姜黄素、丹参单体和苦参碱抑制IL-1β诱导下兔RPE细细胞增生的体外实验研究

Inhibitory effect of curcumin, salvia miltiorrhiza and matrine on IL-1β-induced proliferation of rabbit RPE cells in vitro

背景 增生性玻璃体视网膜病变（PVR）是临床上常见的致盲眼病.视网膜色素上皮（RPE）细胞是PVR发生和发展中的关键细胞,研究中药对体外培养RPE细胞的作用及原理对于防治PVR并揭示其发病机制具有重要意义. 目的 研究姜黄素、丹参单体、苦参碱对白细胞介素-1β（IL-1β）诱导的兔RPE细胞增生的抑制效果.方法 取第3～4代体外培养的有色兔RPE细胞,采用透射电子显微镜鉴定细胞结构.采用MTT法检测2.5、5.0、10.0、20.0 μg/L IL-1β培养后24、48和72 h RPE细胞的增生率;并检测5、10、20 μg/ml姜黄素和丹参单体IH763-4以及100、200、400 μg/ml苦参碱对IL-1β诱导RPE细胞增生的抑制率.采用线性回归分析计算各药物的半数抑制率（IC50）剂量.结果 初分离的兔RPE细胞呈球形,细胞中可见大量黑色素颗粒;第4代细胞色素颗粒明显减少,形态更加狭长.免疫细胞化学染色结果显示,细胞角蛋白（AE1/AE3）在细胞质表达呈阳性.透射电子显微镜下可见细胞顶端有大量的微绒毛,细胞间可见连接复合体.不同质量浓度IL-1β组培养后24、48、72 h细胞增生率总体比较差异均有统计学意义（F时间=30.33,P=0.00;F浓度=9.37,P=0.00）;组内相邻培养时间点间细胞增生率比较差异均有统计学意义（均P＜0.05）,同一培养时间点相邻质量浓度IL-1β组间细胞增生率比较,差异均有统计学意义（均P＜0.05）.10.0μ,g/LIL-1β培养72 h后细胞增生率达峰值.不同质量浓度姜黄素、丹参单体和苦参碱不同培养时间点细胞抑制率总体比较差异均有统计学意义（姜黄素：F时间=128.75,P=0.00;F质量浓度=334.05,P=0.00.丹参单体：F时间=39.32,P=0.00;F质量浓度=165.57,P=0.00.苦参碱：F时间=267.76,P=0.00;F质量浓度=912.34,P=0.00）.3种药物对RPE细胞的抑制作用均呈明显的剂量和时间依赖性,组内相邻培养时间点间细胞抑制率比较差异均有统计学意义（均P＜0.05）,相邻质量浓度药物组间细胞抑制率的比较差异均有统计学意义（均P＜0.05）.培养后24、48及72 h,姜黄素的IC50分别为26.77、19.01和9.45 μg/ml,丹参单体的IC50分别为33.72、23.47和12.56μg/ml,苦参碱的IC50分别为570.96、352.25和97.50 μg/ml. 结论 IL-1β可促进RPE细胞增生,10.0 μg/L IL-1β促增生作用最显著;姜黄素、丹参单体、苦参碱均可抑制IL-1β诱导的RPE细胞增生,其抑制作用均呈时间和剂量依赖性,其中姜黄素抗增生作用最强.

Background Proliferative vitreoretinopathy （PVR） is a common cause of vision loss clinically,and retinal pigment epithelium （RPE） cells play a major part in this disease.Studying the effect of traditional Chinese medicine on RPE cells are of great importance to reveal the pathogenesis and prevention of PVR,which were rarely reported.Objective This study was to study and compare the inhibition effect among curcumin,salvia miltiorrhiza and matrine on IL-1β-induced proliferation of rabbit RPE cells.Methods RPE cells at passages 3-4 were enrolled for the research and identified by transmission electron microscope.The proliferation effect of IL-1 β （2.5,5.0,10.0,20.0 μg/L） and inhibitory effect of curcumin （5,10,20 μg/ml）,salvia miltiorrhiza （5,10,20 μg/ml）or matrine （100,200,400 μg/ml） on RPE cells 24,48 and 72 hours after cultivation were studied by MTT assay.The 50% inhibitory dose （IC50） of the three medicines were analyzed by regression analysis.The use and feeding of the experimental animals were followed by the ARVO Statement.Results RPE cells isolated from the rabbit eye were in round shape and abundant in melanin;The melanin significantly decreased in the fourth generations of RPE cells.Immunohistochemistry showed that the RPE cells was positive for keratin （AE1/AE3）.The proliferation rates of RPE cells were statistically different among different concentrations of IL-1β 24,48 and 72 hours after cultivation （Ftime =30.33,P =0.00;Fconcentration =9.37,P =0.00）;The proliferation rates of RPE were significantly different among different time points or different concentrations of IL-1β （all at P 〈 0.05）.And the proliferation rate run up to maximum at 10 μg/L after 72 hours of cultivation.The inhibitory rates of the three medicines were statistically different among different time points or different concentrations （curcumin：Ftime =128.75,P =0.00;Fconcentration =334.05,P=0.00.salvia miltiorrhiza：Ftime =39.32,P=0.00;Fconcentration =165.57,P=0.00.matrine：Ftime =267.76,P =0.00;Fconcentration =912.34,P =0.00）.The three medicines dose-dependently and time-dependently inhibit IL-1β-induced proliferation of RPE cells,with significant differences between the adjacent time points and concentrations （all at P〈0.05）.The IC50 were 26.77,19.01 and 9.45 μg/ml for curcumin;33.72,23.47 and 12.56 μg/ml for salvia miltiorrhiza,570.96,352.25 and 97.50μg/ml for matrine 24,48 and 72 hours after cultivation.Conclusions The proliferation of RPE cells can be stimulated by IL-1β,and the maximal proliferation occurred with a concentration of 10.0 μg/L IL-1β.Curcumin,salvia miltiorrhiza and matrine dose-dependently and time-dependently inhibit proliferation of RPE cells induced by IL-1β.Curcumin is the best medicine to inhibit the proliferation of RPE cells.