姜黄素防治增殖性玻璃体视网膜病变的实验研究
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摘要
增殖性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是指在视网膜前、后面及玻璃体内形成可以收缩的细胞性膜,进而引起视网膜牵拉、脱离与固定的一种病变,主要在裂孔源性视网膜脱离后形成或为外伤性、血管性、炎症性视网膜病变的结局,是一种常见的难治性致盲性眼病。视网膜色素上皮(retinal pigment epithelial,RPE)细胞是PVR发生、发展的关键细胞之一。目前,临床上治疗PVR的主要方法就是玻璃体切割术,但手术治疗效果并不理想,仍不能解决PVR的复发问题。所以,利用药物抑制RPE细胞增生成为近年来PVR防治研究的热点和主流趋势。药物研究目前较多集中在西药领域,主要有抗代谢药、皮质类固醇、维生素及其衍生物、细胞外基质合成抑制剂和细胞信号转导抑制剂等几类。虽然西药防治PVR研究已有多年,但因为其眼内用药毒副作用较大、药理作用单一、价格昂贵等固有的局限性而导致迄今仍无一种药物能够成功应用于临床。在西药研究无法取得重大突破时,中药防治PVR研究就成为让我们充满期待的新的突破方向。姜黄素是从姜科姜黄属植物根茎中提取的一种中药单体成分,具有抗增生、抗炎症、抗微生物等多种药理作用,其药理活性可以在多方面满足PVR防治药物的条件,并且安全低毒、药源广泛、价格低廉。因此,姜黄素有望成为一种理想的防治PVR的天然药物。本课题将首先观察姜黄素对体外培养的兔眼RPE细胞的抑制作用并探讨其作用机制,进而研究其在兔眼玻璃体腔内的毒副作用和代谢过程,最后观察其对兔眼PVR模型的防治效果,从而全面评价其防治PVR的可行性问题。
第一部分姜黄素抑制兔视网膜色素上皮细胞增生的体外实验研究
目的以苏拉明为对照药,对比研究姜黄素和苏拉明对兔RPE细胞增生的抑制作用,并探讨姜黄素抑制RPE细胞增生的作用机制,以期初步评价姜黄素防治PVR的可行性问题。
方法提取、培养青紫蓝兔RPE细胞,传至第四代并鉴定后,选取生长状态良好的第4代RPE细胞进行实验。设姜黄素组和苏拉明组两组,姜黄素分为20、15、10μg/mL组及空白对照组(10% FBS-DMEM含0.5‰二甲基亚砜(DMSO))等4组;苏拉明分为400、200、100μg/mL组及空白对照组(10% FBS-DMEM)等4组;每组各设6个复孔,共接种4块培养板,加药后24、48、72及96 h时随机抽取1块培养板行,噻唑蓝比色(MTT)法检测两种药物对RPE细胞增生的抑制率。相关回归分析计算各时间点的半数抑制率(IC50)剂量。流式细胞仪(FCM)检测姜黄素(15μg/mL)和苏拉明(200μg/mL)分别作用72 h后对RPE细胞增殖周期的影响。FCM检测姜黄素(15μg/mL)作用24、48、72 h后RPE细胞增生细胞核抗原(PCNA)的表达量。FCM检测姜黄素(15μg/mL)作用8、24、48、72 h后RPE细胞胞质内钙离子(Ca~(2+))和线粒体膜电位(△Ψm)的变化及RPE细胞凋亡率。透射电子显微镜观察姜黄素(15μg/mL)作用48 h后RPE细胞的形态学改变。RT-PCR和Western-blot分别检测姜黄素(15μg/mL)作用8、24、72 h后RPE细胞内凋亡相关基因Bcl-2和P53的mRNA和蛋白表达量的变化。
结果姜黄素和苏拉明对RPE细胞增生均有明显地抑制作用,呈时间和剂量依赖性,两种药物在各时间点的抑制率与对照组相比均明显升高,差异有统计学意义(P<0.05)。在24、48、72及96 h,姜黄素的IC50分别是29.31、17.50、13.24及10.99μg/mL,苏拉明的IC_(50)分别是974.52、309.11、193.58及116.89μg/mL。姜黄素使RPE细胞阻滞在G_0/G_1期,苏拉明使RPE细胞阻滞在G2/M期。姜黄素(15μg/mL)作用24、48、72 h后,RPE细胞的PCNA表达量分别为(565.04±23.60),(473.61±36.88),(396.15±32.45),与对照组相比明显降低,差异均有统计学意义(P<0.05)。