analysis was used to detect the transcriptome of macrophages (Macs). Dorsomorphin was used to inhibit the AMPK signaling pathway. Small interfering RNA (siRNA) was used to knock down Gys1 in raw246.7 macrophages. Results: STS- and UV- induced hucMSC-ABs effectively alleviated abortion in both models, depended on Macs. However, excessive dosage of hucMSC-ABs induced pro-inflammatory Macs (pro-inflam Macs), worsening fetal loss. Appropriate doses suppressed the NF-κB pathway in Macs, while high doses activated it. Neither RNase nor trypsin fully abolished hucMSC-ABs' regulatory effects on macrophage polarization. Metabolomics identified linoleamide and palmitic acid (PA) as the dominant hucMSC-AB metabolites (~1:1 ratio). RNA-seq revealed hucMSC-ABs modulated Nfkb1 and Gys1 in Macs, linked to linoleamide. Appropriate doses of the 1:1 mixture of linoleamide and PA induced anti-inflam Macs, while high dosages of the mixture induced pro-inflam Macs. Linoleamide alone activated AMPK, suppressed NF-κB via Gys1, and protected pregnancy, whereas PA induced pro-inflam Macs and promoted abortion. Conclusion: Appropriate doses of hucMSC-ABs induce anti-inflam Macs in the uterus and prevent fetal loss, while high doses promote the polarization of pro-inflam Macs and lead to fetal loss. The effects of hucMSC-ABs in regulating Macs and treating URSA are related to different doses of linoleamide and PA on Macs. Linoleamide activates AMPK and suppresses NF-κB via Gys1. This study revealed the effect and mechanism of hucMSC-ABs intervention in fetal loss and provided a new idea for the treatment of URSA.

ORGANISM(S): Mus musculus

PROVIDER: GSE303694 | GEO | 2025/08/17

REPOSITORIES: GEO

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Project description:Background and aim: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. Synovial fibroblasts (SFs) play a key role in the pathogenesis of RA. Dysregulation of MicroRNAs (miRNAs) expression in SFs mediates the development of RA. Exosomes (Exos) can transfer miRNAs between cells and have been validated as carriers for the delivery of therapeutic molecules.This study aimed toinvestigate the therapeutic potential of human umbilical cord mesenchymal stem cells (hUCMSC)-Exos deliveringmiR-451a to regulate ATF2 for RA both in vivo and in vitro. Methods: In this study, hUCMSC and RASF were isolated and identified, and hUCMSC-EXOs were extracted and characterized. The influence of hUCMSC-Exos on RASF was detected. hUCMSC-Exos RNA was labeled and hUCMSC-Exos targeting RA-SFs was traced. AGO2 promoted miRNA maturation, and hUCMSCKD-AGO2 was prepared by knocking down AGO2 in hUCMSC. hUCMSCKD-AGO2-Exos was extracted and characterized,and their influence on RASFs was detected. mRNA and miRNA profiles before and after hUCMSC-Exos intervention in RASFs were mapped to identify differential miRNAs. RT-qPCR was used to verify the differential miRNAs, with miR-451a finally selected as the target gene.The effect of miR-451a on SFs was detected. The latent binding of miR-451a to activating transcription factor 2 (ATF2)was analyzed. The effect of hUCMSC-ExosmiR-451a on SFs was detected, and the expression of miR-451a and ATF2 was measured by RT-PCR.In vivo, hUCMSC-ExosmiR-451a was injected into the ankle joint of CIA rats, and arthritis index, joint imaging and synovial pathology were assessed. The expression of miR-451a and ATF2 in synovial tissue was detected. Finally, the safety of hUCMSC-ExosmiR-451a in CIA rats was evaluated. Results: This study revealed that hUCMSC-Exos can inhibit RASF proliferation, migration and invasion through miRNAs. High throughput sequencing detected 13 miRNAs that could be transmitted from hUCMSC to RASF via hUCMSC-EXOs. MiR-451a inhibited RASF proliferation, migration and invasion by regulating ATF2. hUCMSC-Exos loaded with miR-451a targeted ATF2 to inhibit RASF proliferation, migration and invasion, and improve joint inflammation and imaging findings in CIA rats. Conclusions: This study demonstrates that miR-451a carried by hUCMSC-Exos canplay a role ininhibiting RASF biological traits and improving arthritis in CIA rats by inhibiting ATF2. The findings study suggest a promising treatment for RA and provide insights into the mechanism of action of hUCMSC-Exo in RA.

Project description:Background: The therapeutic and protective efects of human umbilical cord mesenchymal stem cells-exosomes (hucMSC-Exs) on traumatic pancreatitis (TP) remain unknown. Here, we established a rat model of TP and evaluated and compared the therapeutic efects of hucMSC-Exs. Methods: HucMSC-Exs were obtained by ultracentrifugation and identifed using transmission electron microscopy and western blot analysis. TP rats were treated by tail vein injection of hUC-MSCs and hucMSC-Exs. Their homing in rats was observed by performing fuorescence microscopy. Rat pancreatic tissue was subjected to high-throughput sequence to determine transcriptome expression levels. The degree of pancreatic tissue damage was assessed by HE staining, the expression levels of amylase, lipase, and infammatory cytokines were detected by ELISA, apoptosis was detected by TUNEL assay, and the expression levels of various apoptosis-related proteins were detected by western-blot. The expression levels of apoptosis-related molecular markers were detected by RT-qPCR. Results: The colonization of exosomes was observed in pancreatic tissue. Compared to TP group, the histopathological score of pancreas was signifcantly decreased in the TP+hucMSC-Exs group (P<0.05). Compared to TP group, the activity of serum amylase and lipase was signifcantly decreased (P<0.05). The expression levels of IL-6 and TNF-α were signifcantly decreased, while those of IL-10 and TGF-β were signifcantly increased (P<0.05). The apoptosis index of the TP group was signifcantly increased (P<0.05), whereas that of the TP+hucMSC-Exs groups was signifcantly decreased (P<0.05). Compared to TP group, the expression levels of Bax, Bcl-2, and Caspase-3 were signifcantly decreased in the TP+hUC-MSCs group and TP+hucMSC-Exs group (P<0.05). Conclusion: HucMSC-Exs can colonize injured pancreatic tissue, inhibit the apoptosis of acinar cells, and control the systemic infammatory response to facilitate the repair of pancreatic tissue.

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Linolamide in Apoptotic Bodies: A Key Factor in Mesenchymal Stem Cell Immunotherapy for Two Abortion Models

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