Anti-Inflammatory CDGSH Iron-Sulfur Domain 2: A Biomarker of Central Nervous System Insult in Cellular, Animal Models and Patients.

Spinal cord injury (SCI) promotes brain inflammation; conversely, brain injury promotes spinal neuron loss. There is a need to identify molecular biomarkers and therapeutic targets for central nervous system (CNS) injury. CDGSH iron-sulfur structural domain 2 (CISD2), an NF-κB antagonist, is downregulated after injury in vivo and in vitro. We aimed to examine the diagnostic value of CISD2 in patients with CNS insult. Plasma and cerebrospinal fluid (CSF) CISD2 levels were decreased in 13 patients with CNS insult and were negatively correlated with plasma IL6 levels (associated with disease severity; r = −0.7062; p < 0.01). SCI-induced inflammatory mediators delivered through CSF promoted mouse brain inflammation at 1 h post-SCI. Anti-CISD2 antibody treatment exacerbated SCI-induced inflammation in mouse spine and brain. Lipopolysaccharide-stimulated siCISD2-transfected EOC microglial cells exhibited proinflammatory phenotypes (enhanced M1 polarization, decreased M2 polarization, and increased intranuclear NF-κB p65 translocation). Plasma and CSF CISD2 levels were increased in three patients with CNS insult post-therapeutic hypothermia. CISD2 levels were negatively correlated with plasma and CSF levels of inflammatory mediators. CISD2 inhibition and potentiation experiments in cells, animals, and humans revealed CISD2 as a biomarker for CNS insult and upregulation of CISD2 anti-inflammatory properties as a potential therapeutic strategy for CNS insult.

Freshwater Clam Extract Mitigates Neuroinflammation and Amplifies Neurotrophic Activity of Glia: Insights from In Vitro Model of Neurodegenerative Pathomechanism.

BACKGROUND: An extensive body of research suggests that brain inflammation and oxidative stress are the underlying causes of Parkinson's disease (PD), for which no potent therapeutic approach exists to mitigate the degradation of dopamine neurons. Freshwater clams, an ancient health food of Chinese origin, have been documented to exhibit anti-inflammatory and antioxidant effects. We previously reported that freshwater clam extract (FCE) can attenuate astrocytic activation and subsequent proinflammatory cytokine production from substantia nigra in an MPTP-induced PD mouse model. This article provides insight into the potential mechanisms through which FCE regulates neuroinflammation in a glia model of injury. MATERIALS AND METHODS: In total, 1 µg/mL lipopolysaccharide (LPS) and 200 µM rotenone were conducted in primary glial cell cultures to mimic the respective neuroinflammation and oxidative stress during injury-induced glial cell reactivation, which is relevant to the pathological process of PD. RESULTS: FCE markedly reduced LPS-induced neuroinflammation by suppressing NO and TNF-α production and the expression of pro-inflammatory cytokines. In addition, FCE was effective at reducing rotenone-induced toxicity by diminishing ROS production, promoting antioxidant enzymes (SOD, catalase, and GPx) and minimizing the decline in glial-cell-secreted neurotrophic factors (GDNF, BDNF). These impacts ultimately led to a decrease in glial apoptosis. CONCLUSIONS: Evidence reveals that FCE is capable of stabilizing reactive glia, as demonstrated by reduced neuroinflammation, oxidative stress, the increased release of neurotrophic factors and the inhibition of apoptosis, which provides therapeutic insight into neurodegenerative diseases, including PD.

Ultrarapid Inflammation of the Olfactory Bulb After Spinal Cord Injury: Protective Effects of the Granulocyte Colony-Stimulating Factor on Early Neurodegeneration in the Brain.

