| Part one:The effect of artesunate on CoCl2-induced hypoxia in ARPE-19 cells.Objective To investigate the effects of ART on HIF-1? and VEGF factor mRNA and the expression of NF-?B and ICAM-1 proteins in ARPE-19 cells induced by CoCl2.Methods1.The chemical hypoxia model of ARPE-19 cells was induced by CoCl2.The concentrations of modeling reagent CoCl2,experimental drug ART and positive control drug TSA were screened by MTT method.2.ARPE-19 cells were divided into normal group,model group,high,medium,low and very low concentration groups of ART,high,medium and low concentration groups of TSA.Immunofluorescence staining was used to detect the fluorescence expression of HIF-la and VEGF in different groups.QRT-PCR was used to detect the relative expression of HIF-la and VEGF factor mRNA in different groups.Western-blot was used to detect the relative expression of NF-?B and ICAM-1 protein in different groups.Results1.MTT cell activity test:at the same time,with the increase of CoCl2 concentration,the cell activity increased at first and then decreased.At 24 h,48 h and 72 h,the concentration of CoCl2 at 400 ?mol/L was significantly lower than that of normal cultured cells(P<0.05).At the same time,with the increase of the concentration of ART,the cell activity decreased gradually,and the concentration of ART>80 ?mol/L at 24 h was significantly lower than that of normal cultured cells(P<0.05),and the concentration of ART>40 ?mol/L at 48 h and 72 h was significantly lower than that of normal cultured cells(P<0.05).At 48 h,the concentration of TSA>10 ?mol/L was significantly lower than that of normal cultured cells,and at 72 h,the concentration of TSA?2.5 ?mol/L was significantly lower than that of normal cultured cells.Therefore,we chose 200 pmol/L CoCl2 to make the model,40,20,10 and 5 ?mol/L ART as high,medium,low and very low concentration groups,and 10,5 and 2.5 ?mol/L TSA as high,medium and low concentration groups of positive control drug,and intervened for 24 hours for follow-up experimental study.2.Immunofluorescence expression:The expression of HIF-1? in the model group was significantly higher than that in the normal group(P<0.05).The expression level of ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration group was significantly lower than that of model group(P<0.05).The expression level of ART low concentration group,ART very low concentration group and TSA high concentration group was also significantly lower than that of normal group(P<0.05).The immunofluorescence expression of VEGF in the model group was significantly higher than that in the normal group(P<0.05).The expression level of ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration group was significantly lower than that of model group(P<0.05).3.qRT-PCR detection:the relative expression of HIF-la mRNA in the model group was significantly higher than that in the normal group(P<0.05).The relative expression of HIF-1? mRNA in ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration group was significantly lower than that in the model group(P<0.05),and also lower than that in the normal group(P<0.05).The expression of VEGF mRNA in the model group was significantly higher than that in the normal group(P<0.05).The relative expression of VEGF mRNA in ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration groupwas significantly lower than that in the model group(P<0.05).4.Western-blot detection:From the relative expression of NF-?B protein,it can be seen that the model group was significantly higher than the normal group(P<0.05).ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration group were significantly lower than the model group(P<0.05).From the relative expression of ICAM-1 protein,it can be seen that the model group was significantly higher than the normal group(P<0.05).ART high concentration group,ART medium concentration group,ART low concentration group,ART very low concentration group,TSA high concentration group,TSA medium concentration group,and TSA low concentration group are significantly lower than the model group(P<0.05).Conclusions1.CoCl2 can successfully establish the model of chemical hypoxia in ARPE-19 cells.2.CoCl2 could promote the expression of HIF-la,VEGF mRNA and fluorescent staining,as well as the expression of NF-?B and ICAM-1 protein in ARPE-19 cells.3.ART could inhibit the expression of HIF-la and VEGF mRNA in ARPE-19 cells induced by CoCl2,as well as the expression of NF-?B and ICAM-1 protein,which had protective effect.Part two:Establishing the model of branch retinal vein occlusion in Wistar rats induced by photochemical method.ObjectiveTo establish a Wistar rat model of BRVO