Comparation And Mechanism Study Of Curcumin, Salvia Miltiorrhiza And Matrine In The Prevention And Treatment Of Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a disease characterized by formation of fabric membrane in the vitreous and on the inner or the outer surface of the retina cause traction retinal detachment after rhegmatogenous retinal detachment (RD). PVR is the most serious complication after retinal detachment surgery, traumatic disease, retinal vascular disease and inflammatory disease. Retinal pigment epithelium (RPE) cells are the main one related to the development of PVR. The pathologic condition can be separated into three distinct phase: inflammatory phase, proliferation phase and fibrosis phase. The first phase involves an infiltration of RPE cells into the preretinal and subretinal place, where the cells begin to proliferate. The next includes the continuation of RPE cell proliferation and the production of an extracellular matrix. The final phase involves of fabric membrane formation and contraction resulting in RD. The inflammatory phase as an initial phase, play an important role in the occurrence of PVR. the disruption of blood-retinal barrier resulting in release of numerous cytokines to vitreous, include of the main inflammatory cytokine- interleukin-1β(IL-1β) which can stimulate the secretion of other inflammatory cytokines and adhesion molecules in RPE cells. These cytokines together promote the migration, differentiation and proliferation of RPE cells. In the research, we chose IL-1βas a stimulated factor of inflammatory in RPE cells in vitro and PVR model in vivo which is much closer to the pathologic condition of PVR.There have been numerous technical advances in the surgical management of PVR so that the retina can almost always be reattached at the time of surgery. However, recurrent detachment is common and is usually caused by recurrent proliferation, resulting in the deterioration of visual acuity. Therefore, pharmaceutical prevention and treatment of PVR are being evaluated to be the practical and promising methods. So far dozens of western medicine have been used in the prevention and treatment of PVR. Exist of cytotoxicity and lack of multiple effect limits the clinical apply of western medicine in PVR though effective on anti-proliferation in vitro cells. The traditional Chinese medicine has promising prospect because of its properties of abundance, multiple-effect and low toxicity. The traditional Chinese medicine curcumin, salvia miltiorrhiza and matrine , which separated and extracted from natural plants, have significant anti-oxidant,anti-inflammatory, anti-proliferation and anti-microorganism effect. These medicines have been extensively research in internal medicine disease, but rarely in ophthalmology disease. Therefore, our study is to assess and compare the effect of three drugs on RPE cells in vitro and PVR model in vivo, discuss the mechanism of drug action.PartⅠInhibition effect of curcumin, salvia miltiorrhiza and matrine on IL-1βinduced proliferation of rabbit RPE cells in vitroObjective:To study the effect of IL-1βwith different dose stimulate proliferation of RPE cells. To assess and make a comparison of inhibition effect among curcumin, salvia miltiorrhiza and matrine on IL-1βinduced proliferation of rabbit RPE cells.Methods:RPE cells were isolated by trypsinization in pigmented rabbits, then cultured and passaged in DMEM containing 10% fetal bovine serum. Cultured RPE cells were identified by immunochemistry and transmission electron microscopy. The fourth generations of cells were used in this experiment. The MTT assay was used to detect the effect of different concentration of IL-1β(2.5, 5, 10, 20μg/L) stimulate proliferation of RPE cells after 24, 48 and 72h. Then we chose the best concentration of IL-1βto detect the inhibition effect in curcumin (5, 10, 20μg/ml), salvia miltiorrhiza (5, 10, 20μg/ml) or matrine (100, 200, 400μg/ml) on IL-1βinduced proliferation of RPE cells after different times. The median inhibitory concentration (IC50) of curcumin, salvia miltiorrhiza and matrine in different times were analyzed by Line Regression.Results:RPE cells just isolated from the rabbit eye were in round shape and abundant in melanin. The adherence time of