Ischemic stroke is a sudden acute brain disease. At present, the only therapeutic drug for ischemic stroke is tissue-type plasminogen activator, but its therapeutic time window is only 4.5 h (Nogueira et al., 2018). ICT has a low molecular weight and high lipophilicity can easily cross the blood‒brain barrier and has a long half-life in the body (Huang et al., 2019). We found that ICT improved the neurological function of tMCAO rats and significantly reduced the number of degenerated neurons, proving that ICT exerts a neuroprotective effect in rats subjected to ischemia/reperfusion.
Inflammation is now recognized as a principal factor in brain injury and neurological dysfunction after CI/RI. After acute cerebral ischemia, damaged neurons and glial cells secrete proinflammatory factors such as IL-1β and TNF-α, which increase the permeability of the blood‒brain barrier by directly affecting multi-capillaries, leading to cerebral edema and allowing peripheral immune cells and cytokines to enter brain tissue, further aggravating inflammation and brain injury (Fu et al., 2015). In a rat model of chronic cerebral ischemia, Radix Salviae Miltiorrhizae extract was found to decrease the levels of TNF-α and IL-6, thereby inhibiting the inflammatory response (Zhang et al., 2013). ICT can effectively inhibit the DNA-binding activity of P65-NF-κB and the expression of IL-1β and TNF-α in brain tissue in mice subjected to ischemia/reperfusion injury (Sun et al., 2018). In our study, ICT treatment decreased the expression of IL-1β and TNF-α while increasing the level of IL-10, indicating that ICT inhibited neuroinflammation and cerebral edema induced by tMCAO in rats by decreasing proinflammatory cytokine levels and increasing anti-inflammatory cytokine levels.
Microglia have been recognized as important contributors to the occurrence and development of neuroinflammation induced by ischemic stroke. Microglia can be rapidly activated and polarized when cerebral ischemia occurs; M1 microglia release proinflammatory factors, whereas M2 microglia produce anti-inflammatory factors and function in tissue repair (Qiu et al., 2020; Thomas et al., 2017). Our present research found that ICT could effectively inhibit the activation of microglia in tMCAO rats. Thus, we evaluated the effect of ICT on microglial M1/M2 polarization. The results showed that ICT reduced the expression of M1 microglial markers in the ischemic penumbra in tMCAO rats and increased the expression of M2 microglial markers. These results indicated that the anti-neuroinflammatory effect of ICT after ischemic stroke can be achieved by restraining microglial M1 polarization and promoting microglial M2 polarization.
It is well known that NF-κB is a key transcription factor in the regulation of the inflammatory response. When NF-κB is activated, it is phosphorylated and translocates to the nucleus, where it mediates inflammatory brain injury by inducing the transcription of many proinflammatory genes, such as IL-1β and TNF-α (Zhang et al., 2019). In vivo, genistein-3′-sodium sulfonate, a structural modifier of the phytoestrogen genistein, was shown to inhibit the activation and nuclear translocation of NF-κB and reduce the M1 polarization of microglia, thereby inhibiting neuroinflammation in tMCAO rats (Liu et al., 2021). Multiple in vitro studies have also demonstrated that NF-κB is involved in microglial activation and M1 polarization (Kong et al., 2022; Liu et al., 2019b). Interfering with the activation of NF-κB inhibits M1 polarization and reduces neuroinflammation induced by ischemia (Su et al., 2022). Our results demonstrated that ICT treatment reduced the activation of NF-κB in tMCAO rats, indicating that it is involved in the regulation of microglial polarization and the inhibition of neuroinflammation induced by ICT.
Activation of ERK signaling can reduce the lipopolysaccharide-induced nuclear translocation of NF-κB in BV2 microglia, thereby inhibiting M1 polarization (Mohanraj et al., 2019; Ni et al., 2015). In addition, ERK signaling may also promote the polarization of microglia toward the M2 phenotype (Wang et al., 2018). Our study found that ICT treatment improved ERK activation, indicating that ICT may exert a regulatory effect on microglial polarization through ERK-mediated inhibition of NF-κB.
GPER is an estrogen-specific membrane receptor that can exert the rapid non-genomic effects of estrogen (Guan et al., 2017). In ovariectomized female and male rats, the GPER agonist G1 inhibits the upregulation of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), increases the expression of the anti-inflammatory cytokine IL-4, and induces the polarization of microglial toward the M2 phenotype, thereby relieving cerebral ischemic injury (Lu et al., 2016). Therefore, GPER is an important target for the regulation of microglial polarization, inhibition of neuroinflammation and neuroprotection. Our study found that there was no change in GPER protein expression in rats after 24 h of ischemia/reperfusion and that ICT, U0126 and G1 did not affect GPER expression, which is consistent with existing reports (Lu and Herndon, 2017). Our study also demonstrated that administration of a GPER or ERK inhibitor reversed the effects of ICT in ischemic stroke rats, including its neuroprotective and anti-neuroinflammatory effects and its ability to regulate the activation and polarization of microglia, inhibit NF-κB, and so on. This result indicated that these effects of ICT were achieved via activation of GPER and ERK. In addition, the GPER inhibitor influenced the activation of ERK, but the ERK inhibitor did not regulate the expression of GPER, indicating that ERK may be one of the downstream signaling molecules of GPER. This finding suggested that ICT might regulate the polarization of microglia through the GPER-ERK-NF-kB pathway, thereby exerting a neuroprotective effect by reducing neuroinflammation in ischemic stroke rats.