姜黄素(15μg/mL)作用8、24、48、72 h后,RPE细胞凋亡率分别为(2.27±0.49)%,(12.83±0.13)%,(32.27±4.51)%,(56.81±8.67)%,除8h组与对照组相比差异无统计学意义外(P>0.05),其余各组与对照组相比均明显升高,差异有统计学意义(P<0.05);RPE细胞质内Ca2 +分别为( 394.16±22.44 )、( 421.64±24.85 )、( 469.43±25.11 )、(516.67±15.06),与对照组相比均明显升高,差异有统计学意义(P<0.05 ); RPE细胞△Ψm分别为( 660.58±21.61 )、( 594.31±23.22 )、(525.96±32.18)、(397.08±44.27),与对照组相比均明显降低,差异有统计学意义(P<0.05)。姜黄素(15μg/mL)作用48h后,透射电子显微镜观察显示RPE细胞出现典型的凋亡特征:细胞核内染色质边集、分布于核膜下,或凝聚成块,呈不规则形,核仁多消失,细胞外形尚规则,胞膜尚完整。姜黄素(15μg/mL)作用8、24、72后,RPE细胞内Bcl-2的mRNA表达量分别为(0.49±0.09)、(0.39±0.06)、(0.20±0.03),与对照组相比均明显降低,差异有统计学意义(P<0.05);P53的mRNA表达量分别为(0.45±0.05)、(0.62±0.06)、(0.64±0.06),与对照组相比均明显升高,差异有统计学意义(P<0.05);RPE细胞内Bcl-2蛋白的表达量分别为(0.58±0.06)、(0.36±0.04)、(0.25±0.04),与对照组相比均明显降低,差异有统计学意义(P<0.05);P53蛋白的表达量分别为(0.25±0.03)、(0.38±0.04)、(0.59±0.05),与对照组相比均明显升高,差异有统计学意义(P<0.05)。
结论姜黄素和苏拉明对体外培养的兔眼RPE细胞均有抑制作用,均呈剂量和时间依赖性,姜黄素抑制效果优于苏拉明。姜黄素将RPE细胞增生阻滞在G_0/G_1期,苏拉明将RPE细胞增生阻滞在G2/M期。姜黄素通过细胞核和细胞质两种途径诱导RPE细胞凋亡进而抑制其增生。细胞核途径为上调P53基因的表达,干扰细胞增生周期,影响DNA合成,从而诱导细胞凋亡;细胞质途径为下调Bcl-2基因的表达,使细胞质内Ca~(2+)超载从而造成线粒体内Ca~(2+)超载,而后导致线粒体△Ψm耗散,进而引起细胞凋亡。
第二部分姜黄素兔眼玻璃体内注射后眼内毒副作用研究
目的姜黄素对体外培养的兔RPE细胞有明显抑制作用,但防治PVR需要在活体玻璃体内用药,因而眼内用药的安全性问题必须给予高度重视。本部分将观察不同剂量姜黄素在兔眼玻璃体内的毒副作用,寻求玻璃体用药的最大安全剂量。
方法40只新西兰白兔,其中24只随机分为姜黄素0.05mg/0.1mL组,0.1mg/0.1mL组,0.2mg/0.1mL组及DMSO组(生理盐水中含有终浓度为0.5‰的DMSO)等4组,每组6只。所用实验兔玻璃体注射前,前房穿刺抽出0.1mL房水。随机选取每只兔的一眼为实验眼,在玻璃体内分别注射0.1mL不同剂量的药物及DMSO;对侧眼为对照眼,在玻璃体内注射0.1mL生理盐水,本组兔供电生理检查使用。其余16只兔随机分成上述4组,每组4只,双眼使用方法同上,本组供3d、7d时摘取眼球作组织学检查使用。注射后1d、3d、7d、14d使用裂隙灯显微镜进行眼前节检查、使用间接检眼镜检查眼底情况,使用暗适应视网膜电图(ERG)检查a波和b波振幅的变化;在3d、7d、14d分别摘取2只眼球做光学和透射电子显微镜检查,观察视网膜组织结构的改变。
结果0.2mg组在注药后3d和7d,实验组眼暗适应ERG a波振幅分别为(129±9)μV和(131±11)μV,与对照组眼的(145±13μV)和(146±11)μV相比明显降低,差异有统计学意义(P<0.05);实验组眼暗适应ERG b波振幅分别为(259±9)μV和(257±7)μV,与对照组眼的(283±13)μV和(276±8)μV相比明显降低,差异有统计学意义(P<0.05)(P<0.05)。14d时a波和b波振幅又恢复正常。其余各组各时间点,实验组眼a波和b波振幅与对照组眼相比差异无统计学意义。各时间点常规眼部检查和视网膜组织学检查正常。
结论0.2mg以下剂量姜黄素对视网膜功能和组织结构无明显损伤,该剂量范围内姜黄素兔眼玻璃体内注射用药毒副作用较小,安全可行。
第三部分姜黄素在兔眼玻璃体内的药代动力学研究
目的姜黄素0.1mg兔眼玻璃体内注射安全可行,本部分将研究该剂量姜黄素在玻璃体内的代谢过程,以期掌握其药代动力学参数,为临床应用提供参考依据。
