| ObjectiveTo explore the role and regulatory mechanism of "Zhixue Huayu Lishui Decoction" in treating retinal vein obstructive macular edema and its neovascular-related complications from the hypoxia-VEGF pathway through in vitro cell experiments and in vivo animal experiments,which provides a preliminary theoretical basis for the treatment of RVO-macular edema and other neovascular-related complications by "discussing the treatment of collateral disease"and "regulating blood and treating water" in the traditional Chinese medicine(TCM),and enriches the theory of TCM treatment of fundus blood syndrome.Methods1.To observe the effect of "Zhixue Huayu Lishui Decoction" on the expression of VEGF and its related factors in ARPE-19 cells induced by hypoxia through in vitro cell experiments,and explore its mechanism of action:ARPE-19 cells were cultured in vitro,and logarithmically well-growing cells were cultured for 24h,48h and 72h with different concentrations of CoCl2.Cell proliferation activity was detected by the CCK-8 method,and the optimal concentration of CoCl2 was selected for the establishment of the hypoxia model.The cells were divided into normal group,blank group of traditional Chinese medicine,hypoxia model group and drug intervention group.The concentration of VEGF protein was detected by ELISA method in the cell supernatant;The expression levels of HIF-1α,NF-κB p65,NF-κB p-p65 protein were detected by Western Blot method in cells;The expression levels of VEGFmRNA and HIF-lamRNA were measured by Realtime-RT-PCR method in cells.For details,please refer to the first chapter of experiment.2.To observe the effect of "Zhixue Huayu Lishui Decoction" on the expression of VEGF and its related factors in retinal tissue of retinal vein occlusion model rabbits through in vivo animal experiments,and explore its mechanism of action:Photochemical method induced RVO model in New Zealand rabbits.After successful modeling,RVO rabbits were randomly divided into model group and traditional Chinese medicine group,and the normal control group was from normal New Zealand rabbits of the same age without modeling.The test time nodes of each indicator are respectively 7d,14d,21d and 28d after modeling.The distribution of VEGF,HIF-1α,NF-κB p65 protein in rabbit retina was detected by immunohistochemistry;The expression levels of VEGF,HIF-1α,NF-κB p65 protein in rabbit retina were detected by Western Blot method;The expression levels of VEGFmRNA and HIF-1αmRNA in rabbit retina were detected by Realtime-RT-PCR.For details,please refer to the second chapter of experiment.Results1.In vitro cell experiments(1)Establishment of cell hypoxia model:in the experiment,CCK-8 method was used to detect the proliferation activity of ARPE-19 cells.The results confirmed CoCl2 could induce hypoxia in cells,and filtered out the optimal concentration of CoCl2 used for modeling was 200umol/L and the optical concentration of drug-containing serum was 20%,which were used in the following experimental studies.(2)Results of cell ELISA test:the VEGF protein concentration in each cells group showed an increasing trend over time at 24h,48h and 72h.Compared with the normal group,the hypoxia model group was increased,the difference was statistically significant(t24h=9.794,t48h-7.447,t72h=10.466,P24,48,72h(0.001).Compared with the hypoxia model group,there was no significant difference between the blank control group and it(P>0.05),the positive control group was significantly decreased(t24h=2.897,P24h<0.01;t48h=2.308,P48h<0.05;t72h=4.748,P72h<0.001),the traditional Chinese medicine group was significantly reduced(t24h=4.242,t48h=3.430,P24,48h<0.001;t72h=2.501,P72h<0.01).Comparing the traditional Chinese medicine group with the positive control group,there was significant difference between the two groups at 48h and 72h(t48h=2.429,t72h=1.933,P48,72h<0.05).(3)Results of cell Western blot test:①The relative expression results of HIF-1αprotein in each group showed that,compared with the normal group,there was no significant difference in the blank group of traditional Chinese medicine(P>0.05).The hypoxia model group was increased at all time points,the difference was statistically significant at 72h(t=4.593,P<0.01).Compared with the hypoxia model group,the traditional Chinese medicine group was reduced at all time points,the difference was statistically significant at 72h(t=2.873,P<0.05).②NF-KB p65 and NF-KB p-p65 protein were not expressed in the hypoxia model group,and were slightly expressed in the traditional Chinese medicine group.(4)Results of cell Realtime-RT-PCR test:①The expression of VEGFmRNA showed that,compared with the normal group,the hypoxia model group was increased at all time points,the difference was statistically significant(t24h=4.946,t48h=12.863,P24,48h<0.001;t72h=5.561,P72h<0.05).Compared with the hypoxia model group,the positive control group and the traditional Chinese medicine group were decreased at 24h and 48h,increased at 72h.The difference at 24h,48h,and 72h in the positive control group was statistically significant(t24h=3.437,P24h<0.01;t48h=13,554,P48h<0.001:t72h=2.219,P72h<0.05),and the difference in the traditional Chinese medicine group was statistically significant(t24h=3.084,t72h=6.115,P24h.72h<0.01;t48h=14.837,P48h<0.001).But there was no significant difference between the two groups(P>0.05).