益气活血法对糖尿病视网膜神经细胞保护作用的实验研究
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摘要
目的：新的观点认为：DR除了视网膜微血管损害以外,还是一种神经组织的慢性退行性变,神经元和神经胶质的改变可能是导致视网膜血管病变的一个主要因素,糖尿病引起的RGCs和Muller细胞的改变要早于出现有证据的血管病变。我们曾观察到益气类中药(红参)对糖尿病大鼠RGCs具有神经保护作用,在此基础上,欲进一步研究益气活血法对DM视网膜神经细胞损害的干预保护作用。本实验欲通过观察DM视网膜神经胶质细胞(Muller细胞)GFAP、GLAST、GS表达的变化、视网膜神经细胞的损害,神经营养因子NT-3的表达,探讨DM对视网膜神经细胞的影响,观察益气活血类中药对视网膜神经细胞的神经保护作用,分析其可能的作用机理,为中药防治DR开辟新思路。
方法：选用SPF级六周龄雄性SD大鼠82只,通过腹腔注射链脲佐菌素(65mg/kg体重)诱导实验性DM大鼠模型,尾静脉血糖≥16.7mmol/L者为DM大鼠模型。造模成功的糖尿病大鼠随机分为模型组20只,治疗组30只,对照组20只。正常组大鼠12只。治疗组和对照组大鼠在糖尿病造模成功后即开始给药,每日上午9-10时灌胃给药一次,治疗组药物为芪参益气滴丸,对照组为多贝斯,剂量均为0.5g/kg体重/日(正常成人剂量的20倍)。定期检测大鼠血糖、体重,实验周期为300天。颈动脉放血处死大鼠,摘取眼球,制备视网膜组织切片,通过光镜、电镜检查,从组织病理水平观察糖尿病大鼠视网膜的改变；RT-PCR法检测视网膜GFAP mRNA表达水平；TUNEL法检测RGCs及内核层细胞的凋亡并记数细胞凋亡数量；免疫组织化学技术LSAB法检测GFAP、GLAST、GS、NT-3在视网膜的表达,从分子生物学水平观察糖尿病大鼠视网膜RGCs及内核层细胞功能的改变,用图象分析仪测量免疫组化的显色强度,进行定量分析。采用SPSS13.0统计软件对数据进行分析处理。
结果：(1)成模后,与正常组相比,DM大鼠各时期体重明显减轻(P<0.01),治疗组、对照组、模型组大鼠体重无显著差异(P>0.05)。(2)成模后,与正常组相比,DM大鼠各时期血糖显著升高(P<0.01),治疗组、对照组、模型组大鼠血糖无显著差异(P>0.05)。(3)正常大鼠视网膜各层组织层次清楚,RGCs单层排列,内核层由3-5层细胞构成,细胞排列整齐,胞核较大,染色稍深。视细胞外节膜盘结构清晰,内节椭圆体内线粒体沿周边排列,结构规整,外核层核染色质分布均匀,内核层排列整齐,核染色质均匀,细胞器结构清楚,RGCs胞体呈卵圆形,胞浆中有较多的粗面内质网、多聚核糖体、线粒体,细胞表面有较多的突起。DM模型组大鼠视网膜神经纤维层水肿、增厚,胶质细胞增生,空泡样变,RGCs空泡变性,核固缩,部分坏死,内核层、外核层细胞排列极度紊乱。视细胞外节膜盘模糊不清,间隙扩大,部分溶解、断裂,内节椭圆体内线粒体肿胀、空泡样变,排列紊乱,外核层细胞核固缩,内核层深染细胞明显增多,细胞表面的微绒毛减少,细胞核固缩,部分崩解,核内染色质呈大小不等的团块,边集明显,粗面内质网高度扩张,线粒体肿胀,嵴断裂,空泡样变,RGCs胞体变形,核固缩,部分溶解、核膜消失,胞浆较多大小不等的空泡,线粒体肿胀,细胞表面突起明显减少(4)RT-PCR检测大鼠视网膜GFAP mRNA表达水平结果显示：与正常组相比,糖尿病大鼠视网膜GFAP mRNA表达明显增加(P<0.01)；与模型组相比,治疗组、对照组表达量明显降低(P<0.01)；与对照组相比,治疗组表达量明显降低(P<0.05)。(5)用TUNEL法检测,大鼠视网膜凋亡阳性细胞仅见于RGCs层和内核层。与正常组相比,治疗组、对照组、模型组大鼠视网膜内核层细胞凋亡及RGCs凋亡数量显著增加(P<0.01)；与模型组相比,治疗组、对照组细胞凋亡数量明显减少(P<0.01)；与对照组相比,治疗组细胞凋亡数量明显减少(P<0.01)。