姜黄素对碱烧伤诱导的兔眼角膜新生血管抑制作用的实验研究

doi:10.3877/cma.j.issn.2095-2007.2019.02.008

目的

探讨姜黄素对碱烧伤诱导的大耳兔眼角膜新生血管(CNV)的影响及其机制。

方法

采用碱烧伤法制备CNV模型。模型制作成功后，采用数字表法将健康的成年雄性日本大耳兔48只随机平均分为4组，对照组给予二甲基亚砜，实验1组给予40 μmol/L姜黄素，实验2组给予80 μmol/L姜黄素，实验3组给予160 μmol/L姜黄素。右眼分别滴眼相应药物，所有滴眼药物用量均为20 μl，4次/d，连续用药14 d。用裂隙灯显微镜观察CNV的生长情况。采用酶联免疫吸附试验检测大耳兔眼碱烧伤2 d、4 d、7 d及14 d后的房水中血管内皮生长因子(VEGF)、4E结合蛋白(4EBP1)因子的蛋白含量；采用逆转录-聚合酶链反应检测眼角膜组织中4EBP1、S6蛋白激酶(P70S6K)、雷帕霉素靶蛋白(mTOR)的信使核糖核酸(mRNA)的相对表达量。组间比较采用两因素重复测量方差分析，当差异有统计学意义时，进一步采用SNK法两两比较。计算Spearman相关系数判断4EBP1与VEGF蛋白之间的相关性。

结果

大耳兔眼角膜碱烧伤后4 d时，各实验组与对照组的平均CNV面积无统计学差异(F＝0.592，P>0.05)；眼角膜碱烧伤后7 d、14 d时，则各实验组与对照组平均CNV面积差异有统计学意义(F＝27.5，28.64；P<0.05)。大耳兔眼角膜碱烧伤后2 d时，各实验组房水中的4EBP1、VEGF蛋白含量与对照组的差异无统计学意义(F＝2.46，3.62；P>0.05)。大耳兔眼角膜碱烧伤后4 d、7 d、14 d时，各实验组房水中的4EBP1、VEGF蛋白含量与对照组相比，差异具有统计学意义(F＝10.73，49.15，62.37，17.55，106.61，202.13；P<0.05)，且4EBP1与VEGF的蛋白表达呈线性正相关(r＝0.969，0.803，0.67，0.972；P<0.05)。大耳兔眼角膜碱烧伤后7 d时，实验1组的角膜组织中mTOR、4EBP1及P70S6K的mRNA相对表达量为(0.73±0.26)、(0.58±0.19)和(0.52±0.29)；实验2组的相对表达量分别为(0.48±0.13)、(0.39±0.21)和(0.41±0.18)；实验3组的相对表达量分别为(0.36±0.09)、(0.22±0.09)和(0.18±0.07)。大耳兔眼角膜碱烧伤后14 d时，各实验组mTOR、4EBP1和P70S6K的mRNA表达量较眼角膜碱烧伤后7 d时的mRNA表达量降低；且随着姜黄素浓度的增高，mTOR、4EBP1和P70S6K在mRNA水平的相对表达量依次降低。

结论

姜黄素可通过抑制mTOR信号传导通路降低VEGF蛋白的表达，从而抑制眼角膜碱烧伤后新生血管的生长。

关键词: 姜黄素, 碱烧伤, 角膜新生血管, 机制

Objective

The aim of this study was to investigate the effects and its mechanism of curcumin on corneal neovascularization (CNV) after alkali burn in rabbit corneal.

