| BackgroundRetinopathy of prematurity?ROP?becomes an important cause of childhood blindness all over the world.Retinal neovascularization?RNV?is the major pathological feature of ROP.Besides premature birth,low birth weight and supplemental oxygen,it's recently been reported that inflammation makes a great contribution to the process of ROP.Microglia,a kind of resident monocytes/macrophages in retina,participates in the process of immune,inflammation and wound healing.It has been identified that microglia plays an important role in the development of retinal vessel and the process of angiogenesis.What's more,microglia can be activated by inflammation to promote RNV,however,the mechanism remains unknown.Caspase-1 is a kind of cysteine protease for activation and secretion of IL-1?and IL-18 playing central roles in inflammatory signaling.It is reported that Caspase-1 is up-regulated in OIR and involved in the process of retinal neovascularization diseases by its downstream signals,the mechanism of which is related to microglia.In vitro,the activation of microglia is regulated by Caspase-1.However,it is unknown whether Caspase-1 participates in the process of RNV in OIR by the regulation of microglia,which remains under study.ObjectiveTo investigate the effect and the mechanism of Caspase-1 on microglia in oxygen-induced retinal neovascularization in mice.MethodsResearch about OIR of mice:1.Animals groups:7-day-old?P7?C57BL/6J mice were randomly divided into normal group,OIR group and OIR+VX-765 group.OIR group and OIR+VX-765 group were exposed to 75%O2 for 5 days.From P12 to P16,OIR+VX-765 group and OIR group were intraperitoneally injected with Caspase-1 inhibitor VX-765?4mg/kg:dissolved in 0.4%polyethylene glycol?and the solvent respectively every day.2.Measurement of weight:From P12 to P17,weight change of three groups was observed.3.Immunofluorescence double staining:The frozen sections of retina of normal group and OIR group in P12 and three groups in P17 were stained with Caspase-1 and Iba-1 to examine the expression of Caspase-1 and the distribution of the activated microglia of normal group and OIR group in P12 and P17 and the activation of microglia of three groups in P17 in retina.4.Western blot:The levels of Caspase-1,p20,IL-1?and VEGF were detected in the normal group and OIR group of P12 and P17.The levels of TLR4,NLRP3,Caspase-1,p20,IL-1?,TNF-?and VEGF were assayed in three groups of P17.5.Retinal flatmounts staining:Retinal flatmounts of normal group and OIR group in P12 and three groups in P17 were stained by Lectin to evaluate the model and measure the ratio of avascular and neovascularization area respectively.Research about microglia and vascular endothelial cells:1.Cells groups:BV-2 cells were divided into control group,hypoxia group and inhibitor group.The inhibitor group and hypoxia group were respectly pre-treated with VX-765 or polyethylene glycol for 3 hours,and then incubated in hypoxic condition for 24h.RF/6A cells were cultured in conventional conditions.2.Western blot:The levels of Caspase-1,p20,TLR4,NLRP3,IL-1?,TNF-?and VEGF in BV-2 groups were detected by Western blot.3.Tube formation assays:RF/6A cells were incubated by the supernatant of the BV-2 groups to perform tube formation assays.4.Transwell experiments:RF/6A cells were incubated by the supernatant of the BV-2 groups to perform Transwell experiments.5.Immunocytochemistry:BV-2 cells were stained by immunofluorescence to examine the activation of BV-2 and the level of Caspase-1.6.RT-PCR:The level of Iba-1 in BV-2 groups were examined by RT-PCR.7.ELISA:The level of IL-1?in the supernatant of the BV-2 groups were detected by ELISA.Results:Research about OIR of mice:1.The retinal vascularization was almost completed in P12 of normal group and the retinal blood vessels were well developed in P17 of normal group.A large avascular area was found in P12 of OIR group and both of avascular and new blood vessel cluster were found in P17 of OIR group.2.Immunofluorescence results showed that Caspase-1 was up-regulated in OIR groups in comparison to that in normal groups.In contrast with P12,Caspase-1 was overexpressed in P17 and mainly co-located with activated microglia in the ganglion cell layer and the inner plexiform layer.Up-regulation of Caspase-1,p20,IL-1? and VEGF in OIR groups was compared to that of normal groups(P12d<0.0001;P17d<0.0001).The levels of such proteins of OIR in P17 were higher than that in P12?P<0.05?,whereas there was no significant difference between that of normal groups?P>0.05?.3.The distribution and morphology of retinal blood vessels were normal in P17 mice of the normal group,and avascular and new blood vessel cluster were found in OIR group and OIR+VX-765 group.The ratio of avascular area was?12.23±1.02?%and that of the new blood vessel area was?2.16±0.52?%in the OIR+VX-765 group,which were decreased in comparison with?16.58±1.14?%and?4.00±0.41?%of the OIR group?P<0.01?.4.Immunofluorescence results displayed few Iba-1?+?microglia in the retina of normal group in P17 whereas the cells were significantly increased and located in the ganglion cell layer and the inner plexiform layer in OIR group.Besides,there were less Iba-1?+? microglia in the ganglion cell layer in OIR+VX-765 group.5.Western blot showed that the levels of TLR4,NLRP3,Caspase-1,p20,IL-1?,TNF-? and VEGF were very low in normal group,and the levels of them were significantly higer in OIR group in P17.In OIR+VX-765 group,there was a lower expression of TLR4 signaling and VEGF,except Caspase-1?P<0.05?.Research about microglia and vascular endothelial cells:1.Western blot showed that the expression of Caspase-1 and p20 were increased in BV-2 cells of the hypoxia group in comparison to control group,which were down-regulated by VX-765?P<0.05?,except Caspase-1.2.The tube length in normal group and RF/6A cells cultured with supernatant of BV-2 cells in the control group were 100 and 162±5 respectively.What's more,the tube length was 271±12 in that of the hypoxia group but it was significantly decreased to 171±22 with VX-765?P<0.05?.3.The number of migrated cells in normal group and RF/6A cells cultured with supernatant of BV-2 cells in the control group were 112±22 and 179±24 respectively.Besides,the number of migrated cells was 347±34 in that of the hypoxia group,which was significantly decreased to 212±27 with VX-765?P<0.05?.4.Immunocytochemistry results showed that the expression of Iba-1 and Caspase-1 in BV-2 cells of hypoxia group were higher than that of control group,which were significantly reduced with VX-765?P<0.05?.RT-PCR showed few expression of Iba-1 in BV-2 cells of control group,which was much more in that of hypoxia group.In addition,it was reduced with VX-765?P<0.05?.5.Results of ELISA displayed that the level of IL-1?in the supernatant of BV-2 cells in the control group was?137±28?pg/ml,whereas it was?459±47?pg/ml and much higher in hypoxia.What's more,it was significantly decreased to?302±39?pg/ml with the treatment of VX-765?P<0.05?.6.Results of western blot showed that the levels of TLR4,NLRP3,IL-1?,TNF-? and VEGF in BV-2 cells of hypoxia group were much higher than that of control group,which were significantly reduced by VX-765?P<0.05?.Conclusion:Caspase-1 promotes retinal neovascularization in the mice of OIR,probably by the activation of TLR4 signaling and activating the downstream inflammatory factor IL-1?and TNF-?in microglia and accelerating the release of VEGF. |
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