Xiao-dong Pan

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of β‐amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimers disease (AD). In this study, tripchlorolide (T4), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia‐mediated oligomeric Aβ toxicity. T4 significantly attenuated oligomeric Aβ(1‐42)‐induced release of inflammatory productions such as tumor necrosis factor‐α, interleukin‐1β, nitric oxide (NO), and prostaglandin E2. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in microglial cells. Further molecular mechanism study demonstrated that T4 inhibited the nuclear translocation of nuclear factor‐κB (NF‐κB) without affecting I‐κBα phosphorylation. It repressed Aβ‐induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF‐κB and JNK by T4 is correlated with the suppression of inflammatory mediators in Aβ‐stimulated microglial cells. These results suggest that T4 protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Aβ(1‐42) and that T4 acts on the signaling of NF‐κB and JNK, which are involved in the modulation of inflammatory response. Therefore, T4 may be an effective agent in modulating neuroinflammatory process in AD.

Ginsenoside Rg1 Attenuates Amyloid-β Content, Regulates PKA/CREB Activity, and Improves Cognitive Performance in SAMP8 Mice

It is well established that the presence of soluble amyloid-beta protein (Abeta) correlates with the severity of dementia in Alzheimers disease (AD). Several lines of evidence indicate that cyclic AMP responsive element binding protein (CREB) and protein kinase A (PKA) are involved in soluble Abeta-trigged disruption of synaptic plasticity in early AD. Previously we demonstrated the beneficial effects of ginsenoside Rg1 on Abeta-induced neuronal insult. Therefore, in the present study, we examined the effects of long-term consumption of Rg1 on the cerebral Abeta content and PKA/CREB signaling molecules, as well as cognitive performance in senescence-accelerated mouse prone 8 (SAMP8). Notably, a significant dose-dependent reduction of soluble Abeta(1-40) was shown in the hippocampus of SAMP8 mice after administration with ginsenoside Rg1 for 3 months. Furthermore, Rg1 treatment resulted in a significant decrease of hippocampal PKA RIIalpha level (isoform IIalpha of the regulatory subunit of PKA). In contrast, phospho-CREB and brain derived neurotrophic factor (BDNF) levels were dramatically increased in the hippocampus of SAMP8 treated with Rg1. Additionally, administration of ginsenoside Rg1 consequently improved learning and memory outcomes in SAMP8 mice. These data suggest that long-term consumption of ginsenoside Rg1 may delay cognitive decline, associated with significant effects on Abeta generation, PKA/CREB activity, as well as BDNF content in the brain. These data provide further support for the therapeutic or intervention potency of ginsenoside Rg1 in the early stage of AD.

Ginsenoside Rg1 attenuates β-amyloid generation via suppressing PPARγ-regulated BACE1 activity in N2a-APP695 cells

The level of β-site APP-cleaving enzyme 1 (BACE1) has been documented to increase in the brains of patients with Alzheimers disease, which has resulted in elevation of β-amyloid (Aβ) peptides. As a transcription factor binding site of the BACE1 promoter, peroxisome proliferator-activated receptor-γ (PPARγ) response element regulates the activity of the BACE1 promoter activity, indicating that PPARγ may become a potential target for Alzheimers disease treatment. Recent studies have demonstrated that ginsenoside Rg1 which is an effective component of extracts of ginseng can prevent memory loss and improve cognitive function in a variety of animal models. However, the underlying mechanism remains unclear. In the present study, we found that Rg1 decreased the levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ secreted in N2a-APP695 cells. The expression levels of both BACE1 mRNA and protein as well as β-CTFs, a cleavaged C-terminal fragment of APP by BACE1, were reduced in cells treated with Rg1. Moreover, Rg1 treatment led to a translocation of PPARγ from cytoplasm to nuclear. Intriguingly, Rg1, like pioglitazone (a PPARγ agonist), suppressed BACE1 activity in N2a-APP695 cells, while its effect on BACE1 activity was attenuated by GW9662 (a PPARγ antagonist). These results indicate that Rg1 may be a PPARγ agonist to enhance the binding of nuclear PPARγ to the BACE1 promoter, which may in turn inhibit the transcription and translation of BACE1, suppress the activity of BACE1, and ultimately attenuate Aβ generation. Therefore, ginsenoside Rg1 may serve as a promising agent in modulating Aβ-related pathology in Alzheimers disease.

Neuroprotective role of tripchlorolide on inflammatory neurotoxicity induced by lipopolysaccharide-activated microglia

