Melatonin ameliorates spatial memory and motor deficits via preserving the integrity of cortical and hippocampal dendritic spine morphology in mice with neurotrauma.

We aimed to elucidate the role of cortical and hippocampal dendritic spines on neurological deficits associated with hippocampal microgliosis, hippocampal neurogenesis, and neuroinflammation in mice with cortical compact impact (CCI) injury. In the present study, we found that CCI reduced spatial memory mean latency (10 s. vs 50 s) and motor dysfunction (130 s. vs 150 s.) in mice, as determined by Morris water maze and rotarod test, respectively. Golgi staining of cortical pyramidal neurons revealed that, compared to the controls, the CCI group treated with vehicle solution had significantly lower values of dendritic order (or dendritic branch number) (4.0 vs 6.2), total spine length (400 µm vs 620 µm) and spine density (40 spines/µm vs 60 spines/µm), but had significantly higher values of dendritic beading (40 beadings/mm vs 20 beadings/mm). Additionally, Sholl analysis showed that, compared to controls, the CCI + NS group mice had significantly lower values of dendritic intersections (1.0 vs 2.0). Immunofluorescence assay also revealed that, compared to controls, the CCI + NS group mice had significantly higher values of the newly formed hippocampal cells (1250/mm2 vs 1000/mm2) but significantly lower values of dendritic order (2.0 branch # vs 4.2 branch #), total spine length (180 µm vs 320 µm) and intersection (1.0 vs 3.0). The CCI + NS group mice further showed significantly higher numbers of microglia in the dentate gyrus of the hippocampus and higher concentrations of pro-inflammatory cytokines in the cerebrospinal fluids. All the CCI-induced spatial memory (40 s) and motor (150 s) dysfunction, deranged dendritic and spine morphology of cortical pyramidal neurons or hippocampal newly formed cells, hippocampal microgliosis, and central neuroinflammation were all significantly reduced by melatonin administration during post-CCI. Simultaneously, melatonin therapy caused an enhancement in the compensatory hippocampal neurogenesis and neurotrophic growth factors (e.g., doublecortin-1) and compensatory central anti-inflammatory cytokines. Our results indicate that melatonin attenuates the spatial memory and motor deficits via the modification of cortical and hippocampal dendritic spine morphology, hippocampal microgliosis and neurogenesis, and neuroinflammation in mice with traumatic brain injury.

The less weight loss due to modest food restriction drove more fat accumulation in striped hamsters refed with high-fat diet.

Food restriction (FR) has been commonly used to decrease body fat, reducing the risk of overweight in humans and animals. However, the lost weight has been shown to be followed by overweight when food restriction ends. It remains uncertain whether the weight loss drives the overweight, or not. In the present study, striped hamsters were restricted by 15%, 30% and 40% of ad libitum food intake for 2 weeks, followed by high-fat refeeding for 6 weeks (FR15%-Re, FR30%-Re and FR40%-Re). The hamsters in FR15%, FR30% and FR40% groups decreased by 21.1%, 37.8% and 50.0% in fat mass (P < 0.01), and 16.8%, 42.8% and 53.4% in leptin levels (P < 0.01) compared with the hamsters fed ad libitum. The FR15%-Re, FR30%-Re and FR40%-Re groups showed 77.0%, 37.2% and 23.7% more body fat than ad libitum group (P < 0.01). The FR15%-Re group showed considerable decreases in gene expression of arcuate nucleus co-expressing proopiomelanocortin (POMC), cocaine - and amphetamineregulated transcript (CART) and the long isoform of leptin receptor (LepRb) in the hypothalamus and of several genes associated with fatty acid transport to mitochondria and ß-oxidation in brown adipose tissue and liver. It suggests that less weight loss is likely to drive more fat accumulation when food restriction ends, in which the impaired function of LepRb, POMC and CART in the brain and fatty acid oxidation in brown adipose tissue and liver may be involved.

Anti-tumor and anti-angiogenic effects of Fucoidan on prostate cancer: possible JAK-STAT3 pathway.

