4. Discussion
In this study, we found that NSC-Exos transplantation has an inhibitory effect on the inflammatory response mediated by microglia. The effect of NSC-Exos may be as follows: (1) NSC-Exos can improve the brain histopathological changes of MCAO model rats and alleviate the neurological function deficit. (2) NSC-Exos decreased the expression of M2-related inflammatory factors and increased the expression of M2-related inflammatory factors, respectively, thus alleviating the microglia-mediated inflammatory response. (3) NSC-Exos exerts neuroprotective effects by inhibiting the activation of TLR4/NF-κB signaling pathway and regulating the polarization of microglia toward anti-inflammatory phenotype, which is consistent with previous studies (Cai et al., 2021; Liu et al., 2020).
The pathogenesis of cerebral ischemia-reperfusion injury is complex and induced by multiple factors, which inflammation plays an important role (Mao et al., 2022). During ischemia, resident microglia in the brain are activated and release a variety of cytokines to participate in the inflammatory response (Liu et al., 2021). Therefore, inhibition of neuroinflammation and regulation of microglial polarization may be important approaches to prevent and treat stroke. In this study, we observed that NSC-Exos inhibited the release of pro-inflammatory factors, decreased the expression of CD86/Iba1 in type M1, and increased the expression of anti-inflammatory factors and CD206/Iba1 in type M2. These data indicate that exosomes therapy can promote the polarization of microglia towards anti-inflammatory phenotype, which has comprehensive anti-neuroinflammatory potential, and these results are consistent with previous studies (L. Li et al., 2021; T. Zhou et al., 2017).
A growing body of evidence suggests that TLR4 is a key target in the regulation of inflammation induced by cerebral ischemic injury (Dong et al., 2021). ​During cerebral ischemia, TLR4 is stimulated and further affects the activation of downstream NF-κB. TAK-242 is a specific antagonist of TLR4, which can block the TLR4/NF-κB signaling pathway and inhibit the activation of NF-κB, thereby protecting the brain from cerebral ischemia induced injury(Zhang et al., 2021; L. Zhou et al., 2017). However, few studies have focused on whether the TLR4/NF-κB pathway regulates the polarization of NSC-Exos on microglia during cerebral ischemia, and whether the inhibitor TAK-242 affects the anti-inflammatory effect of NSC-Exos. Based on the ability of TAK-242 to inhibit the TLR4/NF-κB signaling pathway, we speculate that TAK-242 may play an important role in reducing inflammation and improving cognitive function. The results showed that NSC-Exos treatment significantly down-regulated the expression of TLR4 and NF-κB, while the inhibitor TAK-242 reversed the expression of these proteins. These results strongly suggest that the TLR4/NF-κB signaling pathway is involved in the anti-inflammatory effects of exosomes in cerebral ischemia-reperfusion injury. This is consistent with the results of previous studies (Peng et al., 2023; K. Wang et al., 2020).
However, there are still some limitations in this study. First, inflammation is an ongoing process in the progression of ischemic stroke. We only discuss the short-term protective effects and potential mechanisms of exosomes, but do not study their spatio-temporal expression. Therefore, long-term studies of exosomes during stroke are necessary in the future. Secondly, the components of exosomes are complex, including a variety of miRNAs and proteins etc (B. Li et al., 2021; Yue et al., 2020), and the targets of these components are also different. In future studies, we will further investigate the mechanism of action of specific components in exosomes.