Ginsenoside Re protects against kainate-induced neurotoxicity in mice by attenuating mitochondrial dysfunction through activation of the signal transducers and activators of transcription 3 signaling.

It was demonstrated that ginsenosides exert anti-convulsive potentials and interleukin-6 (IL-6) is protective from excitotoxicity induced by kainate (KA), a model of temporal lobe epilepsy. Ginsenosides-mediated mitochondrial recovery is essential for attenuating KA-induced neurotoxicity, however, little is known about the effects of ginsenoside Re (GRe), one of the major ginsenosides. In this study, GRe significantly attenuated KA-induced seizures in mice. KA-induced redox changes were more evident in mitochondrial fraction than in cytosolic fraction in the hippocampus of mice. GRe significantly attenuated KA-induced mitochondrial oxidative stress (i.e. increases in reactive oxygen species, 4-hydroxynonenal, and protein carbonyl) and mitochondrial dysfunction (i.e. the increase in intra-mitochondrial Ca2+ and the decrease in mitochondrial membrane potential). GRe or mitochondrial protectant cyclosporin A restored phospho-signal transducers and activators of transcription 3 (STAT3) and IL-6 levels reduced by KA, and the effects of GRe were reversed by the JAK2 inhibitor AG490 and the mitochondrial toxin 3-nitropropionic acid (3-NP). Thus, we used IL-6 knockout (KO) mice to investigate whether the interaction between STAT3 and IL-6 is involved in the GRe effects. Importantly, KA-induced reduction of manganese superoxide dismutase (SOD-2) levels and neurodegeneration (i.e. astroglial inhibition, microglial activation, and neuronal loss) were more prominent in IL-6 KO than in wild-type (WT) mice. These KA-induced detrimental effects were attenuated by GRe in WT and, unexpectedly, IL-6 KO mice, which were counteracted by AG490 and 3-NP. Our results suggest that GRe attenuates KA-induced neurodegeneration via modulating mitochondrial oxidative burden, mitochondrial dysfunction, and STAT3 signaling in mice.

Ginsenoside Re blocks Bay k-8644-induced neurotoxicity via attenuating mitochondrial dysfunction and PKCδ activation in the hippocampus of mice: Involvement of antioxidant potential.

Although the anticonvulsant effects of ginsenosides are recognized, little is known about their effects on the convulsive behaviors induced by the activation of L-type Ca2+ channels. Here, we investigated whether ginsenoside Re (GRe) modulates excitotoxicity induced by the L-type Ca2+ channel activator Bay k-8644. GRe significantly attenuated Bay k-8644-induced convulsive behaviors and hippocampal oxidative stress in mice. GRe-mediated antioxidant potential was more pronounced in the mitochondrial fraction than cytosolic fraction. As L-type Ca2+ channels are thought to be targets of protein kinase C (PKC), we investigated the role of PKC under excitotoxic conditions. GRe attenuated Bay k-8644-induced mitochondrial dysfunction, PKCδ activation, and neuronal loss. The PKCδ inhibition and neuroprotection mediated by GRe were comparable to those by the ROS inhibitor N-acetylcysteine, the mitochondrial protectant cyclosporin A, the microglial inhibitor minocycline, or the PKCδ inhibitor rottlerin. Consistently, the GRe-mediated PKCδ inhibition and neuroprotection were counteracted by the mitochondrial toxin 3-nitropropionic acid or the PKC activator bryostatin-1. GRe treatment did not have additional effects on PKCδ gene knockout-mediated neuroprotection, suggesting that PKCδ is a molecular target of GRe. Collectively, our results suggest that GRe-mediated anticonvulsive/neuroprotective effects require the attenuation of mitochondrial dysfunction and altered redox status and inactivation of PKCδ.

Ginsenoside Re Protects Trimethyltin-Induced Neurotoxicity via Activation of IL-6-Mediated Phosphoinositol 3-Kinase/Akt Signaling in Mice.