(6)LSAB法标记染色的GFAP在视网膜的表达结果显示：与正常组相比,治疗组、模型组和对照组GFAP阳性表达IOD值明显增加(P<0.01)；与模型组相比,治疗组、对照组表达明显降低(P<0.05)；与对照组相比,治疗组表达明显降低(P<0.05)。(7)LSAB法标记染色的GLAST在视网膜的表达结果显示：与正常组相比,治疗组、模型组、对照组大鼠视网膜GLAST阳性表达IOD值明显降低(P<0.05)；与模型组相比,治疗组表达明显增加(P<0.05),而对照组无明显增加(P>0.05)；与对照组相比,治疗组表达明显增加(P<0.05)。(8)LSAB法标记染色的GS在视网膜的表达结果显示：与正常组相比,治疗组、模型组、对照组大鼠视网膜GS阳性表达IOD值明显降低(P<0.01)；与模型组相比,治疗组、对照组表达明显增加(P<0.01)；与对照组相比,治疗组表达明显增加(P<0.01)。(9)LSAB法标记染色的NT-3在视网膜的表达结果显示：与正常组相比,治疗组、对照组、模型组视网膜NT-3阳性表达IOD值明显减少(P<0.01)；与模型组相比,治疗组、对照组表达明显增加(P<0.01)；与对照组相比,治疗组表达明显增加(P<0.01)。(10)GFAP阳性表达IOD值与内核层细胞凋亡数及RGCs凋亡数呈正相关；GLAST、GS、NT-3阳性表达IOD值与内核层细胞凋亡数及RGCs凋亡数呈负相关。
结论：
1糖尿病大鼠视网膜RGCs凋亡数及内核层细胞凋亡数显著增加；
2糖尿病大鼠视网膜Muller细胞GFAP表达增强,处于活跃的胶质反应性增生状态；
3糖尿病大鼠视网膜Muller细胞GLAST及GS功能减弱,转运谷氨酸能力下降；4益气活血法(芪参益气滴丸)对糖尿病大鼠视网膜神经细胞具有保护作用,其作用机制：(1)抑制Muller细胞GFAP的过度表达,抑制胶质细胞反应性增生,减轻DM对Muller细胞功能的影响,减轻视网膜神经元的损害；
(2)促进Muller细胞GLAST及GS的表达,及时有效清除细胞外过量谷氨酸,减轻高浓度谷氨酸的兴奋性毒性作用；(3)促进神经营养因子NT-3的表达,减少神经细胞的凋亡。
Purpose:A new view points that DR is not only a retinal microvascular damage but also a chronic degeneration of nerve tissue. The changes of neurons and glial might be the major factors for retinal vascular disease. The change of ganglion cells and Muller cells caused by diabetes should occur as early as there is evidence of vascular lesions. My research was designed to assess the expression of GFAP、GLAST、GS、NT-3 in glial cells(Muller cells) and the damage of RGCs in rats by DM and discuss the effection to retinal neurons by DM, observe the neurologically protective role of Chinese medicine, analyzes its nerve protective mechanism and find a way to prevent and cure DR by Chinese medicine..