②The expression of HIF-1αmRNA showed that,compared with the normal group,the hypoxia model group was increased at all time points,and the difference was statistically significant(t24h=2.658,P24h<0.05;t48h=6.150,P48h<0.001;t72h=3.203,P72h<0.01).Compared with the hypoxia model group,the positive control group and the traditional Chinese medicine group were reduced at all time points,and the difference at 48h was statistically significant(t 阳性对照组=14.918,t 中药组=15.358,P阳性对照,中药组<0.001).The pairwise comparison between the two groups showed no significant difference(P>0.05).2.1n vivo animal experiments(1)Establishment of RVO rabbit model:in the experiment,the observation under the fundus microscope and the inspection results of FFA and fundus photos showed all the experimental rabbits appeared the performance of photocoagulated vein occlusion after modeling,and the animal mortality was relatively low,which confirmed the RVO animal model established by this method was safe and reliable.(2)Results of rabbit retina immunohistochemistry text:①VEGF:a small amount of VEGF was expressed in normal rabbit retinal vascular endothelial cells,ganglion cell layers,inner and outer nuclear layer,RPE cells and choroidal vascular endothelial cells.The positive expression of VEGF in the model group and the traditional Chinese medicine group was mainly found in the retinal vascular endothelial cell cytoplasm,ganglion cell nucleus,RPE cell cytoplasm and choroidal vascular endothelial cell cytoplasm in the model area.AOD results showed that,the positive expression of VEGF on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,the difference was statistically significant(t7d=10.232,t14d=9.895,t21d=10.952,t28d=12.772,P7-28d<0.001).Compared with the model group,it was weakened in the traditional Chinese medicine group after intervention with traditional Chinese medicine,and the difference was statistically significant(t7d=2.233,P7d<0.05;Z14d=2.873,P14d<0.01;t21d=6.648,t28d=7.710,P21d.28d<0.001).AG results showed that,the positive expression of VEGF on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,and the difference was statistically significant(t7d=6.847,t14d=8.751,t21d=9.637,t28d=8.417,P7-28d<0.001).Comparedwith the model group,it was weakened in the traditional Chinese medicine group on 21d and 28d,and the difference was statistically significant(t21d=3.967,P21d<0.001;t28d=3.532,P28d<0.01).The results of AOD and AG were generally consistent,the expression level in the model group was relatively stable,and the positive expression in the traditional Chinese medicine group gradually weakened with time.②HIF-1α:A small amount of HIF-1α was expressed in normal rabbit retinal vascular endothelial cells,ganglion cell layers,inner and outer nuclear layer,RPE cells and choroidal vascular endothelial cells.The positive expression of HIF-1α in the model group and the traditional Chinese medicine group was mainly found in the retinal vascular endothelial cell cytoplasm,ganglion cell nucleus,inner and outer nuclear layer,RPE cell cytoplasm and choroidal vascular endothelial cell cytoplasm.AOD results showed that,the positive expression of HIF-1α on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,and the difference was statistically significant(Z7-28=3.780,P7-28d<0.001).Compared with the model group,it was weakened in the traditional Chinese medicine group,and the difference was statistically significant(t7d=5.041,t14d=10.218,t28d=4.766,P7d,14d,28d<0.001;t21d=3.451,P21d<0.01).AG results showed that,the positive expression of HIF-1α on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,and the difference was statistically significant(t7d=11.795,t14d=8.751,P7d.14d<0.001;t21d=2.674,Z28d=2.419,P21d,28d<0.05).Compared with the model group,it was weakened in the traditional Chinese medicine group on 7d,14d and 28d,but the difference was statistically significant on 14d(t=4.358,P<0.001).The results of AOD and AG showed the expression trend of the model group was basically the same,with the strongest expression on 14d;the positive expression in the traditional Chinese medicine group was generally lower than that in the model group,and its expression gradually weakened with time.③NF-KB p65:In the retina of normal group rabbits,the ganglion cell nuclei and the inner and outer nuclear layer cell nuclei had brownish yellow positive staining,which was scattered.The positive expression of NF-KB p65 in the model group and the traditional Chinese medicine group was mainly found in the retinal vascular endothelial cell cytoplasm,ganglion cell nuclei,inner and outer nuclear layer cell nuclei.AOD results showed that,the positive expression of NF-KB p65 on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,and the difference was statistically significant(t7d=4.264,t14d=15.027,t21d=11.026,t28d=8.073,P7-28d<0.001).Compared with the model group,it was weakened in the traditional Chinese medicine group,the difference was statistically significant on 14d,21d,28d(t14d=4.579,t28d=5.232,P14d,28d<0.001;t21d=3.266,P21d<0.01).AG results showed that,the positive expression of NF-KB p65 on 7d-28d after modeling,compared with the normal group,was enhanced in the model group,and the difference was statistically significant(t7d=8.026,t14d=8.426,t21d=8.013,t28d=4.478,P7-28d<0.001).Compared with the model group,it was weakened in the traditional Chinese medicine group,and the difference was statistically significant(t7d=4.579,t14d-3.769,P7d,14d<0.001;t21d=3.176,P21d<0.01;t28d-2.531,P28d<0.05).T-he results of AOD and AG were generally consistent.The expression in the model group was highest on 14d,and the expression in the traditional Chinese medicine group was gradually weakened with time.