Neuroprotective effects of polysaccharides from wolfberry, the fruits of Lycium barbarum, against homocysteine-induced toxicity in rat cortical neurons.

Previous clinical and epidemiological studies have suggested that elevated plasma homocysteine (Hcy) levels increased the risk of Alzheime's disease (AD). Although the underlying mechanisms of its toxicity are elusive, it has been shown that Hcy damages neurons by inducing apoptosis, DNA fragmentation, and tau hyperphosphorylation. Wolfberry (Lycium barbarum) is a fruit that is known for its eye-protective and anti-aging properties in Asian countries. Previous studies from our laboratory have demonstrated that polysaccharides derived from wolfberry (LBA) have the ability to protect neurons from amyloid-beta (Abeta) peptide neurotoxicity. We hypothesize that the neuroprotective effects of wolfberry is not limited to Abeta and can also provide protection against other AD risk factors. In this study, we aim to elucidate the neuroprotective effects of wolfberry against Hcy-induced neuronal damage. Our data showed that LBA treatment significantly attenuated Hcy-induced neuronal cell death and apoptosis in primary cortical neurons as demonstrated by LDH and caspase-3 like activity assay. LBA also significantly reduced Hcy-induced tau phosphorylation at tau-1 (Ser198/199/202), pS396 (Ser396), and pS214 (Ser214) epitopes as well as cleavage of tau. At the same time, we also found that the phosphorylation level of p-GSK3beta (Ser9/Tyr 216) remained unchanged among different treatment groups at all detected time points. LBA treatment suppressed elevation of both p-ERK and p-JNK. In summary, our data demonstrated that LBA exerted neuroprotective effects on cortical neurons exposed to Hcy. Therefore, LBA has the potential to be a diseasemodifying agent for the prevention of AD.

Polysaccharides from wolfberry antagonizes glutamate excitotoxicity in rat cortical neurons.

Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer's disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 microg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-D-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H(2)O(2)) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H(2)O(2). Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate.

Dietary oxyresveratrol prevents parkinsonian mimetic 6-hydroxydopamine neurotoxicity.

Oxyresveratrol (OXY) is a polyhydroxylated stilbene existing in mulberry. Increasing lines of evidence have shown its neuroprotective effects against Alzheimer disease and stroke. However, little is known about its neuroprotective effect in Parkinson disease (PD). Owing to its antioxidant activity, blood-brain barrier permeativity, and water solubility, we hypothesized that OXY may exert neuroprotective effects against parkinsonian mimetic 6-hydroxydopamine (6-OHDA) neurotoxicity. Neuroblastoma SH-SY5Y cells have long been used as dopaminergic neurons in PD research. We found that both pretreatment and posttreatment with OXY on SH-SY5Y cells significantly reduced the release of lactate dehydrogenase, the activity of caspase-3, and the generation of intracellular reactive oxygen species triggered by 6-OHDA. Compared to resveratrol, OXY exhibited a wider effective dosage range. We proved that OXY could penetrate the cell membrane by HPLC analysis of cell extracts. These results suggest that OXY may act as an intracellular antioxidant to reduce oxidative stress induced by 6-OHDA. Western blot analysis demonstrated that OXY markedly attenuated 6-OHDA-induced phosphorylation of JNK and c-Jun. Furthermore, we proved that OXY increased the basal levels of SIRT1, which may disclose new pathways accounting for the neuroprotective effects of OXY. Taken together, our results suggest OXY, a dietary phenolic compound, as a potential nutritional candidate for protection against neurodegeneration in PD.

Antagonizing beta-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum.

Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in many Asian countries to promote health and longevity. Synaptic degeneration is another key mode of neurodegeneration in Alzheimer's disease (AD). Recent studies have shown the loss of synaptic density proteins in each individual neuron during the progression of AD. It was recently reported that beta-amyloid (Abeta) could cause synaptic dysfunction and contribute to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly attenuated Abeta-induced synaptotoxicity by preserving the synaptic density protein, synaptophysin. In addition, G. lucidum aqueous extract antagonized Abeta-triggered DEVD cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by G. lucidum in Abeta-stressed neurons. Taken together, the results prove a hypothesis that anti-aging G. lucidum can prevent harmful effects of the exterminating toxin Abeta in AD.

