Yongming Bao

Vitamin E renders protection to PC12 cells against oxidative damage and apoptosis induced by single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) are potential candidates in many biomedical applications. However, many reports demonstrated its potential toxicity to human and other biological systems. Our study has demonstrated that SWCNTs can induce apoptosis and oxidative damage on PC12 cells, an in vitro model of neuronal cells. In the present study, we for the first time investigated the neuroprotective effects of vitamin E (VE) on SWCNT-induced neurotoxicity in cultured PC12 cells. Vitamin E (0.01-2mM) increased PC12 cells viability and significantly attenuated SWCNTs-induced apoptotic cell death in a time and dose-dependent manner, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. The presence of VE inhibited the formation of reactive oxygen species (ROS), decreased the level of lipid peroxide, elevated the level of glutathione (GSH) and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Additionally, VE blocked the reduction in the mitochondrial membrane potential and the activation of caspase-3. VE prevented the down-regulation of Bcl-2 expression and up-regulation of Bax expression induced by SWCNTs in PC12 cells. In summary, VE might protect PC12 cells from the injury induced by SWCNTs through the down-regulation of oxidative stress and prevention of mitochondrial-mediated apoptosis.

Nimodipine protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation

Nimodipine, a calcium channel blocker, has been used mainly in the therapy of cardiovascular diseases. Recently, its indications have been extended experimentally to a wider range of disorders especially some central nervous system (CNS) disorders. In this study, we investigated whether nimodipine is neuroprotective to inflammation-mediated neurodegenerative diseases. Pretreatment with nimodipine reduced the degeneration of dopaminergic (DA) neurons induced by LPS in mesencephalic neuron-glia cultures in a dose-dependent manner. The neuroprotective effect of nimodipine was attributed to the inhibition of microglial activation, since nimodipine significantly inhibited the production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)) from LPS-stimulated microglia. Moreover, nimodipine was not neuroprotective to 1-methyi-4-phenylpyridinium (MPP(+))-induced DA neurotoxicity in the absence of microglia. Mechanistic study showed that nimodipine failed to protect the degeneration of neurons in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells. Taken together these results suggest that nimodipine is protective to DA neurodegeneration via inhibiting the microglial-mediated oxidative stress and inflammatory response. Thus, nimodipine may be a potential therapeutic agent for the treatment of inflammation-related neurodegenerative disorders such as Parkinsons disease.

Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage.

Catalpol, an iridoid glycoside abundant in the roots of Rehmannia glutinosa, has been previously found to prevent the loss of CA1 hippocampal neurons and to reduce working errors in gerbils after ischemia‐reperfusion injury. In the present study, we investigated the effects of catalpol on astrocytes in an ischemic model to further characterize its neuroprotective mechanisms. Primary cultured astrocytes exposed to oxygen‐glucose deprivation (OGD) followed by reperfusion (adding back oxygen and glucose, OGD‐R), were used as an in vitro ischemic model. Treatment of the astrocytes with catalpol during ischemia‐reperfusion increased astrocyte survival significantly in a concentration‐dependent manner, as demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. In addition, catalpol prevented the decrease in mitochondrial membrane potential, inhibited the formation of reactive oxygen species (ROS) and the production of nitric oxide (NO), decreased the level of lipid peroxide and the activity of inducible nitric oxide synthase (iNOS), and elevated the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and the content of glutathione (GSH). Our results suggest that catalpol exerts the most significant cytoprotective effect on astrocytes by suppressing the production of free radicals and elevating antioxidant capacity.

Further pharmacological evidence of the neuroprotective effect of catalpol from Rehmannia glutinosa.

We have previously evaluated the neuroprotective effect of catalpol on aging mice induced by d-galactose, in which catalpol treatment ameliorated cognition deficits and attenuated oxidative damage in mice brain. To thoroughly elucidate the anti-aging effects of catalpol, the liver and spleen antioxidative systems and energy metabolism in senescent mice induced by d-galactose have been studied. Except control group, mice were subcutaneously injected with d-galactose (150mgkg(-1)body weight) for 6 weeks. Meanwhile, drug group mice were treated with catalpol (2.5, 5, 10mgkg(-1)body weight) and piracetam (300mgkg(-1)body weight) for the last 2 weeks. The activities of endogenous antioxidants and the level of glutathione (GSH) and lipid peroxide in the liver and spleen were assayed. Compared to control group, model group mice had significantly lower spleen index (spleen weight/body weight), lower level of GSH, lower activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), higher level of malondialdehyde (MDA) in the liver and spleen. However, catalpol administration markedly reversed these effects of senescence induced by d-galactose. Simultaneously, catalpol noticeably elevated the decreased activities of lactate dehydrogenase (LDH), glutamine synthetase (GS), Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase and decreased the elevated activity of creatine kinase (CK) in mice liver or spleen. These results implied that the anti-aging effects of catalpol were achieved at least partly by promoting endogenous antioxidant enzyme activities and normalizing energy disturbance. Catalpol may be a potential anti-aging agent and worth testing for further preclinical study aimed for senescence or neurodegenerative diseases such as Alzheimers and Parkinsons diseases.