The correlation among olfactory dysfunction, spinal cord injury (SCI), subjective cognitive decline, and neurodegenerative dementia has been established. Impaired olfaction is considered a marker for neurodegeneration. Hence, there is a need to examine if SCI leads to olfactory dysfunction. In this study, the brain tissue of mice with spinal cord hemisection injury was subjected to microarray analysis. The mRNA expression levels of olfactory receptors in the brain began to decline at 8 h post-SCI. SCI promoted neuroinflammation, downregulated the expression of olfactory receptors, decreased the number of neural stem cells (NSCs), and inhibited the production of neurotrophic factors in the olfactory bulbs at 8 h post-SCI. In particular, the SCI group had upregulated mRNA and protein expression levels of glial fibrillary acidic protein (GFAP; a marker of astrocyte reactivation) and pro-inflammatory mediators [IL-1ß, IL-6, and Nestin (marker of NSCs)] in the olfactory bulb compared to levels in the sham control group. The mRNA expression levels of olfactory receptors (Olfr1494, Olfr1324, Olfr1241, and Olfr979) and neurotrophic factors [brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF)] were downregulated in the olfactory bulb of the SCI group mice at 8 h post-SCI. The administration of granulocyte colony-stimulating factor (G-CSF) mitigated these SCI-induced pathological changes in the olfactory bulb at 8 h post-SCI. These results indicate that the olfactory bulb is vulnerable to environmental damage even if the lesion is located at sites distant from the brain, such as the spinal cord. Additionally, SCI initiated pathological processes, including inflammatory response, and impaired neurogenesis, at an early stage. The findings of this study will provide a basis for future studies on pathological mechanisms of early neurodegenerative diseases involving the olfactory bulb and enable early clinical drug intervention.

The effect of different colored light emitting diode illumination on egg laying performance, egg qualities, blood hormone levels and behavior patterns in Brown Tsaiya duck.

OBJECTIVE: The objective of this experiment was to investigate the effects of different colors produced by light emitting diode (LED) on Brown Tsaiya ducks. METHODS: A total of 144 female Brown Tsaiya ducks were randomly allocated into three individual cage rearing chambers with different LED illumination colors as treatments. Three different treatments were: i) white color, ii) blue color, and iii) red color. The experiment periods were from ducks 21 to 49 weeks of age, determined traits included i) egg laying performance, ii) feed intake, iii) egg shell breaking strength, iv) egg shell thickness, v) egg Haugh unit, vi) egg weight, vii) serum Estradiol and Progesterone concentration, and viii) behavior pattern. RESULTS: The results indicated that when compared with white and blue color, red color could stimulate ducks sexual maturation and raised the egg laying performance. The red light group was also observed to have the highest feed intake among three treatments. The blue treatment had the lowest egg shell breaking strength and the highest egg weight among three treatments, nevertheless, no significant difference was observed among three treatments on egg shell thickness and egg Haugh unit. The red light group had higher serum estradiol concentration than the white and blue groups, but no significant difference among treatments on the serum Progesterone concentration was found. The results of behavior pattern indicated that red light group showed more feeding and less resting behavior compared to the blue light group. CONCLUSION: We found a potential of applying red light illumination in the indoor laying duck raising system with positive results on egg laying performance and acceptable egg weight, equivalent egg qualities compared to white and blue light.

Wild Bitter Melon Exerts Anti-Inflammatory Effects by Upregulating Injury-Attenuated CISD2 Expression following Spinal Cord Injury.

BACKGROUND: Spinal cord injuries (SCIs) induce secondary neuroinflammation through astrocyte reactivation, which adversely affects neuronal survival and eventually causes long-term disability. CDGSH iron sulfur domain 2 (CISD2), which has been reported to be involved in mediating the anti-inflammatory responses, can serve as a target in SCI therapy. Wild bitter melon (WBM; Momordica charantia Linn. var. abbreviata Ser.) contains an anti-inflammatory agent called alpha-eleostearic acid (α-ESA), a peroxisome proliferator-activated receptor-ß (PPAR-ß) ligand. Activated PPAR-ß inhibits the nuclear factor κB (NF-κB) signaling pathway via the inhibition of IκB (inhibitor of NF-κB) degradation. The role of astrocyte deactivation and CISD2 in anti-inflammatory mechanisms of WBM in acute SCIs is unknown. MATERIALS AND METHODS: A mouse model of SCI was generated via spinal cord hemisection. The SCI mice were administered WBM intraperitoneally (500 mg/kg bodyweight). Lipopolysaccharide- (LPS-) stimulated ALT cells (astrocytes) were used as an in vitro model for studying astrocyte-mediated inflammation post-SCI. The roles of CISD2 and PPAR-ß in inflammatory signaling were examined using LPS-stimulated SH-SY5Y cells transfected with si-CISD2 or scramble RNA. RESULTS: WBM mitigated the SCI-induced downregulation of CISD2, PPAR-ß, and IκB and upregulation of glial fibrillary acidic protein (GFAP; marker of astrocyte reactivation) in the spinal cord of SCI mice. Additionally, WBM (1 µg/mL) mitigated LPS-induced CISD2 downregulation. Furthermore, SH-SY5Y neural cells with CISD2 knockdown exhibited decreased PPAR-ß expression and augmented NF-κB signaling. CONCLUSION: To the best of our knowledge, this is the first study to report that CISD2 is an upstream modulator of the PPAR-ß/NF-κB proinflammatory signaling pathway in neural cells, and that WBM can mitigate the injury-induced downregulation of CISD2 in SCI mice and LPS-stimulated ALT astrocytes.