BACKGROUND: Prostate cancer is the most common cancer in men in the United States. Fucoidan is a bioactive polysaccharide extracted mainly from algae. The aim of this study was to investigate anti-tumor and anti-angiogenic effects of fucoidan in both cell-based assays and mouse xenograft model, as well as to clarify possible role of JAK-STAT3 pathway in the protection. METHODS: DU-145 human prostate cancer cells were treated with 100-1000 µg/mL of fucoidan. Cell viability, proliferation, migration and tube formation were studied using MTT, EdU, Transwell and Matrigel assays, respectively. Athymic nude mice were subcutaneously injected with DU-145 cells to induce xenograft model, and treated by oral gavage with 20 mg/kg of fucoidan for 28 days. Tumor volume and weight were recorded. Vascular density in tumor tissue was determined by hemoglobin assay and endothelium biomarker analysis. Protein expression and phosphorylation of JAK and STAT3 were determined by Western blot. Activation of gene promoters was investigated by chromatin Immunoprecipitation. RESULTS: Fucoidan could dose-dependently inhibit cell viability and proliferation of DU-145 cells. Besides, fucoidan also inhibited cell migration in Transwell and tube formation in Matrigel. In animal study, 28-day treatment of fucoidan significantly hindered the tumor growth and inhibited angiogenesis, with decreased hemoglobin content and reduced mRNA expression of CD31 and CD105 in tumor tissue. Furthermore, phosphorylated JAK and STAT3 in tumor tissue were both reduced after fucoidan treatment, and promoter activation of STAT3-regulated genes, such as VEGF, Bcl-xL and Cyclin D1, was also significantly reduced after treatment. CONCLUSIONS: All these findings provided novel complementary and alternative strategies to treat prostate cancer.

[Danlou Tablet Fought against Inflammatory Reaction in Atherosclerosis Rats with Intermingled Phlegm and Blood Stasis Syndrome and Its Mechanism Study].

OBJECTIVE: To observe the effects of Danlou Tablet (DT) on inflammatory reaction, and expressions of lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2), and to analyze potential mechanisms. METHODS: Forty male Wistar rats were randomly and equally divided into five groups, i.e., the normal control group, the model group, the Western medicine (WM) group, the low dose DT group, the high dose DT group, 8 in each group. Rats in the normal control group were fed with basic forage for 12 successive weeks, while AS rat model was established in rats of the other four groups by feeding high fat and sugar forage plus intraperitoneal injection of vitamin D3. Normal saline, atorvastatin calcium suspension (at the daily dose of 1.8 mg/kg), low dose DT suspension (at the daily dose of 450 mg/kg), and high dose DT suspension (at the daily dose of 900 mg/kg) were administered to rats in the model group, the WM group, the low dose DT group, the high dose DT group respectively by gastragavage for 8 successive weeks. The general condition of all rats was observed. Rats were sacrificed after gastric administration and their serum collected. Serum levels of lipids (TC, TG, HDL-C, LDL-C) and inflammatory factors [IL-6, TNF-α, monocyte chemoattractant protein 1 (MCP-1), oxidized low-density lipoprotein (ox-LDL), lipoprotein-associated phospholipase A2 (LP-PLA2), secretory phospholipase A2 (sPLA2)] were detected. Pathological changes of thoracic aorta were observed by HE staining. Protein and gene expressions of LP-PLA2 and sPLA2 in thoracic aorta were measured by Western blot and real-time fluorescent quantitative PCR respectively. RESULTS: Compared with the normal control group, rats in the model group were in low spirits and responded poorly. Typical atherosclerotic plaque could be seen in thoracic aorta of rats in the model group. Serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, LP-PLA2, and sPLA2 significantly increased (P < 0.05); protein and gene expressions of LP-PLA2 and sPLA2 in rat thoracic aorta increased (P < 0.05) in the model group. After 8 weeks of intervention, rats in 3 medication groups appeared active, and HE staining showed subsidence of plaque in rat thoracic aorta. Compared with the model group, serum levels of TC, TG, LDL-C, IL-6, TNF-α, MCP-1, ox-LDL, and LP-PLA2 decreased in 3 medication groups (P < 0.01, P < 0.05); serum sPLA2 level decreased, protein and mRNA expressions of LP-PLA2 and sPLA2 in rat thoracic aorta decreased in the WM group (P < 0.01, P < 0.05); protein and mRNA expressions of LP-PLA2 in rat thoracic aorta significantly decreased in the low dose DT group (P < 0.01, P < 0.05), and those of LP-PLA2 and sPLA2 decreased in the high dose DT group (P < 0.01, P < 0.05). CONCLUSION: DT could fight against inflammatory reaction and AS possibly through inhibiting LP-PLA2 expression and reducing ox-LDL production.

