明目“五子”对视网膜光化学损伤的防治作用及其机理的实验研究
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摘要
目的：观察光照后大鼠视网膜的病理形态、生化及电生理方面的改变；探讨中药明目“五子”对大鼠视网膜光损伤的保护作用及其机理。
方法：采用绿色荧光灯持续照射24小时造成大鼠视网膜光损伤。分别于光照前、光照后即刻、光照后14天，进行视网膜光镜、电镜形态学观察和外核层厚度测量，同时用TUNEL法标记细胞凋亡；使用高效液相色谱仪测定视网膜游离氨基酸的变化；并于光照前、光照后即刻、光照后3、7、14天行多焦视网膜电图(mfERG)的检测。用同样的方法造模，同时给予明目“五子”高、低剂量灌胃，连续给药14天后，作mfERG检查，进行视网膜光镜形态学观察、外核层厚度测量，并用TUNEL法标记细胞凋亡；免疫组织化学法观察视网膜碱性成纤维细胞生长因子(bFGF)及胶质纤维酸性蛋白(GFAP)的表达；采用硝酸还原酶法测定视网膜NO的含量；进行氨基酸的检测。
结果：(1)光照前视网膜形态正常。光照后视网膜出现大量细胞凋亡，外核层变薄，与光照前比较有显著性差异(P＜0.01)，内外节排列紊乱，分界不清，细胞核肿胀，内节线粒体肿胀，外节盘膜水肿、断裂、空泡变，RPE细胞微绒毛几乎消失，溶酶体增多。14天后各种损伤均有所减轻，但外核层继续变薄；明目“五子”高剂量组视网膜外核层厚度显著高于模型对照组，细胞凋亡率显著低于模型对照组，二者在统计学上均有显著性差异(P＜0.01)。(2)“五子”高剂量组能显著减低视网膜GFAP的表达，增强bFGF的表达，与模型对照组比较统计学上有显著性差异(P＜0.01)；且二者的负相关关系具有显著性意义(P＜0.01)。(3)光照后视网膜天门冬氨酸(ASP)、谷氨酸(GLU)、γ-氨基丁酸(GABA)含量升高，与正常对照组比较有显著性统计学上有显著性差异(P＜0.01)；14天后有所降低，但与正常对照组比较，仍有显著性意义(P＜0.01)；高剂量组视网膜氨基酸含量与模型对照组比较明显降低，统计学上有显著性意义(P＜0.05)。(4)光照后，大鼠视网膜NO含量升高，与正常对照组比较，
统计学上有显著性差异(P<0.01);“五子”高、低剂量组能减低视网膜NO的
含量，与模型对照组比较有显著性差异(P<0.05~0.01)。(5)光照后视网膜
m犯RG显示，大鼠视网膜Nl、Pl波各环反应密度明显降低，与光照前比较统
计学上有显著性差异(P<0.01)，14天后仍未恢复，与模型对照组比较，“五子妙
高剂量组Nl、Pl波反应密度明显升高，具有显著性差异(P<0 .05一0.01)。
结论:明目“五子”具有减轻视网膜光损伤，以及促进光损伤后视网膜的
修复和视功能的恢复的作用。其作用机理与减轻NO对视网膜的毒性、调控视
网膜神经递质的异常，增强视网膜神bFGF表达、抑制神经胶质细胞增生，从
而抑制视网膜细胞凋亡有关。
Objective: To observe the morphologic, biochemical and electrophysiologic changes of the retina in rats after exposure to light, and study the protective effect and mechanism of MingMuWuZi against photic injury of retina.
Methods: Sprague-Dawley (SD) rats were exposed to green fluorescent light for 24 hours. The phathological changes of the retina were examined with light and eletron microscope, and the thickness of the outer nucleus layer (ONL) was measured before and immediately, 14 days after exposure. Simultaneously the apoptotic cells were detected with TUNEL method. The level of aspartate (ASP), glutamate (GLU), glycine (GLY)and Y -aminobutyric acid (GABA) were measured by amino acid automatic analytical apparatus in the retina of rats. Muhifocal electroretinograms (mfERG) examination was given in rats before and immediately, 3, 7, 14 days after exposure. In the second experiment, MingMuWuZi was given at high, low dosage before light exposure in the same model and lasted for 14 days. The phathological changes of the retina were examined with light and eletron microscope, and the ONL was measured, and the apoptotic cells were detected with TUNEL method after mfERG examination before and 14 days after exposure. The expression of GFAP and bFGF was observed by irnmunohistochemical methods. Content of nitric oxide (NO) in retina were examined by nitric acid reductase method. The level of amino acid was measured in retina.