An investigation of the correlation between the S-glutathionylated GAPDH levels in blood and Alzheimer's disease progression.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by two aggregates, namely, amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein (tau-p), which are released into the blood in a very small amount and cannot be easily detected. An increasing number of recent studies have suggested that S-glutathionylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is highly correlated with Aß in patients with AD and that S-glutathionylated GAPDH plays a role as a proapoptotic factor in AD. We found that S-glutathionylated GAPDH is abundant in the blood of AD patients, which is unusual because S-glutathionylated GAPDH cannot exist in the blood under normal conditions. The aim of this study was to further explore the correlation between the S-glutathionylated GAPDH levels in blood plasma and AD progression. As controls, we recruited 191 people without AD, which included 111 healthy individuals and 37 patients with depression and insomnia, in the psychosomatic clinic. Moreover, 47 patients with AD (aged 40-89 years) were recruited at the neurology clinic. The blood S-glutathionylated GAPDH levels in the AD patients were significantly (p < 0.001) higher (752.7 ± 301.7 ng/dL) than those in the controls (59.92 ± 122.4 ng/dL), irrespective of gender and age. For AD diagnosis, the criterion blood S-glutathionylated GAPDH level > 251.62 ng/dL exhibited 95.74% sensitivity and 92.67% specificity. In fact, the individuals aged 70-89 years, namely, 37 patients from the psychosomatic clinic and 42 healthy individuals, showed significant blood S-glutathionylated GAPDH levels (230.5 ± 79.3 and 8.05 ± 20.51 ng/dL, respectively). This finding might indicate neurodegenerative AD progression in psychosomatic patients and suggests that the degree of neuronal apoptosis during AD progression might be sensitively evaluated based on the level of S-glutathionylated GAPDH in blood.

Protective Effects of CISD2 and Influence of Curcumin on CISD2 Expression in Aged Animals and Inflammatory Cell Model.

BACKGROUND: Inflammation and mitochondrial dysfunction have been linked to trauma, neurodegeneration, and aging. Impairment of CISD2 expression may trigger the aforementioned pathological conditions in neural cells. We previously reported that curcumin attenuates the downregulation of CISD2 in animal models of spinal cord injury and lipopolysaccharide (LPS)-treated neuronal cells. In this study, we investigate (1) the role of CISD2 and (2) how curcumin regulates CISD2 in the aging process. MATERIALS AND METHODS: The serial expression of CISD2 and the efficacy of curcumin treatment were evaluated in old (104 weeks) mice and long-term cultures of neural cells (35 days in vitro, DIV). LPS-challenged neural cells (with or without siCISD2 transfection) were used to verify the role of curcumin on CISD2 underlying mitochondrial dysfunction. RESULTS: In the brain and spinal cord of mice aged P2, 8, 25, and 104 weeks, we observed a significant decrease in CISD2 expression with age. Curcumin treatment in vivo and in vitro was shown to upregulate CISD2 expression; attenuate inflammatory response in neural cells. Moreover, curcumin treatment elevated CISD2 expression levels and prevented mitochondrial dysfunction in LPS-challenged neural cells. The beneficial effects of curcumin in either non-stressed or LPS-challenged cells that underwent siCISD2 transfection were significantly lower than in respective groups of cells that underwent scrambled siRNA-transfection. CONCLUSIONS: We hypothesize that the protective effects of curcumin treatment in reducing cellular inflammation associated trauma, degenerative, and aging processes can be partially attributed to elevated CISD2 expression. We observed a reduction in the protective effects of curcumin against injury-induced inflammation and mitochondrial dysfunction in cells where CISD2 expression was reduced by siCISD2.

Growing pigs developed different types of diabetes induced by streptozotocin depending on their transcription factor 7-like 2 gene polymorphisms.

