RESUMO
This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.
Assuntos
Infecções por Circoviridae , Circovirus , Vacinas de Partículas Semelhantes a Vírus , Vacinas Virais , Animais , Anticorpos Antivirais , Proteínas do Capsídeo , Cromatografia em Gel , Infecções por Circoviridae/prevenção & controle , Lasers , Suínos , Vacinas de Produtos InativadosRESUMO
BACKGROUND: Zeaxanthin, a carotenoid commonly found in plants, has a variety of biological functions including anti-cancer activity. PURPOSE: This study aimed to investigate the potential mechanisms of zeaxanthin in human gastric cancer cells. METHODS: CCK-8 assay was used to examine the cytotoxic effect of zeaxanthin on human gastric cancer cells. Flow cytometry was used to analyse AGS cell cycle distribution and apoptosis status. Western blot analysis was used to detect the expression levels of cycle-related proteins (Cyclin A, Cyclin B1, CDK1/2, p21, and p27), apoptosis-related proteins (Bcl-2, Bad, caspase-3, PARP), MAPK, AKT, STAT3, and NF-κB. RESULTS: CCK-8 assay showed that zeaxanthin has obvious cytotoxic effects on 12 types of human gastric cancer cells, but no obvious toxic effect on normal cells. In addition, flow cytometry and Western blotting results showed that zeaxanthin induces apoptosis by reducing mitochondrial membrane potential; increasing Cytochrome C, Bax, cleaved-caspase-3 (cle-cas-3), and cleaved-PARP (cle-PARP) expression levels; and decreasing Bcl-2, pro-caspase-3 (pro-cas-3), and pro-PARP expression levels. Additionally, zeaxanthin caused cell cycle arrest at the G2/M phase by increasing the levels of p21 and p27 and reduced the levels of AKT, Cyclin A, Cyclin B1, and Cyclin-dependent kinase 1/2 (CDK1/2). Furthermore, after zeaxanthin treatment, the expression levels of reactive oxygen species (ROS), p-JNK, p-p38, and I-κB increased, and the expression levels of p-ERK, p-AKT, STAT3, and NF-κB decreased. However, the ROS scavenger N-acetylcysteine (NAC) and MAPK inhibitors inhibited zeaxanthin-induced apoptosis, and under the action of zeaxanthin, MAPK regulated NF-κB and STAT3, and reduced their protein expression levels. CONCLUSION: Zeaxanthin has a potential effect against gastric cancer cells through the ROS-mediated MAPK, AKT, NF-κB, and STAT3 signaling pathways, and it is expected to become a new drug for the treatment of human gastric cancer.
RESUMO
Quinalizarin has been demonstrated to exhibit potent antitumor activities in lung cancer and gastric cancer cells, but currently, little is known regarding its anticancer mechanisms in human breast cancer cells. The aim of the present study was to investigate the apoptotic effects of quinalizarin in MCF7 cells and to analyze its molecular mechanisms. The MTT assay was used to evaluate the viability of human breast cancer cells that had been treated with quinalizarin and 5fluorouracil. Flow cytometric analyses and western blotting were used to investigate the effects of quinalizarin on apoptosis and cycle arrest in MCF7 cells with focus on reactive oxygen species (ROS) production. The results demonstrated that quinalizarin exhibited significant cytotoxic effects on human breast cancer cells in a dosedependent manner. Accompanying ROS, quinalizarin induced MCF7 cell mitochondrialassociated apoptosis by regulating mitochondrialassociated apoptosis, and caused cell cycle arrest at the G2/M phase in a timedependent manner. Furthermore, quinalizarin can activate p38 kinase and JNK, and inhibit the extracellular signalregulated kinase, signal transducer and activator of transcription 3 (STAT3) and NFκB signaling pathways. These effects were blocked by mitogenactivated protein kinase (MAPK) inhibitor and NacetylLcysteine. The results from the present study suggested that quinalizarin induced G2/M phase cell cycle arrest and apoptosis in MCF7 cells through ROSmediated MAPK, STAT3 and NFκB signaling pathways. Thus, quinalizarin may be useful for human breast cancer treatment, as well as the treatment of other cancer types.