| Proliferative vitreoretinopathy (PVR),following the rhegmatogenous retinal detachment or the termination of traumatic disease,vascular disease and inflamed disease, is a common blind-grown oculopathy.It is characterized by the formation of fibrous epiretinal membranes at the vitreoretinal interface in the vitreous cavity. These membranes result from inappropriate proliferation, migration, and differentiation of several cell types, retinal pigment epithelial(RPE) cells is the main one, and may cause tractional retinal detachment. At the current time, the management of proliferative vitreoretinopathy still remains a surgical skill. After successful vitreoretinal surgery, the functional prognosis remains poor in retinal detachment complicated with PVR. Vitreoretinal surgery is also a common inducement of PVR. So pharmaceutical prevention and treatment are being evaluated to be the practical and promising methods.The reasearchs of western medicine for prevention and treatment of PVR have been done for many years,but there is no clinically safe and effective drug as yet.Curcumin,which have been separated and extracted from curcuma,is a simple substance of the traditional Chinese medicine,has significant anti-proliferation,anti-inflamation and anti-microorganism effect.Its pharmacal activity can satisfy the requests for prevention and treatment of PVR from several aspects. Therefore,curcumin may be a good natural durg in the prevention and treatment of PVR.This study is to evaluate the potential possibility of curcumin from several aspects: inhibition effect on RPE cells in vitro and PVR model in vivo, and toxicity and pharmacokinetics in vitreousPartâ… Inhibition effect of curcumin on proliferation of rabbit retinal pigment epithelial cells in vitroObjective The heat research of prevention and treatment of PVR was to use durg to inhibit RPE cells proliferation at present. In this study, inhibition effect of curcumin on proliferation of rabbit retinal pigment epithelial cells in vitro and its mechanism were studied, and the value of curcumin in the prevention and treatment of PVR was investigated. Methods The RPE cells were isolated and collected from thepigmented rabbit and cultured and passaged in 10%FBS-DMEM and identified by anti-human keratin. Cultured RPE cells were divided into 20, 15, 10μg/mL curcumin group and 400, 200, 100μg/mL suramin group and blank control group according to the utilization of drug. The MTT assay was used to evaluate the inhibition effect of different doses of curcumin and suramin in different time. The IC50 value of curcumin and suramin in different time were analyzed by Linear Regression. Cell cycle, proliferating cell nuclear antigen(PCNA), mitochondrial transmembrane potential(△Ψm), Ca2 + and apoptosis of RPE cells cultured with 15μg/mL curcumin were detected using flow cytometry in defferent time. Expression of mRNA and protein of Bcl-2 and P53 of RPE cells cultured with 15μg/mL curcumin were detected with RT-PCR and Western-blot in defferent time.Results RPE cells were significantly inhibited by curcumin and suramin in a dose-dependent and time-dependent manner,there was significant difference comparied with control group(P<0.05). The IC50 value in curcumin groups in 24, 48, 72 and 96 hours was 29.31μg/mL, 17.50μg/mL, 13.24μg/mL and 10.99μg/mL respectively, showing the significantly reduced level in comparison with suramin groups(974.52μg/mL, 309.11μg/mL, 193.58μg/mL and 116.89μg/mL). Cell cycel analysis indicated that curcumin blocked cultured RPE cells in G0/G1 phase, while the cells in suramin were in G2/M phase(P<0.05). The PCNA in curcumin groups in 24, 48 and 72 hours was (565.04±23.60),(473.61±36.88) and (396.15±32.45) respectively, showing the significantly reduced level in comparison with control group(P<0.05). The apoptosis rate of RPE cells was (2.27%±0.49%),(12.83%±0.13%), (32.27%±4.51%) and (56.81%±8.67%) respectively at 8, 24, 48 and 72 hours after cultured with 15μg/mL of curcumin, except for the group in 8 hours,there was significant difference comparied with control group(P<0.05), however, no apoptotic PRE cell was found in the group cultured with suramin. Ca2+ was (394.16±22.44),(421.64±24.85),(469.43±25.11) and (516.67±15.06) at 8,24,48 and 72 hours after cultured with 15μg/mL of curcumin, significantly increased than that of control group respectively(P<0.05).