Methods:82 male SD rats (weight between 180g and 220g) are randomly divided into 4 groups:model group,control group, treatment group and normal group. The rats were injected STZ (65 mg/kg weight) peritoneanouly and those with a blood glucose level higher than or equal to 16.7 mmol/L are DM model rats. Rats in treatment group and normal group were with QiShenYiQI pills and DuoBeiShi pills (1.5g/kg weight) each day. Blood glucose level and body weights were measured regularly. Experiment lasts for 300 days. Carotid bloodletting executed rats.The eyeballs were removed and retinal slides were made. Retinal structure was observed by optical microscope and electron microscopy. The expression level of GFAP mRNA was detected by the method of RT-PCR. Apoptosis of RGCs and cells in retinal kernel layer were measured by Tunel and the apoptotic cells were counted. LSAB method was used to measure the expression level of GFAP,GLAST,GS,NT-3. Immunological histochemistry assay was used for quantitative analysis. SPSS was used for statistical analysis..
Results:(1) Weight of diabetic rats were significantly lower than normal group (P<0.01) and there is no significant differences in weight of treatment group, control group and the model of rats (P>0.05). (2) Glucose in diabetic rats were markedly higher than normal group (P<0.01) and there is no significant differences in glucose in treatment group, control group and the model of rats(P> 0.05). (3) Compared to normal group, the expression of GFAP mRNA by RT-PCR in diabetic rats was enhanced significantly. Compared to model group, the expression in treatment group and control group was decreased. Compared to control group, the expression in treatment group was decreased. (4) The positive apoptotic retinal cell were only expressed in RGCs and inner nuclear layer. The number of apoptotic RGCs and cells in inner nuclear layer in model group,control group and treatment group was significantly higher than in normal group. Compared to model group, the number of apoptotic cells in control group and treatment group decreased obviously. Compared to control group, the number of apoptotic cells in treatment group decreased obviously. (5) Compared to normal group, the positive expression of GFAP in diabetic rats by LSAB was enhanced significantly. Compared to model group,the expression in treatment group and control group was decreased. Compared to control group, the expression in treatment group was decreased (6) Compared to normal group, the positive expression of GLAST by LSAB in model group,control group and treatment group was decreased significantly. Compared to model group and control group, the expression in treatment group was enhanced. (7) Compared to normal group, the positive expression of GS by LSAB in model group,control group and treatment group was decreased significantly. Compared to model group, the expression in treatment group and control group was enhanced. Compared to control group, the expression in treatment group was increased. (8) Compared to normal group, the positive expression of NT-3 by LSAB in control group, model group and treatment group was decreased significantly. Compared to model group, the expression in treatment group and control group was enhanced. Compared to control group, the expression in treatment group was increased. (9) The number of apoptotic RGCs and cells in inner nuclear layer was correlated positively with the positive expression level of GFAP, while the expression of GLAST,GS and NT-3 was negatively.
Conclusion:1 The apoptotic cells number in RGCs and the inner nuclear layer in diabetic rats retina were significantly increased.2 The expression of GFAP in Retinal Muller cells in diabetic rats increases and Muller cells are in active proliferation of glial reactive state.3 The function of GLAST and GS iin diabetic retinal Muller cells is weakened and its ability of transporter glutamate is declined.4 Qishenyiqi pills has a protective effect to retinal nerve cells in diabetic rats. Its mechanism is as follows.(1)Qishenyiqi pills can inhibit the overexpression of GFAP and glial cell reactive hyperplasia in Muller cells and reduce the damage to the Muller cell function and retinal neurons by DM.
(2)Qishenyiqi pills can promote the expression of GLAST and GS in Muller cells and remove the excessive extracellular glutamate effectively and reduce the high concentrations of the role of glutamate excitotoxicity. (3) Qishenyiqi pills can promote the expression of neurotrophic factors (NT-3) and reduce the the number of apoptotic neural cells.