Results: (1) The retina was morphologically normal before light exposure. A significant number of TUNEL-positive nuclei occurred in retina after light exposure, and the ONL was much thinner than before light exposure, compared with before light exposure the differences of both were significant in statistically (P<0.01). regular arrangement of outer and inner segments was disordered, dividing line wasn't distinct, the nucleus and the mitochondrion of inner segment were swollen, discmembrane of outer segment were swollen, vacuolation, disorganization, the apical microvilli of RPE cell almost disappeared with an increase of lysosomes. The damages of all became lessened after 14 days. But the ONL was more attenuate. Compared with the model group, the thickness of the ONL in high dosage group of MingMuWuZi was increased and ratio of apoptotic cell was decreased obviously and the differences were evident(P<0.01). (2) Compared with the model group, the expression of GFAP was decreased and bFGF was increased evidently in high dosage group of MingMuWuZi, which showed significant difference (P<0.01). Both had distinctly negative relativity in statistically (P<0.01). (3) Compared with the normal group, the level of ASP, GLU and GABA in retina after light exposure immediately
and after 14 days were increased evidently (P<0.01). Compared with the model group, the level of amino acid in high dosage group was decreased, which showed significant difference (P<0.05). (4) The content of NO in retina increased distinctly after light exposure, compared with the control group the difference was significant. Compared with the model group, the content of NO in high and low dosage group was decreased significantly, the difference was obvious (P<0.05~0.01). (5)The Nl, PI waves response densities of 1~5 rings markedly decreased in rats after light exposure, compared with before light exposure the difference was significant. Compared with the model group, the Nl, PI waves response densities of 1-5 rings were increased statistically in high dosage group, which showed significant difference in statistically (P<0.05).
Conclusion: MingMuWuZi could release the photic injury of retina, accelerate the renovation of retina after light exposure, and ameliorate the function of retina. The mechanism might relate to inhibit the apoptosis and gliosis, enhance the expression of bFGF in retina, and regulate the neural transmitter in retina.
方法：采用绿色荧光灯持续照射24小时造成大鼠视网膜光损伤。分别于光照前、光照后即刻、光照后14天，进行视网膜光镜、电镜形态学观察和外核层厚度测量，同时用TUNEL法标记细胞凋亡；使用高效液相色谱仪测定视网膜游离氨基酸的变化；并于光照前、光照后即刻、光照后3、7、14天行多焦视网膜电图(mfERG)的检测。用同样的方法造模，同时给予明目“五子”高、低剂量灌胃，连续给药14天后，作mfERG检查，进行视网膜光镜形态学观察、外核层厚度测量，并用TUNEL法标记细胞凋亡；免疫组织化学法观察视网膜碱性成纤维细胞生长因子(bFGF)及胶质纤维酸性蛋白(GFAP)的表达；采用硝酸还原酶法测定视网膜NO的含量；进行氨基酸的检测。
结果：(1)光照前视网膜形态正常。光照后视网膜出现大量细胞凋亡，外核层变薄，与光照前比较有显著性差异(P＜0.01)，内外节排列紊乱，分界不清，细胞核肿胀，内节线粒体肿胀，外节盘膜水肿、断裂、空泡变，RPE细胞微绒毛几乎消失，溶酶体增多。14天后各种损伤均有所减轻，但外核层继续变薄；明目“五子”高剂量组视网膜外核层厚度显著高于模型对照组，细胞凋亡率显著低于模型对照组，二者在统计学上均有显著性差异(P＜0.01)。(2)“五子”高剂量组能显著减低视网膜GFAP的表达，增强bFGF的表达，与模型对照组比较统计学上有显著性差异(P＜0.01)；且二者的负相关关系具有显著性意义(P＜0.01)。(3)光照后视网膜天门冬氨酸(ASP)、谷氨酸(GLU)、γ-氨基丁酸(GABA)含量升高，与正常对照组比较有显著性统计学上有显著性差异(P＜0.01)；14天后有所降低，但与正常对照组比较，仍有显著性意义(P＜0.01)；高剂量组视网膜氨基酸含量与模型对照组比较明显降低，统计学上有显著性意义(P＜0.05)。(4)光照后，大鼠视网膜NO含量升高，与正常对照组比较，
统计学上有显著性差异(P<0.01);“五子”高、低剂量组能减低视网膜NO的
含量，与模型对照组比较有显著性差异(P<0.05~0.01)。(5)光照后视网膜
m犯RG显示，大鼠视网膜Nl、Pl波各环反应密度明显降低，与光照前比较统
计学上有显著性差异(P<0.01)，14天后仍未恢复，与模型对照组比较，“五子妙
高剂量组Nl、Pl波反应密度明显升高，具有显著性差异(P<0 .05一0.01)。
结论:明目“五子”具有减轻视网膜光损伤，以及促进光损伤后视网膜的
修复和视功能的恢复的作用。其作用机理与减轻NO对视网膜的毒性、调控视
网膜神经递质的异常，增强视网膜神bFGF表达、抑制神经胶质细胞增生，从
而抑制视网膜细胞凋亡有关。
Objective: To observe the morphologic, biochemical and electrophysiologic changes of the retina in rats after exposure to light, and study the protective effect and mechanism of MingMuWuZi against photic injury of retina.