A large body of evidence has suggested a strong association between neuroinflammation and the pathogenesis of many neurodegenerative diseases. Therefore, it is a good target for therapeutic treatment. So far, studies have proven anti-inflammatory herbal medicine and its constituents to be effective in slowing down the neurodegenerative process. The present study tested tripchlorolide, an extract of Tripterygium wilfordii Hook F (TWHF), as a novel agent to suppress inflammatory process in microglia. It showed this novel agent to be cytotoxic at a dose of 20-40 nM to primary microglia and BV-2 microglial cells but not to primary cortical neurons and Neuro-2A cells in vitro. Moreover, tripchlorolide protected primary cortical neurons and Neuro-2A cells from neuroinflammatory toxicity induced by the conditioned media from lipopolysaccharide (LPS)-stimulated microglia, which resulted in a significant decrease in their cell survival. The changes of the inflammatory mediators in this process were further investigated. In the LPS-stimulated microglia, the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), nitric oxide (NO), prostaglandin E(2) (PGE(2)), and intracellular superoxide anion (SOA) was markedly attenuated by tripchlorolide at a dose of 1.25-10 nM in a dose-dependent manner. Furthermore, the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was also significantly inhibited by tripchlorolide in both mRNA and protein levels. These results suggest that tripchlorolide can protect neuronal cells via a mechanism involving inhibition of inflammatory responses of microglia to pathological stimulations. Therefore, it is potentially a highly effective therapeutic agent in treating neuroninflammatory diseases.

Tripchlorolide improves age-associated cognitive deficits by reversing hippocampal synaptic plasticity impairment and NMDA receptor dysfunction in SAMP8 mice

Deficits in cognition and performance accompanying age-related neurodegenerative diseases such as Alzheimers disease (AD) are closely associated with the impairment of synaptic plasticity. Here, using a mouse model of senescence-accelerated P8 (SAMP8), we reported the role of tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, in improving cognitive deficits and promoting the long-term potentiation (LTP) of hippocampal slices via the N-methyl-D-aspartate receptor (NMDAR)-dependent signaling pathway. Our results demonstrated that chronic administration of T4 at low doses (0.25, 1.0, or 4.0 μg/kg per day, injected intraperitoneally for 75 days) significantly improved learning and memory function in aged SAMP8 mice, as indicated by a chain of behavioral tests including the Y-maze and Morris water maze. Additionally, T4 reversed the impaired LTP in hippocampal CA1 regions of SAMP8 mice in a dose-dependent manner. Moreover, it upregulated the levels of phospho-NMDAR1, postsynaptic density-95 (PSD-95), phospho-calcium-calmodulin dependent kinase II (CaMKII), phospho-CREB and brain derived neurotrophic factor (BDNF) in the hippocampus. This indicates that T4 prevents the impairment of NMDAR-mediated synaptic plasticity-related signal molecules. At optimal doses, T4 did not show significant side-effects on blood counts, blood biochemical measures, or survival of the mice. This novel mechanism in reversing age-related synaptic dysfunction and NMDAR functional deficits suggests that T4 can halt the manifestation of a key early-stage event in AD. With the consideration of SAMP8 mice as a model to develop therapeutic interventions for AD, our findings provide new insight into the clinical application of tripchlorolide in AD treatment.

Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons.

Aim:To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism.Methods:Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos. Whole-cell configuration of the patch-clamp technique was used to record the voltage-gated calcium currents (VGCCs) from the hippocampal neurons,and the effect of Rb1 was examined.Results:Rb1 (2–100 μmol/L) inhibited VGCCs in a concentration-dependent manner, and the current was mostly recovered upon wash-out. The specific L-type Ca2+ channel inhibitor nifedipine (10 μmol/L) occluded Rb1-induced inhibition on VGCCs. Neither the selective N-type Ca2+ channel blocker ω-conotoxin-GVIA (1 μmol/L), nor the selective P/Q-type Ca2+ channel blocker ω-agatoxin IVA (30 nmol/L) diminished Rb1-sensitive VGCCs. Rb1 induced a leftward shift of the steady-state inactivation curve of ICa to a negative potential without affecting its activation kinetics or reversal potential in the I–V curve. The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 μmol/L), nor by the PKA inhibitor H-89 (10 μmol/L).Conclusion:Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels, without affecting the N-type or P/Q-type Ca2+ channels in hippocampal neurons. cAMP-PKA signaling pathway is not involved in this effect.

Tripchlorolide improves cognitive deficits by reducing amyloid β and upregulating synapse-related proteins in a transgenic model of Alzheimer's Disease.