方法：选用SPF级六周龄雄性SD大鼠82只,通过腹腔注射链脲佐菌素(65mg/kg体重)诱导实验性DM大鼠模型,尾静脉血糖≥16.7mmol/L者为DM大鼠模型。造模成功的糖尿病大鼠随机分为模型组20只,治疗组30只,对照组20只。正常组大鼠12只。治疗组和对照组大鼠在糖尿病造模成功后即开始给药,每日上午9-10时灌胃给药一次,治疗组药物为芪参益气滴丸,对照组为多贝斯,剂量均为0.5g/kg体重/日(正常成人剂量的20倍)。定期检测大鼠血糖、体重,实验周期为300天。颈动脉放血处死大鼠,摘取眼球,制备视网膜组织切片,通过光镜、电镜检查,从组织病理水平观察糖尿病大鼠视网膜的改变；RT-PCR法检测视网膜GFAP mRNA表达水平；TUNEL法检测RGCs及内核层细胞的凋亡并记数细胞凋亡数量；免疫组织化学技术LSAB法检测GFAP、GLAST、GS、NT-3在视网膜的表达,从分子生物学水平观察糖尿病大鼠视网膜RGCs及内核层细胞功能的改变,用图象分析仪测量免疫组化的显色强度,进行定量分析。采用SPSS13.0统计软件对数据进行分析处理。
结果：(1)成模后,与正常组相比,DM大鼠各时期体重明显减轻(P<0.01),治疗组、对照组、模型组大鼠体重无显著差异(P>0.05)。(2)成模后,与正常组相比,DM大鼠各时期血糖显著升高(P<0.01),治疗组、对照组、模型组大鼠血糖无显著差异(P>0.05)。(3)正常大鼠视网膜各层组织层次清楚,RGCs单层排列,内核层由3-5层细胞构成,细胞排列整齐,胞核较大,染色稍深。视细胞外节膜盘结构清晰,内节椭圆体内线粒体沿周边排列,结构规整,外核层核染色质分布均匀,内核层排列整齐,核染色质均匀,细胞器结构清楚,RGCs胞体呈卵圆形,胞浆中有较多的粗面内质网、多聚核糖体、线粒体,细胞表面有较多的突起。DM模型组大鼠视网膜神经纤维层水肿、增厚,胶质细胞增生,空泡样变,RGCs空泡变性,核固缩,部分坏死,内核层、外核层细胞排列极度紊乱。视细胞外节膜盘模糊不清,间隙扩大,部分溶解、断裂,内节椭圆体内线粒体肿胀、空泡样变,排列紊乱,外核层细胞核固缩,内核层深染细胞明显增多,细胞表面的微绒毛减少,细胞核固缩,部分崩解,核内染色质呈大小不等的团块,边集明显,粗面内质网高度扩张,线粒体肿胀,嵴断裂,空泡样变,RGCs胞体变形,核固缩,部分溶解、核膜消失,胞浆较多大小不等的空泡,线粒体肿胀,细胞表面突起明显减少(4)RT-PCR检测大鼠视网膜GFAP mRNA表达水平结果显示：与正常组相比,糖尿病大鼠视网膜GFAP mRNA表达明显增加(P<0.01)；与模型组相比,治疗组、对照组表达量明显降低(P<0.01)；与对照组相比,治疗组表达量明显降低(P<0.05)。(5)用TUNEL法检测,大鼠视网膜凋亡阳性细胞仅见于RGCs层和内核层。与正常组相比,治疗组、对照组、模型组大鼠视网膜内核层细胞凋亡及RGCs凋亡数量显著增加(P<0.01)；与模型组相比,治疗组、对照组细胞凋亡数量明显减少(P<0.01)；与对照组相比,治疗组细胞凋亡数量明显减少(P<0.01)。(6)LSAB法标记染色的GFAP在视网膜的表达结果显示：与正常组相比,治疗组、模型组和对照组GFAP阳性表达IOD值明显增加(P<0.01)；与模型组相比,治疗组、对照组表达明显降低(P<0.05)；与对照组相比,治疗组表达明显降低(P<0.05)。(7)LSAB法标记染色的GLAST在视网膜的表达结果显示：与正常组相比,治疗组、模型组、对照组大鼠视网膜GLAST阳性表达IOD值明显降低(P<0.05)；与模型组相比,治疗组表达明显增加(P<0.05),而对照组无明显增加(P>0.05)；与对照组相比,治疗组表达明显增加(P<0.05)。(8)LSAB法标记染色的GS在视网膜的表达结果显示：与正常组相比,治疗组、模型组、对照组大鼠视网膜GS阳性表达IOD值明显降低(P<0.01)；与模型组相比,治疗组、对照组表达明显增加(P<0.01)；与对照组相比,治疗组表达明显增加(P<0.01)。(9)LSAB法标记染色的NT-3在视网膜的表达结果显示：与正常组相比,治疗组、对照组、模型组视网膜NT-3阳性表达IOD值明显减少(P<0.01)；与模型组相比,治疗组、对照组表达明显增加(P<0.01)；与对照组相比,治疗组表达明显增加(P<0.01)。(10)GFAP阳性表达IOD值与内核层细胞凋亡数及RGCs凋亡数呈正相关；GLAST、GS、NT-3阳性表达IOD值与内核层细胞凋亡数及RGCs凋亡数呈负相关。
结论：
1糖尿病大鼠视网膜RGCs凋亡数及内核层细胞凋亡数显著增加；
2糖尿病大鼠视网膜Muller细胞GFAP表达增强,处于活跃的胶质反应性增生状态；
