Total Flavonoids of Astragalus Inhibit Activated CD4[Formula: see text] T Cells and Regulate Differentiation of Th17/Th1/Treg Cells in Experimental Autoimmune Encephalomyelitis Mice by JAK/STAT and NF[Formula: see text]B Signaling Pathways.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by CD4[Formula: see text] T cell-mediated immune cell infiltration and demyelination in the central nervous system (CNS). The subtypes of CD4[Formula: see text] T cells are T helper cells 1 (Th1), Th2, Th17, and regulatory T cells (Treg), while three other types of cells besides Th2 play a key role in MS and its classic animal model, experimental autoimmune encephalomyelitis (EAE). Tregs are responsible for immunosuppression, while pathogenic Th1 and Th17 cells cause autoimmune-associated demyelination. Therefore, suppressing Th1 and Th17 cell differentiation and increasing the percentage of Treg cells may contribute to the treatment of EAE/MS. Astragali Radix (AR) is a representative medicine with immunoregulatory, anti-inflammatory, antitumor, and neuroprotective effects.The active ingredients in AR include astragalus flavones, polysaccharides, and saponins. In this study, it was found that the total flavonoids of Astragus (TFA) could effectively treat EAE in mice by ameliorating EAE motor disorders, reducing inflammatory damage and demyelination, inhibiting the proportion of Th17 and Th1 cells, and promoting Tregs differentiation by regulating the JAK/STAT and NF[Formula: see text]B signaling pathways. This novel finding may increase the possibility of using AR or TFA as a drug with immunomodulatory effects for the treatment of autoimmune diseases.

Synthesis and Bioactivity Evaluation of Nepetaefolin F and Its Analogues.

Nepetaefolin F (5), an abietane diterpenoid, showed significant inhibitory activity against human cancer cells in vitro with an IC50 value of 6.3 µM. The syntheses of nepetaefolin F and its analogues are presented herein. The cytotoxicity against various cancer cell lines was evaluated; notably, the cyclopropanecarboxylate ester 42 displayed significant antitumor activity against MGC 803 cells with an IC50 value of 20.9 µM. Further studies revealed that 42 could upregulate the expression of p62, microtubule-associated protein 1 light-chain 3 ß (LC3 B-I), cleaved caspase-3, and cleaved caspase-9 and downregulate the expression of Beclin-1 and LC3B-II. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 42 could modulate multiple signaling pathways, especially for peroxisome proliferator-activated receptor (PPAR) and AMP-activated protein kinase (AMPK), which are closely related to autophagy. These results suggested that compound 42 is a promising lead by inhibiting cell proliferation and autophagy, as inducing cell apoptosis in MGC 803 cells.

Astragaloside IV ameliorates metabolic disorder in db/db obese mice as a PPARγ antagonist.

Metabolic disorder is highly related to obesity, insulin resistance, hypertension, and hyperlipidemia. The present study found that astragaloside IV (ASI) attenuated metabolic disorder related symptoms and modulated hepatic lipid metabolism associated gene mRNA expression in db/db mice. ASI inhibited rosiglitazone-induced adipocyte differentiation of 3T3-L1 cells, and lipid accumulation in palmitic acid (PA)-induced HepG2 cells with down-regulated mRNA expression of lipogenesis-related genes. In addition, it was predicted to bind to the ligand binding domain (LBD) of PPARγ and inhibit its transactivity. Collectively, our study suggested that ASI improves lipid metabolism in obese mice probably through suppressing PPARγ activity.

Helichrysetin inhibits gastric cancer growth by targeting c-Myc/PDHK1 axis-mediated energy metabolism reprogramming.