D-galactose administration induces memory loss and energy metabolism disturbance in mice : Protective effects of catalpol

The neuroprotective effects of catalpol, an iridoid glycoside isolated from the fresh rehmannia roots, on the behavior and brain energy metabolism in senescent mice induced by d-galactose were assessed. Except control group, mice were subcutaneously injected with d-galactose (150 mg/kg body weight) for 6 weeks. From the fifth week, drug group mice were treated with catalpol (2.5, 5, 10 mg/kg body weight) and piracetam (300 mg/kg body weight) for the last 2 weeks. Behavioral changes including open field test and passive avoidance were examined after drug administration. To determine the brain damage, pathological alterations were measured by hematoxylin and eosin (HE) staining. The activities of lactate dehydrogenase (LDH), glutathione S-transferase (GSH-ST), glutamine synthetase (GS), creatine kinase (CK) in brain cortex and hippocampus were determined using different biochemical methods. Consistent with the cognition deficits, the activities of GSH-ST, GS and CK decreased while the activity of LDH increased in aging mice brain. Administration of catalpol for 2-weeks not only ameliorated cognition deficit, but also reversed the biochemical markers mentioned above and reduced the histological lesions in mouse brain. These results suggest that catalpol has protective effects on memory damage and energy metabolism failure in aging model mice and is worth testing for further preclinical study aimed for senescence or neurodegenerative diseases such as Alzheimers disease (AD) and Parkinsons disease (PD).

Catalpol attenuates the neurotoxicity induced by β-amyloid1–42 in cortical neuron–glia cultures

A glia-mediated inflammation plays an important role in the pathogenesis of Alzheimers disease (AD). In vitro, besides a direct neurotoxic effect on neurons, Abeta activates glia to produce an array of inflammatory factors including tumor necrosis factor-alpha (TNF-alpha), reactive oxygen species (ROS), nitric oxide (NO) and inducible nitric oxide synthase (iNOS), which accelerate the progression of AD. Catalpol, an iridoid glycoside, isolated from the root of Rehmannia glutinosa, protects neuronal cells from damage caused by a variety of toxic stimulus. In the present study, the effect of catalpol against Abeta(1-42)-induced neurotoxicity in primary cortical neuron-glia cultures as well as its mechanism acting on cells was further investigated. Pretreatment with catalpol at the dosage of 500 microM for 30 min prior to 5 microM Abeta(1-42) not only attenuated the Abeta(1-42)-triggered neurotoxicity to neurons but also inhibited the glial activation to some extent, which was examined by inspecting the morphological changes and measuring the release of the above mentioned inflammatory factors. Therefore, the results demonstrated that catalpol might be a promising anti-inflammatory agent in the therapy or prevention of neurodegenerative diseases associated with inflammation.

Efficient targeted pDNA/siRNA delivery with folate–low-molecular-weight polyethyleneimine–modified pullulan as non-viral carrier

Folate receptor (FR)-mediated gene/short interfering RNA (siRNA) targeting shows advantage for the delivery of gene/siRNA into specific FR-overexpressing cancer cells. In this study, the non-targeted gene vector P-PEI was synthesized by grafting low-molecular-weight (1kDa) branched polyethyleneimine (PEI) to succinylated pullulan, and the targeted gene vector P-PEI-FA was synthesized by coupling the carboxyl of folate (FA) to the amino of PEI. Gel electrophoresis retardation assay demonstrated that both P-PEI and P-PEI-FA can efficiently wrap pDNA and siRNA with electrostatic interaction at N/P ratios higher than 1.56 and can protect pDNA from degradation by DNase I and serum. Compared with PEI/pDNA, P-PEI/pDNA and P-PEI-FA/pDNA showed lower cytotoxicity against different cells. Under serum-containing conditions, compared with Lipofamine 2000/DNA and Lipofamine2000/siRNA, P-PEI-FA/DNA at N/P ratio of 6.25 displayed higher gene transfection efficiency, whereas P-PEI-FA/siRNA at N/P ratio of 12.5 demonstrated better enhanced gene silencing effect. P-PEI-FA/siRNA can also deliver FAM-labeled siRNA to endosomes and escape. Moreover, the gene transfection and silencing effects of P-PEI-FA were higher than those of P-PEI, and were dependent on the dose of FA in FR(+) HeLa cells. Thus, P-PEI-FA can assist DNA or siRNA targeting to FR-overexpressing cells, and the uptake pathway of P-PEI-FA/siRNA was FR-mediated endocytosis. These results indicate that P-PEI-FA is a potential candidate for safe and targeted gene delivery applications.

