Neuroprotective effect of mesenchymal stem cell-derived miR-143-3p on dopaminergic neurons via the TLR3/NF-κB/p53 signaling pathway

BACKGROUND: Transplantation of midbrain dopaminergic neurons (mDANs) derived from human pluripotent stem cells (hPSCs) represents a promising therapeutic approach for Parkinson's disease (PD), with several clinical trials currently in progress. However, the poor survival of grafted mDANs, in part due to inflammation triggered by surgical injury, poses significant challenges to both therapeutic efficacy and safety. In recent years, mesenchymal stem cells (MSCs) and their derivatives have played an important role in the field of stem cell therapy due to their easy accessibility and immunomodulatory properties. However, their potential role as an adjunct in cell therapy remains unclear. Therefore, this study aims to investigate the protective effects of conditioned medium (CM) derived from MSCs and the mechanisms of action by which it attenuates surgery-induced neuroinflammation and enhances the survival of transplanted mDANs. METHODS: To mimic the neuroinflammatory response caused by the surgical aspect of cell transplantation, transplantation medium alone (i.e. without cells) was injected into the striatum of male NOD SCID mice. The anti-inflammatory effects of MSC-CM in vivo were evaluated by assessing the activation of microglia and expression levels of pro-inflammatory cytokines. Next, an in vitro cell death model was established using IFN-γ and TNF-α stimulation. The protective effects of MSC-CM against IFN-γ and TNF-α-induced inflammatory injury in mDANs were assessed using immunofluorescence staining and TUNEL assay. To identify the active components and underlying mechanisms for the protective effects of MSC-CM, inhibitory treatments, miRNA sequencing, dual-luciferase reporter assay, and subsequent validation by qPCR and Western blotting were performed. Finally, MSC-CM was administered intranasally into male NOD SCID mice following mDAN transplantation to evaluate its protective effects on the grafted mDANs in vivo. RESULTS: MSC-CM suppressed neuroinflammation and significantly protected mDANs from inflammation-induced cell death via inhibition of TLR3/NF-κB/p53 signaling pathway in this model system. This protective effect was largely reproduced by treatment with a specific component of the MSC-CM, miR-143-3p. Overexpression of miR-143-3p further enhanced the protective effects and mechanisms of action of MSC-CM. CONCLUSIONS: This study demonstrates that MSC-CM or its component miR-143-3p exert protective effects on mDANs against inflammatory injury, primarily by inhibiting the TLR3/NFκB/p53 signaling pathway.