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AIM: To investigate the effects of antitumor drug paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, cell morphology, and related protein expression of Müller cells, and to evaluate its potential toxicity to the retina.METHODS:Müller cells were cultured in vitro and divided into two groups: control group(normal medium)and PTX group. Retinal Müller cells were treated with different concentrations of PTX(0.005, 0.05, 0.5 and 5mg/L)for varying durations(12, 24, 36, 48 and 72h). The CCK8 method was used to assess the effects of different concentrations of PTX and treatment duration on the proliferation Müller cells. Flow cytometry was employed to investigate the impact of different concentrations of PTX on Müller cells apoptosis and cell cycle arrest. Immunofluorescence was used to observe morphological changes in Müller cells. The effects of PTX on the expression of apoptosis-related proteins and aquaporins were analyzed by Western blot and qRT-PCR.RESULTS: PTX exhibits the ability to inhibit the proliferation of Müller cells when cultured in vitro. The efficacy of this inhibition was found to be dependent on both the concentration of the drug and the duration of the stimulation. Higher concentrations of the drug and longer stimulation times resulted in a weaker ability of the cells to proliferate. Additionally, PTX also induces apoptosis in Müller cells, with increased drug concentrations and longer stimulation times leading to higher apoptosis rates. Flow cytometry analysis demonstrates that PTX arrests Müller cells in the G2-M phase of the cell cycle. Moreover, there is a distinct change in cell morphology, with a shift from the typical appearance characterized by clear and slender fibrous structures to a rounder morphology, accompanied by a significant decrease in cell numbers. Further, our findings reveal that there is a transient increase in the expression of cytoinflammatory factors following drug treatment compared to the control group. However, discontinuation of drug stimulation can alleviate this heightened expression. In treated cells, the expression of the CA XIV protein is upregulated compared to the control group, while the expression of vascular endothelial growth factor(VEGF)is downregulated(P<0.05). Additionally, the levels of inflammatory factors in the PTX group are significantly higher than those in the control group(P<0.05), suggesting that PTX has the potential to disrupt the retinal barrier function.CONCLUSION: PTX affects the proliferation and apoptosis of Müller cells, with the effects dependent on stimulation duration and drug concentration. In addition, PTX blocks the Müller cell cycle at the G2-M phase and alters cell morphology, leading to a transient upregulation of inflammatory factors and affecting the integrity of the retinal barrier. These findings indicate the potential toxicity of the antitumor drug PTX to the retina.
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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.
