Study on regulation mechanism of glaucocalyxin A on the autophagy and apoptosis of HCCLM 3 hepatocellular carcinoma cells through Bcl- 2/Beclin1 target / 中国药房

OBJECTIVE To study the regulator y mech anism of glaucocalyxin A （GLA） on autophagy and apoptosis of HCCLM3 hepatocellular carcinoma cells. METHODS HCCLM3 cells were taken ，and control group ，GLA 2.5 μg/mL group，GLA 5 μg/mL group and GLA 10 μg/mL group were mainly set according to different experimental purposes. In control group，only complete medium was added ；in each administration group ，complete medium containing the corresponding final concentration of GLA was added. Cell cycle distribution and apoptosis were detected by flow cytometry ；mitochondrial morphology and autophagy were observed by transmission electron microscope （only control group ，GLA 5 μg/mL group）；JC-1 staining and fluorescence inverted microscope were used to observe and detect the mitochondrial membrane potential of the cells ；Western blot assay was used to detect the protein expression of Bcl- 2， Bax， Beclin1 and cleaved caspase- 3 proteins in the cells ； the co-immunoprecipitation method was used to detect the binding and dissociation of Bcl- 2 and Beclin 1（only GLA 5 μg/mL group， GLA 10 μg/mL group）. RESULTS Compared with control group ，GLA 5 μg/mL and GLA 10 μg/mL could induce a significant arrest of the cell cycle in the G 2-M phase for HCCLM 3，a significant decrease in mitochondrial membrane potential ，an increase in apoptosis as well as significant promotion of the protein expression of Bax ，cleaved caspase- 3 and Beclin 1，and significant inhibition of the protein expression of Bcl- 2（P＜0.01）. GLA 5 μg/mL also significantly changed mitochondrial morphology and increased autophagosomes. The results of co-immunoprecipitation showed that compared with GLA 5 μg/mL，GLA 10 μg/mL could enhance the binding of Bcl- 2 and Beclin 1. CONCLUSIONS GLA can regulate the autophagy and apoptosis of HCCLM 3 cells by Bcl-2/Beclin1 target. The effect is closely related to the dose of GLA.

Glaucocalyxin A attenuates allergic responses by inhibiting mast cell degranulation through HMGB1/TLR4/NF-κB signaling pathways / 药学学报

The study is to investigate the effect of glaucocalyxin A (GLA) on mast cell-mediated anaphylaxis. The animal welfare and experimental process of this experiment followed the regulations of the Animal Ethics Committee of Yanbian University. BALB/c mice were used in the animal experiment and randomly divided into five groups, control group, model group, and GLA low, medium, and high dose groups (10, 20, and 40 mg·kg-1). Mice were sensitized by intradermal injection of anti-dinitrophenyl-immunoglobulin E (DNP-IgE) into the ears and challenged with a mixture of DNP-human serum albumin (HSA) and 4% evans blue into the tail veins to prepare an animal skin passive cutaneous anaphylaxis (PCA) model, which was collected from both ears for measurement of dye staining and histology. Rat peritoneal mast cells (RPMCs) were used in the cell experiment and divided into control, IgE + antigen (Ag), and IgE + Ag + GLA groups to determine histamine release as well as calcium influx levels. High-affinity IgE receptor (FcεRI)-mediated signaling pathway proteins and HMGB1/TLR4/NF-κB (high mobility group box 1/toll like receptor 4/nuclear transcription factor kappa B) signaling proteins were detected by Western blot. The results of animal experiments suggest that GLA inhibits PCA, reduces evans blue dye exudation, and reduces ear inflammation and ear thickness in mice. The results of cellular experiments suggested that GLA could reduce histamine release and calcium influx, and inhibit tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-13, and IL-1β production; Western blot results showed that GLA inhibited FcεRI-mediated phosphorylation levels of spleen tyrosine kinase (Syk), Lck/Yes novel tyrosine kinase (Lyn), tyrosine kinase Fyn (Fyn), growth-factor receptor-bound protein 2 (Gab2), and phospholipase C (PLC) γ1, while GLA inhibited HMGB1/TLR4 signaling pathway to limit NF-κB p65 nuclear metastasis. The results indicate that GLA inhibits mast cell degranulation and attenuates allergic inflammation through the HMGB1/TLR4/NF-κB signaling pathway.