Ginseng (Panax ginseng), an herbal medicine, has been used to prevent neurodegenerative disorders. Ginsenosides (e.g., Re, Rb1, or Rg1) were obtained from Korean mountain cultivated ginseng. The anticonvulsant activity of ginsenoside Re (20 mg/kg/day × 3) against trimethyltin (TMT) insult was the most pronounced out of ginsenosides (e.g., Re, Rb1, and Rg1). Re itself did not significantly alter tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-ϒ), and interleukin-1ß (IL-1ß) expression, however, it significantly increases the interleukin-6 (IL-6) expression. In addition, Re attenuated the TMT-induced decreases in IL-6 protein level. Therefore, IL-6 knockout (-/-) mice were employed to investigate whether Re requires IL-6-dependent neuroprotective activity against TMT toxicity. Re significantly attenuated TMT-induced lipid peroxidation, protein peroxidation, and reactive oxygen species in the hippocampus. Re-mediated antioxidant effects were more pronounced in IL-6 (-/-) mice than in WT mice. Consistently, TMT-induced increase in c-Fos-immunoreactivity (c-Fos-IR), TUNEL-positive cells, and nuclear chromatin clumping in the dentate gyrus of the hippocampus were significantly attenuated by Re. Furthermore, Re attenuated TMT-induced proapoptotic changes. Protective potentials by Re were comparable to those by recombinant IL-6 protein (rIL-6) against TMT-insult in IL-6 (-/-) mice. Moreover, treatment with a phosphoinositol 3-kinase (PI3K) inhibitor, LY294002 (1.6 µg, i.c.v) counteracted the protective potential mediated by Re or rIL-6 against TMT insult. The results suggest that ginsenoside Re requires IL-6-dependent PI3K/Akt signaling for its protective potential against TMT-induced neurotoxicity.

Treatment with Mountain-Cultivated Ginseng Alleviates Trimethyltin-Induced Cognitive Impairments in Mice via IL-6-Dependent JAK2/STAT3/ERK Signaling.

Panax ginseng is the most widely used herbal medicine for improving cognitive functions. The pharmacological activity and underlying mechanisms of mountain-cultivated ginseng, however, have yet to be clearly elucidated, in particular, against trimethyltin-induced cognitive dysfunction. We previously reported that interleukin-6 plays a protective role against trimethyltin-induced cognitive dysfunction. Because of this, we have implemented a study system that uses interleukin-6 null (-/-) and wild-type mice. Interestingly, mountain-cultivated ginseng significantly upregulated interleukin-6 expression. With this study, we sought to determine whether the interleukin-6-dependent modulation of the Janus kinase 2/signal transducer activator of transcription 3 and extracellular signal-regulated kinase signaling network is also associated with the pharmacological activity of mountain-cultivated ginseng against trimethyltin-induced cognitive dysfunction. Trimethyltin treatment (2.4 mg/kg, intraperitoneal) causes the downregulation of Janus kinase 2/signal transducer activator of transcription 3, extracellular signal-regulated kinase signaling, and impairment of the cholinergic system. We found that mountain-cultivated ginseng treatment (50 mg/kg, intraperitoneal) significantly attenuated cognitive impairment normally induced by trimethyltin by upregulating p-Janus kinase 2/signal transducer activator of transcription 3, p-extracellular signal-regulated kinase signaling, and the cholinergic system. Trimethyltin-induced cognitive impairments were more pronounced in interleukin-6 (-/-) mice than wild-type mice, and they were markedly reduced by treatment with either mountain-cultivated ginseng or recombinant interleukin-6 protein (6 ng, intracerebroventricular). Additionally, treatment with either AG490 (20 mg/kg, intraperitoneal), a Janus kinase 2/signal transducer activator of transcription 3 inhibitor, or U0126 (2 µg/head, intracerebroventricular), an extracellular signal-regulated kinase inhibitor, reversed the effects of mountain-cultivated ginseng treatment. The effects of mountain-cultivated ginseng treatment were comparable to those of recombinant interleukin-6 protein in interleukin-6 (-/-) mice. Our results, therefore, suggest that mountain-cultivated ginseng acts through interleukin-6-dependent activation of Janus kinase 2/signal transducer activator of transcription 3/extracellular signal-regulated kinase signaling in order to reverse cognitive impairment caused by trimethyltin treatment.

