ABSTRACT
Objective To explore the role of integrin αvβ3 in the promotion of the development of choroidal neovascularization (CNV) by SDF-1/CXCR4.Methods This study was divided into two parts in vitro and in vivo.As for the in vivo study,a CNV model was induced by laser on C57BL/6J mice,and then assigned into 4 groups:mice with solely CNV modeling as control group,with intravitreal injection of SDF-1 after immediate CNV modeling as SDF-1 group,with intravitreal injection of SDF-1 + CXCR4 inhibitor (AMD3100) after CNV modeling as SDF-1 + AMD3100 group,and mice with intravitreal injection of SDF-1 + αvβ3 inhibitor (SB273005) after modeling as SDF-1 + SB273005 group.CXCR4 and αvβ3 expression levels in laser-induced eyes were quantified by qRT-PCR at time points of day 1,3,5,7,10 and 14 after modeling,and immunofluorescence staining was applied to detect αvβ3 expression in regional CNV and its endothelial cells in the four groups.Finally,OCT was used to observe the height of retinal pigment epithelial (RPE) layers in CNV after treatment in the four groups.Moreover,in the experiment in vitro,Western blot was used to measure the expression of CXCR4 protein of RF/6A cells in normal control group,Si-CXCR4 knockdown group and Si-NC knockdown model group.Meanwhile,the expression of integrin subunit β3 protein was determined in the normal control group,SDF-1 group,SDF-1 + AMD3100group,SDF-1 + Si-NC group and SDF-1 + Si-CXCR4 group.Transwell assay was conducted to detect the migration ability of RF/6A cells in the normal control group,SDF-1group,SDF-1 +AMD3100 group,SDF-1 + SB273005 group.Results On the one hand,the study in vivo,qRT-PCR showed that the expression of CXCR4 and integrin subunit β3 mRNA was up-regulated at first,and then down-regulated with time passed after CNV induction,with the highest expression level of CXCR4 mRNA (4.263 ± 0.464) on day 3,and the peak expression of β3 mRNA (3.678 ±0.364) on day 7 after CNV modeling.The results of immunofluorescence staining showed that the β3 fluorescence intensity of SDF-1 group was significantly enhanced,and the ratio of β3/CD31 was also significantly increased,which both were significantly higher than those of the control group (P ＜ 0.01).However,the β3 fluorescence intensity and β3/CD31 ratio of SDF-1 +AMD3100 group and SDF-1 + SB273005 group were significantly weakened and decreased,respectively (P ＜0.05).OCT showed that the elevation level of RPE layer inSDF-1 group was significantly higher than that in the control group [(135.503 ± 10.301) μm vs.(94.443 ± 12.156) μm](P＜0.05).The height of RPE uplift in SDF-1 + AMD3100 group [(95.283 ±20.062) μm] and SDF-1 + SB273005 group [(99.807 ± 10.403) μm] was significantly decreased (P ＜ 0.05).On the other hand,in experiment in vitro,Western blot showed that the expression levels of integrin β3 in SDF-1 group and SDF-1 + Si-NC group were significantly higher than those in the control group [(1.301 ± 0.043) and (1.273 ± 0.077) vs.(0.244 ± 0.069)] (P ＜ 0.01).The levels of integrin subunit β3 protein in SDF-1 + si-CXCR4 group (0.322 ± 0.042) and SDF-1 + AMD3100 group (0.336 ± 0.077) were significantly down-regulated (P ＜ 0.01).Transwell assay showed that the amount of migrating cells in SDF-1 group increased,which was significantly higher than that of the control group (P ＜ 0.01),while the number of migrating cells in SDF-1 +AMD3100 group and SDF-1 + SB273005 group was significantly decreased.Conclusion Integrin αvβ3 can promote the development of CNV by mediating SDF-1/CXCR4 signaling in endothelial cells.
ABSTRACT
Objective To establish a high performance liquid chromatography (HPLC) method for determining the content and dissolution of soluble guanylate cyclase(sGC) - 003 tablets. Methods Content and dissolution of sGC- 003 tablets were determined by HPLC. Phenomenex Luna C18 column(250 mm×4.6 mm, 5 μm) was used. The mobile phase was acetonitrile-water(40∶60, V/V), with a flow rate of 1.0 ml/min, and the detection wavelength was set at 214 nm. The column temperature was 40 ℃ and the injection volume was 20 μl(injection volume of dissolution was 80 μl). Dissolution of sGC-003 tablets was determined by the second method described in Chinese Pharmacopoeia(ChP) 2015. 900 ml of pH 4.5 acetic acid buffer, pH 6.8 phosphoric acid buffer and water were used as dissolution media at the rotation speeds of 50 and 75 r/min to select the dissolution condition. Results This method had high specificity. The average recovery was about 99.58%(RSD=0.75%, n=9). And the working solution was stable within 12 h. The calibration curves were had good linearity (R2=1) within the concentration range of 0.25-50 μg/ml. The method of dissolution tests for sGC-003 tablets was established that 900 ml pH 6.8 phosphoric acid buffer was used as dissolution medium and paddle rotation speed was 50 r/min. The dissolution would be 80% at 30 min. Conclusion The dissolution condition can be used to determine the dissolution of sGC-003 tablets. The HPLC method is convenient, fast, sensitive and accurate in determining the content and dissolution of sGC-003 tablets.