Helichrysetin (HEL), a chalcone isolated from Alpinia katsumadai Hayata, has an antitumor activity in human lung and cervical cancers. However, the inhibitory effect and underlying mechanism of HEL in gastric cancer have not been elucidated. Here, HEL significantly inhibited the growth of gastric cancer MGC803 cells in vitro and in vivo. HEL decreased expression and transcriptional regulatory activity of c-Myc and mRNA expression of c-Myc target genes. HEL enhanced mitochondrial oxidative phosphorylation (OXPHOS) and reduced glycolysis as evidenced by increased mitochondrial adenosine triphosphate (ATP) production and excessive reactive oxygen species (ROS) accumulation, and decreased the pPDHA1/PDHA1 ratio and Glyco-ATP production. Pyruvate enhanced OXPHOS after HEL treatment. c-Myc overexpression abolished HEL-induced inhibition of cell viability, glycolysis, and protein expression of PDHK1 and LDHA. PDHK1 overexpression also counteracted inhibitory effect of HEL on cell viability. Conversely, c-Myc siRNA decreased cell viability, glycolysis, and PDHK1 expression. NAC rescued the decrease in viability of HEL-treated cells. Additionally, HEL inhibited the overactivated mTOR/p70S6K pathway in vitro and in vivo. HEL-induced cell viability inhibition was counteracted by an mTOR agonist. mTOR inhibitor also decreased cell viability. Similar results were obtained in SGC7901 cells. HEL repressed lactate production and efflux in MGC803 cells. These results revealed that HEL inhibits gastric cancer growth by targeting mTOR/p70S6K/c-Myc/PDHK1-mediated energy metabolism reprogramming in cancer cells. Therefore, HEL may be a potential agent for gastric cancer treatment by modulating cancer energy metabolism reprogramming.

[Galangin enhances autophagy by inhibiting NF-κB pathway in gastric cancer MGC-803 cells].

This study aimed to explore the effects of galangin on energy metabolism and autophagy in gastric cancer MGC803 cells and the underlying mechanism. Cell counting kit-8(CCK-8) was used to detect the effects of galangin at different concentrations on via-bility of MGC803 cells after 48 h intervention. Western blot was carried out to measure the effects of galangin on expression of proteins related to autophagy, nuclear factor-κB(NF-κB) pathway and energy metabolism, followed by the determination of its effects on mRNA expression of energy metabolism-related proteins by Real-time quantitative PCR(qPCR). The impact of galangin on autophagy was explored using AutophagyGreen dye reagent, with autophagosomes and lysosomes observed under the transmission electron microscope(TEM). Nude mice transplanted with gastric cancer MGC803 cells via subcutaneous injection were randomly divided into the following three groups: control(0.5% sodium carboxymethyl cellulose, once a day), 5-fluorouracil(5-FU, 50 mg·kg~(-1), twice a week), and galangin(120 mg·kg~(-1), once a day) groups. The body weight and tumor volume were measured once every three days with a vernier caliper at the same time point by the same person. After 21-d treatment, the tumor tissue was isolated and weighed for the calculation of the tumor-suppressing rate. The comparison with the control group revealed that galangin inhibited the viability of MGC803 cells, up-regulated the protein expression of microtuble-associated protein 1 light chain 3 B(LC3 B) Ⅱ, inhibited the phosphorylation of NF-κB pathway-related proteins, and promoted the formation of autophagosomes in MGC803 cells. However, it did not obviously affect the expression of energy metabolism-related proteins. Furthermore, galangin at 120 mg·kg~(-1) significantly reduced the tumor weight and volume in mice, enhanced LC3 BⅡ protein expression, and inhibited the phosphorylation of NF-κB pathway-related proteins. All these have suggested that galangin inhibited the growth of gastric cancer MGC803 cells both in vivo and in vitro, possibly by inhibiting the NF-κB pathway and enhancing autophagy.

Alteration of Gut Microbiota and Inflammatory Cytokine/Chemokine Profiles in 5-Fluorouracil Induced Intestinal Mucositis.