方法将36只新西兰白兔随机分为标准及质控组3只兔(6只眼)和实验组33只兔(66只眼)。实验组再随机分为11组,每组3只兔6只眼。标准及质控组动物无需手术操作,实验组动物穿刺抽取玻璃体0.1 mL后玻璃体腔内注入姜黄素0.1 mL(含0.1 mg)。注药后0、1、3、7、12、24、36、48、72、96及120 h分别摘除眼球并制备玻璃体待检样品。应用高效液相色谱技术(HPLC)检测玻璃体腔内药物质量浓度。用DAS软件计算主要的药代动力学参数。
结果姜黄素与玻璃体内源性物质之间的色谱峰分离良好,姜黄素保留时间为4.4 min。提取回收率约90%,日内和日间变异较小。玻璃体内姜黄素质量浓度在0.01～4μg/mL范围内呈线性关系,标准曲线为y=143 973x+603.32(r= 0.9991)。在注药后0、1、3、7、12、24、36、48、72、96、120 h等时间点测得的清除率分别为1.06%、4.02%、9.44%、19.77%、23.90%、35.44%、57.54%、70.63%、84.65%、91.76%及96.34%。半衰期t_(1/2)=(26.68±2.66)h。
结论HPLC法检测玻璃体内姜黄素含量特异性好。姜黄素在玻璃体内稳定存在,无代谢产物。姜黄素在玻璃体内半衰期较长约为27h,0.1 mg注射后整个代谢过程先为零级动力学代谢,48 h后为一级动力学代谢。
第四部分兔眼增殖性玻璃体视网膜病变模型的建立
目的探讨兔眼PVR模型的建立方法,以期为防治PVR研究提供合理可靠的动物模型。
方法新西兰白兔9只,随机分为A、B、C 3组,每组3只兔6只眼。所用实验兔首先抽取玻璃体液0.1mL,然后A组玻璃体内注入0.1mL生理盐水,B组注入1×10~6 /0.1mL的RPE细胞0.1mL,C组注入2×10~6 /0.1mL的RPE细胞0.1mL。在注入细胞后1、3、7、14、21和28d进行裂隙灯显微镜、间接眼底镜等常规眼科检查;利用眼底照像机进行眼底照像;眼科B超检查玻璃体和视网膜情况。眼后节PVR分级:0级:视网膜在位,玻璃体无混浊;I级:视网膜在位,玻璃体混浊并见增殖条带;II级:玻璃体条带牵拉至髓线抬高,或有局限性视网膜脱离;III级:视网膜全脱离或有漏斗状脱离。II～III级的累计和占总数的百分率为视网膜脱离的发生率。
结果注射后28d,生理盐水组成模0眼;1×10~6细胞组成模5眼,其中I级眼1只,II级眼3只,III级眼1只;2×10~6细胞组成模6眼,其中II级眼2只,III级眼4只。
结论兔眼玻璃体内注射2×10~6同种RPE细胞建立PVR模型,符合病变发展规律,而且稳定可靠,成模较快,简单易行。
第五部分姜黄素防治兔眼增殖性玻璃体视网膜病变的体内实验研究
目的本课题前期的系列研究发现,姜黄素可以诱导体外培养的兔RPE细胞凋亡,进而抑制其增生;0.1mg姜黄素兔眼玻璃体内注射无毒副作用,并且有效抑制浓度能够维持较长时间;因而本部分将进一步探讨该剂量姜黄素玻璃体内注射对RPE细胞诱导的兔眼PVR模型的防治效果。
方法新西兰白兔20只,40只眼,所有兔眼玻璃体注射前首先抽取0.2mL玻璃体液,然后所有兔眼玻璃体内注射0.1mL(2×10~6)同种RPE细胞,每只兔的双眼中随机选取1只眼立即注入质量浓度为0.1 mg/0.1 mL的姜黄素0.1mL,此组20只眼作为姜黄素组,另一只眼注入含0.5‰DMSO的生理盐水0.1mL,此组20只眼作为对照组。注药后1、3、7、14、21和28d使用裂隙灯显微镜观察角膜、房水、晶状体是否清亮,前节炎症反应情况;使用间接检眼镜、眼底彩色照像和B超检查玻璃体视网膜情况。眼后节PVR分级:0级:视网膜在位,玻璃体无混浊;I级:视网膜在位,玻璃体混浊并见增殖条带;II级:玻璃体条带牵拉至髓线抬高,或有局限性视网膜脱离;III级:视网膜全脱离或有漏斗状脱离。II～III级的累计和占总数的百分率为视网膜脱离的发生率。以视网膜脱落率为判断标准,评价姜黄素的防治效果。
结果注药后1、3d所有兔眼前节可见中度至轻度的炎症反应,玻璃体轻、中度混浊,眼底隐约可见,未见增殖条带,未见视网膜脱离。注药后7d,所有兔眼前节炎症反应基本消退,对照组75%眼玻璃体出现增殖条带,姜黄素组10%眼玻璃体内出现增殖条带,两组均未见视网膜脱离。注药后14d,对照组55%眼出现视网膜脱离,姜黄素组10%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。注药后21d,对照组80%眼出现视网膜脱离,姜黄素组15%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。注药后28d,对照组95%眼出现视网膜脱离,姜黄素组15%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。
