[Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway].

Notoginsenoside R1(NGR1),a critical compound in traditional herb Panax notoginseng, is a kind of estrogen receptor agonist.It is reported to exhibit anti-apoptotic,anti-oxidative and anti-inflammatory properties activity, so it is widely used for treatment of various diseases.In order to investigate the potential neuroprotective effect of NGR1 in hypoxic-ischemic brain damage(HIBD), primary cortical neurons were used in this study to establish oxygen-glucose deprivation/reoxygenation(OGD/R) injury models. They were treated with NGR1 and estrogen receptor inhibitor ICI-182780 respectively, then the neuronal survival, cell membrane integrity and apoptosis were assessed by MTT assay,lactate dehydrogenase test(LDH) and Hoechst 33342 stain respectively, while the protein expression levels of ATF6α,p-Akt,Akt,Bax and Cleaved Caspase-3 were measured by Western blotting. Results indicated that as compared with the blank control group,OGD/R could induce cell injury and apoptosis(P<0.05), reduce relative integrity of cell membrane(P<0.05), decrease protein expression of ATF6α,p-Akt(P<0.05), and increase protein expression of Bax and Cleaved Caspase-3(P<0.05) in the primary cortical cells. After NGR1 treatment, the expression levels of ATF6α,p-Akt were obviously increased, and the expression levels of Bax and Cleaved Caspase-3 and the apoptosis of neuron were decreased(P<0.05). However, these neuroprotective properties of NGR1 against ODG/R-induced cell damage could be blocked by ICI-182780. This finding indicated that NGR1 may protect the primary cortical neurons against OGD/R induced injury,and the mechanism may be associated with accelerating the activation of the ATF6/Akt signaling pathway via estrogen receptors.

Transcriptome analysis reveals translational regulation in barley microspore-derived embryogenic callus under salt stress.

KEY MESSAGE: Transcriptome analysis of barley embryogenic callus from isolated microspore culture under salt stress uncovered a role of translation inhibition and selective activation of stress-specific proteins in cellular defense. Soil salinity is one of the major abiotic stresses which constrains the plant growth and reduces the productivity of field crops. In this study, it was observed that the salt stress in barley isolated microspore culture impacted not only on the quantity of embryogenic callus but also on the quality for later differentiation. The barley microspore-derived embryogenic callus, a transient intermediate form linked cells and plants, was employed for a global transcriptome analysis by RNA sequencing to provide new insights into the cellular adaptation or acclimation to stress. A total of 596 differentially expressed genes (DEGs) were identified, in which 123 DEGs were up-regulated and 473 DEGs were down-regulated in the embryogenic callus produced from microspore culture under salt stress as compared to the control conditions. KEGG pathway analysis identified 'translation' (27 DEGs; 12.56 %) as the largest group and followed by 'folding, sorting and degradation' (25 DEGs; 11.63 %) in 215 mapped metabolic pathways. The results of RNA-Seq data and quantitative real-time polymerase chain reaction validation showed that the genes related to translation regulation (such as eIF1A, RPLP0, RPLP2, VARS) were down-regulated to control general protein synthesis, and the genes related to endoplasmic reticulum stress response (such as small heat shock protein genes) were selectively up-regulated against protein denaturing during microspore embryogenesis under continuous salt stress. These transcriptional remodeling might affect the essential protein synthesis for the cell development to fulfill totipotency under salt stress.

[Study on molecular target promoting human neural stem cells of ginsenoside Rg1 by gene chip].

OBJECTIVE: To screen out main molecular target promoting human neural stem cells (NSCs) of ginsenoside Rg1 by using the gene chip technology. METHOD: First, MTT assay was adopted to screen out the optimal concentration of Rg1-promoted NSC proliferation (120 mg x L(-1)). Then, on the 7th day after the Rg1-promoted NSC proliferation, the expression of target genes was observed by the gene chip technology. The most important target gene and signal transduction pathways were screened out through the data calculations. RESULT: On the 7th day after the Rg1-promoted NSC proliferation, obtained 440 differential genes, 266 significantly upregulated genes and 174 significantly down-regulated genes. HES1 gene, CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol 3 kinase)-AKT signal transduction pathways were closely related to the NSC proliferation. CONCLUSION: The differentially expressed genes screened out by gene chip may provide new clues for studies on molecular mechanism of ginsenoside Rg1-promoted NSCs proliferation.

Bisdemethoxycurcumin Increases Sirt1 to Antagonize t-BHP-Induced Premature Senescence in WI38 Fibroblast Cells.