Methods: Sprague-Dawley (SD) rats were exposed to green fluorescent light for 24 hours. The phathological changes of the retina were examined with light and eletron microscope, and the thickness of the outer nucleus layer (ONL) was measured before and immediately, 14 days after exposure. Simultaneously the apoptotic cells were detected with TUNEL method. The level of aspartate (ASP), glutamate (GLU), glycine (GLY)and Y -aminobutyric acid (GABA) were measured by amino acid automatic analytical apparatus in the retina of rats. Muhifocal electroretinograms (mfERG) examination was given in rats before and immediately, 3, 7, 14 days after exposure. In the second experiment, MingMuWuZi was given at high, low dosage before light exposure in the same model and lasted for 14 days. The phathological changes of the retina were examined with light and eletron microscope, and the ONL was measured, and the apoptotic cells were detected with TUNEL method after mfERG examination before and 14 days after exposure. The expression of GFAP and bFGF was observed by irnmunohistochemical methods. Content of nitric oxide (NO) in retina were examined by nitric acid reductase method. The level of amino acid was measured in retina.
Results: (1) The retina was morphologically normal before light exposure. A significant number of TUNEL-positive nuclei occurred in retina after light exposure, and the ONL was much thinner than before light exposure, compared with before light exposure the differences of both were significant in statistically (P<0.01). regular arrangement of outer and inner segments was disordered, dividing line wasn't distinct, the nucleus and the mitochondrion of inner segment were swollen, discmembrane of outer segment were swollen, vacuolation, disorganization, the apical microvilli of RPE cell almost disappeared with an increase of lysosomes. The damages of all became lessened after 14 days. But the ONL was more attenuate. Compared with the model group, the thickness of the ONL in high dosage group of MingMuWuZi was increased and ratio of apoptotic cell was decreased obviously and the differences were evident(P<0.01). (2) Compared with the model group, the expression of GFAP was decreased and bFGF was increased evidently in high dosage group of MingMuWuZi, which showed significant difference (P<0.01). Both had distinctly negative relativity in statistically (P<0.01). (3) Compared with the normal group, the level of ASP, GLU and GABA in retina after light exposure immediately
and after 14 days were increased evidently (P<0.01). Compared with the model group, the level of amino acid in high dosage group was decreased, which showed significant difference (P<0.05). (4) The content of NO in retina increased distinctly after light exposure, compared with the control group the difference was significant. Compared with the model group, the content of NO in high and low dosage group was decreased significantly, the difference was obvious (P<0.05~0.01). (5)The Nl, PI waves response densities of 1~5 rings markedly decreased in rats after light exposure, compared with before light exposure the difference was significant. Compared with the model group, the Nl, PI waves response densities of 1-5 rings were increased statistically in high dosage group, which showed significant difference in statistically (P<0.05).
Conclusion: MingMuWuZi could release the photic injury of retina, accelerate the renovation of retina after light exposure, and ameliorate the function of retina. The mechanism might relate to inhibit the apoptosis and gliosis, enhance the expression of bFGF in retina, and regulate the neural transmitter in retina.
引文
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56. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991; 43(2):109-142
57. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994; 269(19): 13725-13728
58．严兵，周初松，刘萍等．脊髓伤后脊髓组织中一氧化氮合酶活性变化与兴奋性氨基酸释放间的联系．现代康复，2000；4(12)：1838-1839
59．刘晓缺，丁正平，吕源淑．大鼠视网膜急性光损伤后一氧化氮酶阳性神经元的改变．中华眼底病杂志，2000；16(3)：209-210
60．徐超，张绍东，崔雯等．一氧化氮合成酶抑制剂(L-NNA)对缺血纹体兴奋性和抑制性氨基酸释放的影响．中华神经外科杂志，1998；14(4)：321-324
61. Sutter EE. Field topography of the visual evoked response. Invest Ophthalmol Vis Sci, 1988,29(Suppl):433.
62. Sutter EE, Vaegan. Lateral interation component and local luminance nonlinearities in the human pattern reversal ERG. Vision Res, 1990; 30(5): 659-671.