结论姜黄素玻璃体腔内注射可以有效抑制RPE细胞诱导的兔眼实验性PVR的发生、发展。
基于上述研究结果可以得出以下结论:姜黄素可以诱导体外培养的兔RPE细胞凋亡,进而抑制其增生;兔眼玻璃体内注射毒副作用较小,半衰期和维持有效抑制浓度的时间较长,并且可以有效抑制兔眼实验性PVR的发生、发展。因此,姜黄素有望成为较为理想的防治PVR的天然药物,值得进一步研究、开发和利用。
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 vitreous
PartⅠInhibition effect of curcumin on proliferation of rabbit retinal pigment epithelial cells in vitro
Objective 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 the
pigmented 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. Ca~(2+) 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 G_0/G_1 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 Ca~(2+) overloading in intracytoplasm,and resulting in Ca~(2+) overloading in mitochondrion fellowing△Ψm dissipation, apoptosis of RPE cell occurs,this is the cytoplasmic pathway.
PartⅡThe study on intraocular toxicity and side effect of curcumin after intravitreal injection in adult rabbits
Objective 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.
PartⅢPharmacokinetics of curcumin in vitreous of rabbit
Objective 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.
PartⅣEstablishment of proliferative vitreoretinopathy in rabbit eye
Objective 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, 1×10~6 cells(0.1ml )and 2×10~6 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 1×10~6 cells group included 1eye in stage I,3 eyes in stage II and 1eye in stage III, and 6 PVR eyes in 2×10~6 cells group included 2 eyes in stage II and 4 eyes in stage III.
Conclusion This PVR model established by the 2×10~6 homogeneity RPE cells injection into rabbit vitreous is consistent with PVR clinical process.It is found in short time, with good stablity and facility.
PartⅤExprimental study on the effect of curcumin in inhibition of proliferative vitreoretinopathy in rabbit eye
Objective 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 2×10~6)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.
