PURPOSE: To investigate the regulatory role of nerve injury-induced protein 1 (NINJ1) in the anti-inflammatory function of human umbilical cord mesenchymal stem cells (hUC-MSCs) co-stimulated by interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). METHODS: hUC-MSCs were expanded in vitro using standard protocols, with stem cell characteristics confirmed by flow cytometry and multilineage differentiation assays. The immunomodulatory properties and cellular activity of cytokine-co-pretreated hUC-MSCs were systematically evaluated via quantitative reverse transcription RT-qPCR, lymphocyte proliferation suppression assays, and Cell Counting Kit-8 viability tests. Transcriptome sequencing, Western blotting and small interfering RNA interference were integrated to analyze the regulatory mechanisms of NINJ1 expression. Functional roles of NINJ1 in pretreated hUC-MSCs were elucidated through gene silencing combined with lactate dehydrogenase release assays, Annexin V/Propidium Iodide apoptosis analysis, macrophage co-culture models, and cytokine Enzyme-Linked Immunosorbent Assay. Therapeutic efficacy was validated in a cecal ligation and puncture-induced septic mouse model: 80 mice were randomly allocated into 4 experimental groups (n=20/group): sham group (laparotomy without cecal ligation); phosphate-buffered saline-treated group (cecal ligation and puncture (CLP) + 0.1 mL phosphate-buffered saline); hUC-MSCs (small interfering RNA (siRNA)-interferon-gamma and tumor necrosis factor-alpha co-stimulation (IT))-treated group (CLP + hUC-MSCs transfected with scrambled siRNA); and hUC-MSCs (siNINJ1-IT)-treated group (CLP + hUC-MSCs with NINJ1-targeting siRNA). RESULTS: hUC-MSCs demonstrated compliance with International Society for Cellular Therapy criteria, confirming their stem cell identity. IFN-γ/TNF-α co-pretreatment enhanced the immunosuppressive capacity of hUC-MSCs, accompanied by the reduction of cellular viability, while concurrently upregulating pro-inflammatory cytokines such as interleukin-6 and interleukin-1β. This co-stimulation significantly elevated NINJ1 expression in hUC-MSCs, whereas genetic silencing of NINJ1 effectively suppressed pro-inflammatory cytokine production and attenuated damage-associated molecular patterns release through inhibition of programmed plasma membrane rupture. Furthermore, the NINJ1 interference potentiated the ability of cytokine-pretreated hUC-MSCs to suppress LPS-induced pro-inflammatory responses in RAW264.7 macrophages. In cecal ligation and puncture-induced sepsis model, NINJ1-silenced hUC-MSCs exhibited enhanced therapeutic efficacy, manifested by reduced systemic inflammation and multi-organ damage. CONCLUSION Our findings shed new light on the immunomodulatory functions of cytokine-primed MSCs, offering groundbreaking insights for developing MSC-based therapies against inflammatory diseases via interfering the expression of NINJ1.
Mesenchymal Stem Cells/drug effects* ; Animals ; Interferon-gamma/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Humans ; Mice ; Umbilical Cord/cytology* ; Cells, Cultured ; Apoptosis ; Male

2019 Novel coronavirus (2019-nCoV) infection has caused a global pandemic. Although researchers have carried out a lot of research on 2019-nCoV, analyzed the molecular structure and conducted evolutionary tree analysis, there is still insufficient understanding of its specific pathogenic mechanism, resulting in the lack of specific and effective therapeutic drugs and method. 2019-nCoV infection can cause inflammation and may deteriorate to acute respiratory distress syndrome (ARDS) and sepsis, which have become the main complication of its death. Therefore, using antiviral and symptomatic treatment with inflammation reduction can have a better therapeutic effect. Mesenchymal stem cells (MSCs) not only have a significant immune-regulation function, but also play a role in regeneration and repair, repairing damaged lungs, so they can be considered as a new effective method for the treatment of coronavirus disease 2019 (COVID-19). This article analyzes the main pathogenic mechanism of 2019-nCoV, and the process of developing into ARDS, combined with the research status of MSCs, to explore its significance and feasibility for the treatment of COVID-19. Finally, it will provide a substantial theoretical basis for clinical treatment now and in the future.