| Objective:To evaluate the effect of puerarin and extract of Ginkgo biloba improved retinal and choroidal blood flow, study the effection of puerarin and Ginkgo biloba extract on retinal vascular endothelial cells and choroidal vascular endothelial cells secretion of eNOS and ET-1from the perspective of molecular biology. Investgate the molecular biological mechanism of puerarin and Ginkgo biloba extract to improve the retina and choroidal blood flow, thereby to provide an objective basis and guidance for clinical application, also to provide new ideas and methods for future research of Huoxue Recipe.Methods:1. Fouty one Anterior ischemic optic neuropathy patients were randomly divided into two groups, and accepted puerarin or Ginkgo biloba extract injection of intravenous therapy combined oral Buyanghaiwu decoction.Colordoppler flow imaging technique was used to detect the PSV,EDV,RI of ophthalmic artery and central retinal artery of the two groups before and after treatment. All data were dealt with SPSS17.0statistical software.2. Fouty one Anterior ischemic optic neuropathy patients were randomly divided into two groups, and accepted puerarin or Ginkgo biloba extract injection of intravenous therapy. Bulbar conjunctiva microcirculation monitoring technology was used to detect the change in the bulbar conjunctiva blood flow of the two groups before and after treatment. All data were dealt with SPSS17.0statistical software.3. Retinal vascular endothelial cells and choroidal vascular endothelial cells Were cultured, and the effect of Puerarin and Ginkgo biloba extract on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay. Using western blotting assay to detect the effection of puerarin and Ginkgo biloba extract on retinal vascular endothelial cells and choroidal vascular endothelial cells secretion of eNOS and ET-1.Results:l.The PSV of ophthalmic artery was significantly increased after treatmen and there was a statistically significant difference between before and after treatment(P<0.05). The EDV of ophthalmic artery was increased after treatmen, and there was no statistically significant difference between before and after treatment(P>0.05). The RI of ophthalmic artery was decreased after treatmen, and there was no statistically significant difference between before and after treatment(P>0.05). The PSV of central retinal artery was significantly increased after treatmen, and there was a statistically significant difference between before and after treatment(P<0.05). The EDV of central retinal artery was increased after treatmen, and there was no statistically significant difference between before and after treatment(P>0.05). The RI of central retinal artery was decreased after treatmen, and there was no statistically significant difference between before and after treatment(P<0.05).2.The weighted score of bulbar conjunctiva microcirculation in Puerarin group after the infusion is less than before infusion, the difference was statistically significant (P<0.05). The weighted score of bulbar conjunctiva microcirculation in Ginkgo biloba extract group after the infusion is less than before infusion, the difference was statistically significant (P<0.05).3.Endothelial cells identification:factor Ⅷ is located in cytoplasm. Immunofluorescence chemistry shows the culture cells cytoplasm with yellow-green fluorescent coloring, nucleus was dark green, the contour of cells are clear, and it indicated these cells are endothelial cells.4. the key factor of Endothelial cells culture:20g L-1gelatin can obtain higher cell adherent rate;in addition, endothelial cell medium is the key to maintaining culture. This study uses DMEM and Ham’F12as basic culture medium, addition of fetal bovine serum, ECGF and heparin, the results showed that it can meet the growth of endothelial cells. Fetal bovine serum concentration and quality also have a crucial role on the growth of endothelial cells. Endothelial cells are density dependent growth cells and if the cells were too thin, it will be detrimental to cells growth. The results showed3×104/ml density is conducive to the growth of endothelial cells. 5.The effect of Puerarin on the proliferation of RVECs was measured by methyl thiazolyl thiazolium (MTT) assay. The results showed Puerarin has apparent stimulative effects on proliferation of RVECs. The OD values of24and48h five concentration of Puerarin group were higher than those in blank control group (P<0.05), RVECs proliferation was markedly increased with the drug concentration increased and the OD values also increased. It showed Puerarin has obvious promoting proliferation effect on RVECs. The effect of EGB on the proliferation of RVECs was also measured by methyl thiazolyl thiazolium (MTT) assay. The results showed EGB has apparent stimulative effects on proliferation of RVECs. The OD values of24h five concentration of EGB group were higher than those in blank control group (P<0.05), RVECs proliferation was markedly increased with the drug concentration increased and the OD values also increased. In48h the OD values of0.001,0.01,0.1g/ml were higher than blank control group (P<0.05); OD values of0.0001u g/ml group were higher than those in blank control group, but has no statistical difference (P>0.05); OD values of1u g/ml group were lower than those in blank control group (P<0.05). It showed EGB has obvious promoting proliferation effect on RVECs.6. The effect of Puerarin on the proliferation of CVECs was measured by methyl thiazolyl thiazolium (MTT) assay. The results showed Puerarin has apparent stimulative effects on proliferation of CVECs. The OD values of24and48h four concentration of Puerarin group were higher than those in blank control group (P<0.05), CVECs proliferation was markedly increased with the drug concentration increased and the OD values also increased. It showed Puerarin has obvious promoting proliferation effect on CVECs. The effect of EGB on the proliferation of CVECs was also measured by methyl thiazolyl thiazolium (MTT) assay. The results showed EGB has apparent stimulative effects on proliferation of CVECs. The OD values of24and48h four concentration of EGB group were higher than those in blank control group (P<0.05), CVECs proliferation was markedly increased with the drug concentration increased and the OD values also increased. It showed EGB has obvious promoting proliferation effect on CVECs.7. Compared with the negative control group, eNOS relative expressions in EGB and puerarin low concentration and high concentration group were increased (P<0.01), and eNOS expression were also increased with the increase of the concentration of drugs; the ET-1 relative expressions of EGB and puerarin in low concentration and high concentration group were decreased (P<0.01). ET-1expression between low concentration and high concentration of EGB has no significant difference(P>0.05), while ET-1expression in puerarin were increased with the increase of the concentration of puerarin.(P<0.05)。Conclusion:1.This study found that the puerarin and Ginkgo biloba extract combined oral Buyanghaiwu decoction can improve AION patients with eye ophthalmic artery and central retinal artery perfusion pressure, improve blood flow velocity of ophthalmic artery and central retinal artery, thereby improving optic nerve ischemia.In order to observe the single effect to the ocular blood flow of the puerarin and EGB, we detected the blood flow of the bulbar conjunctiva before and30minites after infusion and found that puerarin and Ginkgo biloba extract can also improve the blood flow of the bulbar conjunctiva. And thus we Speculate that puerarin and Ginkgo biloba extract can improve the ocular artery and anterior ciliary artery blood flow. So the comprehensive analysis of colordoppler flow imaging and conjunctival blood flow observation, we can speculate that puerarin and Ginkgo biloba extract can improve ocular blood flow of AION patients, can not only improve the retinal blood supply, but also improve the choroidal blood supply.2.Whether in RVECs or CVECs, eNOS expression were increased and ET-1expression are reduced in the Experimental group, and thus we can speculate that puerarin and Ginkgo biloba extract may regulate vascular nitric oxide endothelin (NO/ET) ratio, Maintenance of endothelium-derived relaxing factor (EDRF) and endothelium dependent contraction factor (EDCF) function of dynamic balance, and thus play a relaxation of retinal and choroidal vascular smooth muscle, improvement of retinal and choroidal blood flow. |
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