3糖尿病大鼠视网膜Muller细胞GLAST及GS功能减弱,转运谷氨酸能力下降；4益气活血法(芪参益气滴丸)对糖尿病大鼠视网膜神经细胞具有保护作用,其作用机制：(1)抑制Muller细胞GFAP的过度表达,抑制胶质细胞反应性增生,减轻DM对Muller细胞功能的影响,减轻视网膜神经元的损害；
(2)促进Muller细胞GLAST及GS的表达,及时有效清除细胞外过量谷氨酸,减轻高浓度谷氨酸的兴奋性毒性作用；(3)促进神经营养因子NT-3的表达,减少神经细胞的凋亡。
Purpose:A new view points that DR is not only a retinal microvascular damage but also a chronic degeneration of nerve tissue. The changes of neurons and glial might be the major factors for retinal vascular disease. The change of ganglion cells and Muller cells caused by diabetes should occur as early as there is evidence of vascular lesions. My research was designed to assess the expression of GFAP、GLAST、GS、NT-3 in glial cells(Muller cells) and the damage of RGCs in rats by DM and discuss the effection to retinal neurons by DM, observe the neurologically protective role of Chinese medicine, analyzes its nerve protective mechanism and find a way to prevent and cure DR by Chinese medicine..
Methods:82 male SD rats (weight between 180g and 220g) are randomly divided into 4 groups:model group,control group, treatment group and normal group. The rats were injected STZ (65 mg/kg weight) peritoneanouly and those with a blood glucose level higher than or equal to 16.7 mmol/L are DM model rats. Rats in treatment group and normal group were with QiShenYiQI pills and DuoBeiShi pills (1.5g/kg weight) each day. Blood glucose level and body weights were measured regularly. Experiment lasts for 300 days. Carotid bloodletting executed rats.The eyeballs were removed and retinal slides were made. Retinal structure was observed by optical microscope and electron microscopy. The expression level of GFAP mRNA was detected by the method of RT-PCR. Apoptosis of RGCs and cells in retinal kernel layer were measured by Tunel and the apoptotic cells were counted. LSAB method was used to measure the expression level of GFAP,GLAST,GS,NT-3. Immunological histochemistry assay was used for quantitative analysis. SPSS was used for statistical analysis..