Curcuminoids are well known for their capabilities to combat risk factors that are associated with ageing and cellular senescence. Recent reports have demonstrated that curcuminoids can extend the lifespan of model organisms. However, the underlying mechanisms by which these polyphenic compounds exert these beneficial effects remain unknown. In this study, t-BHP-induced premature senescence model in human fibroblasts was chosen to explore the protective effects of a curcuminoid, bisdemethoxycurcumin (BDMC), on cellular senescence. The results demonstrated that BDMC attenuated oxidative stress-induced senescence-like features which include the induction of an enlarged cellular appearance, higher frequency of senescence-associated ß -galactosidase staining activity, appearance of senescence-associated heterochromatic foci in nuclei, decrease in proliferation capability, and alteration in related molecules such as p16 and retinoblastoma protein. Notably, we found that BDMC treatment activated Sirt1/AMPK signaling pathway. Moreover, downregulating Sirt1 by the pharmacological inhibitor nicotianamine or small interfering RNA blocked BDMC-mediated protection against t-BHP-mediated decrease in proliferation. These results suggested that BDMC prevented t-BHP-induced cellular senescence, and BDMC-induced Sirt1 may be a mechanism mediating its beneficial effects.

A new ganoderic acid from Ganoderma lucidum mycelia and its stability.

A new ganoderic acid (GA), 3α,22ß-diacetoxy-7α-hydroxyl-5α-lanost-8,24E-dien-26-oic acid (1), together with four known compounds GA-Mk (2), -Mc (3), -S (4) and -Mf (5), was isolated and characterized from Ganoderma lucidum mycelia. The structure of compound 1 was elucidated on the basis of interpretation of extensive spectroscopic data (HRMS, IR, UV, 1D and 2D NMR). Due to its apparent degradation during preparation procedures, the stability of compound 1 was assessed in several solvents in a short-term study that demonstrated the optimal stability in aproptic environment. A possible mechanism of acid-catalyzed degradation of compound 1 in methanol was proposed, consisting of a fast protonation, followed by a committed step of hydroxyl group removal. In addition, all isolated compounds were tested in vitro for their cytotoxic activities against 95D and HeLa tumor cell lines, with IC(50) values ranging from14.7 to 38.5µM. The results may improve the understanding of chemical stability of GAs and provide valuable information on their separation, analysis and application.

[Effect of ginsenoside Rg1 on functional expression of human neural stem cells: a patch clamp study].

OBJECTIVE: To observe the effects of ginsenoside Rg1 on the functional expression of human neural stem cells (hNSCs). METHOD: The membrane electrophysiological properties and sodium and potassium ion channels in the hNSCs induced by Rg1 were analyzed using the whole-cell patch-clamp. RESULT: On the 7th day, the neuron-like cells derived from ginsenoside Rg1 (20 mg x L(-1))-induced NSCs show: (1) The resting membrane potential: (-45.70 +/- 2.63) mV, the membrane capacitance: (26.89 +/- 1.91) pF, the membrane input impedance: (877.51 +/- 20.44) MH (P < 0.05 compared with the control group, respectively); (2) The detection rate of inward sodium current which is rapidly activated and inactivated in voltage-dependence was 50%, and its average peak value was (711.48 +/- 158.03) pA (P < 0.05 compared with the control group); (3) The outward potassium currents were composed of rapidly activated and inactivated transient outward potassium current and delayed rectifier outward potassium current, and its average peak value was (1 070.42 +/- 177.18) pA (P < 0.05 compared with the control group). CONCLUSION: Ginsenoside Rg1 can promote the functional expression and maturity of hNSCs.

Ganoderic acid DM, a natural triterpenoid, induces DNA damage, G1 cell cycle arrest and apoptosis in human breast cancer cells.

Ganoderic acid DM (GADM) is a triterpenoid isolated from Ganoderma lucidum, a well-known edible medicinal mushroom. In the present study, we found that GADM effectively inhibited cell proliferation and colony formation in MCF-7 human breast cancer cells, which was much stronger than that of MDA-MB-231 breast cancer cells. GADM both concentration- and time-dependently mediated G1 cell cycle arrest and significantly decreased the protein level of CDK2, CDK6, cycle D1, p-Rb and c-Myc in MCF-7 cells. Moreover, GADM obviously induced DNA fragmentation and cleavage of PARP which are the characteristics of apoptosis and decreased the mitochondrial membrane potential in MCF-7 cells. Besides, we also showed that GADM elicited DNA damage as measured by comet assay which is a sensitive method for DNA damage detection. γ-H2AX, a marker of DNA damage, was also slightly up-regulated after treated with GADM for 6h, suggesting that the G1 cell cycle arrest and apoptosis induced by GADM may be partially resulted from GADM-induced DNA damage. These results have advanced our current understandings of the anti-cancer mechanisms of GADM.