[Effects of Xuefu Zhuyu Granule and Danlou Tablet on Anti-atherosclerosis Rats and Potential Mechanisms].

OBJECTIVE: To observe anti-atherosclerotic effect of Xuefu Zhuyu Granule (XZU) and Danlou Tablet (DT) on blood lipids, platelet derived growth factor (PDGF), vascular smooth muscle cells (VSMCs) proliferation, extracellular signal-regulated kinase (ERK) signal pathway in atherosclerosis (AS) model rats, and to explore their potential mechanisms. METHODS: Forty male Wistar rats were randomly divided into five groups, i.e., the normal control group, the model group, the Atorvastatin group, the DT group, the XZG group, 8 in each group. Rats in the normal control group were fed with basic forage for 12 weeks, while rats in the other four groups were fed with high fat forage plus intraperitoneal injection of vitamin D3 to build AS model. Then rats in the model control group, the Atorvastatin group, the DT group, the XZG group were administered with normal saline, Atorvastatin suspension (0.18 mg/mL), DT suspension (45 mg/mL), and XZG (1 g/mL) by gastrogavage for 8 successive weeks, respectively. After intervention serum levels of TC, TG, LDL-C, HDL-C, and PDGF were detected by ELISA. Pathological changes in thoracic aorta were observed by HE staining. Protein expression levels of ERK1/2 and pERK1/2 in thoracic aorta were measured by Western blot. RESULTS: Compared with the normal group, serum TC, TG, LDL-C, PDGF levels, and expression levels of ERK1/2 and pERK1/2 significantly increased (P <0. 05) in the model control group. HE staining showed irregular intimal thickness, accumulated endothelial foam cells, lipids deposited, disarranged media VSMCs, forming typical AS plaque. Compared with the model group, TC and PDGF levels decreased in all medicated groups (P < 0.05, P < 0.01). Serum levels of TG and LDL-C significantly decreased in the Atorvastatin group and the DT group (P < 0.01, P < 0.05). Expressions of ERK1/2 and pERK1/2 significantly decreased in the Atorvastatin group, the DT group, and the XZG group (P < 0.01). HE staining also showed typical AS plaque in three medicated groups, but with reduced pathological degree of endometrial hyperplasia and plaque area. CONCLUSIONS: XZG and DT could reduce the plaque area and attenuate pathological degree of AS in model rats, thereby postponing the progress of AS. Its mechanism might be achieved through reducing serum lipids and release of PDGF, inhibiting ERK signal pathway activation and VSMC proliferation.

[Meta-analysis on effect of Shenfu injection in treating angina pectoris].

To systematically evaluate the efficiency and safety of Shenfu injection in treating patients with angina pectoris. Retrievals were made in Embase, Pubmed, Cochrane Library, Clinical Trials.gov, CNKI, CBM, VIP and Wanfang (before September 2015) for randomized or semi-randomized controlled trials reporting data of Shenfu injection in the adjuvant treatment of angina pectoris. The quality of included trials was evaluated according to tool evaluation at cochrane.org. STATA version 12.0 was applied for Meta analysis after quality assessment of included studies. Finally, a total of 17 studies, including 16 randomized controlled trials (RCTs) and 1 controlled clinical trial (CCT) involving 1 309 patients, met the inclusion criteria, of which 659 patients received Shenfu injection treatment. Meta-analysis results showed that Shenfu injection treatment group significantly improved angina pectoris symptoms (OR=3.38, 95%CI： 2.47-4.64, P=0.000) and ischemic ST-T changes in electrocardiogram (OR=3.30, 95%CI： 2.22-4.90, P=0.000), compared with control group. In the Meta-regression analysis, the average age of patients was positively correlated with the improved clinical (ß=0.17) and electrocardiogram (ß=1.15) efficacies. Major complication rate of Shenfu injection was 3.4%, and no serious adverse events were reported. Current clinical evidence in this study proved that Shenfu injection could significantly improve clinical symptoms and ECG ischemic changes for angina pectoris patients, with a good safety.

Morphological Observations and Fatty Acid Composition of Indoor-Cultivated Cordyceps sinensis at a High-Altitude Laboratory on Sejila Mountain, Tibet.