第一部分姜黄素抑制兔视网膜色素上皮细胞增生的体外实验研究
目的以苏拉明为对照药,对比研究姜黄素和苏拉明对兔RPE细胞增生的抑制作用,并探讨姜黄素抑制RPE细胞增生的作用机制,以期初步评价姜黄素防治PVR的可行性问题。
方法提取、培养青紫蓝兔RPE细胞,传至第四代并鉴定后,选取生长状态良好的第4代RPE细胞进行实验。设姜黄素组和苏拉明组两组,姜黄素分为20、15、10μg/mL组及空白对照组(10% FBS-DMEM含0.5‰二甲基亚砜(DMSO))等4组;苏拉明分为400、200、100μg/mL组及空白对照组(10% FBS-DMEM)等4组;每组各设6个复孔,共接种4块培养板,加药后24、48、72及96 h时随机抽取1块培养板行,噻唑蓝比色(MTT)法检测两种药物对RPE细胞增生的抑制率。相关回归分析计算各时间点的半数抑制率(IC50)剂量。流式细胞仪(FCM)检测姜黄素(15μg/mL)和苏拉明(200μg/mL)分别作用72 h后对RPE细胞增殖周期的影响。FCM检测姜黄素(15μg/mL)作用24、48、72 h后RPE细胞增生细胞核抗原(PCNA)的表达量。FCM检测姜黄素(15μg/mL)作用8、24、48、72 h后RPE细胞胞质内钙离子(Ca~(2+))和线粒体膜电位(△Ψm)的变化及RPE细胞凋亡率。透射电子显微镜观察姜黄素(15μg/mL)作用48 h后RPE细胞的形态学改变。RT-PCR和Western-blot分别检测姜黄素(15μg/mL)作用8、24、72 h后RPE细胞内凋亡相关基因Bcl-2和P53的mRNA和蛋白表达量的变化。
结果姜黄素和苏拉明对RPE细胞增生均有明显地抑制作用,呈时间和剂量依赖性,两种药物在各时间点的抑制率与对照组相比均明显升高,差异有统计学意义(P<0.05)。在24、48、72及96 h,姜黄素的IC50分别是29.31、17.50、13.24及10.99μg/mL,苏拉明的IC_(50)分别是974.52、309.11、193.58及116.89μg/mL。姜黄素使RPE细胞阻滞在G_0/G_1期,苏拉明使RPE细胞阻滞在G2/M期。姜黄素(15μg/mL)作用24、48、72 h后,RPE细胞的PCNA表达量分别为(565.04±23.60),(473.61±36.88),(396.15±32.45),与对照组相比明显降低,差异均有统计学意义(P<0.05)。姜黄素(15μg/mL)作用8、24、48、72 h后,RPE细胞凋亡率分别为(2.27±0.49)%,(12.83±0.13)%,(32.27±4.51)%,(56.81±8.67)%,除8h组与对照组相比差异无统计学意义外(P>0.05),其余各组与对照组相比均明显升高,差异有统计学意义(P<0.05);RPE细胞质内Ca2 +分别为( 394.16±22.44 )、( 421.64±24.85 )、( 469.43±25.11 )、(516.67±15.06),与对照组相比均明显升高,差异有统计学意义(P<0.05 ); RPE细胞△Ψm分别为( 660.58±21.61 )、( 594.31±23.22 )、(525.96±32.18)、(397.08±44.27),与对照组相比均明显降低,差异有统计学意义(P<0.05)。姜黄素(15μg/mL)作用48h后,透射电子显微镜观察显示RPE细胞出现典型的凋亡特征:细胞核内染色质边集、分布于核膜下,或凝聚成块,呈不规则形,核仁多消失,细胞外形尚规则,胞膜尚完整。姜黄素(15μg/mL)作用8、24、72后,RPE细胞内Bcl-2的mRNA表达量分别为(0.49±0.09)、(0.39±0.06)、(0.20±0.03),与对照组相比均明显降低,差异有统计学意义(P<0.05);P53的mRNA表达量分别为(0.45±0.05)、(0.62±0.06)、(0.64±0.06),与对照组相比均明显升高,差异有统计学意义(P<0.05);RPE细胞内Bcl-2蛋白的表达量分别为(0.58±0.06)、(0.36±0.04)、(0.25±0.04),与对照组相比均明显降低,差异有统计学意义(P<0.05);P53蛋白的表达量分别为(0.25±0.03)、(0.38±0.04)、(0.59±0.05),与对照组相比均明显升高,差异有统计学意义(P<0.05)。
结论姜黄素和苏拉明对体外培养的兔眼RPE细胞均有抑制作用,均呈剂量和时间依赖性,姜黄素抑制效果优于苏拉明。姜黄素将RPE细胞增生阻滞在G_0/G_1期,苏拉明将RPE细胞增生阻滞在G2/M期。姜黄素通过细胞核和细胞质两种途径诱导RPE细胞凋亡进而抑制其增生。细胞核途径为上调P53基因的表达,干扰细胞增生周期,影响DNA合成,从而诱导细胞凋亡;细胞质途径为下调Bcl-2基因的表达,使细胞质内Ca~(2+)超载从而造成线粒体内Ca~(2+)超载,而后导致线粒体△Ψm耗散,进而引起细胞凋亡。
第二部分姜黄素兔眼玻璃体内注射后眼内毒副作用研究
目的姜黄素对体外培养的兔RPE细胞有明显抑制作用,但防治PVR需要在活体玻璃体内用药,因而眼内用药的安全性问题必须给予高度重视。本部分将观察不同剂量姜黄素在兔眼玻璃体内的毒副作用,寻求玻璃体用药的最大安全剂量。