63. Wu S, Sutter EE. A topographic study of oscillatory potentials in man. Vis Neurosci. 1995; 12(6):1013-25.
64. SutterEE, Tran D. The field topography of ERG components in man--Ⅰ. The photopic luminance response. Vision Res. 1992; 32(3):433-446
65. Sutter EE. The interpretation of multifocal binary kernels. Doc ophthalmol, 2000; 100(2-3):49-75
66. Curcio CA, Sloan KP, Kalina RE, et al. Human photoreceptor topography. J Comp Neurol, 1990; 292(4): 497-523
67. Hood DC, Seiple W, Holopigian M, et al. A comparison.of the components of the multifocal and full-field ERGs. Vis Neurosci, 1997; 14(3): 533-544
68. Kondo M, Miyake Y, Horiguchi M, et al. Clinical evaluation of multifocal electroretinogram
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72． Horiguchi M, Suzuki S, Kondo M, et al. Effect of glutamate analogues and inhibitory neurotransmitters on the electroretmograms elicited by random sequence stimuli in rabbits. Invest Ophthalmol Vis Sci, 1998; 39(11) : 2171-2176
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54. Hardy P, Nuyt AM, Abran D,et al. Nitric oxide in retinal and choroidal blood flow autoregulation in newborn pigs: interactions with prostaglandins. Pediatr Res. 1996; 39(3): 487-493.
55. Maynard KI, Yanez P, Ogilvyc S. Nitric oxide modulates light-evoked compound action potentials in the intact rabbit retina. Neuroreport 1995; 6(6): 850-852
56. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991; 43(2):109-142
57. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994; 269(19): 13725-13728
58．严兵，周初松，刘萍等．脊髓伤后脊髓组织中一氧化氮合酶活性变化与兴奋性氨基酸释放间的联系．现代康复，2000；4(12)：1838-1839
59．刘晓缺，丁正平，吕源淑．大鼠视网膜急性光损伤后一氧化氮酶阳性神经元的改变．中华眼底病杂志，2000；16(3)：209-210
60．徐超，张绍东，崔雯等．一氧化氮合成酶抑制剂(L-NNA)对缺血纹体兴奋性和抑制性氨基酸释放的影响．中华神经外科杂志，1998；14(4)：321-324
61. Sutter EE. Field topography of the visual evoked response. Invest Ophthalmol Vis Sci, 1988,29(Suppl):433.
62. Sutter EE, Vaegan. Lateral interation component and local luminance nonlinearities in the human pattern reversal ERG. Vision Res, 1990; 30(5): 659-671.
63. Wu S, Sutter EE. A topographic study of oscillatory potentials in man. Vis Neurosci. 1995; 12(6):1013-25.
64. SutterEE, Tran D. The field topography of ERG components in man--Ⅰ. The photopic luminance response. Vision Res. 1992; 32(3):433-446
65. Sutter EE. The interpretation of multifocal binary kernels. Doc ophthalmol, 2000; 100(2-3):49-75
66. Curcio CA, Sloan KP, Kalina RE, et al. Human photoreceptor topography. J Comp Neurol, 1990; 292(4): 497-523
67. Hood DC, Seiple W, Holopigian M, et al. A comparison.of the components of the multifocal and full-field ERGs. Vis Neurosci, 1997; 14(3): 533-544
68. Kondo M, Miyake Y, Horiguchi M, et al. Clinical evaluation of multifocal electroretinogram
Invest Ophthalmol Vis Sci. 1995; 36(10) :2146-2150
69． Vaegan, Buckland L. The spatial distribution of ERG loses across the posterior pole of glauoomatous eyes multifocal recodings. Aust N J Ophthalmic, 1996, 24(2suppl): 28-31
70． Pahnowski AM, Sutter EE, Bearse MA, et al. Mapping of retina function in diabetic retinopathy using rnultifocal eletroretinogram Invest Ophthalmol Vis Sci, 1997;38(12) : 2586-2596
71． Klistorner A, Crewther DP, Crewther SG Temporal analysis of the topographic ERG: Chromatic versus achromatic stimulation. Vision Res, 1998; 38(7) . 1047-1062
72． Horiguchi M, Suzuki S, Kondo M, et al. Effect of glutamate analogues and inhibitory neurotransmitters on the electroretmograms elicited by random sequence stimuli in rabbits. Invest Ophthalmol Vis Sci, 1998; 39(11) : 2171-2176
73． Seeliger MW, Kretschmarm UH, Apfeltedt-Sylla, et al. Implicit time topography of multifocal eletrorctinograms. Invest Vis Sci, 1998; 39(5) : 718-723
74． Hood DC, Holopigian K, Greenstain V, et al. Assessment of local retinal function in patients with retrnitis pigmentosa using the multifocal ERG technique. Vision Res, 1998; 38(1) : 163-179
75． Chan HL, Brown B. Investigation of retinitis pigmentosa using the multifocal electroretinogram Ophthal Physiol Opt, 1998; 18(4) : 335-350
76． Hood DQ Wladis EJ, Sbady S, et al. Multifocal rod electroretmograms. Invest Ophthahnol Vis Sci. 1998; 39(7) : 1152-1162