Disturbed homeostasis of gut microbiota has been suggested to be closely associated with 5-fluorouracil (5-Fu) induced mucositis. However, current knowledge of the overall profiles of 5-Fu-disturbed gut microbiota is limited, and so far there is no direct convincing evidence proving the causality between 5-Fu-disturbed microbiota and colonic mucositis. In mice, in agreement with previous reports, 5-Fu resulted in severe colonic mucositis indicated by weight loss, diarrhea, bloody stool, shortened colon, and infiltration of inflammatory cells. It significantly changed the profiles of inflammatory cytokines/chemokines in serum and colon. Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and VE-Cadherin were increased. While tight junction protein occludin was reduced, however, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were increased in colonic tissues of 5-Fu treated mice. Meanwhile, inflammation related signaling pathways including NF-κB and mitogen activated protein kinase (MAPKs) in the colon were activated. Further study disclosed that 5-Fu diminished bacterial community richness and diversity, leading to the relative lower abundance of Firmicutes and decreased Firmicutes/Bacteroidetes (F/B) ratio in feces and cecum contents. 5-Fu also reduced the proportion of Proteobacteria, Tenericutes, Cyanobacteria, and Candidate division TM7, but increased that of Verrucomicrobia and Actinobacteria in feces and/or cecum contents. The fecal transplant from healthy mice prevented body weight loss and colon shortening of 5-Fu treated mice. In addition, the fecal transplant from 5-Fu treated mice reduced body weight and colon length of vancomycin-pretreated mice. Taken together, our study demonstrated that gut microbiota was actively involved in the pathological process of 5-Fu induced intestinal mucositis, suggesting potential attenuation of 5-Fu induced intestinal mucositis by manipulating gut microbiota homeostasis.

20(S)-Protopanaxadiol enhances angiogenesis via HIF-1α-mediated VEGF secretion by activating p70S6 kinase and benefits wound healing in genetically diabetic mice.

Impaired angiogenesis is one of the crucial factors that impede the wound healing process in diabetic foot ulcers (DFUs). In this study, we found that 20(S)-protopanaxadiol (PPD), an aglycone of ginsenosides in Panax notoginseng, stimulated angiogenesis and benefited wound healing in genetically diabetic mice. In HUVECs, PPD promoted cell proliferation, tube formation and VEGF secretion accompanied by increased nuclear translocalization of HIF-1α, which led to elevated VEGF mRNA expression. PPD activated both PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways in HUVECs, which were abrogated by LY294002 and PD98059. Furthermore, these two pathways had crosstalk through p70S6K, as LY294002, PD98059 and p70S6K siRNA abolished the angiogenic responses of PPD. In the excisional wound splinting model established in db/db diabetic mice, PPD (0.6, 6 and 60 mg ml-1) accelerated wound closure, which was reflected by a significantly reduced wound area and epithelial gaps, as well as elevated VEGF expression and capillary formation. In addition, PPD activated PI3K/Akt/ERK signaling pathways, as well as enhanced p70S6K activity and HIF-1α synthesis in the wounds. Overall, our results revealed that PPD stimulated angiogenesis via HIF-1α-mediated VEGF expression by activating p70S6K through PI3K/Akt/mTOR and Raf/MEK/ERK signaling cascades, which suggests that the compound has potential use in wound healing therapy in patients suffering from DFUs.

Astragaloside IV improves lipid metabolism in obese mice by alleviation of leptin resistance and regulation of thermogenic network.

Obesity is a worldwide threat to public health in modern society, which may result from leptin resistance and disorder of thermogenesis. The present study investigated whether astragaloside IV (ASI) could prevent obesity in high-fat diet (HFD)-fed and db/db mice. In HFD-fed mice, ASI prevented body weight gain, lowered serum triglyceride and total cholesterol levels, mitigated liver lipid accumulation, reduced fat tissues and decreased the enlargement of adipose cells. In metabolic chambers, ASI lessened appetite of the mice, decreased their respiratory exchange ratio and elevated VCO2 and VO2 without altering circadian motor activity. Moreover, ASI modulated thermogenesis associated gene expressions in liver and brawn fat tissues, as well as leptin resistance evidenced by altered expressions of leptin, leptin receptor (ObR) or appetite associated genes. In SH-SY5Y cells, ASI enhanced leptin signaling transduction. However, in db/db mice, ASI did not change body weight gain and appetite associated genes. But it decreased serum triglyceride and total cholesterol levels as well as liver triglyceride. Meanwhile, it significantly modulated gene expressions of PPARα, PGC1-α, UCP2, ACC, SCD1, LPL, AP2, CD36 and SREBP-1c. Collectively, our study suggested that ASI could efficiently improve lipid metabolism in obese mice probably through enhancing leptin sensitivity and modulating thermogenic network.

MAPK pathways are involved in the inhibitory effect of berberine hydrochloride on gastric cancer MGC 803 cell proliferation and IL-8 secretion in vitro and in vivo.