primary RPE cells is 48-72h, and the time of cells near confluence is 7d after culture. The adherence time of subculture cells was cut to 12h. The melanin significantly decreased in the forth generations of RPE cells. Immunohistochemistry demonstrated the strong positive response of RPE cells for keratin (AE1/AE3).All of the different concentration of IL-1βcan induced proliferation of RPE cells compared with control group (serum free medium) (p<0.05).Treatment of RPE cells with IL-1β(2.5-10μg/L) resulted in both time-dependent and dose-dependent in the effect of stimulate proliferation, and the maximal proliferation rate occurred with a concentration of 10μg/L IL-1βin 72h (p<0.05). The proliferation rates of 20μg/L were lower than 10μg/L IL-1βduring different times (p<0.05). Treatment of RPE cells with IL-1β(10μg/L) and different concentration of three drugs resulted in both time-dependent and dose-dependent in the effect of inhibit proliferation (p<0.05). The IC50 in 24, 48 and 72h: curcumin were 26.77, 19.01 and 9.45μg/ml,salvia miltiorrhiza were 33.72, 23.47 and 12.56μg/ml,matrine were 570.96, 352.25 and 97.50μg/ml。Conclusion:Amount of rabbit RPE cells cultured in vitro provide stabilized cell source for research in vitro and vivo. Proliferation of rabbit RPE cells can be stimulated by IL-1β, and maximal proliferation rate occurred with a concentration of 10μg/L IL-1β. curcumin, salvia miltiorrhiza and matrine resulted in both time-dependent and dose-dependent in inhibit of RPE cell proliferation. The best effect of inhibit proliferation of RPE cells is curcumin follows by salvia miltiorrhiza and matrine.PartⅡEffect of curcumin on interleukin-1β-induced nuclear factor-κB-dependent transcription in rabbit retinal pigment epithelium cellsObjective:we observed the effect of curcumin on IL-1β-induced migration and NF-κBp65, IκB-α, COX-2 protein and mRNA expression by Western blot, RT-PCR analysis and immunocytochemistry staining in rabbit RPE cells. The effect and mechanism of curcumin inhibition on IL-1β-induced inflammatory in RPE cells was studied in this part.Methods:The fourth generations of cells were used in this experiment. In migration research, a wound model was used to count the number of cells which entered to injure area after dealt with IL-1β(10μg/L) /and curcumin (10μg/ml) . COX-2: Protein and mRNA of RPE cells dealt with IL-1β(0.1,1,10μg/L) for 24h and IL-1β(1μg/L) /and curcumin (10μg/ml) for 24-72h were examined by Western blot and RT-PCR analysis. NF-κBp65 and IκB-α:Protein of RPE cells dealt with IL-1β(1μg/L) /and curcumin (10μg/ml) for 24-72h were examined by Western blot analysis. NF-κBp65, IκB-αand COX-2 in RPE cells dealt with IL-1β(10μg/L) /and curcumin (10μg/ml) were examined by immunocytochemistry staining.Results:1 RPE cells dealt with IL-1β(10μg/L) significantly migrate to injure area, the number of cells was more numerous than control group (serum free medium), and the migration rate was time-dependent (p<0.05). The number of cells significantly declined after dealt with curcumin (10μg/ml) and IL-1β(10μg/L), and the effect of inhibition on migration was time-dependent (p<0.05). 2 Protein and mRNA of COX-2: Different concentration of IL-1βsignificantly increase the expression of COX-2 than control group (p<0.05), and expression of COX-2 reached a maximum by 1.0μg/L IL-1β(p<0.05). RPE cells dealt with 1.0μg/L IL-1βafter 48h resulted in increase of expression of COX-2 than 24h (p<0.05), no statistically significant was found between 48 and 72h (p>0.05). Curcumin combined with IL-1βsignificantly decrease of expression of COX-2 than IL-1βsingle (p<0.05), and the effect of inhibition was time-dependent (p<0.05). 3 Protein of NF-κBp65 and IκB-α:Increase significantly of NF-κBp65 and decrease significantly of IκB-αwere detected in IL-1β-induced RPE cells (p<0.05). At 48h, expression of NF-κBp65 and IκB-αreached to maximum and minimum respectively, while no statistically significant was found between 48 and 72h (p>0.05). Treatment of RPE cells with Curcumin and IL-1βresulted in a time-dependent decrease in NF-κBp65 and a time-dependent increase in IκB-αexpression (p<0.05). 