Protective, antioxidative and antiapoptotic effects of 2-methoxy-6-acetyl-7-methyljuglone from Polygonum cuspidatum in PC12 cells.

Much correlative evidence indicates that the oxidative modification of protein by reactive oxygen species (ROS) is involved in normal aging as well as the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. In this study, we explored the antioxidative and neuroprotective effects of a naphthoquinone, 2-methoxy-6-acetyl-7-methyljuglone (MAM), purified from the dried rhizome of POLYGONUM CUSPIDATUM (Chinese name Hu-Zhang). Pretreatments with MAM (24 h) were investigated for their protective effects against apoptosis induced by the oxidizing agent TERT-butyl hydroperoxide ( T-BHP) in PC12 cells. The results indicated that MAM pretreatments could effectively protect PC12 cells against cytotoxicity induced by T-BHP in a dose-dependent manner. Cell viability was determined by both MTT and LDH assays. Increasing concentrations of MAM enhanced cell viability significantly and completely prevented cell death induced by T-BHP at 2.5 µM. The corresponding extracellular lactate dehydrogenase (LDH) levels were also attenuated significantly by various concentrations of MAM. In addition, it was found that the antioxidative effect of MAM was stronger than those of resveratrol and lipoic acid. The antiapoptotic property of MAM was further investigated with Hoechst 33342 nuclear staining and TUNEL assay. Pretreatments of MAM were able to prevent the T-BHP-induced nucleus fragmentation and accumulation of apoptotic bodies (commonly accepted as markers of apoptosis) inside the cells in a dose-dependent manner. T-BHP induced the phosphorylation of ERK 1/2, JNK and p38 MAPK, which were all impeded by pretreatments with MAM, indicating that MAM may act as a potent antioxidant which significantly interferes with the MAPK apoptotic cascades, probably rescuing cells by inhibiting the death pathways.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

Curcumin produces neuroprotective effects via activating brain-derived neurotrophic factor/TrkB-dependent MAPK and PI-3K cascades in rodent cortical neurons.

Curcumin is a major constituent of curcuma longa, a traditional medicine used to manage mental disorders effectively in China. The neuroprotective effects of curcumin have been demonstrated in our previous studies. In the present research, we confirmed this effect by showing that curcumin application promoted the viability of cultured rodent cortical neurons. Moreover, when neurons were pretreated with tyrosine kinase B (TrkB) antibody, known to inhibit the activity of brain-derived neurotrophic factor (BDNF), the protective effect of curcumin was blocked. Additionally, treatment of curcumin increased BDNF and phosphor-TrkB and both of these enhancements can be suppressed by ERK and PI-3K inhibitors. The administration of curcumin led to increased levels of phosphor-ERK and AKT, which were each blocked by MAPK and PI-3K inhibitors. Furthermore, the curcumin-induced increase in phosphorylated cyclic AMP response element binding protein (CREB), which has been implicated as a possible mediator of antidepressant actions, was prevented by MAPK and PI-3K inhibitors. Therefore, we hypothesize the neuroprotection of curcumin might be mediated via BDNF/TrkB-MAPK/PI-3K-CREB signaling pathway.

A traditional Chinese medicine formulation consisting of Rhizoma Corydalis and Rhizoma Curcumae exerts synergistic anti-tumor activity.

Synergy analysis of anticancer agents is an important approach to determining the ratio and/or dose of drugs for clinical combination therapy. However, this method is rarely used to evaluate the composition of traditional Chinese medicine formulation. 'Yanhusuo San' (YHSS), which consists of yanhusuo (Rhizoma Corydalis) and Ezhu (Rhizoma Curcumae), has been an archaic Chinese medicine prescription since the Song dynasty (960-1279 AD). We previously demonstrated that either yanhusuo or ezhu has strong anticancer effect. Herein, we sought to determine the possible synergic effect between these two Chinese herbs. We measured the IC50 of each herb extract and both extracts at different ratios of doses by MTT assay. Isobologram and combination index (CI) analyses were used to evaluate the synergistic effect of yanhusuo and ezhu in different fixed ratios. Our results indicated that a combination of two herbal extracts exhibits the strongest anticancer cell proliferation effect at the ratio of 3:2 (ezhu to yanhusuo; referred to as E3Y2). Using Boyden Chamber assay, flow cytometry, and fluorescence microscopy analysis, we found that E3Y2 could markedly reduce the cell invasion ability and induce cytochrome c release rather than single use, but E3Y2 could not influence the cell cycle distribution. When the levels of ERK1/2, p-ERK1/2 and p-Rb were determined by Western blot analysis, we found that the E3Y2 significantly suppresses the level of p-ERK. Thus, our studies provide a plausible molecular basis of the synergistic anti-tumor effect of ezhu and yanhusuo.