Gastric cancer is the second leading cause of cancer-associated mortality worldwide. This investigation aimed to identify whether the mitogen­activated protein kinase (MAPK) signaling pathways are involved in the inhibitory effect of berberine hydrochloride (BER) on MGC 803 cells in vitro and in vivo. BER time­ and dose­dependently inhibited proliferation of MGC 803 cells. It also suppressed tumorigenesis in nude mice xenografted with MGC 803 cells. Additionally, BER reduced interleukin­8 (IL­8) secretion in vitro and in vivo. Further investigation demonstrated that inactivation of p38 MAPK, extracellular-signal regulated kinase 1/2 and c­Jun N­terminal kinase by BER contributed to the decreased proliferation and tumorigenesis, and the change in IL­8 expression levels. However, there was no significant synergistic inhibitory effect of combined BER and evodiamine (EVO) treatment on tumorigenesis, and BER reduced the upregulation of IL­8 induced by EVO in vivo. The results of the current study suggested that BER may be an effective and safe drug candidate for treating gastric cancer via modulation of the MAPK signaling pathways.

Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway.

Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases.

[Notoginsenoside Ft1 down-regulates HIF-1α, inhibits cell proliferation, decreases migration and promotes apoptosis in breast cancer cells].

To investigate the effect of notoginsenoside Ft1 (Ft1) on proliferation, migration and apoptosis of breast cancer cells, we conducted several assays including CCK-8 assay, Ed U staining, single cell migration assay and Hoechst 33258 staining. The effect of Ft1 on expression of apoptosis related proteins, HIF-1α, PI3K/Akt/mTOR/p70S6 K and MAPK pathways was examined with Western blot. Ft1 could significantly reduce cell survival and inhibit cell proliferation in breast cancer cells in a dose-dependent manner. Ft1 also increased chromatin condensation of MDA-MB-231 cells. Furthermore, Ft1 decreased protein expression of Bcl-2 and HIF-1α and increased expression of cleaved caspase 3 in MDA-MB-231 cells after 12 h treatment. Ft1 significantly down-regulated the levels of p-Akt, p-mTOR and p-p70S6 K as well as p-ERK1/2, but up-regulated that of p-JNK. Ft1 significantly inhibited the level of p-EGFR (Tyr1068) and p-EGFR (Ser1046/1047) in MDA-MB-231 cells. Finally, Ft1 significantly inhibited the migration path length and velocity of HS578 T cells when used at the concentration without affecting cell viability. Thus, Ft1 exhibited multiple antitumor effects including inhibition of cell survival and migration, promotion of cell apoptosis in breast cancer cells. Suppression of HIF-1α via Akt/mTOR/p70S6K and MAPK pathways may be involved in the pharmacological effect of Ft1 on cell proliferation and apoptosis of breast cancer cells.

[Inhibitory effects of vina-ginsenoside R7 on activation of rat C6 astrocytes induced by LPS and TNF-α combination].

To investigate the inhibitory effect and mechanism of vina-ginsenoside R7 (R7) on the activation of rat C6 astrocytes cells induced by LPS/TNF-α, cells in logarithmic growth phase were cultured in DMEM medium without FBS for 24 h. After dissociated using 0.25% EDTA-trypsin, the cells were seeded into respective plates at the density of 1.5×106 cells per mL and cultured overnight. The cells were divided into the following groups： control group (no treatment), model group (treated with LPS 1 µg•mL⁻¹ and TNF-α 10 µg•L⁻¹ treated for 24 h), R7 groups (pre-treated with 6.25, 12.5, 25, 50, and 75 µmol•L⁻¹ R7, 4 µmol•L⁻¹ L-NMMA for 2 h and then stimulated with LPS 1 mg•L⁻¹ and TNF-α 10 µg•L⁻¹ for 24 h). Cell viability was analyzed by CCK-8 kit. Secretion of nitric oxide (NO) in the medium was measured by Greiss method. Concentrations of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) were assayed by ELISA kits. Gene expressions of inflammatory factors were examined by quantitative-PCR analysis. Activation of NF-κB was detected by dual luciferase reporter gene assay kit. The results showed that R7 could significantly inhibit the secretion of NO from C6 cells in a dose-effect manner, with an IC50 of 34 µmol•L⁻¹. And it could reduce cell proliferation induced by LPS/TNF-α stimulation. Furthermore, R7 at 50 µmol•L⁻¹ significantly down-regulated gene expressions of iNOS (P<0.001), TNF-α (P<0.001), IL-1ß(P<0.05), and COX-2 (P<0.001), but could not change gene expression of IL-6. However, R7 reduced the secretion of TNF-α (P<0.001) and IL-6 (P<0.001). Further studies disclosed that, different concentrations of R7 (25, 50, 100 µmol•L⁻¹) could significantly inhibit the transcription activity of NF-κB(P<0.05, P<0.01, and P<0.001). In conclusion, R7 could inhibit inflammatory responses in C6 cells induced by LPS/TNF-α probably by inhibiting the transcription activity of NF-κB, which indicates its possible therapeutic effect in neurological diseases related to neuroinflammation.

Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction.

AIM: To determine the molecular mechanisms of Shugan decoction (SGD) in the regulation of colonic motility and visceral hyperalgesia (VHL) in irritable bowel syndrome (IBS). METHODS: The chemical compounds contained in SGD were measured by high-performance liquid chromatography. A rat model of IBS was induced by chronic water avoidance stress (WAS). The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension. Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining. The contents of tumor necrosis factor (TNF)-α in colonic tissue and calcitonin-gene-related peptide (CGRP) in serum were measured by ELISA. The protein expression of serotonin [5-hydroxytryptamide (5-HT)], serotonin transporter (SERT), chromogranin A (CgA) and CGRP in colon tissue was measured by immunohistochemistry. RESULTS: SGD inhibited colonic motility dysfunction and VHL in rats with IBS. Blockers of transient receptor potential (TRP) vanilloid 1 (TRPV1) (Ruthenium Red) and TRP ankyrin-1 (TRPA1) (HC-030031) and activator of protease-activated receptor (PAR)4 increased the pain pressure threshold, whereas activators of PAR2 and TRPV4 decreased the pain pressure threshold in rats with IBS. The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1 (capsaicin), TRPV4 (RN1747), TRPA1 (Polygodial) and PAR2 (AC55541). In addition, CGRP levels in serum and colonic tissue were both increased in these rats. TNF-α level in colonic tissue was also significantly upregulated. However, the levels of 5-HT, SERT and CgA in colonic tissue were decreased. All these pathological changes in rats with IBS were attenuated by SGD. CONCLUSION: SGD alleviated VHL and attenuated colon motility in IBS, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, CgA and SERT, and reducing CGRP and TNF-α level.

[Astragaloside IV regulates STAT1/IκB/NF-κB signaling pathway to inhibit activation of BV-2 cells].

OBJECTIVE: The study was aimed to investigate the inhibitory effect and mechanism of astragaloside IV (ASI) on the activation of microglial cells. METHOD: After pre-incubated with ASI for 2 h, microglial cells BV-2 were stimulated with interferon-γ (IFN-γ) for 1. 5 h and 24 h, respectively. Secretion of nitric oxide (NO) in the medium was measured by Griess method. Production of tumor necrosis factor alpha (TNF-α) was detected by ELISA approach. Cellular gene expressions of CD11b, TNF-α, interleukin 1ß (IL-1ß) and induced nitric oxide synthase (iNOS) were examined by quantitative-PCR analysis. Total and phosphorylation of STAT1, IκB and NF-κB was analyzed by Western blot method. RESULT: ASI could significantly inhibit the increased secretion of TNF-α and NO from BV-2 cells upon IFN-γ stimulation (P < 0.001). Further study showed that ASI significantly down-regulated gene expression of IL-1ß and TNF-α (P < 0.01, P < 0.05) and exhibited a trend to reduce that of iNOS. IFN-γ and ASI have no obvious effect on gene expression of CD11b. Moreover, ASI inhibited the phosphorylation of STAT1, IκB and NF-κB elicited by IFN-γ stimulation. CONCLUSION: ASI could restrain microglial activation through interfering STAT1/IκB/NF-κB signaling pathway, reducing gene expres- sion of IL-1ß and TNF-α, and thus inhibiting the production of proinflammatory mediators such as NO and TNF-α.