Alzheimers disease (AD) is characterized by early impairments in memory and progressive neurodegeneration. Disruption of synaptic plasticity processes that underlie learning and memory contribute partly to this pathophysiology. Tripchlorolide (T4), an extract from a traditional Chinese herbal Tripterygium wilfordii Hook F, has been shown to be neuroprotective in animal models of Parkinsons disease and to improve cognitive deficits in senescence‐accelerated mouse P8. In this study, we investigated the effect of T4 on cognitive decline and synaptic plasticity in five times familial AD (5XFAD) mice co‐expressing mutated amyloid precursor protein and presenilin‐1. Five‐month‐old 5XFAD mice and wild type littermates were intraperitoneally injected with T4, 5 μg/kg or 25 μg/kg, every other day for 60 days. T4 treatment significantly improved spatial learning and memory, alleviated synaptic ultrastructure degradation, up‐regulated expression of synapse‐related proteins, including synaptophysin, post‐synaptic density‐95, N‐methyl‐D‐aspartate receptor subunit 1, phosphorylation of calcium/calmodulin dependent protein kinase II α, and phosphorylation of cyclic AMP‐response element binding protein, and promoted activation of the phophoinositide‐3‐kinase‐Akt‐mammalian target of rapamycin signaling pathway in 5XFAD mice. Accumulation of amyloid β (Aβ) may contribute to synapse dysfunction and memory impairment in AD. We found that T4 treatment significantly reduced cerebral Aβ deposits and lowered Aβ levels in brain homogenates. These effects coincided with a reduction in cleavage of β‐carboxyl‐terminal amyloid precursor protein (APP) fragment, levels of soluble APPβ, and protein expression of β‐site APP cleaving enzyme 1. Taken together, our findings identify T4 as a potent negative regulator of brain Aβ levels and show that it significantly ameliorates synaptic degeneration and cognitive deficits in a mouse model of AD. Tripchlorolide (T4), a traditional Chinese herbal compound, was shown to be neuroprotective, but the underlying mechanisms remain unclear. The present study demonstrates that T4 alleviates cognitive decline in five times familial AD (5XFAD) mice. T4 acts against cognition impairment through downregulating BACE1 activity, reducing Aβ deposits, upregulating synapse‐related proteins expression, in particular the PI3K‐Akt‐mTOR signaling pathway. The findings suggest that T4 may be a promising novel therapeutic option for treating AD. Akt = protein kinase B; Aβ = amyloid β; AD = Alzheimeŕs disease; BACE1 = beta‐secretase 1; PI3K = phosphatidylinositol 3‐kinase; mTOR = mammalian target of rapamycin

Aging-related changes in RP3V kisspeptin neurons predate the reduced activation of GnRH neurons during the early reproductive decline in female mice

Kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) play a key role in relaying the positive feedback effects of estradiol that activate gonadotropin-releasing hormone (GnRH) neurons and drive a surge in the GnRH/luteinizing hormone (LH) level. However, the precise role of kisspeptin neurons during female reproductive senescence remains unclear. Focusing on middle-aged intact female mice with irregular estrous cycles, we found a parallel decline in c-Fos-positive kisspeptin neurons and c-Fos-positive GnRH neurons at the time of the GnRH/LH surge. Furthermore, in kisspeptin neurons, the expression of estrogen receptor α (ERα), but not progesterone receptor (PR), decreased with age. Interestingly, some kisspeptin neurons in the RP3V, but none of the GnRH neurons in the rostral preoptic area (rPOA), had a characteristic cellular senescence in middle-aged mice and old mice. These data suggest that, among the groups of neurons involved in reproductive control, the kisspeptin neurons in the RP3V are likely among the earliest to undergo aging processes and thus participate in initiating the early reproductive decline.

Tripchlorolide ameliorates experimental autoimmune encephalomyelitis by down-regulating ERK1/2-NF-κB and JAK/STAT signaling pathways

Tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, has been found to possess anti‐inflammatory and immunosuppressive actions. In the current study, these actions were evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis by scoring the clinical signs, observing the infiltration of inflammatory cells and myelin sheath in the lumbar spinal cord of EAE mice. The results demonstrated that T4 (at a dose of 40 μg/kg) significantly reduced the severity of EAE and slowed down the ongoing EAE. Further analysis showed that T4 suppressed the mRNA and protein levels of the transcription factors T‐bet and RoRrt and mRNA levels of IFN‐γ and IL‐17 in the spinal cords. Furthermore, T4 down‐regulated the ERK1/2‐NF‐κB and JAK/STAT signaling pathways. At 40 μg/kg, T4 did not induce side effects on hematological parameters. These findings suggest that T4 ameliorates EAE by immunosuppression, providing a new insight into T4 application in multiple sclerosis treatment.

Tripchlorolide Attenuates β-amyloid Generation via Suppressing PPARγ-Regulated BACE1 Activity in N2a/APP695 Cells

Due to its apparent rate-limiting function, BACE1 (β-secretase) appears to be a prime target for prevention of amyloid-β (Aβ) generation in brains with Alzheimer’s disease (AD). The activity of BACE1 is regulated by peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor binding site of the BACE1 promoter, indicating that PPARγ may be a potential target for AD treatment. Several studies have demonstrated that PPARγ activation is involved in the immunostimulation of amyloid-β precursor protein processing by nonsteroidal anti-inflammatory drugs (NSAIDs). The present study found that tripchlorolide (T4), with a similar chemical structure to that of NSAIDs, decreased the levels of Aβ secreted in N2a-APP695 cells. T4 treatment reduced the mRNA and protein levels of BACE1 and the protein level of sAPPβ, a cleaved N-terminal fragment of APP by BACE1. The treatment also translocated PPARγ from cytoplasm to nuclear. Intriguingly, T4, like pioglitazone (a PPARγ agonist), suppressed the BACE1 activity in N2a-APP695 cells, which was attenuated by GW9662 (a PPARγ antagonist). These results indicate that T4 may be a PPARγ agonist to enhance the binding of nuclear PPARγ to the BACE1 promoter, which may in turn inhibit the transcription and translation of BACE1, suppress the activity of BACE1, and ultimately attenuate the generation of Aβ. Due to its capability to alter Aβ generation and to protect central neural system against the neurotoxicity of Aβ, T4 may serve as a promising agent in modulating Aβ-related pathology in Alzheimer’s disease.