Methods

The alkali burn models were established by alkali. After that, forty-eight rabbits were distributed randomly into 4 groups. The rabbits of DMSO group were treated with dimethyl sulfoxide; those of experimental group 1st were treated with 40 μmol/L curcumin; those of experimental group 2nd were treated with 80 μmol/L curcumin and those of experimental group 3rd were treated with 160 μmol/L curcumin. Right eye was dripped with the drug, 1 drop/time, 4 times/day, for 14 days. The corneal neovascularization was observed by the slit-lamp microscopy after alkali burn. The enzyme-linked immunoassay was used to detect VEGF, 4EBP1 factor of protein content from the aqueous humor of rabitts burn by alkali after 2, 4, 7, and 14 days. The reverse transcription polymerase chain reaction was used to detect relative expression of 4EBP1-P70S6K-mTOR factor gene in corneal tissue of rabbits burn by alkali after 7 and 14 days. To compare the two groups, two-factor repeated measurement analysis of variance was used. SNK method was used to compare when the difference was statistically significant. Spearman correlation coefficient was calculated to determine the correlation between 4EBP1 and VEGF protein.

Results

The area of CNV among expertmental groups and DMSO group had no significant difference when cornea alkali was burned after 4 days (F=0.592, P>0.05). The area of CNV after cornea alkali burned for 7 days and 14 days had significant difference between them (F=27.5, 28.64; P<0.05). Additionally, there was no significant difference on the amount of 4 EBP1 and expression of VEGF protein in the aqueous humor (F=2.462, 3.617; P>0.05). When cornea alkali was burned after 4 d, 7 d, 14 d , there was significant difference on the amount of 4 EBP1 and expression of VEGF protein in the aqueous humor (F=10.734, 49.154, 62.367, 17.552, 106.607, 202.130; P<0.05). Line Pearson correlation analysis found that 4EBP1 in aqueous humor is linear positive correlation with VEGF protein expression (r=0.969, 0.803, 0.67, 0.972). The expression of mTOR, 4EBP1, P70S6K in corneal tissue at mRNA level on 7 days after alkali burn in experimental group 1st were (0.73±0.26), (0.58±0.19), (0.52±0.29); those of experimental group 2nd were (0.48±0.13), (0.39±0.21), (0.41±0.18); those of experimental group 3rd were (0.36±0.09), (0.22±0.09), (0.18±0.07). The expression of mTOR, 4EBP1, P70S6K in corneal tissue at mRNA level after alkali burn for 14 days was lower than that of after alkali burn for 7 days. With the concentration of curcumin increasing, the expression quantity of mTOR, 4EBP1, P70S6K in corneal tissue at mRNA level reduced in turn.

Conclusion

Curcumin might reduce the expression of VEGF by inhibiting mTOR signaling pathways, and thus could inhibit the CNV growth after alkali burn.

Key words: Curcumin, Alkali burn, Corneal neovascularization, Mechanism

图1 大耳兔碱烧伤后各组不同时间点角膜新生血管的形态　图A~D依次为大耳兔碱烧伤后2 d，二甲基亚砜组、实验1组、实验2组及实验3组角膜新生血管的形态变化，图示碱烧伤后2 d各组均无角膜新生血管；图E~H依次为大耳兔碱烧伤后4 d，二甲基亚砜组、实验1组、实验2组及实验3组角膜新生血管的形态变化；图I~L依次为大耳兔碱烧伤后7 d，二甲基亚砜组、实验1组、实验2组及实验3组角膜新生血管的形态变化；图M~P依次为大耳兔碱烧伤后14 d，二甲基亚砜组、实验1组、实验2组及实验3组角膜新生血管的形态变化。图示大耳兔碱烧伤后4 d、7 d和14 d时对照组角膜新生血管致密粗大；随着时间延长，实验1组和实验2组角膜新生血管管腔小、稀疏；实验3组角膜新生血管稀疏细小，周边部角膜新生血管大部分已消退萎缩

表1 大耳兔眼角膜碱烧伤后各组不同时间点角膜新生血管面积的比较(mm²)

组别	眼数(只)	时间点
组别	眼数(只)	4 d	7 d	14 d
对照组	12	20.24±0.89	24.22±2.70	25.96±2.44
实验1组	12	20.38±2.18	19.04±1.33^a	18.71±1.94^a
实验2组	12	19.43±1.01	5.88±0.97^ab	13.54±1.44^ab
实验3组	12	21.04±1.53	12.66±0.75^abc	9.32±1.16^abc
F值		0.59	27.35	28.64
P值		>0.05	<0.05	<0.05