Berberine hydrochloride IL-8 dependently inhibits invasion and IL-8-independently promotes cell apoptosis in MDA-MB-231 cells.

Breast cancer, the leading cause of cancer-related mortality worldwide in females, has high metastastic and recurrence rates. The aim of the present study was to evaluate the anti-metastatic and anticancer in situ effect of berberine hydrochloride (BER) in MDA-MB-231 cells. BER dose-dependently inhibited proliferation and the IL-8 secretion of MDA-MB-231 cells. Additional experiments revealed that the inactivation of PI3K, JAK2, NF-κB and AP-1 by BER contributed to the decreased IL-8 secretion. BER abrogated cell invasion induced by IL-8 accompanied with the downregulation of the gene expression of MMP-2, EGF, E-cadherin, bFGF and fibronectin. In addition, BER reduced cell motility but induced G2/M arrest and cell apoptosis in an IL-8­independent manner. BER modulated multiple signaling pathway molecules involved in the regulation of cell apoptosis, including activation of p38 MAPK and JNK and deactivation of JAK2, p85 PI3K, Akt and NF-κB. The enhanced cell apoptosis induced by BER was eliminated by inhibitors of p38 MAPK and JNK but was strengthened by activator of p38 MAPK. Thus, BER inhibited cell metastasis partly through the IL-8 mediated pathway while it induced G2/M arrest and promoted cell apoptosis through the IL-8 independent pathway. Apoptosis induced by BER was mediated by crosstalks of various pathways including activation of p38 MAPK and JNK pathways and inactivation of Jak2/PI3K/NF-κB/AP-1 pathways. The results suggested that BER may be an efficient and safe drug candidate for treating highly metastatic breast cancer.

Astragalosides from Radix Astragali benefits experimental autoimmune encephalomyelitis in C57BL /6 mice at multiple levels.

BACKGROUND: Radix Astragali is famous for its beneficial effect on inflammation associated diseases. This study was to assess the efficacy of astragalosides (AST) extracted from Radix Astragali, on the progression of experimental autoimmune encephalomyelitis (EAE), and explore its possible underlying molecular mechanisms. METHODS: EAE was induced by subcutaneous immunization of MOG35-55. Infiltration of inflammatory cells was examined by HE staining. ROS level was detected by measuring infiltrated hydroethidine. Leakage of blood brain barrier (BBB) was assessed using Evan's blue dye extravasation method. Levels of inflammatory cytokines were measured using ELISA kits. Activities of total-SOD, GSH-Px, and iNOS and MDA concentration were measured using biochemical analytic kits. Gene expression was detected using real-time PCR method. Protein expression was assayed using western blotting approach. RESULTS: AST administration attenuated the progression of EAE in mice remarkably. Further studies manifested that AST treatment inhibited infiltration of inflammatory cells, lessened ROS production and decreased BBB leakage. In peripheral immune-systems, AST up-regulated mRNA expression of transcriptional factors T-bet and Foxp3 but decreased that of RORγt to modulate T cell differentiation. In CNS, AST stopped BBB leakage, reduced ROS production by up-regulation of T-SOD, and reduced neuroinflammation by inhibition of iNOS and other inflammatory cytokines. Moreover, AST inhibited production of p53 and phosphorylation of tau by modulation of the Bcl-2/Bax ratio. CONCLUSIONS: AST orchestrated multiple pathways, including immuno-regulation, anti-oxidative stress, anti-neuroinflammation and anti-neuroapoptosis involved in the MS pathogenesis, to prevent the deterioration of EAE, which paves the way for the application of it in clinical prevention/therapy of MS.

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells.

AIMS: This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells. MAIN METHODS: CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit. KEY FINDINGS: Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC50 of 45µM. Ft1 not only arrested the cell cycle at S, G2/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1. SIGNIFICANCE: Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.

Simultaneous quantification of seven hippocampal neurotransmitters in depression mice by LC-MS/MS.

