RESUMO
Purpose: To evaluate keratocyte viability and proinflammatory cytokine secretion induced by HSV-1 infection. Methods: Keratocytes were separated from corneal tissues obtained with the SMILE procedure, and an in vitro system was established to study HSV-1 infection in human keratocytes. Cell viability, HSV-1 genomic DNA copy number, and the expression levels of α-SMA, ALDH1A1, phospho-p38, p38, phospho-IRF3, and IRF3 were evaluated. Antibody array and ELISA kits were used to measure the production of proinflammatory cytokines and chemokines. Results: We found that HSV-1 infection reduced cell viability and activated keratocyte transdifferentiation into corneal fibroblast-like cells. Furthermore, p38 inhibition improved cell viability and IFN-ß production and played an anti-inï¬ammatory role by reducing the production of proinflammatory cytokines and chemokines. Conclusions: Our study reveals an important role played by keratocytes during innate immune responses and identifies p38 inhibition as a potential therapeutic approach to control ocular HSV-1 infection.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ceratócitos da Córnea/efeitos dos fármacos , Herpes Simples/prevenção & controle , Herpesvirus Humano 1/fisiologia , Imidazóis/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Chlorocebus aethiops , Ceratócitos da Córnea/virologia , Citocinas/metabolismo , Variações do Número de Cópias de DNA , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Fatores de Tempo , Células VeroRESUMO
OBJECTIVE: To establish an HPLC-MS method for the analysis of the impurity profile of cefotaxime sodium. METHODS: Shimadzu-LCMS-IT-TOF was used with Waters XBridge Shield (RP18, 4.6 mm×250 mm, 5 μm) column. Mobile phase A was 20 mmol·L-1 ammonium acetate (pH adjusted to 6.25)-methanol (92: 8), and mobile phase B was set at 20 mmol·L-1 ammonium acetate-methanol (60: 40) (pH adjusted to 6.25).Gradient elution was performed at a flow rate of 1.0 mL·min-1. ESI source was used.Positive and negative ion scanning was conducted in the range of m/z 150-900.The heating temperature was 200℃, CDL temperature was maintained at 200℃, atomization gas flow rate was 1.5 L·min-1, dry gas pressure was 94.0 kPa, and the post-column diversion ratio was 1: 4.Some related substances in cefotaxime sodium were identified by comparing the retention time in chromatography, [M+H]+ spectrum and MS2 spectrum with those of reference substances, the others which haven't reference substances were deduced or speculated by analyzing the MS2 or MSn fragmentation with the help of a rule summarized from the MS2 fragmentation of cefotaxime sodium and the reference substances of system suitability impurities. RESULTS: Twenty-six related substances were separated and detected in the sample, all of which were identified or deduced. They were cefotaxime sodium isomeric compounds and homologs generated during the production process or degradation products. CONCLUSION: The method can be applied in the identification and qualitative analysis of the related substances of cefotaxime sodium and the quality control and optimization of the synthesis of cefotaxime sodium.
RESUMO
Paeonia suffruticosa also named Moutan that cultivated in five geographic regions during different growth stages were chosen in this study. Biolog and 454 pyrosequencing technology were used to detect the whole microbial activity and fungal diversity for exploring the relationship between the geo-authentic features of the medicinal plant and the rhizosphere microorganism. The results suggest that the value of average well color development (AWCD) from the rhizosphere soil of P. suffruticosa in the five regions at the fo ur growth stage have an increasing tendency. 9 703 operational taxonomic unit (OTU) were obtained from 272 463 high quality sequences according to the similarity of 97% by the pyrosequencing. Fungi in five phyla, twenty-two classes, seventy orders, one hundred and thirty-nine families and two hundred and sixty-six genera were detected in the five regions excluding twelve percent to fifty-eight percent unidentified fungi. They were divided into four branches, i.e. Blastocladiales, Chytridiomycota, Dikarya and Glomeromycetes. Twenty-four genera such as Leptosphaeria were found in the five regions while six genera such as Curvularia were only detected in the geo-authentic regions. The dominant genera were Guehomyces, Exophiala and Fusarium in geo-authentic regions, whereas genus Leptosphaeria, Cryptococcus, Exophiala, Fusarium and Ceratobasidium in non-authentic regions. The results from principal co-ordinates analysis (PCoA) showed that the fungi formations were similar in Tongling and Nanling region at four different growth stages, and the same in Heze at the leaf bud and fruiting stage. According to heatmap analysis, Tongling and Nanling region showed a close similarity in fungal community structures on phylogenetic analysis, while Bozhou, Heze and Luoyang showed the same. In brief, the whole microbial activity was higher in geo-authentic regions than the non-authentic. Fungi in rhizosphere soil of the medicinal peony presented diversity and region specificity. We found not only the abundant new species in the five regions, but also the phylogenetic similarity in the geo-authentic regions.