The different polymorphisms of the transcription factor 7-like 2 (TCF7L2) gene promote variances in diabetes susceptibility in humans. We investigated whether these genotypes also promote differences in diabetic susceptibility in commercial pigs. Growing pigs (Landrace, both sex, 50-60 kg) with the C/C (n=4) and T/T (n=5) TCF7L2 genotypes were identified and intravenously injected with streptozotocin (STZ, 40 mg/kg) twice in weekly intervals, then a high-energy diet was offered. Oral glucose tolerance tests, blood analyses and the homeostasis model assessment-insulin resistance (HOMA-IR) index calculations were performed. The animals were sacrificed at the end of 12 weeks of treatment to reveal the pancreas histomorphometry. The results showed that all of the treated pigs grew normally despite exhibiting hyperglycemia at two weeks after the induction. The glycemic level of the fasting or postprandial pigs gradually returned to normal. The fasting insulin concentration was significantly decreased for the T/T carriers but not for the C/C carriers, and the resulting HOMA-IR index was significantly increased for the C/C genotype, indicating that the models of insulin dependence and resistance were respectively developed by T/T and C/C carriers. The histopathological results illustrated a significant reduction in the pancreas mass and insulin active sites, which suggested increased damage. The results obtained here could not be compared with previous studies because the TCF7L2 background has not been reported. Growing pigs may be an excellent model for diabetic in children if the animals are genetically pre-selected.

Neuroprotection by freshwater clam extract against the neurotoxin MPTP in C57BL/6 mice.

Freshwater clams are a popular health food in Asia and are traditionally used to prevent hepatic inflammation. Freshwater clam extract (FCE) inhibits inflammatory responses in activated macrophages by reducing the activation of mitogen-activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells. In this study, we used a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP; a neurotoxin)-induced Parkinson's disease (PD) to demonstrate the protective effect of FCE on dopamine neurons in the substantia nigra pars compacta (SNpc). Locomotor behavior and tyrosine hydroxylase immunohistochemical staining indicated that FCE significantly inhibited MPTP-induced dopaminergic cell loss in the SNpc. Glial fibrillary acidic protein immunohistochemistry and quantitative polymerase chain reaction analysis revealed that astroglial activation and tumor necrosis factor alpha, inducible nitric oxide synthase, and interleukin 1 beta production were significantly inhibited by FCE. The expressions of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and nerve growth factor were markedly increased by FCE action against MPTP-induced toxicity. FCE showed a neuroprotective effect in a MPTP-induced PD model, which might be correlated with anti-inflammation and the stimulation of neurotrophic factors.

Quantitative assessment of brain shifting in the late phase postevacuation in unilateral and bilateral chronic subdural hematomas.

AIM OF THE STUDY: Recurrence is more common in bilateral chronic subdural hematomas (CSDHs) than in unilateral. Our aim was to quantitatively compare the late phase of brain shifting postevacuation in unilateral and bilateral CSDHs. MATERIALS AND METHODS: We reviewed computed tomography (CT) scans and medical records of consecutive patients with CSDHs who underwent burr hole drainage. CT scan images (preoperative and postoperative days [PODs] 30 and 60) were imported to Adobe Photoshop, and temporal and spatial changes in brain shifting between PODs 30 and 60, and also the subdural space on POD 60, were analyzed. RESULTS: The bilateral group exhibited a significantly greater late phase of brain shifting than the unilateral group between PODs 30 and 60 (P < 0.001). The median late phase of brain shifting of the bilateral group was 8.9 mm (interquartile range [IQR]: 8.3-9.0 mm) between PODs 30 and 60, while that of the unilateral group was 1.8 mm (IQR: 1.3-2.5 mm). CONCLUSIONS: The postevacuation late phase of brain shifting is statistically greater in bilateral CSDHs than in unilateral CSDHs, which might facilitate bridging vein tearing and consequent rebleeding. This may be one factor accounting for the higher recurrence rate of bilateral CSDHs.

Sperm Quality Parameters and Reproductive Efficiency in Muscovy Duck (Cairina moschata).

The in vitro sperm quality parameters (motility, M; viability, V; normal morphology, NM; plasma membrane integrity, PMI; mitochondrial function, MF) in Muscovy drakes (Cairina moschata) were evaluated by using microscopy and flow cytometry, the correlation among sperm quality parameters and results of artificial insemination was also assessed in present study. M, V and NM were detected by phase contrast microscopy assisted with eosinnigrosin staining, and PMI and MF were detected by using flow cytometry within appropriate fluorescence staining (SYBR-14/PI and R123/PI, respectively). Fertility (F), early embryonic mortality (EEM) and the survival embryo rate (SER) were assessed after the artificial insemination of Muscovy or Kaiya duck (Anas platyrhynchos) females. Sperm PMI and MF, the parameters detected by flow cytometry were positively correlated with sperm M, V, and NM, those were detected by phase contrast microscopy (P<0.05). Sperm V and PMI were negatively correlated with the percentage of early embryo mortality of Muscovy duck fertile eggs (P<0.05). The results of the present study showed the relationships among the AI results and the sperm quality parameters detected by microscopy as well as flow cytometry. In conclusion, flow cytometry assisted with microscopy can be an effective tool to evaluate in vitro sperm quality and may contribute to predict the reproductive performances of individual Muscovy drakes, which helps to improve duck production efficiency.