△Ψm was (660.58±21.61), (594.31±23.22),(525.96±32.18) and (397.08±44.27) significantly decreased than that of the control group respectively(P<0.05).Under transmission electron microscope,RPE cells showed apoptosis at 48 hours after cultured with 15μg/mL of curcumin. At 8,24 and 72 hours after cultured with 15μg/mL of curcumin, mRNA and protein expression of Bcl-2 in RPE cells was (0.49±0.09),(0.39±0.06),(0.20±0.03) and (0.58±0.06),(0.36±0.04),(0.25±0.04) respectively, showing the significantly reduced level in comparison with control group(P<0.05). At 8,24 and 72 hours, mRNA and protein expression of P53 was (0.45±0.05),(0.62±0.06),(0.64±0.06) and (0.25±0.03), (0.38±0.04),(0.59±0.05) respectively, showing the significantly increased level in comparison with control group(P<0.05).Conclusion RPE cells can be significantly inhibited by curcumin and suramin in a dose-dependent and time-dependent manner,inhibition effect of curcumin is better than that of suramin. Curcumin can block cultured RPE cells in G0/G1 phase, while the cells in suramin are in G2/M phase. RPE cells apoptosis induced by curcumin may pass through two pathway:nuclear pathway and cytoplasmic pathway.To elevate the expression of P53,block cell cycle and inhibit DNA production is the nuclear pathway.To degrade the expression of Bcl-2,and leading to Ca2+ overloading in intracytoplasm,and resulting in Ca2+ overloading in mitochondrion fellowing△Ψm dissipation, apoptosis of RPE cell occurs,this is the cytoplasmic pathway.Partâ…¡The study on intraocular toxicity and side effect of curcumin after intravitreal injection in adult rabbitsObjective To study the pharmacal toxicities and side effects to rabbits'eyes received by intravitreal injection of various dose of curcumin.Methods 40 rabbits were randomly divided into 4 groups,10 eyes in each group were intravitreal injected curcumin at different concentration (0.05mg/0.1ml,0.1mg/0.1ml,0.2mg/0.1ml) and 0.5‰DMSO in 0.1 ml respectively,10 control eyes were intravitreal injected 0.9% salt water 0.1 ml. Ophthalmoscopy and ERG were performed on 1d,3d,7d and 14d after injection respectively.Additionally,on 3d,7d and 14d after injection, 2 eyeballs of each group were removed for the examination of light microscope and transmission electron microscope.Results The a-wave amplitudes of dark adaptation ERG were reduced in 0.2mg group(129μV±9μV,131μV±11μV) than those of control group(145μV±13μV,146μV±11μV) on 3d and 7d respectively,there was significant difference(P<0.05). The b-wave amplitudes were reduced in 0.2mg group (259μV±9μV, 257μV±7μV) than those of control group(283μV±13μV,276μV±8μV) on 3d and 7d respectively,there was significant difference(P<0.05). But the a-wave and b-wave amplitudes recovered at 14d.In other groups, the a-wave and b-wave amplitudes were no significant difference compared with control group on all stages. The ophthalmic examination and the retinal tissue structure examined by light microscope and transmission electron microscope were normal on all stages.Conclusion It is safe that curcumin is injected into the rabbit vitreous if the dose less than 0.2mg.Partâ…¢Pharmacokinetics of curcumin in vitreous of rabbitObjective Researches have showed that curcumin presents a antiproliferation, antitumor and antiinflammation effect. Our previous in vitro study also demonstrated that curcumin can induce the apoptosis of retinal pigment epithelial cells and further inhibit the proliferative vitreoretinopathy. This study was to observe the pharmacokinetical process and determine the clearance rate and halflife time of curcumin in the vitreous of rabbit.Methods Thirty-six healthy and mature New Zealand albino rabbits were randomly divided into standard and quality control group (3 rabbits, 6 eyes) and experimental group (33 rabbits, 66 eyes). Rabbits in experimental group were subdivided into 11 groups and 6 eyes of 3 animals for each. 0.1 mL curcumin(containing 0.1 mg) were injected into the vitreous of experimental rabbits and drug did not be administrated in the standard and quality control group. The both eyes from experimental animals were enucleated at the 0,1, 3, 7, 12, 24, 36, 48, 72, 96 and 120 hours after injection of curcumin respectively. The vitreous specimens were collected immediately for the detection of curcumin concentration by high performance liquid chromatography(HPLC). The main parameters of pharmacokinetics were calculated through DAS pharmacokinetics software.Results The chromatographic peak of curcumin and endogenous substance were separated well with the lasting time 4.4 min. The extract recovery of