Cordyceps sinensis, a caterpillar entomopathogenic fungus-host larva complex, is a rare medicinal herb found in the Qinghai-Tibetan Plateau and its surrounding high-altitude areas. The alternation of generations in the life cycle, whatever the fungus or its host insect, requires special growth conditions. However, it is difficult to simulate the growth conditions of C. sinensis, which hinders its artificial cultivation. In this work, the life cycle from the host larva to C. sinensis was observed in an indoor-cultivation laboratory at 4,200 m a.s.l. on Sejila Mountain, Tibet. Comparative examinations between indoor-cultivated and wild C. sinensis demonstrated that the indoor-cultivated C. sinensis preferred to germinate multiple long, slim stromata at diverse positions on dead larvae, including but not limited to their heads. Their fatty acid composition shows a significant difference in the levels of polyunsaturated fatty acids (PUFAs). In indoor-cultivated C. sinensis, PUFAs constituted 24.59% and 49.43%, respectively, of neutral and polar lipids; meanwhile, in wild C. sinensis, PUFAs represented 34.34% and 61.25% of neutral and polar lipids, respectively. These observations and fatty acid data suggest that environmental factors, particularly temperature, soil pressure and light intensity, strongly affect the growth of C. sinensis. Our new findings may provide important information for improving techniques for the large-scale artificial cultivation of C. sinensis.

Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp. HZ01 from the crude oil-contaminated seawater at the Daya Bay, southern China.

Microorganisms play an important role in the biodegradation of petroleum contaminants, which have attracted great concern due to their persistent toxicity and difficult biodegradation. In this paper, a novel hydrocarbon-degrading bacterium HZ01 was isolated from the crude oil-contaminated seawater at the Daya Bay, South China Sea, and identified as Achromobacter sp. Under the conditions of pH 7.0, NaCl 3% (w/v), temperature 28 °C and rotary speed 150 rpm, its degradability of the total n-alkanes reached up to 96.6% after 10 days of incubation for the evaporated diesel oil. Furthermore, Achromobacter sp. HZ01 could effectively utilize polycyclic aromatic hydrocarbons (PAHs) as its sole carbon source, and could remove anthracene, phenanthrene and pyrence about 29.8%, 50.6% and 38.4% respectively after 30 days of incubation. Therefore, Achromobacter sp. HZ01 may employed as an excellent degrader to develop one cost-effective and eco-friendly method for the bioremediation of marine environments polluted by crude oil.

Inhibitory effect of cryptotanshinone on angiogenesis and Wnt/ß-catenin signaling pathway in human umbilical vein endothelial cells.

OBJECTIVE: To investigate the anti-angiogenic effect of cryptotanshinone (CPT) on human umbilical vein endothelial cells (HUVECs) and the effect of CPT on Wnt/ß-catenin signaling pathway. METHODS: HUVECs were incubated with 0, 2.5, 5, 10, and 20 µ mol/L CPT for detecting cell viability with dimethyl thiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Then, HUVECs were incubated with 0, 2.5, 5, and 10 µ mol/L CPT for detecting endothelial cell migration, invasion, and tubular-like structure formation with wound healing, transwell invasion and matrigel tube formation assays, respectively. To gain insight into CPT-mediated signaling, the effects of CPT on T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factors were detected by the Dual-luciferase reporter assay. Next, the nuclear expression of ß-catenin was evaluated using Western blot and immunochemistry. Finally, vascular endothelial growth factor (VEGF) and cyclin D1, downstream proteins of the Wnt pathway were examined with Western blot. RESULTS: CPT dose-dependently suppressed endothelial cell viability, migration, invasion, and tubular-like structure formation. In particular, CPT blocked ß-catenindependent transcription in HUVECs in a dose-dependent manner. In Western bolt, 10 µ mol/L CPT decreased expression of ß-catenin in nucleus of HUVECs (P<0.01). In immunohistochemistry, ß-catenin was more potent in response to LiCl (an activator of the pathway) treatment. However, the signals were weaker in the nucleus of the CPT (10 µ mol/L) group, compared to the positive control. Also, VEGF and cyclin D1 were both eliminated by CPT in 5 and 10 µ mol/L doses (P<0.05). CONCLUSION: Our study supported the role of CPT as an angiogenic inhibitor, which may impact on the Wnt/ß-catenin signaling pathway.

Two new compounds from Comastoma pedunlulatum.

Two new compounds, a xanthonoid and a flavonoid C-glycoside, were isolated from the ethyl acetate extract of the dried herb of Comastoma pedunlulatum. The structures of the new compounds were elucidated, respectively, as 1,8-dihydroxy-3,5-dimethoxyxanthone 1-O-[2-(4'-hydroxy-3',5'-dimethoxy-E-cinnamoyl)]-ß-D-xylopyranosyl-(1-6)-ß-D-glucopyranoside (1) and 6″-O-acetylisoorientin (2) on the basis of their spectroscopic and physicochemical properties.

