| BackgroundUnder normal cornea basis of many factors in a stable "immune privilege" state, to maintain the transparency of the cornea, which is the normal physiological function of the corneal play.In certain factors, such as wearing corneal contact lens, infection, trauma, thermal burns or chemical injury, previous surgery, and other autoimmune cornea can "immune privilege" the balance is broken resulting in corneal neovascularization (corneal neovascularization, CNV).Corneal neovascularization in a certain time and the repair process is within the range of normal harmless,but beyond tolerated by normal physiological level, corneal neovascularization will lead to decreased vision, in addition to causing blindness, but also accompanied by a high rate of rejection after corneal transplantation.For the pathogenesis of corneal neovascularization is unclear,recent studies showed that the incidence of angiogenesis and angiogenic factors and anti-angiogenic factor imbalance, limbal vascular barrier damage, hypoxia, inflammation, corneal edema, and other relevant factors are involved.At present the clinical treatment of corneal neovascularization are the anti-angiogenesis and pro-vascular degeneration,there are specific medications such as hormones, non-steroidal anti-inflammatory drugs, VEGF inhibitors,etc;gene therapy such AS ES, pigment epithelium derived factor (PEDF) and angiogenesis inhibition (AS), etc;laser therapy; photodynamic therapy; surgical treatment such as electrolysis needle cauterization, ocular surface reconstruction, etc.Given the in-depth study of CNV pathogenesis, treatment of CNV has also made great progress, but there is no ideal drug for the treatment of corneal neovascularization on current clinical.Looking for strong effect, less side effects, economic price, the more effective method of angiogenesis inhibitors are still hot topics in the study of CNV.Ursolic acid is one of the triterpene compounds in natural plants, there are various biological activities,such as anti-inflammatory, antibacterial, anti diabetes, lower blood sugar, anti-cancer, resistance to promote cancer and so on.In recent years studies have found that ursolic acid on a variety of malignant tumors induce differentiation and anti-angiogenic effect, not only can inhibit the growth of tumor cells, but also inhibit the formation of tumor angiogenesis.VEGF is an angiogenic biological effects of the peptide, is one of the most important factors regulating angiogenesis, it is possible to adjust the development of hematopoietic stem cells, modifications of inflammatory cytokines and extracellular matrix and is a growth factor specificed role in vascular endothelial cell.Matrix metalloproteinases is a kind of Zinc protease,that can degrade the basement membrane and an extracellular matrix,it involved in angiogenesis.Experiments have shown that VEGF can through a variety of cytokines way effects on vascular endothelial cells,to stimulate the degradation of extracellular matrix,induced endothelial cell proliferation, migration, differentiation, and into the role of the tube.In vitro experiments also showed that UA can restrain the migration of endothelial cells, effectively improve the expression of urokinase and MMP-2, inhibit endothelial cell tube formation.Ursolic acid research used for corneal neovascularization lags behind,thus,our experiments by observing the cornea toxic effects of ursolic acid, the topical application of ursolic acid of eye drops to inhibit the corneal neovascularization, and discuss the possible mechanism.Part I Toxicological study of ursolic acid eye drops on rat corneasObjectiveTo observe the toxicity of ursolic acid drops in rat corneas and determine the safe concentration for experimental and clinical use,to lay the foundation about ursolic acid drops for the application of corneal neovascularization.MethodsUrsolic acid was dissolved in PBS solution with DMSO cosolvent under sterile conditions,were made2mg/ml,4mg/ml,6mg/ml,8mg/ml ursolic acid drops respectively.48SPF level female SD rats were randomly divided into PBS solution group, PBS solution with DMSO group,2mg/mlUA group,4mg/mlUA group,6mg/mlUA group,8mg/mlUA group.Eye drops were applied four times daily and one drop per time,seven consecutive days.Rat corneas were checked by slit lamp microscope everyday. Seven days later,histopathological methods were carried out on all right corneas and left the micro structure of the corneal tissue were evaluated under light microscope.ResultsNo significant changes were observed among PBS solution group, PBS solution with DMSO group,2mg/ml,4mg/ml,6mg/ml UA group.On the forth day swelled corneal epithelia was observed in the8mg/ml UA group,and no further damages were found as time went by.Histological examination showed that epithelial basal cell layer of the cornea were disorganized, cell polarity were