curcumin was 90%, and the relative standard difference (RSD) of intra-day and inter-day was low. The calibration curve of curcumin presented linear through 0.01μg/mL to 4μg/mL with a regression formation as follow: y=143973x+603.32(r=0.9991). The total eliminated amount of curcumin was 1.06%, 4.02%, 9.44%, 19.77%, 23.90%, 35.44%, 57.54%, 70.63%, 84.65%, 91.76% and 96.34% at 0, 1, 3, 7, 12, 24, 36, 48, 72, 96 and 120 hours after injection respectively, showing a halflife time of 26.68±2.66 hours.Conclusion 0.1mg curcumin has an effective mass concentration for a long-standing time after its intravitreal injection. The pharmacokinetical process is zero-order kinetics in the early stage and first-order kinetics 48 hours later.Partâ…£Establishment of proliferative vitreoretinopathy in rabbit eyeObjective To establish an animal model of proliferative vitreoretinopathy(PVR) in rabbit eyes.Methods The retinal pigment epithelial(RPE) cells were dissociated from rabbit eyes and cultured.18 rabbit eyes were divided randomly into 3 groups,0.1ml normal saline, 1×106 cells(0.1ml )and 2×106 cells(0.1ml )were injected into the vitreous of 3 groups respectively.All eyes were examined by slit–lamp biomicroscope,indirect ophthalmoscope,fundus color camera and B ultrasonograph at different stages.Results On 28d after cells injection,0 PVR eye was found in normal saline group,5 PVR eyes were found in 1×106 cells group included 1eye in stage I,3 eyes in stage II and 1eye in stage III, and 6 PVR eyes in 2×106 cells group included 2 eyes in stage II and 4 eyes in stage III.Conclusion This PVR model established by the 2×106 homogeneity RPE cells injection into rabbit vitreous is consistent with PVR clinical process.It is found in short time, with good stablity and facility.Partâ…¤Exprimental study on the effect of curcumin in inhibition of proliferative vitreoretinopathy in rabbit eyeObjective Our previous in vitro study demonstrated that curcumin can induce the apoptosis of retinal pigment epithelial(RPE) cells. 0.1mg curcumin is safe and has an effective mass concentration for a long-standing time after its intravitreal injection. In this part, the effectiveness of curcumin on the prevention and treatment of experimental proliferative vitreoretinopathy(PVR) in rabbit eyes was evaluated.Methods The PVR model was induced by injection of 0.1mL(containing 2×106)RPE cells into vitreous cavity of 20 healthy and mature New Zealand albino rabbits(40 eyes). 0.1mL curcumin(containing 0.1mg) was injected into one eye of each rabbit at random after injection of RPE cells,this group was curcumin containing 20 eyes. 0.1mL normal sodium(containing 0.5‰DMSO) was injected into the other eye of each rabbit,this group was taken as control group containing 20 eyes. On 1, 3, 7, 14, 21 and 28d after injection,the changes of cornea, aqueous humor, lens, vitreous and fundus were examined and recorded by slit lamp biomicroscope, indirect ophthalmoscope, fundus color camera and B ultrasonograph.The effectiveness of curcumin was evaluated by the incidence rate of retinal detachment of the two group.Results On 1d and 3d after injection, inflammatory reaction was found in anterior chamber of all rabbits'eyes, misty opacity of vitreous occurred, fundus may be seen indistinctly,and there was no proliferative strap and retinal detachment. On 7d after injection, inflammatory reaction was extinct in anterior chamber of all eyes, proliferative strap occurred in 75% eyes of control group and in 10% eyes of curcumin grpup,no retinal detachment occurred in the two group. On 14,21 and 28d after injection,the incidence rate of retinal detachment of control group and curcumin group were 55%,80%, 95% and 10%,15%,15% respectively, there was significant statistical difference between the two group on the 3 time-points(P<0.05).Conclusion Injection of curcumin into vitreous cavity can effectively inhibit the occurrence and development of PVR induced by RPE cells in rabbit model.According to the researches in those five parts above, it is concluded that curcumin can inhibit the proliferation of RPE cells by induce its apoptosis,have slighter toxicity to retina and longer time with effective drug concentration after intravitreal injection, and can inhibit the occurrence and development of experimental PVR.Therefore curcumin may be a good natural durg in the prevention and treatment of PVR,and deserve further study,exploitation and application. |
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