[Significance of high expression of C5orf46 in gastric cancer and potential intervention of tarditional Chinese medicine based on bioinformatics, molecular docking, and cell experiments].

This study aims to investigate the expression, prognosis, and clinical significance of C5orf46 in gastric cancer and to study the interaction between the active components of C5orf46 and tarditional Chinese medicine. The ggplot2 package was utilized for differential expression analysis of C5orf46 in gastric cancer tissues and normal tissues. The survival package was used for survival analysis, univariate regression analysis, and multivariate regression analysis. Nomogram analysis was used to assess the connection between C5orf46 expression in gastric cancer and overall survival. The abundance of tumor-infiltrating lymphocytes was calculated by GSVA package. Coremine database, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and PubChem database were used to search the potential components corresponding to C5orf46 gene and tarditional Chinese medicine. Molecular docking was performed to explore the binding affinity of potential components to C5orf46. Cell experiments were performed to explore the expression of C5orf46 gene in cells of the blank group, model group, and drug administration groups. As compared with normal tissues, C5orf46 expression was higher in gastric cancer tissues, which had more significant predictive effects in the early stages(T2, N0, and M0). The more advanced the tumor node metastasis(TNM) stage, the higher the C5orf46 expression and the lower the probability of survival of patients with gastric cancer. The expression of C5orf46 positively correlated with the helper T cells1 in gastric cancer and the macrophage infiltration level in gastric cancer, and negatively correlated with B cells, central memory T cells, helper T cells 17, and follicular helper T cells. Seven potential components of C5orf46 were obtained, and three active components were obtained after the screening, which matched five tarditional Chinese medicines, namely, Sojae Semen Nigrum, Jujubae Fructus, Trichosanthis Fructus, Silybi Fructus, and Bambusae Concretio Silicea. Molecular docking revealed that sialic acid and adeno-sine monophosphate(AMP) had a good binding ability to C5orf46. The results of real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot showed that, as compared with the model group, the mRNA and protein expression levels of C5orf46 were significantly lower in the drug administration groups. The lowest expression level was found at the concentration of 40 µmol·L~(-1). The results of this study provide ideas for the clinical development of traditional Chinese medicine compounds for the treatment of gastric cancer as well as other cancers.

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons.

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion. Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion, but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear. Therefore, in the current study, we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons. Hippocampal neurons were treated with Mg2+-free extracellular solution containing glutamate (300 µM) for 3 hours as a model of glutamate-mediated excitotoxicity (glutamate group). In the normal group, hippocampal neurons were incubated in Mg2+-free extracellular solution. In the Achyranthes bidentata polypeptide group, hippocampal neurons were incubated in Mg2+-free extracellular solution containing glutamate (300 µM) and Achyranthes bidentata polypeptide at different concentrations. At 24 hours after exposure to the agents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear morphology, respectively. Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay, respectively. At various time points after glutamate treatment, reactive oxygen species in cells were detected by H2DCF-DA, and mitochondrial membrane potential was detected by rhodamine 123 staining. To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors, electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons. Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate. In addition, Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current. Furthermore, the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment. These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway. All animal studies were approved by the Animal Care and Use Committee, Nantong University, China (approval No. 20120216-001) on February 16, 2012.

A herbal formula, SYKT, reverses doxorubicin­induced myelosuppression and cardiotoxicity by inhibiting ROS­mediated apoptosis.

Doxorubicin (DOX) is an antineoplastic drug widely used for the treatment of various types of cancer; however, it can induce severe side effects, such as myelosuppression and cardiotoxicity. Sanyang Xuedai (SYKT) is a natural medicine originating from an ancient prescription of the Dai nationality in Southwest China. With eight Chinese herbal medicines, including sanguis draconis, radix et rhizoma notoginseng, radix et rhizoma glycyrrhizae and radix angelicae sinensis as the primary ingredients, SYKT has been reported to possess numerous biological functions. The present study investigated whether SYKT can confer protection against DOX­induced myelosuppression and cardiotoxicity, and explored the potential mechanism involved. Mice were treated with DOX, SYKT or a combination of the two; hematopoietic functions were assessed by measuring the number of peripheral blood cells, cluster of differentiation CD34+/CD44+ bone marrow cells and apoptotic cells. Myocardial enzymes, including aspartate aminotransferase, lactate dehydrogenase, creatine kinase (CK) and its isoform CK­MB, were assessed using a biochemical analyzer. The apoptotic rate of cardiomyocytes was assessed using flow cytometry. Histopathological analysis was conducted using hematoxylin­eosin staining. Intracellular reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein intensity assay. The mice treated with DOX exhibited a reduced survival rate, reduced peripheral blood and CD34+/CD44+ cell counts, elevated myocardial enzymes and apoptotic indices in bone marrow cells and cardiomyocytes, all of which were effectively prevented by SYKT co­administration. Furthermore, bone marrow cells and myocytes from mice treated with DOX demonstrated increased dichlorofluorescein intensity, which was attenuated by SYKT. Notably, SYKT did not interfere with the effects of DOX on tumor volume or the induction of tumor cell apoptosis in tumor­bearing mice. The present study indicated that SYKT may counteract DOX­induced myelosuppression and cardiotoxicity through inhibiting ROS­mediated apoptosis. These findings suggested that SYKT may have potential as a means to counteract the potentially fatal hematopoietic and cardiac complications associated with DOX treatment.

