Human umbilical cord mesenchymal stem cell-derived exosomes inhibit inflammation and fibrotic scar formation after intracerebral hemorrhage.

Abstract

Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-ex) have emerged as a promising alternative to whole-cell therapies due to their minimal immunogenicity and tumorigenicity. Pentraxin 3 (PTX3) acts as an inflammatory marker and pattern recognition receptor, playing a critical role in promoting tumor progression and inflammatory diseases. Fibrotic scars resulting from cerebral hemorrhage can impair motor and sensory functions, leading to poor prognosis. This study aimed to investigate whether hUCMSC-ex regulate matrix metalloproteinase-3 (MMP3) expression via the PTX3/Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway, thereby inhibiting inflammation and fibrotic scar formation following intracerebral hemorrhage and ultimately promoting the recovery of nerve function. A stereotactic technique was used to inject type IV collagenase (1 μL) into the striatum of rats, establishing an animal model of hemorrhagic stroke. Concurrently, hUCMSC-ex were administered via the tail vein at a dosage of 200 μg. In vitro, primary astrocytes were treated with hUCMSC-ex and subsequently stimulated with Hemin (20 μmol/mL) to create a cellular model of cerebral hemorrhage. The expression levels of PTX3, TLR4/NF-κB/MMP3 pathway proteins, and inflammatory factors, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-10 (IL-10), were assessed both in vivo and in vitro to investigate the effects of hUCMSC-ex on the inflammatory response and fibroblast migration. Neurological function in rats with cerebral hemorrhage was evaluated on days 1, 3, and 5 using the corner turn test, forelimb placement test, Longa score, and Bederson score. Additionally, real-time PCR was utilized to measure PTX3 mRNA expression following treatment with hUCMSC-ex. hUCMSC-ex inhibited MMP3 expression by downregulating the protein levels of PTX3, TLR4, NF-κB/P65, and p-P65. This action resulted in a reduction of pro-inflammatory cytokines TNF-α and IL-1β while simultaneously increasing the expression of the anti-inflammatory cytokine IL-10. Furthermore, hUCMSC-ex suppressed the inflammatory response, prevented fibroblast migration, and decreased MMP3 expression in the conditioned medium derived from primary astrocytes. Importantly, hUCMSC-ex improved behavioral performance in rats with intracerebral hemorrhage (ICH). hUCMSC-ex modulated the expression of MMP3 through the downregulation of PTX3, TLR4, NF-κB/P65, and p-P65. This regulatory mechanism contributed to a decrease in pro-inflammatory cytokines TNF-α and IL-1β, while concurrently enhancing the expression of the anti-inflammatory cytokine IL-10. Additionally, hUCMSC-ex effectively suppressed the inflammatory response, inhibited fibroblast migration, and reduced MMP3 expression in primary astrocyte-conditioned medium. Overall, hUCMSC-ex significantly improved behavioral performance in rats with ICH.