Effects of catalpol on mitochondrial function and working memory in mice after lipopolysaccharide-induced acute systemic inflammation.

The aim of this study was to investigate whether catalpol could facilitate recovery from lipopolysaccharide (LPS)-induced cognitive deficits and protect brain mitochondrial function from LPS-induced acute systemic inflammation. In the study, except control group, mice were challenged with a single dose of LPS (100 microg/mouse, i.p.) to mimic an acute peripheral infection. The results showed that LPS enhanced nuclear factor kappa B (NF-kappaB) activation and induced a loss in mitochondrial integrity as shown by a significant decrease in membrane potential and increase in mitochondrial permeability transition pore opening. Pretreatment with catalpol (10 mg/kg d, i.p.) for 10d before injection of LPS reversed the memory deficits induced by LPS, protected brain mitochondrial function, and attenuated LPS-induced NF-kappaB activation. Taken together, these data indicate that catalpol may possess therapeutic potential against LPS-induced acute systemic inflammation by attenuating NF-kappaB activation and protecting mitochondrial function in cerebral cortex and hippocampus.

Tocopheryl pullulan-based self assembling nanomicelles for anti-cancer drug delivery

Amphiphilic α-tocopherol pullulan polymers (PUTC1, PUTC2, and PUTC3) with different degrees of substitution were synthesized as new carriers for anticancer drugs. The polymers easily self-assembled into nanomicelles through dialysis method. The critical micelle concentrations (CMCs) were 38.0, 8.0, and 4.3mg/L for PUTC1, PUTC2, and PUTC3, respectively. 10-Hydroxycamptothecin (HCPT) used as a model drug was successfully loaded into the PUTC nanomicelles. Transmission electron microscopy images demonstrated that HCPT-loaded PUTC nanomicelles were almost spherical and had sizes ranging within 171.5-257.8 nm that increased with increased HCPT-loading content, as determined by dynamic laser scattering. The highest encapsulation efficiency of HCPT in PUTC nanomicelles reached 98.3%. The in vitro release of HCPT from PUTC micelles demonstrated sustained release for over 80 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays showed that blank PUTC micelles were nontoxic to normal cells and that the HCPT-loaded PUTC2 nanomicelles showed higher cytotoxicity than the free drug, which was attributed to the enhanced cellular uptake of drug-loaded nanomicelles. Biodistribution experiments showed that PUTC micelles provided an excellent approach to rapid drug transport into cell nuclei. Moreover, the cellular uptake of micelles was found to be an energy-dependent and actin polymerization-associated endocytic process by endocytosis inhibition experiments. These results suggested that PUTC nanomicelles had considerable potential as a drug carrier for drug intracellular delivery in cancer therapy.

Apoptosis of BGC823 cell line induced by p-hydroxymethoxybenzobijuglone, a novel compound from Juglans mandshurica.

p‐Hydroxymethoxybenzobijuglone (HMBBJ), a new quinone compound isolated from Juglans mandshurica (by bioassay‐guided fractionation), showed cytotoxic activity in the gastric carcinoma cell line BGC823. The growth of BGC823 cells was inhibited as demonstrated by MTT assay and several cellular characteristic changes, such as cell shrinkage, chromatin condensation and apoptotic body formation with programmed cell death. Flow cytometry analysis revealed that the BGC823 cell cycle was arrested at G2/M phase by HMBBJ, and the apoptotic rate of BGC823 cells increased with respect to HMBBJ in a dose‐dependent manner. HMBBJ also activated caspase‐3, decreased the expression of Bcl‐2 and caused a decrease in the mitochondrial membrane potential (ΔΨm). These findings suggest that HMBBJ could significantly induce apoptosis in BGC823 cells and should be considered as a potential candidate for a chemotherapeutic drug against cancer. Copyright