BACKGROUND: There is no method available to simultaneously detect GABA, Glu, Epi, NE, DA, 5-HT and 5-HIAA in mouse hippocampus. NEW METHOD: A rapid and sensitive LC-MS/MS method has been developed for simultaneously measuring seven neurotransmitters in mouse hippocampus. The analytes were detected in positive mode with multiple reaction monitoring (MRM) and the procedure was completed in less than 9min. RESULTS: This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99, and showed good intra- and inter-day precisions (RSD<15%) with good accuracy (80-120%). Moreover, the method was successfully applied for the quantitative determination of neurotransmitters in a mouse depression model induced by successive methylprednisolone injections. The results indicated that this depression model was closely associated with the decreased level of Epi (p=0.002) and elevated ratio of 5-HIAA/5-HT (p=0.01), which has never been reported elsewhere. COMPARISON WITH EXISTING METHOD(S): Compared with previous methods, current approach is more convenient without any pre-column derivatization of the analytes but enhances detectability with incremental neurotransmitter profile and shortens detection time. CONCLUSIONS: This work represents the first accurate simultaneous determination of seven neurotransmitters in the mouse depression model induced by methylprednisolone. The reliable method will benefit the research of neurological diseases with the altered neurotransmitter profile in brain.

Decichine enhances hemostasis of activated platelets via AMPA receptors.

INTRODUCTION: Dencichine, one of the non-protein amino acids present in the roots of Panax notoginseng, has been found to shorten bleeding time of mice and increase the number of platelets. However, the exact underlying mechanisms have not been elucidated yet. This study was aimed to identify the hemostatic effect of dencichine and uncover its mechanisms. MATERIALS AND METHODS: Hemostatic effect was assessed by measuring tail bleeding time and coagulation indices of rats. PT, APTT, TT and FIB concentration were measured using a Sysmex CA-1500 plasma coagulation analyzer. Platelet aggregation rate was determined by using a platelet aggregometer. Concentration of cyotosolic calcium was evaluated by Fluo-3 and levels of cyclic adenosine monophosphate (cAMP) and thromboxane A2 (TXA2) were measured by ELISA method. RESULTS AND CONCLUSION: Dencichine administered orally shortened tail bleeding time, reduced APTT and TT but increased the concentration of FIB in plasma in a dose-dependent manner. When induced with trap, dencichine could elevate the cytoplasmic concentration of calcium, and secretion of TXA2 as well as the ratio of TXA2 to PGI2 from platelets. Meanwhile, it decreased the level of intracellular cAMP. However, CNQX could block the enhanced hemostatic effect of dencichine. These results suggested that dencichine exerted hemostatic function via AMPA receptors on platelets, therefore, facilitated coagulation cascade in a paracrine fashion by control of platelet cytosolic calcium influx, cAMP production and TXA2 release. Current study may contribute to its clinical use in therapy of hemorrhage.

The ethanol extract isolated from Weiqi Decoction induces G2/M arrest and apoptosis in AGS cells.

OBJECTIVE: To evaluate the effects of the ethanol extract isolated from Weiqi Decoction (WQD-EE) on AGS cell proliferation and apoptosis. METHODS: By using high-performance liquid chromatography with ultraviolet detectors (HPLC-UV) assay and MTT method, the main compounds in WQD-EE and cell viability were detected. And cell cycle distributions were determined by flow cytometry with propidium iodine (PI) staining while apoptosis was detected by flow cytometry with annexin V/PI double staining. Finally, caspase-3 activities were measured by colorimetric method and protein expression was determined by Western blotting. RESULTS: HPLC analysis showed that naringin (35.92 µg/mg), nobiletin (21.98 µg/mg), neohesperidin (17.98 µg/mg) and tangeretin (0.756 µg/mg) may be the main compounds in WQD-EE. WQD-EE not only inhibited AGS and MCF 7 cell proliferation in a dose-dependent manner, but also blocked cell cycle progression at G2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells. While at 0.5 mg/mL, WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h, respectively. Moreover, WQD-EE in one hand reduced protein expressions of p53 and cyclin B1, and in other hand enhanced protein expressions of cytochrome c and Bax. Protein levels of Bcl-2, Fas L and Fas were not significantly affected by WQD-EE. CONCLUSIONS: WQD-EE inhibits AGS cell proliferation through G2/M arrest due to down-regulation of cyclin B1 protein expression, and promotes apoptosis by caspase-3 and mitochondria-dependent pathways, but not by p53-dependent pathway.