方法40只新西兰白兔,其中24只随机分为姜黄素0.05mg/0.1mL组,0.1mg/0.1mL组,0.2mg/0.1mL组及DMSO组(生理盐水中含有终浓度为0.5‰的DMSO)等4组,每组6只。所用实验兔玻璃体注射前,前房穿刺抽出0.1mL房水。随机选取每只兔的一眼为实验眼,在玻璃体内分别注射0.1mL不同剂量的药物及DMSO;对侧眼为对照眼,在玻璃体内注射0.1mL生理盐水,本组兔供电生理检查使用。其余16只兔随机分成上述4组,每组4只,双眼使用方法同上,本组供3d、7d时摘取眼球作组织学检查使用。注射后1d、3d、7d、14d使用裂隙灯显微镜进行眼前节检查、使用间接检眼镜检查眼底情况,使用暗适应视网膜电图(ERG)检查a波和b波振幅的变化;在3d、7d、14d分别摘取2只眼球做光学和透射电子显微镜检查,观察视网膜组织结构的改变。
结果0.2mg组在注药后3d和7d,实验组眼暗适应ERG a波振幅分别为(129±9)μV和(131±11)μV,与对照组眼的(145±13μV)和(146±11)μV相比明显降低,差异有统计学意义(P<0.05);实验组眼暗适应ERG b波振幅分别为(259±9)μV和(257±7)μV,与对照组眼的(283±13)μV和(276±8)μV相比明显降低,差异有统计学意义(P<0.05)(P<0.05)。14d时a波和b波振幅又恢复正常。其余各组各时间点,实验组眼a波和b波振幅与对照组眼相比差异无统计学意义。各时间点常规眼部检查和视网膜组织学检查正常。
结论0.2mg以下剂量姜黄素对视网膜功能和组织结构无明显损伤,该剂量范围内姜黄素兔眼玻璃体内注射用药毒副作用较小,安全可行。
第三部分姜黄素在兔眼玻璃体内的药代动力学研究
目的姜黄素0.1mg兔眼玻璃体内注射安全可行,本部分将研究该剂量姜黄素在玻璃体内的代谢过程,以期掌握其药代动力学参数,为临床应用提供参考依据。
方法将36只新西兰白兔随机分为标准及质控组3只兔(6只眼)和实验组33只兔(66只眼)。实验组再随机分为11组,每组3只兔6只眼。标准及质控组动物无需手术操作,实验组动物穿刺抽取玻璃体0.1 mL后玻璃体腔内注入姜黄素0.1 mL(含0.1 mg)。注药后0、1、3、7、12、24、36、48、72、96及120 h分别摘除眼球并制备玻璃体待检样品。应用高效液相色谱技术(HPLC)检测玻璃体腔内药物质量浓度。用DAS软件计算主要的药代动力学参数。
结果姜黄素与玻璃体内源性物质之间的色谱峰分离良好,姜黄素保留时间为4.4 min。提取回收率约90%,日内和日间变异较小。玻璃体内姜黄素质量浓度在0.01～4μg/mL范围内呈线性关系,标准曲线为y=143 973x+603.32(r= 0.9991)。在注药后0、1、3、7、12、24、36、48、72、96、120 h等时间点测得的清除率分别为1.06%、4.02%、9.44%、19.77%、23.90%、35.44%、57.54%、70.63%、84.65%、91.76%及96.34%。半衰期t_(1/2)=(26.68±2.66)h。
结论HPLC法检测玻璃体内姜黄素含量特异性好。姜黄素在玻璃体内稳定存在,无代谢产物。姜黄素在玻璃体内半衰期较长约为27h,0.1 mg注射后整个代谢过程先为零级动力学代谢,48 h后为一级动力学代谢。
第四部分兔眼增殖性玻璃体视网膜病变模型的建立
目的探讨兔眼PVR模型的建立方法,以期为防治PVR研究提供合理可靠的动物模型。
方法新西兰白兔9只,随机分为A、B、C 3组,每组3只兔6只眼。所用实验兔首先抽取玻璃体液0.1mL,然后A组玻璃体内注入0.1mL生理盐水,B组注入1×10~6 /0.1mL的RPE细胞0.1mL,C组注入2×10~6 /0.1mL的RPE细胞0.1mL。在注入细胞后1、3、7、14、21和28d进行裂隙灯显微镜、间接眼底镜等常规眼科检查;利用眼底照像机进行眼底照像;眼科B超检查玻璃体和视网膜情况。眼后节PVR分级:0级:视网膜在位,玻璃体无混浊;I级:视网膜在位,玻璃体混浊并见增殖条带;II级:玻璃体条带牵拉至髓线抬高,或有局限性视网膜脱离;III级:视网膜全脱离或有漏斗状脱离。II～III级的累计和占总数的百分率为视网膜脱离的发生率。
结果注射后28d,生理盐水组成模0眼;1×10~6细胞组成模5眼,其中I级眼1只,II级眼3只,III级眼1只;2×10~6细胞组成模6眼,其中II级眼2只,III级眼4只。
结论兔眼玻璃体内注射2×10~6同种RPE细胞建立PVR模型,符合病变发展规律,而且稳定可靠,成模较快,简单易行。
第五部分姜黄素防治兔眼增殖性玻璃体视网膜病变的体内实验研究
目的本课题前期的系列研究发现,姜黄素可以诱导体外培养的兔RPE细胞凋亡,进而抑制其增生;0.1mg姜黄素兔眼玻璃体内注射无毒副作用,并且有效抑制浓度能够维持较长时间;因而本部分将进一步探讨该剂量姜黄素玻璃体内注射对RPE细胞诱导的兔眼PVR模型的防治效果。