Inhibition of cPLA2 activation by Ginkgo biloba extract protects spinal cord neurons from glutamate excitotoxicity and oxidative stress-induced cell death.

Ginkgo biloba extract (EGb761) has been shown to be neuroprotective; however, the mechanism by which EGb761 mediates neuroprotection remains unclear. We hypothesized that the neuroprotective effect of EGb761 is mediated by inhibition of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that is known to play a key role in mediating secondary pathogenesis after acute spinal cord injury (SCI). To determine whether EGb761 neuroprotection involves the cPLA(2) pathway, we first investigated the effect of glutamate and hydrogen peroxide on cPLA(2) activation. Results showed that both insults induced an increase in the expression of phosphorylated cPLA(2) (p-cPLA(2)), a marker of cPLA(2) activation, and neuronal death in vitro. Such effects were significantly reversed by EGb761 administration. Additionally, EGb761 significantly decreased prostaglandin E(2) (PGE(2)) release, a downstream metabolite of cPLA(2). Moreover, inhibition of cPLA(2) activity with arachidonyl trifluromethyl ketone improved neuroprotection against glutamate and hydrogen peroxide-induced neuronal death, and reversed Bcl-2/Bax ratio; notably, EGb761 produced greater effects than arachidonyl trifluromethyl ketone. Finally, we showed that the extracellular signal-regulated kinase 1/2 signaling pathway is involved in EGb761's modulation of cPLA(2) phosphorylation. These results collectively suggest that the protective effect of EGb761 is mediated, at least in part, through inhibition of cPLA(2) activation, and that the extracellular signal-regulated kinase 1/2 signaling pathway may play an important role in mediating the EGb761's effect.

Effects of extracellular matrix molecules on the growth properties of oligodendrocyte progenitor cells in vitro.

The extracellular matrix (ECM) is a component of neural cell niches and regulates multiple functions of diverse cell types. To date, limited information is available concerning its biological effects on the growth properties of oligodendrocyte progenitor cells (OPCs). In the present study, we examined effects of several ECM components, i.e., fibronectin, laminin, and Matrigel, on the survival, proliferation, migration, process extension, and purity of OPCs isolated from embryonic day 15 rat spinal cords. All three ECM components enhanced these biological properties of the OPCs compared with a non-ECM substrate, poly-D-lysine. However, the extents of their effects were somewhat different. Among these ECMs, fibronectin showed the strongest effect on almost all aspects of the growth properties of OPCs, implying that this molecule is a better substrate for the growth of OPCs in vitro. Because of its survival- and growth-promoting effects on OPCs, fibronectin may be considered as a candidate substrate for enhancing OPC-mediated repair under conditions when exogenous delivery or endogenous stimulation of OPCs is applied.

EGb761 protects hydrogen peroxide-induced death of spinal cord neurons through inhibition of intracellular ROS production and modulation of apoptotic regulating genes.

The present study was conducted to investigate whether Ginkgo biloba extract (EGb) 761 could protect spinal cord neurons from H(2)O(2)-induced toxicity. In primary spinal cord neurons isolated from embryonic day 14 rats, H(2)O(2) administration resulted in a significant decrease in the survival of spinal cord neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst 33342 nuclear staining showed that these cells die by apoptosis. Such neuronal death, however, was significantly reversed by EGb761 in a dose-dependent manner. Moreover, a marked increase in intracellular free radical generation was found after the H(2)O(2) administration which could be reversed almost completely by EGb761, indicating that inhibition of free radical generation is an important mechanism of the anti-apoptosis action of EGb761. Finally, treatment of cells with H(2)O(2) for 12 h reduced the expression of Bcl-2, an anti-apoptotic gene, by 70% but showed no effect on the level of Bax, a pro-apoptotic gene. EGb76 treatment, however, significantly reversed H(2)O(2)-induced reduction of Bcl-2 expression and inhibited Bax expression by 2.3-fold. Thus, our study provided evidence showing that the protective effect of EGb761 on spinal cord neuronal apoptosis after oxidative stress is mediated, at least in part, by its anti-oxidative action and regulation of apoptosis-related genes Bcl-2 and Bax.