induced by rose bengal and 532 nm laser,so as to provide a model reference for the animal study of BRVO and lay a foundation for follow-up experimental research.Methods1.The experimental BRVO model of Wistar rats was established by injecting rose bengal into tail vein combined with 532 nm laser by photochemical method.Fundus photography,FFA,retinal smear and vascular occlusion were evaluated at 1,3,7,14 and 21 days after modeling.2.Make the histopathological specimens of the BRVO eye of Wistar rats,and observe the changes of the pathological tissues of the retina after modeling by HE staining.Results1.Fundus photography:In the normal group,the retinal arteriovenous vessels were uniformly distributed alternately,and the choroidal vessels were vaguely seen.In the 1-day model group,the blood flow of the retinal vein was interrupted and the terminal vein was dilated.In the 3-day model group,the retinal occlusive vein was uneven in diameter,showing sausage-like changes,partial venous blood flow recanalized and tortuous.In the 7-day model group,most of the retinal occlusive veins recanalized,the veins tortuously dilated,and the diameter was uneven.In the 14-day model group,the end of the retinal vein was tortuous and the blood vessels were radially shaped.While in the 21-day model group,the retinal vessels were radially shaped and uniform in diameter.2.FFA:In the normal group,the retinal filling time was normal,the diameter of the blood vessel was uniform,the diameter of the vein was thicker than that of the artery,and there was no telangiectasia and fluorescent leakage.In the 1-day model group,the filling of the retinal vein was slow,the vessels in the occlusive part showed low fluorescence,accompanied by fluorescent leakage.In the 3-day model group,the diameter of the retinal vein was uneven,partial recanalization,and telangiectasia was seen in the supply area of the occluded vein.In the 7-day model group,the retinal filling time was normal,the diameter of the retinal vein was uneven,the occlusive vein was unobstructed and the terminal blood vessel was tortuous.In the 14-day model group,the retinal filling time was normal,the diameter of the retinal vein was slightly uneven,and the obstruction vein was unobstructed and the terminal blood vessel was tortuous.In the 21-day model group,the retinal filling time was normal,the diameter of the retinal vein was basically uniform,the occlusive vein was unobstructed,and the terminal vessel was tortuous.3.Retinal smears:In the normal group,the retinal vessels were clear and no bleeding spots were seen.In the 1-day model group,the retina was scattered along the vein in a large number of bleeding spots,bright red in color.In the 3-day model group,the retina was scattered along the vein in a large number of bleeding spots,bright red in color.In the model group on the 7-day,and the hemorrhage was absorbed in the peripheral part of the retina in the model group on the 14th day,with a small amount of bleeding points scattered around the retina,the color was dark red.And in the model group,the retina was scattered in a small amount of bleeding points in the periphery of the retina in 14-day.At the 21-day,the retinal hemorrhage in the model group was basically absorbed and the blood vessels were clear.4.Pathological of retina:In the normal group,the structure of the retina was intact and clear,and the ganglion cell layer,inner nuclear layer and outer nuclear layer were arranged closely.While in the model group,the retinal layer was not clear and the retinal edema.The arrangement of ganglion cell layer,inner nuclear layer and outer nuclear layer was loose,and the gap between cells could be seen.Conclusions1.The experimental BRVO model of Wistar rats could be successfully established by photochemical model.2.The BRVO model of Wistar rats developed the most at 3 days after photocoagulation,and the recanalization rate of blood vessels was high,and the condition gradually improved.3.The BRVO model of Wistar rats have high success rate,which can lay the foundation for experimental research.Part three:Experimental study of artesunate inhibiting branch retinal vein occlusion in Wistar rats through HIF-1?/VEGF signal pathway ObjectiveTo observe the inhibitory effect of ART on experimental BRVO in Wistar rats and to regulate the activity of factors related to HIF-1?