| Part Ⅰ:Effects of ginsenoside Rg3 on proliferation,migration and lumen formation of RF/6A cells induced by VEGFObjectiveTo observe the effects of ginsenoside Rg3 on the proliferation,migration and lumen formation of RF/6A cells induced by VEGF,to explore the effect of the Rg3 on on the formation of CNV,so as to provide an in vitro experimental basis for the clinical application of Rg3 in the treatment of CNV.Methods1.The CCK-8 method was used to detect the effects of ginsenoside Rg3 on the RF/6A cells at different concentrations and different times,to determine the safe concentration of ginsenoside Rg3.2.The CCK-8 method was used to detect the effects of VEGF on RF/6A cells at different concentrations and different time,to chose the best concentration of ginsenoside Rg3 and Conbercept.3.The CCK-8 method was used to detect the inhibitory effects of different concentrations of ginsenoside Rg3 and Conbercept on the proliferation of VEGF-induced RF/6A cells.The optimal concentration of ginsenoside Rg3 and Conbercept was determined.4.The RF/6A cells were divided into normal cell group,VEGF induction group,VEGF+Conbercept group,VEGF+ginsenoside Rg3 group and VEGF+Conbercept+ginsenoside Rg3 group.The effect of different groups on cell proliferation activity was observed by CCK-8 assay.5.The effect of different drugs on the migration ability of RF/6A cells was tested by cell scratch test.6.Matrigel lumen formation assay was used to test the effect of different drugs on the ability of RF/6A cells to form 1μMens.Results1.The results of CCK-8 assay showed that the high concentration of ginsenoside Rg3(270μM)interfered with the cultured cells for 24,48,72h,and the OD value was statistically significant(P<0.05).Growth was inhibited;The group induced by different concentrations of VEGF for 48h,50 ng/mL VEGF group compared with other groups,and there was no significant difference(P<0.05),the effect was the best;The10,30,90μM concentration The ginsenoside Rg3 was used to interfere with VEGF-induced RF/6A cell culture.The OD values of the cells in the 90μM group were lower than those in the 10 and 30 μM groups(P<0.05).2.The scratch test results show:24h after scratching,comparison of cell migration rates in each group,the VEGF group was significantly higher than the normal group(P<0.05),and the drug intervention group was significantly lower than the VEGF group(P<0.05).There was no significant difference between the combination group and the Conbercept group(P>0.05),and lower than ginsenoside Rg3 group(P<0.05).After 48 hours of scratching,the cell migration rate of each group was compared:the combination group<Conbercept<ginsenoside Rg3,and was lower than the VEGF group(P<0.05).3.The Matrigel lumen formation test results show:culture for 24h,comparison of the number of lumens,the VEGF group(48±3.61)was significantly higher than the normal group(34.33±3.51),and the drug group was significantly lower than the VEGF group(P<0.05).The tube-forming ability was compared:ginsenoside Rg3>Conbercept group>combination group,the difference between the groups were statistically significant(P<0.05).Conclusion1.Both ginsenoside Rg3 and Conbercept can inhibit VEGF-induced RF/6A cell proliferation,migration and lumen formation,and the combined inhibition is the strongest.2.Ginsenoside Rg3 may inhibit the formation of CNV by inhibiting VEGF-induced proliferation,migration and lumen formation of RF/6A cells.Part :Effect and Mechanism of Ginsenoside Rg3 on Experimental CNV Formation in BN RatsObjective1.To observe the feasibility of 532nm laser to induce CNV formation in BN rats.2.To observe the role of TNF-a/NF-κB and HIF-1α/VEGF signaling pathways in CNV induced in rats induced by laser.3.To observe the effect of ginsenoside Rg3 on the formation of CNV in rats induced,and the effect on the activity of TNF-α/NF-κB and HIF-1α/VEGF signaling pathway,to explore the mechanism of ginsenoside Rg3 intervention in CNV formation,for the clinical application of ginsenoside Rg3 Treatment of CNV provides the basis for in vivo experiments.Methods1.The CNV induced in rats was induced by 532nm laser(300mW power,50μm spot diameter,0.05s Exposure time),At 1 day,3 days.5 days,7 days,14days and 21 days after laser induced,the FFA and ICGA was used to observe the development of CNV.Histopathological changes of the retina and choroid were observed by HE.2.The animal model of CNV in rats were sacrificed at 3,7,and 14 days after photocoagulation,and the eyes were removed to prepare molecular biological specimens,RT-qPCR、Western Blot and ELISA were used to detect the expression changes of TNF-α/NF-κB and HIF-la/VEGF signaling pathways in experimental CNV