Glaucocalyxin A alleviates ovalbumin-induced airway inflammation through HMGB1/TLR4/NF-κB signaling pathway in asthmatic mice / 国际药学研究杂志

Objective: To investigate the therapeutic effect of glaucocalyxin A(GLA)on airway inflammation in a mouse mod- el of ovalbumin(OVA)-induced asthma and whether the mechanism is associated with the HMGB1/TLR4/NF-κB signaling pathway. Methods: Forty BALB/c mice were divided into 5 groups(8 mice in each group):control group, OVA model group, GLA-L group (10 mg/kg), GLA-H group(40 mg/kg)and dexamethasone group(1 mg/kg). HE and PAS staining were used to observe the inflammatory infiltration and the number of goblet cells in mouse lung tissue;Diff-Quick staining was used to count various cell types in mouse bronchoalveolar lavage fluid(BALF);ELISA was used to detect the content of inflammatory and pro-inflammatory cytokines in mouse BALF;Western blotting(WB)was used to detect the expression of HMGB1, TLR4, MyD88, NF-κB(nuclear)and NF-κB(cytosol)in lung tissue;immunohistochemistry was used to detect the expression level of HMGB1, TLR4 and NF-κB p65 protein in mouse lung tissue. Results: GLA reduced inflammatory cell exudation and goblet cell proliferation in OVA-induced asthmatic model;GLA treatment significantly decreased eosinophils, neutrophils and lymphocytes in BALF of asthmatic mice, and reduced the levels of pro-inflammatory cytokines(P<0.05);WB results showed that GLA inhibited the protein expression of HMGB1, TLR4, MyD88 and NF-κB(nuclear)(P<0.05);immunohistochemistry results showed that GLA reduced the expression of HMGB1, TLR4 and NF-κB p65 protein in lung tissue(P<0.05). Conclusion: GLA could ameliorate the OVA-induced airway inflammation in asthmatic mice, possibly via interfering in the HMGB1/TLR4/NF-κB signaling pathway.

Effect of glaucocalyxin A on proliferation and cell cycle of triple-negative breast cancer MDA-MB-231 cells / 中草药

Objective: To investigate the effect of glaucocalyxin A on proliferation and cell cycle of triple-negative breast cancer MDA-MB-231 cells and its mechanism. Methods The proliferation inhibition rates of MDA-MB-231 cells were measured by MTT assay. The cell cycle was analyzed by flow cytometry, and the expression of the protein cyclin B1, cyclin D1, CDK2, CDK4, p53, p21, p27, LSD1, H3K4me2, and H3K9me2 was detected by Western blotting. Results Growth of MDA-MB-231 cells was significantly inhibited by glaucocalyxin A in a dose-dependent and time-dependent manner. Flow cytometric analysis indicated that the percentage of MDA-MB-231 cells at G2/M phase was increased significantly. As the results of Western blotting, the protein expression levels of p53, p21, p27, H3K4me2, and H3K9me2 in MDA-MB-231 cells were increased, while that of cyclin B1, cyclin D1, CDK2, CDK4, and LSD1 were decreased after treated with glaucocalyxin A. Conclusion Glaucocalyxin A could inhibit the proliferation of MDA-MB-231 cells and induce cell cycle arrest at the G2/M phase, and the mechanism may be related to the activation of p53 protein expression and the regulation of histone methylation.

Glaucocalyxin A regulates STAT3 signaling pathway through influencing IL-6 expression in HCCLM3 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of glaucocalyxin A ( GLA) on the expression of signal transduction and activator of transcription 3 (STAT3) signaling pathway in HCCLM3 cells for better understand-ing the role of GLA in tumor metastasis. Methods HCCLM3 cells were stimulated by different concentrations (0. 1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml and 1000 ng/ml) of GLA for 48 h. Expression of IL-6, STAT3/pSTAT3Tyr705 and MMP9 at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Transwell invasion and migration assays were performed to analyze cell invasion and migration. ELISA was used to measure the concentrations of IL-6 in culture media. Results Expression of IL-6 at mRNA and protein lev-els, STAT3Tyr705 phosphorylation, and the migration and invasion of HCCLM3 cells were significantly enhanced after stimulation of HCCLM3 cells with 0. 1 ng/ml of GLA, but inhibited by GLA at the concentration of 1000 ng/ml. Moreover, more IL-6 was secreted out of the HCCLM3 cells treated with 0. 1 ng/ml of GLA. Conclu-sions GLA might affect the metastasis of HCCLM3 cells through regulating the expression of IL-6 and the phos-phorylation of STAT3Tyr705. These regulatory effects were closely related to the concentration of GLA.