/VEGF signal pathway,so as to provide experimental basis for ART treatment of BRVO in vivo.Methods1.Using photochemical method,tail vein injection of rose bengal combined with 532 nm laser to establish experimental BRVO model on Wistar rats.The rats were divided into blank control group,placebo control group,Conbercept group,ART high concentration group,ART medium concentration,and ART low-concentration group.Fundus photographic were performed 1,3,7,14,and 21 days after modeling.2.Make the histopathological specimens of the BRVO eye of Wistar rats,and perform HE staining to observe the changes of the retinal pathological tissues in different groups at 1,3,7,14,and 21 days after modeling.3.The relative expressions of HIF-la and VEGF mRNA in different groups were detected by qRT-PCR.Results1.Fundus photography:The diameter of the retinal arteriovenous vessels of the blank control group is uniform,distributed radially,and choroidal vessels are faintly visible.The diameter of the 1-7 days model group and the drug intervention group of different concentrations is uneven,showing sausage-like changes.Part of the venous blood flow recanalized,and the blood vessels were tortuous.The blood vessels gradually recovered in 14-21 days,and the course and thickness tended to be normal.2.Pathological changes of the retina:In the blank control group,the retina has clear layers,the ganglion cells are clearly arranged and regular,and the inner plexiform layer,inner nuclear layer,and outer nuclear layer are evenly dense and neatly arranged.The cells in each layer of the retina in the model group and the drug intervention group of different concentrations were loosely arranged,partly missing,and showed hollow changes from 1-7 days.No retinal atrophy and degeneration occurred in the Conbercept group and the ART high,medium and low concentration group in 21 days.3.qRT-PCR detection:HIF-la mRNA expression level in the placebo control group was significantly higher than that of the blank control group in 1 day after intravitreal injection(P<0.05).The Conbercept group,ART high concentration group,and ART medium concentration group were all lower than the placebo control group(P<0.05),and the ART low-concentration group was significantly higher than the Conbercept group(P<0.05).3 days after intravitreal injection,the expression of HIF-la mRNA in the placebo control group was significantly higher than that of the blank control group(P<0.05),and the Conbercept group was significantly lower than the blank control group(P<0.05).And the Conbercept group,ART high concentration group,ART medium concentration group,and ART low concentration group are lower than the placebo control group(P<0.05).Regarding the expression of VEGF mRNA,the ART high concentration group,ART medium concentration group,and ART low concentration group were significantly higher than the blank control group(P<0.05).7 days after intravitreal injection,the expression of HIF-la mRNA in the ART low concentration group was significantly higher than that of the blank control group(P<0.05),and both the ART medium concentration group and the ART low concentration group were higher than the placebo control group(P<0.05).And the ART low concentration group was significantly higher than the Conbercept group and the ART high concentration group(P<0.05).For the expression of VEGF mRNA,the placebo control group was significantly higher than the blank control group(P<0.05),and the Conbercept group was significantly higher Lower than the placebo control group(P<0.05).14 days after intravitreal injection,the expression of VEGF mRNA in the placebo control group,the ART medium concentration group,and the ART low concentration group were significantly higher than the blank control group(P<0.05),and the Conbercept group was significantly lower than the blank control group(P<0.05).The Conbercept group,ART high concentration group,ART medium concentration group,and ART low concentration group were significantly lower than the placebo control group(P<0.05),but ART high concentration group,ART medium concentration group,ART low concentration group was significantly higher than the Conbercept group(P<0.05).And the ART medium concentration group and the ART low concentration group were significantly higher than the ART high concentration group(P<0.05).21 days after intravitreal injection,the expression of VEGF mRNA in the Conbercept group was significantly higher than that in the blank control group(P<0.05).Conclusions1.The BRVO model was established by photochemical method,and the HIF-la/VEGF signal pathway was activated,which participated in the formation of BRVO.2.The expressions of HIF-la and VEGF in the model are not completely synchronized.HIF-1? increased in the early stage of the model,but VEGF increased in the later stage.3.ART can inhibit the experimental BRVO of Wistar rats by reducing the activity of HIF-1?/VEGF signal pathway. |
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