4 immunocytochemistry staining: RPE cells treated with IL-1βresulted in significantly increase of NF-κBp65, COX-2 and decrease of IκB-αthan control group (p<0.05). Curcumin combined with IL-1βcould significantly inhibit the expression of NF-κBp65, COX-2 and enhance the expression of IκB-αin RPE cells(p<0.05). Conclusion : Curcumin can significantly inhibit rabbit RPE cellsmigration induced by IL-1β; IL-1β-induced NF-κB-dependent inflammatory gene expression can be blocked by Curcumin.PartⅢExprimental study on the effect of curcumin, salvia miltiorrhiza and matrine in inhibition of proliferative vitreoretinopathyObjective:By comparison of the two different methods of establish PVR model in rabbit eyes to chose the better one for the following research. The different effect among curcumin, salvia miltiorrhiza and matrine in inhibition of proliferative vitreoretinopathy in rabbit eyes was evaluated .Methods:36 natural pigment rabbits (72 eyes) were used in this part. 18 natural pigment rabbits (36eyes) used for establish of PVR model were divided randomly into 3 groups: normal saline was injected into the vitreous in control group(12eyes); RPE cells 2×106 (0.1ml) was injected into the vitreous in RPE group(12eyes); mixed of RPE cells 2×106 and IL-1β250U(0.1 ml)was injected into the vitreous in IL-1βgroup(12eyes). 18 natural pigment rabbits (36eyes) were used for drug experiment were divided randomly into 3 drug groups, 12eyes were contained in each group. AS the same time of PVR model establishment, curcumin (0.1ml), salvia miltiorrhiza (0.1ml) or matrine (0.1ml) was injected into the vitreous respectively in each group. By 1, 3, 7, 14, 21 and 28 d after injection, the changes of anterior segment, vitreous and fundus were examined and recorded by slit lamp biomicroscope, indirect ophthalmoscope, color fundus photography and B ultrasonic examination. Classification of vitreous opacity and PVR is on the basis of fundus and B ultrasonic examination.Results:1 1th day after injection IL-1βgroup showed an intense ?inflammatory response in anterior chamber while both RPE and control group showed little or no reaction. The inflammatory response in anterior chamber of IL-1βgroup gradually peaked, and finally resolved by 14d. Both IL-1βand RPE group showed varying degrees of vitreous opacity, peaked by 14d and 7d respectively, and finally resolved by 21d in the two groups. Control group didn't show any vitreous opacity. PVR occurred earlier in IL-1βgroup, 3 eyes occurred PVR stageⅠby 1d after injection. 3 eyes(25%) in IL-1βgroup developed RD by 3d, while no eye developed RD in RPE group. 7d after injection, occurrence of PVR is 100% in both of groups. IL-1βgroup included 3 eyes of PVR stageⅠ, 2 eyes occurred PVR stageⅡ,3 eyes of PVR stageⅢ, 3eyes of PVR stageⅣand 1eye of PVR stageⅤ, incidence rate of RD is 58%. RPE group included 3 eyes of PVR stageⅠ, 5 eyes occurred PVR stageⅡ, 3 eyes of PVR stageⅢand 1 eye of PVR stageⅣ, incidence rate of RD is 33%. RD incidence rate in IL-1βand RPE group are 75% and 67% by 14d, and 93% and 67% by 28d.2 We chose intravitreal of RPE cells combined with IL-1βto establish PVR model in this research, and the effect of drugs is compared with IL-1βgroup above. Application of drugs in vitreous significantly relieved inflammatory response in anterior chamber compared with IL-1βgroup, and completely disappeared by 7d. Classification of vitreous opacity in drug group is significantly lower than IL-1βgroup, and eyes in curcumin group had the lightest vitreous opacity in the three groups. Classification of PVR in drug group is significantly lower than IL-1βgroup in different times after injection. The time of RD appeared in curcumin, salvia miltiorrhiza and matrine group is 21d, 14d and 7d. By the end of observation, classification of PVR in drug group is much lower than IL-1βgroup. Incidence rate of PVR and RD in curcumin, salvia miltiorrhiza and matrine group is 8%, 17% and 25% respectively.Conclusion:Intravitreal of RPE cells combined with IL-1βis more effective than RPE cells alone to establish PVR model in rabbit eyes model. This method show higher Incidence of PVR and RD, and better reflect ?pathological process of PVR. Curcumin, salvia miltiorrhiza and matrine can significantly reduce Incidence rate of PVR and RD in PVR model, and curcumin is the most effective one of the three drugs in the prevention and treatment of PVR.