Pseudoginsenoside-F11(PF11) Inhibits Microglial Activation Induced By LPS And Exerts Protective Effects On Neuronal Cells

Microglia mediated neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases (NDD), cerebral ischemia and traumatic brain injury. Once activated, microglia release a large number of proinflammatory cytokines, chemokines and reactive oxygen species, which induce neuronal damage and destroyed neurological functions. Therefore, blocking inflammatory signaling cascade in microglia and reducing the release of proinflammatory cytokines are able to alleviate or treatment of these diseases. Pseudoginsenoside-F11(PF11), a component of Panax quinquefolium (American ginseng), has been demonstrated to antagonize morphine and methamphetamine induced increasing of locomotor activity, pain tolerance and conditioned place preference in mice, inhibit learning and memory deficits induced by scopolamine, morphine and methamphetamine in mice, suppress the expression of APP and Aβ1-40in APP/PS1mice, improve the deficits of spatial reference memory and passive avoidance ability in APP/PS1transgenic mice. In the present study, The experimental techniques including confocal microscopy, high content screening (HCS), ELISA, RT-PCR and Western-blot were used to investigated the effect of PF11on microglial activation and neuronal protection, and tried to reveal the underlying mechanisms.First, LPS was used to establish the microglial activation model. Our study shows, PF11inhibited LPS-induced NO and prostaglandin E2production through suppressing mRNA and protein expression of iNOS and COX-2, and restrained IL-1β, IL-6and TNF-α released by LPS-stimulated N9cells through the suppression of those cytokines at the transcriptional level. The mechanism of PF11may lie in attenuating LPS-stimulated formation of the TLR4/MyD88and TLR4/TAK1complexes, inhibiting TAK1, IKKα/β activation, IκBα degradation, and attenuating the translocation and expression of NF-κB. Furthermore, PF11attenuated the phosphorylation of ERK1/2, JNK, p38and Akt induced by LPS. In short, PF11inhibited neuroinflammation induced by LPS in N9microglia through inhibiting the activation of TLR4-mediated TAK1-IKK-NF-κB, MAPK and PI3K/Akt pathways. Moreover, PF11inhibited the expression of gp91phox, p22phox, p40phox and Rac1, which are the subunits of NADPH oxidase. In addition, PF11also significantly impaired the translocation of p47phox and p67phox to the cellular membrane. These findings indicate that PF11decreased the level of ROS through suppressing subunits expression and inhibiting subunit activation of NADPH oxidase.This study further investigated the protective effect of PF11on neurons in vitro. We found that PF11inhibited loss of dendritic processes of primary neurons and exerted significant protective effects against microglial-mediated mouse and rat primary neurons and SH-SY5Y neuroblastoma cells injury. There were no significant changes in the survival ratio of neuronal cells when treated with LPS alone. This suggests that the toxic action of LPS-stimulated microglial-conditioned medium is mostly dependent on microglial secreted products but not LPS. PF11inhibited the decrease of SH-SY5Y cell viability induced by H2O2, which might be caused by the reduction of membrane lipid peroxidation, inhibition of apoptosis in neurons, up-regulation of the Bcl-2expression and down-regulation of Bax. Thus, PF11may play a direct protective effect on neurons by reducing neuronal cells apoptosis and inhibiting oxidative stress.In conclusion, the present study demonstrated that PF11exerts strong neuroprotective and anti-neuroinflammatory effects through blocking the formation of TLR4/MyD88complexes, inhibiting TAK1/DCK-α/β/NF-κB, MAPKs and Akt signaling pathways, down-regulating NADPH oxidase activty in LPS-activated microglial cells. Meanwhile, PF11protected neurons through anti-inflammatory, anti-apoptotic and anti-oxidative stress. Therefore, PF11may have therapeutic potential in the treatment of diseases associated with microglial activation. This study suggests that TLR4may be an important target for the treatment of neurological diseases.