(3)Results of rabbit retina western blot text:①The expression of VEGF protein showed that,its expression in the cytoplasm of retinal cells:compared with the normal group,the model group was decreased on all time points;compared with the model group,the traditional Chinese medicine group was decreased on 7d,14d and 21d,and gradually decreased with time.But its expression in the nucleus of retinal cells:compared with the normal group,the model group increased on 14d,21d and 28d;compared with the model group,the traditional Chinese medicine group was decreased on 14d,21 d and 28d,and gradually decreased with time.However,there was no significant difference between the above groups(P>0.05).②The expression of HIF-1α protein showed that,its expression in the cytoplasm of retinal cells:compared with the normal group,the model group was increased on 7d and 21 d;compared with the model group,the traditional Chinese medicine group was decreased on 21d.But its expression in the nucleus of retinal cells:compared with the normal group,the model group was increased on 14d;compared with the model group,the traditional Chinese medicine group was decreased on 7d,14d and 21d.However,there was no significant difference between the above groups(P>0.05).③The expression of NF-KB p65 protein showed that,its expression in the cytoplasm of retinal cells:compared with the normal group,the model group was increased on 7d,21d and 28d;compared with the model group,the traditional Chinese medicine group was decreased on 7d and 21d.But its expression in the nucleus of retinal cells:compared with the normal group,the model group was increased on all time points;compared with the model group,the traditional Chinese medicine group was decreased on all time points.However,there was no significant difference between the above groups(P>0.05).(4)Results of rabbit retina Realtime-RT-PCR text:①Relative expression of VEGFmRNA:compared with the normal group,the expression level of the model group was increased on 7d,14d and 21d,and the difference was statistically significant on 7d and 14d(t7d=2.935,t14d=3.149,P7.14d<0.05).Its expression level was highest on 14d.Compared with the model group,the expression level of the traditional Chinese medicine group was decreased on each time point,but there was no significant difference(P>0.05).②Relative expression of HIF-1αmRNA:compared with the normal group,the expression level of the model group was increased on each time point,and the difference was statistically significant on 14d(Z=-2.087,P<0.05).Its expression level was highest on 14d;Compared with the model group,the expression level of the traditional Chinese medicine group was decreased on 7d,14d,21d,but there was no significant difference(P>0.05).Conclusion1.CoCl2 could successfully induce the hypoxia model of ARPE-19 cells to better simulate the hypoxia environment in vivo.2.1n vitro cell experiments:the expression of VEGF and HIF-1α increased in hypoxia-induced ARPE-19 cells,suggesting that cells under the hypoxia environment trend the high expression of the above two factors.3."Zhixue Huayu Lishui Decoction" and Fufang Xueshuantong’s drug-containing serum inhibited the proliferation of ARPE-19 cells and the expression of VEGF and HIF-1α after hypoxia injury.Therefore,it is speculated that improving the hypoxia environment and inhibiting VEGF are the key mechanism for the role of traditional Chinese medicine in the treatment of RVO.4.Photochemical method could successfully induce RVO rabbit model,which has the advantage of low animal mortality and high success rate.5.In vivo animal experiments:the expression of VEGF,HIF-1α and NF-KB p65 increased in the retina of RVO rabbit model,especially on 14d,suggesting the above three factors were involved in the pathological process of RVO and its complications and 14d is the turning point in time when correlation factors were maintained.6."Zhixue Huayu Lishui Decoction" had a better inhibitory effect on VEGF,HIF-1α and NF-KB p65 in the retina of RVO rabbit model,indicating the traditional Chinese medicine might improve the retinal ischemia and hypoxia pathway and reduce the production of neovascular growth-related factors such as VEGF,so as to achieve the purpose of preventing and treating ME and neovascular complications caused by RVO.7.Combining the results of in vitro and in vivo experiments showed "Zhixue Huayu Lishui Decoction" might have better prevention and treatment effect on RVO-ME and neovascular complications through hypoxia-VEGF pathway. |
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