Characterization of the effects of anti-aging medicine Fructus lycii on beta-amyloid peptide neurotoxicity.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. There are increasing lines of evidence showing that the molecular signaling pathways in aged cells are altered so that cells are susceptible to injury. We and other laboratories have demonstrated the significant involvement of double-stranded RNA-dependent protein kinase (PKR) in beta-amyloid (A beta) peptide neurotoxicity and in AD. Fructus lycii (the fruit of Lycium barbarum) has long been used in oriental medicine as an anti-aging agent. Our previous studies demonstrated that the aqueous extract isolated from L. barbarum exhibited significant protection on cultured neurons against harmful chemical toxins such as A beta and dithiothreitol. We also showed that the polysaccharide-containing extract (LBP) from L. barbarum exhibited neuroprotective effects in the retina against ocular hypertension in a laser-induced glaucoma animal model. In this study, we aimed to investigate whether LBP can elicit neuroprotection to neurons stressed by A beta peptides. Furthermore, we planned to isolate and identify the neuroprotective agent from LBP using chromatographic methods. Our results showed that pretreatment of LBP effectively protected neurons against A beta-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. A new arabinogalactan-protein (LBP-III) was isolated from LBP and attenuated A beta peptide-activated caspase-3-like activity. LBP-III markedly reduced the phosphorylation of PKR triggered by A beta peptide. Since the phosphorylation state of PKR increased with age, reduction of its phosphorylation triggered by A beta peptide may implicate that LBP-III from Fructus lycii is a potential neuroprotective agent in AD. As herbal medicine has received increasing attention for the treatment of AD, our study will open a window for the development of a neuroprotective agent for anti-aging from Chinese medicine.

Characterizing the neuroprotective effects of alkaline extract of Lycium barbarum on beta-amyloid peptide neurotoxicity.

Lycium barbarum is an oriental medicinal herb that has long been used for its anti-aging and cell-protective properties. Previous studies have shown that aqueous extracts from L. barbarum exhibit neuroprotection via inhibiting pro-apoptotic signaling pathways. Other active components can also be accomplished by novel alkaline extraction method, which may give different profiles of water-soluble components. We hypothesize that another active component obtained by alkaline extraction method exerts different biological mechanisms to protect neurons. In this study, we aim to examine the neuroprotective effects from the alkaline extract of L. barbarum, namely LBB, to attenuate beta-amyloid (Abeta) peptide neurotoxicity. Primary cortical neurons were exposed to Abeta-peptides inducing apoptosis and neuronal cell death. Pretreatment of LBB significantly reduced the level of lactate dehydrogenase (LDH) release and the activity of caspase-3 triggered by Abeta. "Wash-out" procedures did not reduce its neuroprotective effects, suggesting that LBB may not bind directly to Abeta. We have further isolated three subfractions from LBB, namely LBB-0, LBB-I and LBB-II. LBB-I and LBB-II showed differential neuroprotective effects. Western blot analysis demonstrated that LBB-I and LBB-II markedly enhanced the phosphorylation of Akt. Taken together, our results suggested that the glycoconjugate isolated from novel alkaline extraction method can open up a new avenue for drug discovery in neurodegenerative diseases.

New polysaccharide from Nerium indicum protects neurons via stress kinase signaling pathway.

Most of the polysaccharides purified from Chinese medicinal herbs showed anti-tumor and immune-stimulating effects. However, little is known about their effects on neuroprotection. Our previous study has demonstrated that polysaccharides (J2, J3 and J4) isolated from the flowers of Nerium indicum (Oleander) exert partial protection in cortical neurons stressed by beta-amyloid (Abeta) peptides or deprivation of nutrition from serum. In this study, we have isolated and characterized a new polysaccharide from the flowers of N. indicum (named as J6) and aimed to investigate its neuroprotective effects against Abeta-induced apoptosis. Pretreatment of the polysaccharide J6 significantly decreased the activity of caspase-3 as well as the cytotoxicity triggered by Abeta peptides in a dose-dependent manner. In contrast to the activation of survival signaling such as Akt found in J2, J3 and J4 fractions, neuroprotective effects of J6 markedly inhibited Abeta peptide-stimulated phosphorylation of c-Jun N-terminal kinase (JNK-1) as determined by Western blot analysis. Taken together, the polysaccharide J6 isolated from the flowers of N. indicum can serve as potential neuroprotective agent against neuronal death in Alzheimer's disease and the neuroprotective mechanism may primarily rely on inactivation of JNK signaling pathway.

Cytoprotective effects of Lycium barbarum against reducing stress on endoplasmic reticulum.

Chinese medicinal herbs have been consumed for thousands of years for the purpose of healthy aging. Lycium barbarum is valued in Chinese culture for its benefits to anti-aging, vision, kidney and liver. Recent studies showed that extracts from L. barbarum possess biological activities including anti-aging, anti-tumor, immune-stimulatory and cytoprotection. Most of these studies emphasized that the protective function of L. barbarum is due to its anti-oxidative effects. We have previously demonstrated that extract from L. barbarum can protect neurons against beta-amyloid (Abeta) peptide-induced apoptosis. Since Abeta toxicity may be mediated via oxidative stress, it is still unclear whether the extract from L. barbarum is a simple anti-oxidant exhibiting cytoprotective effects. We hypothesized that extract from L. barbarum is not simply an anti-oxidant in order to function as a neuroprotective agent. The aim of this study is to investigate whether the extract from L. barbarum (LBG) protect neurons via mechanisms independent of anti-oxidative effects. Using a reducing agent, dithiothreitol (DTT), we found that LBG exhibits cytoprotective effects against reducing stress by lowering the DTT-induced LDH release and caspase-3 activity. DTT can trigger endoplasmic reticulum (ER) stress leading to PKR-like ER kinase (PERK) activation. We also showed that LBG attenuates DTT-induced PERK phosphorylation. The extract from L. barbarum is not simply an anti-oxidant; it can also exhibit cytoprotective effects against reducing stress by DTT.

