Galangin enhances autophagy by inhibiting NF-κB pathway in gastric cancer MGC-803 cells / 中国中药杂志

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.

Chemical constituents and anti-cancer effect of Semen Alpinia Katsumadai:recent advances / 中国药理学与毒理学杂志

Semen Alpiniae Katsumadai,nearly mature seeds of Alpinia katsumadai Hayata(Zingib-eraceae),mainly contains flavonoids and terpenoids,which have significant anti-tumor effect.The anti-cancer mechanisms of Semen A.katsumadai include anti-proliferation,apoptosis induction,anti-metas-tasis, energy metabolism modulation and anti-inflammation. This paper reviews the latest advances in studies on the anti-tumor components and the underlying mechanisms of Semen A. katsumadai, the purpose of which is to contribute to the study of both chemical constituents and anti-cancer pharmacology of Semen A.katsumadai.

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.

Notoginsenoside Ft1 down-regulates HIF-1α, inhibits cell proliferation, decreases migration and promotes apoptosis in breast cancer cells / 药学学报

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, EdU staining, single cell migration assay and Hoechst 33258 staining. The effect of Ft1 on expression of apoptosis related proteins, HIF-1α, PI3K/Akt/mTOR/p70S6K 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-p70S6K 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 HS578T 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 / 中国中药杂志

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-1β(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.

Astragaloside IV regulates STAT1/IκB/NF-κB signaling pathway to inhibit activation of BV-2 cells / 中国中药杂志

<p><b>OBJECTIVE</b>The study was aimed to investigate the inhibitory effect and mechanism of astragaloside IV (ASI) on the activation of microglial cells.</p><p><b>METHOD</b>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 1β (IL-1β) 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.</p><p><b>RESULT</b>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-1β 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.</p><p><b>CONCLUSION</b>ASI could restrain microglial activation through interfering STAT1/IκB/NF-κB signaling pathway, reducing gene expres- sion of IL-1β and TNF-α, and thus inhibiting the production of proinflammatory mediators such as NO and TNF-α.</p>

The ethanol extract isolated from Weiqi Decoction induces G₂/M arrest and apoptosis in AGS cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of the ethanol extract isolated from Weiqi Decoction (WQD-EE) on AGS cell proliferation and apoptosis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle / 生理学报

Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.

Effect of astragalus on calcium accumulation and SERCA2a gene expression in myocardial tissues in rats with pressure overload-induced left ventricular hypertrophy / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of astragalus (As) on calcium accumulation and SERCA2a gene expression in left ventricular tissues in rats with pressure overload-induced cardiac hypertrophy.</p><p><b>METHOD</b>cardiac hypertrophy was induced by clipping the abdominal aorta in rats. Male SD rats were allocated to six groups: sham-operrated (Sham), aortic stenosis (Model), model +As-L (5 g x kg(-1) x d(-1)), model+As-M (10 g x kg(-1) x d(-1)), model+As-H (20 g x kg(-1) x d(-1)) and model + captopril (0.05 mg x kg(-1) x d(-1), a positive control). The drugs were administered orally from the 13 th week after surgery. Rats were examined after 12 week treatment with drugs. The cardiac hypertrophy was evaluated by left ventricular mass index (LVMI, left ventricular weight/ body weight). The calcium content in left ventricular tissue was measured by atomic absorption spectrometry. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein, respectively.</p><p><b>RESULT</b>The increase of LVMI was dose-dependently lessened by As (P < 0.01, P < 0.001). The effect of As-H was similar to that of Captopril. As markedly attenuated calcium accumulation in myocardial tissure (P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expressions were downregulated (P < 0.05) significantly in model group compared with sham group. As-H upregulated SERCA2a gene expressions (P < 0.05), whereas Captopril had no effect on that.</p><p><b>CONCLUSION</b>The inhibition of As on left ventricular hypertrophy induced by pressure overload in rats may partly contribute to its attenuation of calcium accumulation and up-regulation of SERCA2a gene expressions in left ventricular tissues.</p>