[The effect of TSPG in vivo on transplantation of neural stem cells in treatment of Parkinson's disease mouse].

AIM: To observe the effect for the model of PD and the transplantation of NSCs after injection of TSPG into mouse in advance. METHODS: Firstly, we divided the mouse into 5 groups. For the group of 1-4, we established the model of PD with MPTP. For the group of 5, before the establishment of the model, we injected TSPG into mouse in advance for prevention. And then, we evaluated the effect by paralysis agitans score standard and praxiology marker. Secondly, we obtained the NSCs from the 7-12 week embryo cerebral cortex. Then we transplanted NSCs which pretreated by TSPG into the striatum of the 5 groups. After 60 days, we obtained the brain section, and detected the TH by ICC to analyse the differentiation status of NSCs. RESULTS: The prevention of TSPG could decrease the neural cells damage by MPTP, and could protect the nervous system. After we transplanted NSCs into the striatum of Parkinson' s disease mouse, we found that for the group of 5, the paralysis agitans, auto-activity and memory function had the most distinct amelioration. And the number of dopaminergic neurons increased most transparently in brain section, and the neurons contact was the most enriched with the adjacent nervous cells. CONCLUSION: TSPG can decrease the neural cells damage and can produce a marked effect in treatment of PD by transplanting NSCs invivo.

Salvianolic acid B protects human endothelial cells from oxidative stress damage: a possible protective role of glucose-regulated protein 78 induction.

AIMS: The purposes of the present study were to both evaluate the protective effects of Salvianolic acid B (Sal B) and to determine the possible molecular mechanisms by which Sal B protects endothelial cells from damage caused by oxidative stress. METHODS AND RESULTS: Pretreatment with Sal B markedly attenuated H(2)O(2)-induced viability loss, lactate dehydrogenase leakage and apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism of Sal B protection was studied using two-dimensional gel electrophoresis coupled with hybrid quadrupole time-of-flight mass spectrometry. Database searching implicated that glucose-regulated protein 78 (GRP78), a central regulator for endoplasmic reticulum (ER) stress, was up-regulated in Sal B-exposed HUVECs. GRP78 expression regulation was confirmed by western blot and RT-PCR (reverse transcription-polymerase chain reaction) analyses. Additionally, GRP94, which shares significant sequence homology with GRP78, was also up-regulated in Sal B-treated cells. Sal B caused pancreatic ER kinase (PKR)-like ER kinase (PERK) activation followed by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha) and the expression of activating transcription factor 4 (ATF4). Knockdown of endogenous ATF4 expression partially repressed Sal B-induced GRP78 induction. Further investigation showed that ATF6 was also activated by Sal B. Knockdown of GRP78 by siRNA significantly reduced the protective effects of Sal B. CONCLUSION: The results suggest that Sal B induces the expression of GRP78 by activating ATF6 and the PERK-eIF2 alpha-ATF4 pathway. Furthermore, up-regulation of GRP78 by Sal B may play an important role in protecting human endothelial cells from oxidative stress-induced cellular damage.

The antidepressant effects of curcumin in the forced swimming test involve 5-HT1 and 5-HT2 receptors.