表1 大耳兔眼角膜碱烧伤后各组不同时间点角膜新生血管面积的比较(mm²)

组别	眼数(只)	时间点
组别	眼数(只)	4 d	7 d	14 d
对照组	12	20.24±0.89	24.22±2.70	25.96±2.44
实验1组	12	20.38±2.18	19.04±1.33^a	18.71±1.94^a
实验2组	12	19.43±1.01	5.88±0.97^ab	13.54±1.44^ab
实验3组	12	21.04±1.53	12.66±0.75^abc	9.32±1.16^abc
F值		0.59	27.35	28.64
P值		>0.05	<0.05	<0.05

表2 大耳兔眼角膜碱烧伤后各组不同时间点房水中4E结合蛋白因子蛋白含量的比较(pg/ml)

组别	例数	时间点
组别	例数	2 d	4 d	7 d	14 d
对照组	3	44.24±2.87	54.31±3.75	60.41±3.92	64.49±3.15
实验1组	3	46.75±3.56	55.14±3.52	49.97±2.81^a	40.25±2.65^abc
实验2组	3	45.63±4.14	52.59±2.66	43.58±2.38^ab	31.08±6.44^ab
实验3组	3	51.20±4.20	42.65±1.94^abc	32.54±2.08^abc	22.81±2.01^abc
F值		2.46	10.73	49.15	62.37
P值		>0.05	<0.05	<0.05	<0.05

表2 大耳兔眼角膜碱烧伤后各组不同时间点房水中4E结合蛋白因子蛋白含量的比较(pg/ml)

组别	例数	时间点
组别	例数	2 d	4 d	7 d	14 d
对照组	3	44.24±2.87	54.31±3.75	60.41±3.92	64.49±3.15
实验1组	3	46.75±3.56	55.14±3.52	49.97±2.81^a	40.25±2.65^abc
实验2组	3	45.63±4.14	52.59±2.66	43.58±2.38^ab	31.08±6.44^ab
实验3组	3	51.20±4.20	42.65±1.94^abc	32.54±2.08^abc	22.81±2.01^abc
F值		2.46	10.73	49.15	62.37
P值		>0.05	<0.05	<0.05	<0.05

表3 大耳兔眼角膜碱烧伤后各组不同时间点房水中血管内皮生长因子蛋白含量的比较(pg/ml)

组别	例数	时间点
组别	例数	2 d	4 d	7 d	14 d
对照组	3	275.12±6.59	310.58±9.43	316.57±11.14	346.20±11.97
实验1组	3	292.77±8.39	291.33±10.29^a	260.84±13.36^a	243.54±9.87^a
实验2组	3	289.54±7.02	269.36±8.63^ab	214.15±2.54^ab	213.43±4.35 ^ab
实验3组	3	291.71±7.82	265.36±5.63^ab	192.59±5.58^abc	177.05±7.36^abc
F值		3.62	17.56	106.61	202.13
P值		>0.05	<0.05	<0.05	<0.05

表3 大耳兔眼角膜碱烧伤后各组不同时间点房水中血管内皮生长因子蛋白含量的比较(pg/ml)

组别	例数	时间点
组别	例数	2 d	4 d	7 d	14 d
对照组	3	275.12±6.59	310.58±9.43	316.57±11.14	346.20±11.97
实验1组	3	292.77±8.39	291.33±10.29^a	260.84±13.36^a	243.54±9.87^a
实验2组	3	289.54±7.02	269.36±8.63^ab	214.15±2.54^ab	213.43±4.35 ^ab
实验3组	3	291.71±7.82	265.36±5.63^ab	192.59±5.58^abc	177.05±7.36^abc
F值		3.62	17.56	106.61	202.13
P值		>0.05	<0.05	<0.05	<0.05

图2 大耳兔眼角膜碱烧伤后各组4E结合蛋白和血管内皮生长因子蛋白表达量平均值的变化

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Effects of curcumin on corneal neovascularization induced by alkali burn

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Effects of curcumin on corneal neovascularization induced by alkali burn