CISD2 serves a novel role as a suppressor of nitric oxide signalling and curcumin increases CISD2 expression in spinal cord injuries.

BACKGROUND: CISD2 is known to have roles in calcium metabolism, anti-apoptosis, and longevity. However, whether CISD2 is involved in the inflammatory response associated with injuries of the central nervous system (CNS) remains unclear. This issue is particularly relevant for traumatic spinal cord injuries (SCIs), which lack therapeutic targeting and often cause long-term disability in patients. The authors previously demonstrated the neuroprotective effects of curcumin against RANTES-mediated neuroinflammation. In this study, we investigated (1) the role of CISD2 in injury-induced inflammation and (2) whether curcumin influences CISD2 expression in acute SCI. MATERIALS AND METHODS: The efficacy of curcumin treatment (40 mg/kg i.p.) was evaluated in an animal model of SCI. In a neural cell culture model, lipopolysaccharide (LPS) was administrated to induce inflammation with the aim of mimicking the situation commonly encountered in SCI. Additionally, knockdown of CISD2 expression by siRNA (siCISD2) in LPS-challenged neural cells was performed to verify the causal relationship between CISD2 and SCI-related inflammation. RESULTS: The injuries were shown to reduce CISD2 mRNA and protein expression in vivo, and CISD2-positive cells were upregulated by the curcumin treatment. LPS led to a decrease in CISD2 expression in vitro; however, treatment with 1 µM curcumin attenuated the downregulation of CISD2. Furthermore, in a cellular model of LPS-induced injury, the loss of CISD2 function caused by siCISD2 resulted in a pronounced iNOS increase as well as a decrease in BCL2 expression. CONCLUSIONS: To the best of our knowledge, this is the first study to report the following: (1) CISD2 exerts anti-apoptotic and anti-inflammatory effects in neural cells; and (2) curcumin can attenuate the downregulation of CISD2 in SCI and LPS-treated astrocytes.

Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy.

The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1ß and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome-derived IL-1ß secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy.

Quantitative assessment of post-operative recurrence of chronic subdural haematoma using mean haematoma density.

PURPOSE: The purpose of this study was to establish a quantitative method with which to assess the post-operative recurrence of chronic subdural haematoma (CSDH). METHODS: CT scans were reviewed from 44 consecutive patients with CSDHs who underwent burr hole drainage between July 2008 and January 2012. The area of the haematoma was quantified according to the mean haematoma density (MHD) using computer-based image analysis of pre-operative brain CT scans. MHD as well as other variables of patients with and without post-operative recurrences was statistically compared. RESULTS: Post-operative recurrence was noted in six of the 44 patients that underwent surgical procedures. Among these variables, high MHD, separated type and bilateral and skull base involvement of CSDHs were shown to be significantly related to post-operative recurrence (p < 0.05). Controlling for separated type in logistic regression analysis revealed the OR of MHD as statistically significant indicators with a p value of less than 0.05 (OR = 1.243; 95% CI = 1.003-1.54). CONCLUSION: This study provides statistical proof that MHD is a significant, independent, prognostic factor for the post-operative recurrence of CSDH. As such, consideration of MHD could aid in the prediction of post-operative prognosis of CSDHs.

Rutin, a Flavonoid That Is a Main Component of Saussurea involucrata, Attenuates the Senescence Effect in D-Galactose Aging Mouse Model.

Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in the Tian Shan and A'er Tai areas of China. It has recently been reported that the ethyl acetate extract of S. involucrata (SI-2) can inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells. This study investigated the protective effect of ethyl acetate extract of S. involucrata (SI-2) or rutin, a flavonoid extracted from ethyl acetate extract of S. involucrata (SI-2), on D-galactose- (D-gal-) induced brain injury in mice. Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) for 6 weeks, concomitant with D-gal injection, significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, the result showed that the percentages of cleaved caspase-3 and PARP in the D-gal-treated mice were much higher than those in the control. Pretreatment using SI-2 or rutin decreased the expression of cyclooxygenase-2 via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. Furthermore, our results also showed that oral administration of rutin to these mice significantly improved behavioral performance in a step-through passive avoidance task and these results suggest that SI-2 or rutin exerts potent antiaging effects on D-gal in mice via antioxidative mechanisms.