方法新西兰白兔20只,40只眼,所有兔眼玻璃体注射前首先抽取0.2mL玻璃体液,然后所有兔眼玻璃体内注射0.1mL(2×10~6)同种RPE细胞,每只兔的双眼中随机选取1只眼立即注入质量浓度为0.1 mg/0.1 mL的姜黄素0.1mL,此组20只眼作为姜黄素组,另一只眼注入含0.5‰DMSO的生理盐水0.1mL,此组20只眼作为对照组。注药后1、3、7、14、21和28d使用裂隙灯显微镜观察角膜、房水、晶状体是否清亮,前节炎症反应情况;使用间接检眼镜、眼底彩色照像和B超检查玻璃体视网膜情况。眼后节PVR分级:0级:视网膜在位,玻璃体无混浊;I级:视网膜在位,玻璃体混浊并见增殖条带;II级:玻璃体条带牵拉至髓线抬高,或有局限性视网膜脱离;III级:视网膜全脱离或有漏斗状脱离。II～III级的累计和占总数的百分率为视网膜脱离的发生率。以视网膜脱落率为判断标准,评价姜黄素的防治效果。
结果注药后1、3d所有兔眼前节可见中度至轻度的炎症反应,玻璃体轻、中度混浊,眼底隐约可见,未见增殖条带,未见视网膜脱离。注药后7d,所有兔眼前节炎症反应基本消退,对照组75%眼玻璃体出现增殖条带,姜黄素组10%眼玻璃体内出现增殖条带,两组均未见视网膜脱离。注药后14d,对照组55%眼出现视网膜脱离,姜黄素组10%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。注药后21d,对照组80%眼出现视网膜脱离,姜黄素组15%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。注药后28d,对照组95%眼出现视网膜脱离,姜黄素组15%眼出现视网膜脱离,明显低于对照组,差异有统计学意义(P<0.05)。
结论姜黄素玻璃体腔内注射可以有效抑制RPE细胞诱导的兔眼实验性PVR的发生、发展。
基于上述研究结果可以得出以下结论:姜黄素可以诱导体外培养的兔RPE细胞凋亡,进而抑制其增生;兔眼玻璃体内注射毒副作用较小,半衰期和维持有效抑制浓度的时间较长,并且可以有效抑制兔眼实验性PVR的发生、发展。因此,姜黄素有望成为较为理想的防治PVR的天然药物,值得进一步研究、开发和利用。
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 vitreous
PartⅠInhibition effect of curcumin on proliferation of rabbit retinal pigment epithelial cells in vitro
Objective 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 the
pigmented 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. Ca~(2+) 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 G_0/G_1 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 Ca~(2+) overloading in intracytoplasm,and resulting in Ca~(2+) overloading in mitochondrion fellowing△Ψm dissipation, apoptosis of RPE cell occurs,this is the cytoplasmic pathway.
PartⅡThe study on intraocular toxicity and side effect of curcumin after intravitreal injection in adult rabbits
Objective 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.
PartⅢPharmacokinetics of curcumin in vitreous of rabbit
Objective 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.
PartⅣEstablishment of proliferative vitreoretinopathy in rabbit eye
Objective 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, 1×10~6 cells(0.1ml )and 2×10~6 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 1×10~6 cells group included 1eye in stage I,3 eyes in stage II and 1eye in stage III, and 6 PVR eyes in 2×10~6 cells group included 2 eyes in stage II and 4 eyes in stage III.
Conclusion This PVR model established by the 2×10~6 homogeneity RPE cells injection into rabbit vitreous is consistent with PVR clinical process.It is found in short time, with good stablity and facility.