Curcuma longa is a main constituent of many traditional Chinese medicines, such as Xiaoyao-san, used to manage mental disorders effectively. Curcumin is a major active component of C. longa and its antidepressant-like effect has been previously demonstrated in the forced swimming test. The purpose of this study was to explore the possible contribution of serotonin (5-HT) receptors in the behavioral effects induced by curcumin in this animal model of depression. 5-HT was depleted by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA, 100 mg/kg, i.p.) prior to the administration of curcumin, and the consequent results showed that PCPA blocked the anti-immobility effect of curcumin in forced swimming test, suggesting the involvement of the serotonergic system. Moreover, pre-treatment of pindolol (10 mg/kg, i.p., a beta-adrenoceptors blocker/5-HT(1A/1B) receptor antagonist), 4-(2'-methoxy-phenyl)-1-[2'-(n-2''-pyridinyl)-p-iodobenzamino-]ethyl-piperazine (p-MPPI, 1 mg/kg, s.c., a selective 5-HT(1A) receptor antagonist), or 1-(2-(1-pyrrolyl)-phenoxy)-3-isopropylamino-2-propanol (isamoltane, 2.5 mg/kg, i.p., a 5-HT(1B) receptor antagonist) was found to prevent the effect of curcumin (10 mg/kg) in forced swimming test. On the other hand, a sub-effective dose of curcumin (2.5 mg/kg, p.o.) produced a synergistic effect when given jointly with (+)-8-hydroxy-2-(di-n-propylamino)tetralin, (8-OH-DPAT, 1 mg/kg, i.p., a 5-HT(1A) receptor agonist), anpirtoline (0.25 mg/kg, i.p., a 5-HT(1B) receptor agonist) or ritanserin (4 mg/kg, i.p., a 5-HT(2A/2C) receptor antagonist), but not with ketanserin (5 mg/kg, i.p., a 5-HT(2A/2C) receptor antagonist with higher affinity to 5-HT(2A) receptor) or R(-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1 mg/kg, i.p., a 5-HT(2A) receptor agonist). Taken together, these results indicate that the antidepressant-like effect of curcumin in the forced swimming test is related to serotonergic system and may be mediated by, at least in part, an interaction with 5-HT(1A/1B) and 5-HT(2C) receptors.

[Effect of TSPG on proliferation and differentiation of human embryonic neural stem cell into dopaminergic neuron].

OBJECTIVE: To investigate the effects of total saponins of panax ginseng (TSPG) on proliferation and differentiation of human embryonic neural stem cell (NSC) into dopaminergic neuron. METHOD: Isolation, cultivation and identification of human embryonic NSC from cerebral cortex of 7-12 week abortus. By using flow cytometry and MTT assay, the effects of various concentration of TSPG and TSPG cooperating with cytokines( EGF, bFGF) in NSC culture media for 3 days on proliferation of human embryonic NSC has studied. By employing immunocytochemistry assay of the expression of tyrosine hydroxylase (TH), the effect of different dilution of TSPG and TSPG cooperating with IL-1 on induced differentiation of human embryonic NSC into dopaminergic neuron has researched. RESULT: TSPG can significantly promote the proliferation of NSC. When TSPG cooperating with EGF and bFGF, the proliferation of NSC is much stronger than that of only using FGF and bFGF. TSPG also induces NSC to differentiate into dopaminergic neuron, especially when TSPG is cooperating with IL-1. CONCLUSION: TSPG can not only obviously accelerate the proliferation of NSC, but also significantly induce differentiation of NSC into dopaminergic neuron.

Antifungal activities of major tea leaf volatile constituents toward Colletorichum camelliae Massea.

A crude glycosidic fraction was prepared from fresh tea leaves and treated with the crude tea enzyme, fractions of cis-3-hexenol, linalool oxide I (cis-furanoid), linalool oxide II (trans-furanoid), linalool, methyl salicylate, geraniol, benzyl alcohol, and 2-phenylethanol were monitored to be the major aglycone moieties by analyzing the released volatiles. The amount of the released aglycone moieties is 5.8 times higher than those in free form. For investigation of the functions of the glycosidically bound form aroma constituents in tea leaves, their antifungal activities were determined by antifungal assay. Geraniol, linalool, methyl salicylate, benzyl alcohol, and 2-phenylethanol exhibited significant antifungal activities toward Colletorichum camelliae Massea, although cis-3-hexenol and linalool oxides showed weaker activities by comparison. Among them, geraniol was shown to be the most potential antifungal substance with a MIC value of 440 microg/mL. The crude glycosidic fraction prepared from tea leaves also exhibited significant antifungal activities in a wide range of concentrations from 2 to 25 mg/mL in a PDA medium. It was deduced that the glycosidically bound volatiles are formed and stored in the intact tissue of tea leaf and hydrolyzed by the actions of both the endogenous and the exogenous glycosidases to release volatiles as antifungal substances when exposed to Colletorichum camelliae Massea. The results suggested that the higher content of the bound form geraniol in tea leaves of var. sinensis might be responsible for their stronger antipathogen properties toward tea leaf blight, as opposed to those of var. assamica.