Effect of total alkaloids from Alstonia scholaris on airway inflammation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Alstonia scholaris (Apocynaceae) have been traditionally used for treatment of respiratory diseases in "dai" ethnopharmacy for hundreds years, especially for cough, asthma, phlegm, chronic obstructive pulmonary disease and so on. The formulas including the leaf extract have also been prescribed in hospitals and sold over the retail pharmacies. AIM OF THE STUDY: A. scholaris is used as a traditional herbal medicine for the treatment of respiratory tract inflammation. However, there is no scientific evidence to validate the use of total alkaloids of A. scholaris in the literature. Here, we investigated the protective activity of total alkaloids (TA), extracted from the leaves of Alstonia scholaris, against lipopolysaccharide (LPS)-induced airway inflammation (AI) in rats. MATERIALS AND METHODS: 200 µg/µL LPS was instilled intratracheally in each rat, and then the modeling animals were divided into six groups (n=10, each) randomly: sham group, LPS group, Dexamethasone [1.5mg/kg, intra-gastricly (i.g.)] group, and three different doses (7.5, 15, and 30 mg/kg, i.g.) of total alkaloids-treated groups. Corresponding drugs or vehicles were orally administered once per day for 7 days consecutively. The concentration of albumin (ALB), alkaline phosphatase (AKP), lactate dehydrogenase (LDH), and the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) were determined by fully automatic biochemical analyzer and blood counting instrument. Nitric oxide (NO) level, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activities were examined by multiskan spectrum, and histological change in the lungs was analyzed by H.E. staining. The levels of inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) were measured using ELISA. RESULTS: Total alkaloids decreased the percentage of neutrophil, number of WBC, levels of ALB, AKP and LDH in the BALF, while increased the content of ALB in serum. It also improved SOD activity and increased NO level in the lungs, serum and BALF, and reduced the concentration of MDA in the lungs. Total alkaloids also inhibited the production of inflammatory cytokines TNF-α and IL-8 in the BALF and lung. Finally, histopathological examination indicated that total alkaloids attenuated tissue injury of the lungs in LPS-induced AI. CONCLUSIONS: Total alkaloids have an inhibitory effect against LPS-induced airway inflammation in rats.

Characterization of chemical constituents and rats metabolites of an alkaloidal extract of Alstonia scholaris leaves by liquid chromatography coupled with mass spectrometry.

Alstonia scholaris has been used in "Dai" ethnic medicine to treat chronic respiratory diseases for a long history, and the major bioactive constituents are alkaloids. An alkaloidal extract of A. scholaris leaves (AAS) has been developed into an investigational new drug, and has been approved for phase I/II clinical trials by China Food and Drug Administration. However, little is known on the chemical composition and in vivo metabolism of AAS, thus far. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS) method was established to characterize the chemical constituents of AAS. Samples were separated on an ACQUITY UPLC CSH column (2.1×100mm, 1.7µm) with acetonitrile and water containing 0.3% formic acid as the mobile phase. On the basis of high-accuracy mass spectral analysis, a total of 35 alkaloids were characterized from AAS, including 11 scholaricine-type, 9 vallesamine-type, 12 picrinine-type, and 3 tubotaiwine-type alkaloids. Furthermore, the metabolic pathways of 4 representative alkaloids in rats were studied. They mainly undertook hydroxylation and glucuronidation reactions. Based on the above metabolic pathways, the metabolism of AAS (10mg/kg) in rats after oral administration was studied by LC/MS. A total of 33 compounds in plasma, 40 compounds in urine, and 38 compounds in feces were characterized. The results indicated that scholaricine-type alkaloids could get into circulation more readily than the other types. This is the first systematic study on chemical profiling and metabolites identification of AAS.