PartⅤExprimental study on the effect of curcumin in inhibition of proliferative vitreoretinopathy in rabbit eye
Objective 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 2×10~6)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.
引文
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1 Pastor JC. Proliferative vitreoretinopathy: An overview. Surv Ophthalmol, 1998,43(1):3-18
2 Charteris DG.Proliferative vitreoretinopathy:pathobiology,surgical management and adjunctive treatment.Br J Ophthalmol,1995,79(10):953-960
3 Mietz H, Heimann K.Onset and recurrence of proliferative vitreoretinopathy in various vitreoretinal disease. Br J Ophthalmol,1995,79(10):874-879
4 Baudouin C, Khosrzvi E, Pisella PJ, et al. Inflammation measurement and immunocharacterization of cell proliferation in an experimental model of proliferative vitreoretinopathy. Ophthalmic Res, 1998,30(6):340-350
5 Machemer R. Proliferative Vitreoretinopathy(PVR):A personal account of its pathogenesis and treatment. Invest Ophthalmol Vis Sci, 1998,29(12):1771-1783
6 Yasushi Ikuno, Fee–Lai Leong, Andrius Kazlauskas, et al. Attenuation of Experimental Proliferative Vitreoretinopathy by Inhibiting the Platelet-Derived Growth Factor Receptor. Invest Ophthalmol Vis Sci, 2000, 41(10):3017-3116
7 Sobaci G, Mutliu FM, Vayer A, et al. Deadly weapon-related open-globe injuries:outcome assessment by the ocular trauma classification system. Am J Ophthalmol, 2000,129(1):47-43
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12 Cardillo JA,Farah ME,Mitre J,et al. An intravitreal biodegradable sustained release naproxen and 5-fluorouracil system for the treatment ofexperimental post-traumatic proliferative vitreoretinopathy.Br J Ophthalmol,2004,88(9):1201-1205
13 Hueber A,Weller M,Welsandt G,et al.Characterization of daunorubicin-induced apoptosis in retinal pigment epithelial cells:modulation by CD95L.Invest Ophthalmol Vis Sci,2003,44(7):2851-2857
14许东晖,王胜,金晶,等.姜黄素的药理作用研究进展.中草药,2005,36(11):1737-1740
15盛柳青,颜继忠,梁万根.姜黄素的研究进展及应用概况.中国西部科技,2006,4(1):14-15
16边芳,张名昌.姜黄素抑制翼状胬肉成纤维细胞增生的研究.临床眼科杂志,2005,13(3):198-201
17龚凌,姜德咏,朱晓华,等.姜黄素对培养的人胚胎视网膜色素上皮细胞增殖活性的影响.眼科学报,2004,20(4):246-248
18胡艳红,祁明信,黄秀榕,等.姜黄素对晶状体上皮细胞增殖的抑制作用.眼科研究,2006,24(2):122-124
19黄秀榕,祁明信,康可人.姜黄素诱导牛晶状体上皮细胞凋亡的机制.中华眼科杂志,2006,42(7):649-653
20 Chun KS, Keum YS, Han SS,et al. Curcumin inhibits phorbol ster-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-kappaB activation. Carcinogenesis. 2003,24(9):1515-1519
21 Han SS, Keum YS, Seo HJ, et al. Curcumin suppresses activation of NF-kappaB and AP-1 induced by phorbol ester in cultured human promyelocytic leukemia cells. J Biochem Mol Biol, 2002,35(3):337-342
22 Pan MH, Chang WL, Lin-Shiau SY ,et al. Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells. J Agric Food Chem. 2001,49(3):1464-1470
23 Bush JA, Cheung KJ, Li G. Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent ofp53. Exp Cell Res, 2001,271(2):305-310
24 Anto RJ,Mukhopadhyay A,Denning K,et al.Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID, cleavage and cytochrome c release: its suppression by ectopic expression of bcl-2 and bcl-xl. Carcinogenesis, 2002,23(1):143-147
25 Wu Y, Chen Y, Xu J, et al. Anticancer activities of curcumin on human Burkitt’s lymphoma. Zhonghua Zhong Liu Za Zhi, 2002,24(4):348-352
26 Moragoda L, Jaszewski R, Majumdar AP.Curcumin induced modulation of cellcycle and apoptosis in gastric and colon cancer cells. Anticancer Res, 2001,21(2A):873-878
27 Rashmi R, Kumar S, Karunagaran D. Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downtegulation of Bcl-XL.Carcinogenesis, 2005,26(5):713-723
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