Results:(1) Weight of diabetic rats were significantly lower than normal group (P<0.01) and there is no significant differences in weight of treatment group, control group and the model of rats (P>0.05). (2) Glucose in diabetic rats were markedly higher than normal group (P<0.01) and there is no significant differences in glucose in treatment group, control group and the model of rats(P> 0.05). (3) Compared to normal group, the expression of GFAP mRNA by RT-PCR in diabetic rats was enhanced significantly. Compared to model group, the expression in treatment group and control group was decreased. Compared to control group, the expression in treatment group was decreased. (4) The positive apoptotic retinal cell were only expressed in RGCs and inner nuclear layer. The number of apoptotic RGCs and cells in inner nuclear layer in model group,control group and treatment group was significantly higher than in normal group. Compared to model group, the number of apoptotic cells in control group and treatment group decreased obviously. Compared to control group, the number of apoptotic cells in treatment group decreased obviously. (5) Compared to normal group, the positive expression of GFAP in diabetic rats by LSAB was enhanced significantly. Compared to model group,the expression in treatment group and control group was decreased. Compared to control group, the expression in treatment group was decreased (6) Compared to normal group, the positive expression of GLAST by LSAB in model group,control group and treatment group was decreased significantly. Compared to model group and control group, the expression in treatment group was enhanced. (7) Compared to normal group, the positive expression of GS by LSAB in model group,control group and treatment group was decreased significantly. Compared to model group, the expression in treatment group and control group was enhanced. Compared to control group, the expression in treatment group was increased. (8) Compared to normal group, the positive expression of NT-3 by LSAB in control group, model group and treatment group was decreased significantly. Compared to model group, the expression in treatment group and control group was enhanced. Compared to control group, the expression in treatment group was increased. (9) The number of apoptotic RGCs and cells in inner nuclear layer was correlated positively with the positive expression level of GFAP, while the expression of GLAST,GS and NT-3 was negatively.
Conclusion:1 The apoptotic cells number in RGCs and the inner nuclear layer in diabetic rats retina were significantly increased.2 The expression of GFAP in Retinal Muller cells in diabetic rats increases and Muller cells are in active proliferation of glial reactive state.3 The function of GLAST and GS iin diabetic retinal Muller cells is weakened and its ability of transporter glutamate is declined.4 Qishenyiqi pills has a protective effect to retinal nerve cells in diabetic rats. Its mechanism is as follows.(1)Qishenyiqi pills can inhibit the overexpression of GFAP and glial cell reactive hyperplasia in Muller cells and reduce the damage to the Muller cell function and retinal neurons by DM.
(2)Qishenyiqi pills can promote the expression of GLAST and GS in Muller cells and remove the excessive extracellular glutamate effectively and reduce the high concentrations of the role of glutamate excitotoxicity. (3) Qishenyiqi pills can promote the expression of neurotrophic factors (NT-3) and reduce the the number of apoptotic neural cells.
引文
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4 Srivastava BK,Rema M.Does hypertension play a role in diabetic retinopathy. Assoc Physicians Indian,2005,53:803-808.
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6 Dagher Z, Patio YS, Aenaghi V, et al. Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy. Diabetes, 2004,53:2404-2411.
7 Freedman BI, Wuerth JP, Cartwright K, et al. Design and baseline characteristics for the aminoguanidine Clinical Trial in Overt Type 2 Diabetic Nephropathy(ACTION Ⅱ). Control Clin Trials,1999,20(5):493-510
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9 Way KJ, Chou E, King GL. Identification of PKG-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci,2000,21 (5):181-187.
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13 陈忠平,姜德味.糖尿病性视网膜病变药物治疗进展一PKCβ抑制剂.国外医学眼科学分册,2004,28(3)：205-208
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