formation.3.The animals model of CNV randomly divided into normal group,model group and ginsenoside Rg3 low,medium and high dose groups.After 7days later,The FFA,ICGA,HE,RT-qPCR and Western Blot method were used to observe the changes of corresponding indexes in each group.Results1.The FFA and ICGA test results show:there were only a small amount of photo-condensation fluorescence leakage in the Id,3d,and 5d groups,and a large amount of high fluorescence(63.33%)was observed in the 7d group.In the 14d group,the fluorescence leakage spots increased(76.67%).and the mold 21 d was stable(66.67%).The average optical density values of fluorescence leakage were compared in the 7d group(159.50±16.42)and the model 14d group(127.56±10.50),and the 14d group was compared with the 21 d group(131.49±7.19).There was no significant difference between the two groups(P>0.05).HE was used to observe the maximum central thickness of CNV at different time points:the model 14d group(75.87±7.13)was higher than the model 7d group(56.90±1.03),and the model 21d group(61.98±6.87)was not statistically significant(P>0.05).2.RT-PCR and Western Blot were used to detect the expression of target genes in the choroid complex of rat retina at different time points.The results showed that at the mRNA level,the expression levels of TNF-a and NF-κB mRNA in the model group were normal.Significantly increased(P<0.05),the model 7d group was significantly larger than the model 3d group(P<0.05),and the model 14d group was lower than the model 7d group(P<0.05).The expressions of HIF-la and VEGF mRNA in the model group were significantly higher than those in the normal group(P<0.05),and the model 7d group was significantly larger than the model 3d group(P<0.05).There was no significant difference between the model 14d and the model 7d.At the relative expression level of the protein,the expression trend of each factor protein was the same as that by RT-PCR.The relative protein concentration of TNF-a and NF-κB in rat serμM was detected by ELISA.The results showed that the expression of TNF-α and NF-κB in serum was higher than normal group(P<0.05).It was significantly larger than the model 3d group(P<0.05);the model 14d group was smaller than 7d group(P<0.05).The expression of HIF-la and VEGF in rat serum was significantly higher than that in the normal group(P<0.05),and the model 7d group was larger than the 3d group(P<0.05).The difference between the model 14d and the model 7d was not statistical.Academic significance(P>0.05).3.After 7 days of ginsenoside Rg3 low,mediμM and high dose group intervention,the FFA and ICGA results showed that there was no statistical difference between the fluorescence leakage spot ratio and the fluorescence average optical density value in the low dose group of ginsenoside Rg3 compared with the model group(P>0.05).The formation rate and average optical density of CNV in the ginsenoside Rg3 mediμM dose group were 50.00%and 102.373±6.607,respectively,lower than the low dose ginsenoside Rg3 group(P<0.05),higher than the high dose ginsenoside Rg3 group(P<0.05).).HE staining observation showed that the maximμM central thickness of CNV in each group was from small to large,ginsenoside Rg3 high dose group(32.457 ±2.884),ginsenoside Rg3 mediμM dose group(41.933 ±2.965),ginsenoside Rg3 low dose group.It has no significant difference between the two groups(P>0.05).The RT-PCR and Western Blot assays showed that at the mRNA level,the relative expression levels of the target gene mRNA in the choroidal tissue of the rats in each group were higher than those in the normal group(P<0.05).The model group was compared with the low dose group of ginsenoside Rg3.The difference was not statistically significant(P>0.05).The high dose of ginsenoside Rg3 was higher than that of ginsenoside Rg3,and the difference has statistically significant(P<0.05).At the protein expression level,the expression trends of the target genes in each group were the same as those of RT-PCR.Conclusion1.The experimental model of CNV can be successfully induced by 532nm laser.7 days to 14 days after laser photocoagulation is a period of rapid growth of CNV,which tends to be stable by 21 days.2.TNF-a/NF-KB and HIF-1α/VEGF signaling pathways were activated during laser-induced BN rat CNV formation,and the expression of related pathway factors increased,which promoted the formation of CNV in experimental animals.3.Ginsenoside Rg3 can inhibit the formation of experimental CNV,which may inhibit the expression of pathway-related factors by inhibiting the activation of early TNF-α/NF-kB and HIF-1α/VEGF signaling pathways in CNV. |
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