Mechanism of glaucocalyxin A that induces apoptosis of glioma cell and up-regulates GEF-H1 / 药学学报

Rabdosia japonica(Burm.f.) Hara var.glaucocalyx(Maxim.) Hara is a traditional Chinese medicine, and is known to have anti-tumor effects. This study aims to investigate the effect of glaucocalyxin A (GLA), a diterpenoids extracted from Glaucocalyx Hara, on apoptosis of glioma cells and its mechanism. This study investigated the molecular signaling mechanism of GLA-induced glioma cell apoptosis by analyzing survival rate of C6 rat glioma cells, cell morphology, colony formation ability, interference ribonucleic acid, polymerase chain reaction, and Western blot. The result showed that in the presentce of GLA, the survival rate of C6 rat glioma cells decreased significantly, while the expression of guanine nucleotide-exchange factor-H1 was up-regulated, causing phosphorylation of extracellular regulated protein kinases proteins and apoptosis. Hence, the mechanism of GLA-induced glioma cell apoptosis was the GEF-H1/ERK pathway.

Advances on the research of the pharmacological activity mechanism and toxicolo-gy of Glaucocalyxin A / 药学实践杂志

Glaucocalyxin A (GLA) is a kind of diterpenoid enantiomers with organic chemical structure of en-15-oxo-16-kaurene.It has various pharmacological activities,such as cardiovascular and endothelium cellular protection,anti-blood coagu-lation,anti-Hepatitis B Virus (HBV),anti-tumor,anti-bacteria,anti-inflammation,hypoxic tolerance,immunomodulation and Ca2+ concentration regulation effect,where as its in vivo safety is quite good.As a folk medicine,it is conventionally used to treat hepatitis,gastritis,mastitis,stomachache and arthralgia.It is also used for ischemic and/or hypoxic cardio-cerebrovas-cular diseases such as coronary heart diseases,stenocardia and chronic cerebral circulation insufficiency in clinic.This review gives a summary of the pharmacological effects,biological mechanism,and toxicity of Glaucocalyxin A.

High drug loading glaucocalyxin a nanosuspensions: preparation and in vitro evaluation / 中国药学杂志

OBJECTIVE: To prepare high drug loading glaucocalyxin A (GLA) nanosuspensions and study their properties in vitro. METHODS: Using solvent precipitation combined with high pressure homogenization method, the GLA nanosuspensions were successfully prepared with PLGA-PEG-PLGA block copolymer being a stabilizer. The particle size of the resultant nanosuspensions was determined by dynamic light scattering (DLS), their morphology was observed by transmission electron microscopy (TEM), the crystalline state of GLA in the nanosuspensions was examined by differential scanning calorimetry (DSC), and the in vitro release of GLA nanosuspensions was investigated using dialysis method. The in vitro anticancer cytotoxic activity of free GLA solution and GLA nanosuspensions against a gastric cancer cell line (BGC cells) and a hepatic cancer cell line (HepG-2 cells) were performed using MTT assay. RESULTS: GLA Nanosuspensions showed a mean particle size of (222.6±4.32) nm, a polydispersity index (PDI) of (0.237±0.016) and a Zeta potential of (-11.5±0.954) mV. The average entrapment efficiency was (69.11±3.02)% and the average drug loading was (40.67±2.45)%. The particles in the nanosuspensions were spherical with smooth surface and u-nilorm size distribution, and displayed sustained release in vitro. GLA nanosuspensions showed significant higher cytotoxicity than free GLA against BGC cells (IC50 value 0.784 μg·ml-1 vs. 1.658 μg·mL-1) and HepG2 cells (IC50 value 12.4 μg·mL-1 vs. 6.44 μg·mL-1). CONCLUSION: High drug loading GLA nanosuspensions are successfully prepared and well characterized in vitro. The resultant nanoparticles can efficiently enhance the antitumor activity of GLA and thus can be used as a suitable dosage form in this respect.

Determination of glaucocalyxin A by HPLC and its protein binding rate in plasma of rat / 中草药

Objective To study the protein binding rate of glaucocalyxin A in plasma of rat. Methods The plasma balance dialysis was used. An HPLC for the quantitative determination of glaucocalyxin A was presented and used to calculate the protein binding rate in plasma of rat. Results There were protein binding rate results of 74.46%, 77.87%, and 75.29% at three various concentrations 20, 10, and 1 ?g/ mL of glaucocalyxin A in plasma of rat. Conclusion The HPLC method used to determine glaucocalyxin A is simple, rapid, and sensitive with good specificity, precision and accuracy, and glaucocalyxin A has medium capacities in protein binding rate in plasma of rat.