Relationship between breast cancer and levels of serum thyroid hormones and antibodies: a meta-analysis.

The breast and the thyroid are hormone responsive organs that are closely related with changes of endocrine function and glandular disease. An association between thyroid disorders and breast cancer (BC) risk has been suggested, although the results are inconclusive. The purpose of the present study was to summarize evidence supporting a relationship between BC and the level of thyroid hormones and antibodies. The MEDLINE and EMBASE electronic databases were searched for studies published between 2000 and 2014. The pooled effects were presented as weighted mean differences (WMD) with 95% confidence intervals (CI) using fixed or random effect models. We summarized the results of 8 cross-sectional studies with 4, 189 participants. The overall pooled results showed that the levels of FT3 and FT4 were significantly increased in patients with BC (WMD=1.592 pmol/l; 95% CI: 0.15-3.033 and WMD=0.461 ng/dl; 95% CI: 0.015-0.906; p=0.043). The TPOAb level in patients with BC was higher than that in the control group (WMD=81.4 IU/ml; 95% CI: 78.7-84.0; p=0.000). The overall pooled results of the TgAb with random effects analyses showed that the TgAb level was significantly increased in patients with BC (WMD=101.3 IU/ml; 95% CI: 48.7-153.9; p=0.000). The present results indicated that the serum levels of FT3, TPOAb and TgAb are significantly higher in patients with breast cancer than in healthy controls.

[Effective dose and time window of ginseng total saponins treatment in rat after traumatic brain injury].

OBJECTIVE: To investigate the neuroprotective effect, effective dose and time window of ginseng total saponins (GTS) treatment in rat after traumatic brain injury (TBI). METHODS: The modified Feeney's method was used to establish TBI model in rat. GTS was treated intraperitoneally. The neurological function and histological morphology of brain tissue were observed. RESULTS: Different doses of GTS were used 6 h after TBI. The neurological and histological results showed that: compared with the TBI group, significant efficacy was observed 2 - 14 days after injury with GTS treatment at 10, 20, 40, 60 and 80 mg/kg (P < 0.05); The effects of GTS at 20, 40, and 60 mg/kg were better than those of GTS at 10 and 80 mg/kg. During the research on the time window of GTS intervention, GTS (20 mg/kg) showed significant effect when used at 3 h and 6 h after TBI; however 12 h, 24 h after TBI, application of GTS did not exert any significant effect. CONCLUSION: GTS intervention after TBI could reduce brain damage and promote recovery of the neurological function. Among doses of GTS 5 - 80 mg/kg, 20 - 60 mg/kg is the best dose limit. The effective time window of GTS is 6 h after TBI.

[Therapeutic efficacy of hyperbaric oxygen on traumatic brain injury in the rat and the underlying mechanisms].

OBJECTIVE: To investigate the effects of hyperbaric oxygen (HBO) treatment on the activation of astrocytes and the expression of glia-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in the brain after traumatic brain injury (TBI). METHODS: 54 male SD rats were randomly divided into three groups (n = 18): sham-operated, TBI and HBO treatment groups. TBI was induced with Feeney's method, bone window was opened without strike on the brain tissue in the sham-operated group. HBO group rats received HBO treatment for 60 min in the hyperbaric chamber containing O2 100% at 3 ATA. When neurological functions were measured 48 h after TBI, rats were decapitated, the brain water content of 18 rats was measured, 18 brains were sliced for the morphological observation after Nissl staining and for the immunohistochemistry staining of astrocyte markers glial fibrillary acidic protein (GFAP), vimentin and S100, and the other 18 brains of injured side were used for Western blot analysis of GDNF and NGF. RESULTS: HBO treatment reduced the neurological deficit, brain water content and hippocampal neuronal loss. In the observed cortex and hippocampal area astrocytes were activated, the cell number of positive expression of astrocyte markers GFAP, vimentin and S100 was increased, and the expression of GDNF and NGF was elevated after TBI. However, these indices were all enhanced further after the HBO treatment. CONCLUSION: It is suggested that HBO may be an effective therapy for TBI and upregulation of the expression of GDNF and NGF may underly the effect of HBO.