disappeared.Conclusion2mg/ml,4mg/ml,6mg/ml ursolic acid eye drops do no harm to rat corneas.Part II The inhibitory effect and mechanism research of Ursolic acid eye drops on corneal neovascularization in ratsObjectiveTo study the inhibition and mechanism about the ursolic acid drops on corneal neovascularization and to investigate the expressions of VEGF and MMP-2in rat cornears.MethodsBefore the model were as normal control group randomly5rats, corneal neovascIllarization was induced by suture method,and then50SD rats were randomly assigned to five groups:Drugs were delivered four times daily after operations.Rat corneas were checked by slit lamp microscope everyday on the first week, every three days to observe the rat corneas situation in the second week.The length of corneal neovaseularization was measured at the third,the seventh and the fourteenth day,so the area of corneal neovascularizafion was recorded.4、7、14days later the expressions of VEGF and MMP-2were detected with immunohistochemical techniques. Explore the possible mechanism about ursolic acid inhibited corneal neovascularization.ResultOn4th,7th,14th days, the area of CNV control group were (8.11±0.89) mm2、(25.00±1.75) mm2,(38.00±0.36) mm2respectively; the CNV area of2mg/mlUA eye drops were (4.98±1.20)mm2、(19.97±1.47)mm、(36.74±0.73)mm2;4mg/mlUA eye drops were (2.18±1.08)mm2、(15.63±1.57)mm2、(27.15±1.14)mm2;6mg/mlUA eye drops were (0±0) mm2、(11.35±1.39) mm2、(20.83±0.60) mm2; the CNV area of Dexamethasone Sodium Phosphate Eye Drops were (0±0) mm2、(11.97±1.13) mm2、(21.40±0.68) mm2respectively.From the all time points, PBS control group area of CNV is significantly higher than the rest of the four groups (P<0.05), the difference was statistically significant.On the same time, the area of CNV about PBS control group was significantly higher than the other four groups, the difference was statistically significant (P<0.05).Among the three different doses of UA drops through to the pairwise comparison, the difference was statistically significant CNV area (P<0.05), Therefore, three different doses of ursolic acid can significantly inhibit the growth of CNV in rats, with the increase of dose, inhibiting ability enhancement. The CNV area of6mg/mlUA eye drops group compared with dexamethasone group (P>0.05), there was no statistically significant difference.Corneal tissue biopsy results:Group A, on4th the local hyperplasia cortex, substrate layer visible neovascularization, new blood vessels around the inflammatory cell infiltration of the corneal.On7th and14th the cornea thickening, cortex with inflammatory cells infiltration, the substrate layer has a large number of new blood vessels, with inflammatory cells infiltration, endothelial no new blood vessels;Group B, on the corneal layer with inflammatory cells infiltration, the substrate near the cortex layer has a new blood vessel formation, inflammatory cells infiltration around it on4th;On7th and14th the thickening of the cornea, the cortex is disorder, inflammation cells infiltration, the substrate layer has a large number of new blood vessels, mainly in the majority in the upper, new blood vessels around the visible more inflammatory cells infiltration, endothelial no new blood vessels. On4th the corneal layer with inflammatory cells infiltration, stromal layer accidentally see new blood vessels form, new blood vessels around with inflammatory cells infiltration;On7th and14th a thickening of the cornea, the epithelium with inflammatory cells infiltration, stromal layer visible new blood vessels form, new blood vessels with inflammatory cells infiltration around, did not see new blood vessels in cortex. Group D and grouo E,on4th corneal epithelium is complete, did not see new vascular structure.On7th and14th corneal epithelium is complete, substrate layer is a few of new blood vessels, new blood vessels around with a few inflammatory cells infiltration, did not see new blood vessels in cortexImmunohistochemical staining results:Normal corneal epithelium and basement membrane a small amount of expression of VEGF, MMP-2; group A、B、C on4th the corneal full-thickness expression of VEGF, MMP-2, on7th expression enhancement, on14th expression decreased significantly;group D and E On4th Corneal suture, cortex, stromal layer appear the expression of VEGF, MMP-2on the forth day, on7th the corneal full-thickness are expressed, mainly in cortex and around the new blood vessels, the14th day the expression of VEGF、MMP-2are decreased.ConclusionUrsolic acid eye drops can effectively restrain the growth of corneal neovascularization by the induced sutures.With increasing doses, inhibition of angiogenesis effect is more significant and dose dependent.Ursolic acid may be by reducing the expression of VEGF, MMP-2is to inhibit the growth of corneal neovascularization. |
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