Repeated exposure to far infrared ray attenuates acute restraint stress in mice via inhibition of JAK2/STAT3 signaling pathway by induction of glutathione peroxidase-1.

Exposure to far-infrared ray (FIR) has been shown to exert beneficial effects on cardiovascular and emotional disorders. However, the precise underlying mechanism mediated by FIR remains undetermined. Since restraint stress induces cardiovascular and emotional disorders, the present study investigated whether exposure to FIR affects acute restraint stress (ARS) in mice. c-Fos-immunoreactivity (IR) was significantly increased in the paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) in response to ARS. The increase in c-Fos-IR parallels that in oxidative burdens in the hypothalamus against ARS. Exposure to FIR significantly attenuated increases in the c-Fos-IR, oxidative burdens and corticosterone level. ARS elicited decreases in GSH/GSSG ratio, cytosolic Cu/Zn-superoxide dismutase (SOD-1), glutathione peroxidase (GPx), and glutathione reductase (GR) activities. FIR-mediated attenuation was particularly observed in ARS-induced decrease in GPx, but not in SOD-1 or GR activity. Consistently, ARS-induced decreases in GPx-1-immunoreactivity in PVN and DMH, and decreases in GPx-1 expression in the hypothalamus were significantly attenuated by FIR. ARS-induced significant increases in phosphorylation of JAK2/STAT3, and nuclear translocation and DNA-binding activity of NFκB were observed in the hypothalamus. Exposure to FIR selectively attenuated phosphorylation of JAK2/STAT3, but did not diminish nuclear translocation and DNA-binding activity of NFκB, suggesting that JAK2/STAT3 constitutes a critical target for FIR-mediated pharmacological potential. ARS-induced increase in c-Fos-IR in the PVN and DMH of non-transgenic mice was significantly attenuated by FIR exposure or JAK2/STAT3 inhibitor AG490. GPx-1 overexpressing transgenic mice significantly protected increases in the c-Fos-IR and corticosterone level induced by ARS. However, neither FIR exposure nor AG490 significantly affected attenuations by genetic overexpression of GPx-1. Moreover, AG490 did not exhibit any additional positive effects against the attenuation by genetic overexpression of GPx-1 or FIR exposure. Our results indicate that exposure to FIR significantly protects ARS-induced increases in c-Fos-IR and oxidative burdens via inhibition of JAK2/STAT3 signaling by induction of GPx-1.

Protective potential of IL-6 against trimethyltin-induced neurotoxicity in vivo.

We investigated the role of cytokines in trimethyltin (TMT)-induced convulsive neurotoxicity. Evaluation of TNF-α, interferon-γ, and interleukin (IL)-6 knockout (-/-) mice showed that the IL-6(-/-) mice had the greatest susceptibility to TMT-induced seizures. In both wild-type and IL-6(-/-) mice, TMT treatment increased glutathione oxidation, lipid peroxidation, protein oxidation, and levels of reactive oxygen species in the hippocampus. These effects were more pronounced in the IL-6(-/-) mice than in wild-type controls. In addition, the ability of TMT to induce nuclear translocation of Nrf2 and upregulation of heme oxygenase-1 and γ-glutamylcysteine ligase was significantly decreased in IL-6(-/-) mice. Treatment of IL-6(-/-) mice with recombinant IL-6 protein (rIL-6) restored these effects of TMT. Treatment with rIL-6 also significantly attenuated the TMT-induced inhibition of phosphoinositol 3-kinase (PI3K)/Akt signaling, thereby increasing phosphorylation of Bad (Bcl-xL/Bcl-2-associated death promoter protein), expression of Bcl-xL and Bcl-2, and the interaction between p-Bad and 14-3-3 protein and decreasing Bax expression and caspase-3 cleavage. Furthermore, in IL-6(-/-) mice, rIL-6 provided significant protection against TMT-induced neuronal degeneration; this effect of rIL-6 was counteracted by the PI3K inhibitor LY294002. These results suggest that activation of Nrf2-dependent glutathione homeostasis and PI3K/Akt signaling is required for the neuroprotective effects of IL-6 against TMT.