Inhibition of NMDA receptors underlies the neuroprotective effect of ginsenoside Rb3.

In order to investigate the mechanisms underlying the neuroprotective effect of ginsenoside Rb3, rat hippocampal neurons were primarily cultured, and exposed to 1 mM N-methyl-D-aspartate (NMDA), cell viability and lactate dehydrogenase leakage were measured. Ca2+ influx was determined by calcium imaging with a laser confocal microscopy. The influences of ginsenoside Rb3 on these variables were examined. Patch-clamp technique was used to observe the effects of ginsenoside Rb3 on NMDA-evoked current. The results show that treatment of Rb3 raised the neuronal viability, reduced the leakage of lactate dehydrogenase, and inhibited NMDA-elicited Ca2+ influx in a dose-dependent manner. In the presence of Rb3, NMDA-evoked peak current was inhibited, and Ca2+-induced desensitization of NMDA current was facilitated. It is suggested that ginsenoside Rb3 could exert a neuroprotective role on hippocampal neurons, a role which was partly mediated by the facilitation of Ca2+-dependent deactivation of NMDA receptors, and the resultant reduction of intracellular free Ca2+ level.

[Relationship among sexual differences of susceptibility to motion sickness, AVP levels of plasma and pituitary and V1b receptor expression of pituitary in rats].

AIM: To investigate the relationship among sexual differences of motion sickness (MS), AVP levels of plasma and pituitary and the expression of pituitary V1b receptors for further understanding of the MS mechanisms. METHODS: The conditioned taste aversion (CTA) of 0.15% saccharin sodium solution (SSS) was served as MS model. 98 (49 male and 49 female) rats were used in this experiment, 50 for the detection of the AVP level in plasma and pituitary with radioimmunoassay (RIA), 12 for the observation of the number of V1b receptor-positive neurons in the pituitary with the fluorescence immunohistochemistry method, the rest for the evaluation of the expression of V1b receptor in the pituitary by Western blot. RESULTS: With regard to male rats, decrease of the drinking volume of 0.15% SSS was greater in female rats after rotatory stimulation. The plasma AVP concentration of female rats was significantly higher than that of males under normal conditions, but reduced significantly after rotatory stimulation. However, no significant change was found in male rats. In addition, the pituitary AVP level of the female rats was significantly higher than that of the male rats under normal conditions, but decreased at 8 h and significantly at 24 h after rotation. Similarly, the pituitary AVP level of male rats also decreased significantly at 8 h after rotation, but this decrease was not comparable to that of the females. At 24 h after rotation the pituitary AVP level almost recovered in male rats. In the pituitary, which was related to the stress response, the V1b receptor-positive neurons and the expression level of V1b receptor in female rats were significantly higher than those of the male rats, but they decreased significantly after rotation, while no apparent change was detected in the male rats. CONCLUSION: The changes of plasma and pituitary AVP and V1b receptor level of the pituitary after rotatory stimulation are different between male and female rats and the AVP secretion of the pituitary may be involved in the sexual difference in susceptibility to motion sickness.

Tissue specificity of methylation and expression of human genes coding for neuropeptides and their receptors, and of a human endogenous retrovirus K family.

The purpose of the present study was to understand the tissue specificity of DNA methylation and the relationship between methylation and expression of genes with essential roles in neurodevelopment and brain function. We chose dopamine receptor genes (DRD1 and DRD2), NCAM, and COMT as examples of genes with CpG islands around the promoter region, and serotonin receptor genes (HTR2A and HTR3A), HCRT, and DRD3 as genes without CpG islands. Methylation states were investigated in fetal brain, fetal liver, placenta, and in adult peripheral leukocytes from three individuals by Southern blot and bisulfite-modified DNA sequencing. A repetitive sequence, human endogenous retrovirus (HERV)-K was also examined. All genes examined were almost completely unmethylated in brains. The genes with CpG islands were unmethylated regardless of their expression state. In contrast, genes without CpG islands showed various methylation patterns, which did not necessarily reflect the transcriptional activity of the genes. Most HERV-K loci were methylated, but some loci showed relatively low methylation in the placenta and liver. Interestingly, we found inter-individual differences in methylation levels in HTR2A and HCRT in the placenta and in some loci of HERV-K in the placenta and liver. The sample with the lowest methylation levels in the two unique genes showed higher methylation of HERV-K loci than the other samples. These results provide detailed information about the methylation states of the genes analyzed and evidence for inter-individual variations in methylation in both unique and repetitive sequences.