Novel neuroprotective effects of the aqueous extracts from Verbena officinalis Linn.

Verbena officinialis Linn. (Verbenaceae) is a perennial plant which has been used as herbal medicine or health supplement in both Western and Eastern countries for centuries. It has been used to treat acute dysentery, enteritis, amenorrhea and depression. In view of its wide array of biological effects, we hypothesized that V. officinalis can exert cytoprotective effects on cells of the central nervous system. Pre-treatment of aqueous extracts of V. officinalis significantly attenuated the toxicity of beta-amyloid (Abeta) peptide and reducing agent dithiothreitol in primary cultures of cortical neurons. As extracellular accumulation of Abeta peptide is an important cytotoxic factor involved in Alzheimer's disease (AD), we have further explored its neuroprotective effect against Abeta. Treatment of V. officinalis attenuated Abeta-triggered DEVD- and VDVAD-cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of both interferon-inducing protein kinase (PKR) and c-Jun N-terminal kinase (JNK) was attenuated in Abeta-treated neurons. Taken together, we have proved our hypothesis by showing the novel neuroprotective effects of V. officinalis. As V. officinalis has long been used for many years to be a folk medicine, our study may provide a lead for its potential to be a neuroprotective agent against neuronal loss in AD.

Neuroprotective effects of anti-aging oriental medicine Lycium barbarum against beta-amyloid peptide neurotoxicity.

As aged population dramatically increases in these decades, efforts should be made on the intervention for curing age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Natural plant extracts of Lycium barbarum are well-known to exhibit anti-aging effects. We therefore hypothesized that they exhibit neuroprotective effects against toxins in aging-related neurodegenerative diseases. In this study, we aimed to investigate whether extracts from L. barbarum have neuroprotective effects against toxicity of fibrillar Abeta(1-42) and Abeta(25-35) fragments. Primary rat cortical neurons exposed to Abeta peptides resulted in apoptosis and necrosis. Pre-treatment with extract isolated from L. barbarum significantly reduced the release of lactate dehydrogenase (LDH). In addition, it attenuated Abeta peptide-activated caspases-3-like activity. The extract elicited a typical dose-dependent neuroprotective effect. Effective dosage of this extract was wider than that of a well-known western neuroprotective medicine lithium chloride (LiCl). We have further examined the underlying mechanisms of the neuroprotective effects. In agreement with other laboratories, Abeta peptides induce a rapid activation of c-Jun N-terminal kinase (JNK) by phosphorylation. Pre-treatment of aqueous extract markedly reduced the phosphorylation of JNK-1 (Thr183/Tyr185) and its substrates c-Jun-I (Ser 73) and c-Jun-II (Ser 63). Taken together, we have proved our hypothesis by showing neuroprotective effects of the extract from L. barbarum. Study on anti-aging herbal medicine like L. barbarum may open a new therapeutic window for the prevention of AD.

Characterization of polysaccharides from the flowers of Nerium indicum and their neuroprotective effects.

Degeneration of neurons is a key problem in Alzheimer's disease (AD) and neuroprotection is a possible way to safeguard neurons from neurodegeneration. Polysaccharides isolated from Chinese medicinal herbs have been investigated extensively for their anti-tumor and immune stimulating effects. Yet, little is known about the effects of polysaccharides in neurons. Recently, two pure polysaccharides isolated from the flowers of Nerium indicum were shown to stimulate proliferation and differentiation of PC12 pheochromocytoma cells, an effect similar to that observed from nerve growth factor. In this notion, it is hypothesized that polysaccharides isolated from the flowers of N. indicum could exhibit beneficial effects in neurons. In this study, we isolated, characterized and investigated two new polysaccharides from the flowers of N. indicum for their neuroprotective effects on neurons against serum-deprivation and beta-amyloid (Abeta) peptide toxicity in primary rat cortical neuronal cultures. Pretreatment of the polysaccharides significantly reduced the number of apoptotic neurons revealed by DAPI staining when neurons were exposed to serum-free medium. Besides, the polysaccharides could also decrease the activity of caspase-3 triggered by Abeta peptides. Western blot analysis indicated that polysaccharides stimulated the phosphorylation of PDK-1 (Serine 241) and Akt (Threonine 308). In conclusion, the polysaccharides J2, J3 and J4 isolated from N. indicum provide a lead for future development of neuroprotective agent against neuronal death in neurodegenerative diseases and the neuroprotective mechanism may primarily rely on activation of Akt survival signaling pathway.