Novel cationic antimicrobial peptide GW-H1 induced caspase-dependent apoptosis of hepatocellular carcinoma cell lines.

Due to its malignancy, the development of effective therapeutic strategies for hepatocellular carcinoma (HCC) is of urgent needs. Natural antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), not only act as direct antimicrobial agents, but also represent important regulators of the innate immune system. It has been reported that cationic AMPs may exhibit cancer-selective toxicity. We have designed a series of novel AMPs with potent antimicrobial activity against a broad spectrum of bacterial pathogens. In the current study, we evaluate the antitumor potency of these AMPs toward HCC cell lines J5, Huh7, and Hep3B. Selected AMPs inhibit the viability of HCC cells in a dose-dependent fashion, while the normal 3T3 cells were significantly less susceptible to these AMPs. GW-H1 treatment (20µM) of J5 cells for 24-72h resulted in the induction of apoptosis, as revealed by flow cytometry (increased sub-G1 populations), and western blot analysis for the appearance of activated caspase-3, -7 and -9 cleavages. Two-dimensional gel electrophoresis was applied to further analyze the AMP-responsive protein profiles of HCC, down-regulation of Hsp27, phophoglycerate kinase 1 and triosephosphate isomerase indicated that GW-H1 may induce apoptosis, and further inhibit progression and metastasis of J5 HCC cells. FITC-labeled GW-H1 was found to attach to cell membrane initially, then translocated into the cytoplasm, and eventually membranous organelles or nucleus. GW-H1 induced a marked growth suppression of J5 xenografts in nude mice in a dose dependent manner. These findings provided support for future application of GW-H1 as potential therapeutic agent for HCC.

Freshwater clam extract inhibits inflammatory responses in LPS-activated macrophages by reducing the activation of mitogen-activated protein kinases and NF-kappaB.

Recent studies demonstrated that freshwater clam (Corbicula fluminea) has lipid-lowering and hepatoprotective activities, but its effect on immune responses has not yet been addressed. Here we showed that ethanol extracts of C. fluminea (ECF) reduced nitrite oxide, interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in lipopolysaccharide-activated macrophages. Further, ECF was fractionated into n-hexane, ethyl acetate, ethanol, and water soluble fractions. Of these, the ethyl acetate soluble fraction (EACF) had the highest capacity to inhibit pro-inflammatory mediators expression. The underlying mechanisms for the anti-inflammatory activity of EACF were demonstrated as down-regulation of ERK1/2, JNK1/2, and p38 phosphorylation and NF-kappaB activity. Using gas chromatography-mass spectrometric analysis EACF was found to be composed mainly of fatty acids and steroids. Our results provide evidence that freshwater clam has anti-inflammatory activity, and support the possibility for the development of freshwater clam as a health supplement or adjuvant therapeutic agent for either preventing or treating inflammation related diseases.

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model.

AIM: to investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H(2)O(2)-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12). METHODS: viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo. RESULTS: pretreatment with isochaihulactone (5-10 micromol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H(2)O(2)-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mgxkg(-1)xd(-1), sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control. CONCLUSION: the results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.

Interaction of cationic antimicrobial peptides with phospholipid vesicles and their antibacterial activity.

We have designed and synthesized a series of cationic α-helical AMPs with improved antibacterial activity and selectivity against a broad spectrum of G(+) and G(-) bacteria. In the current study, we intended to gain further insight into the mechanisms of action between AMPs and cellular membranes using model liposomes of various phospholipid compositions. Circular dichroism measurements showed that AMPs adopted amphipathic α-helical conformation in the presence of negatively charged vesicles (DOPC/DOPG=1:3), while they were largely unstructured when incubated with neutral vesicles (DOPC). The interaction of AMPs with phospholipid vesicles were further analyzed by calcein leakage experiments. AMPs exhibited weak dye-leakage activity for DOPC (neutral) vesicles, while they effectively induced calcein leakage when interacted with DOPC/DOPG-entrapped vesicles. These results indicated that our newly designed cationic AMPs did show preferences for bacteria-mimicking anionic membranes. All of them exert their cytolytic activity by folding into an amphipathic helix upon selectively binding and insertion into the target membrane, leading to breakdown of the membrane structure, thus causing leakage of cell contents, resulting finally in cell death. Elucidating the mechanism of the membranolytic activity of AMPs may facilitate the development of more effective antimicrobial agents.