[Protective effects of ginsenoside RB3 on hypoxic/ischemic brain injury and involved mechanisms].

AIM: To observe protective effects and involved mechanism of ginsenoside Rb3 on hypoxic/ischemic brain injury, using cultured hippocampal neurons, rat hippocampal slices and intact animals. METHODS: (1) Mice were tightly closed in glasses of 150 ml, and then survival time of them was observed. (2) During simulated ischemia and after reoxygenation, changes of orthodromic population spikes (OPS) in the area CA1 of hippocampal slice were investigated. (3) By using histochemistry, the expressions of NOS in CA1 area of rat hippocampus after hypoxic exposure were observed. (4) Using LDH detection, tests of total NOS, iNOS and cNOS activity, the protective effects of ginsenoside Rb3 were investigated on cultured hippocampal neurons treated with hypoxia. RESULTS: (1) Given ginsenoside Rb3 (10 mmol/L), mice survived significantly longer than that of control group. (2) The occurrence of HIP (hypoxic injury potentials) decreased after administration of ginsenosides Rb3 (60 micromol/L) in many slices, while the recovery rate and amplitude of OPS after reoxygenation were significantly higher than those of the control group. (3) In CA1 area of rat hippocampus, NOS-positive neurons increased at the end of 24 h hypoxia and further 24 h reoxygenation, while the number of NOS-positive neurons decreased after treatment with ginsenoside Rb3. (4) The LDH leakage rate of cultured rat hippocampal neurons increased at the end of hypoxia, while it decreased after treatment with Rb3. Moreover the total NOS, especially iNOS activity of these neurons also decreased. CONCLUSION: Ginsenoside Rb3 has a significant protective effect on hypoxic-ischemic injury of neurons, and this involves the stabilization of the cell membrane, the inhibition of the expression and activity of NOS, especially iNOS activity.

[Effects and mechanisms of ginsenoside Rb3 on glutamate excitotoxic injury in cultured neurons of rat hippocampus].

AIM: To observe protective effects of ginsenoside Rb3 on glutamate excitotoxic injury in cultured hippocampal neurons and involved mechanisms. METHODS: On cultured rat hippocampal neurons treated with glutamate at toxic concentration, we made the following investigations: by using MTT assay, LDH leakage detection, tests of total NOS, iNOS and cNOS activity, and the protective effects of ginsenoside Rb3. RESULTS: Ginsenoside Rb3 can enhance the hippocampal neuronal viability, decrease the LDH leakage, elevate the viability of cNOS, and in the same time weaken iNOS's viability. CONCLUSION: Ginsenoside Rb3 has the significant protective effects on glutamate excitotoxic injury. The involved mechanism may include antagonizing the injury of neuron membrane, inhibiting the viability of iNOS, and increasing the activity of cNOS.

[Nutritional status survey in 200 of the Korean and Han nationality elderly in Yanji].

OBJECTIVE: To assess the nutritional status and dietary intake of the Korean and Han nationality elderly in Yanji. METHODS: We selected 200 of the Korean and Han nationality adults aged 60 and older and measured their blood pressure. Dietary survey was performed with 24-hour dietary recall method. RESULTS: (1) The daily average intake of energy surpassed recommended nutrient intake (RNI) value in male and amounted RNI in women. In the male, Breakfast energy intake ratio was significantly lower and supper energy intake ratio was significantly higher than female. Supper energy intake in male with hypertension was significantly higher than normal blood pressure. (2) The daily average intake of fat in the Korean was significantly lower than in Han nationality (P < 0.01), and also was lower than RNI value. The daily average intake of carbohydrate in the Korean was significantly higher than in Han nationality (P < 0.01). (3) The daily average intake of protein exceed RNI value in the Korean male and was slightly lower than RNI value in Han male and both nationality women. The daily average intake of protein in the Korean males was significantly higher than in Han males (P < 0.01). The ratio of good protein was 35%-45% and bean protein exceed 15%. (4) The daily average intakes of calcium and vitamin A were only half RNI value and vitamin B2 lower than RNI values. CONCLUSION: The consumption of some nutrients among the Korean and Han nationality is inequality. The daily intake of calcium, vitamin A and vitamin B2 in elderly is seriously inadequate. Distribution of three meal energy is irrational and the high ratio of supper energy in male relates to hypertension.