益精杞菊地黄颗粒对慢性高眼压大鼠视网膜RGCs凋亡相关因子表达的影响
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摘要
目的观察益精杞菊地黄颗粒对慢性高眼压大鼠视网膜RGCs凋亡相关因子Bcl-2、Bax、Caspase-3蛋白表达的影响。方法选用健康雄性的SD大鼠60只,随机平均分为5组,空白组、模型组、低剂量中药组、中剂量中药组、高剂量中药组。除空白组,其余4组均采用烧灼巩膜上静脉法,建立慢性高眼压模型。干预1个月后,分别采用免疫组化染色法检测视网膜RGCs层Bcl-2、Bax、Caspase-3蛋白的表达,RT-PCR法检测Bcl-2 mRNA、Bax mRNA、Caspase-3 mRNA的相对表达量。结果 (1)免疫组化染色法检测,中药治疗组、模型组大鼠RGCs中Bcl-2、Bax、Caspase-3较正常组均有统计学差异(P<0.01);与模型组相比,中剂量中药组的Bcl-2表达显著增多(P<0.01),Bax、Caspase-3的表达显著减少(P<0.01);Bcl-2、Bax、Caspase-3表达,高、低剂量组与模型组比较,无统计学意义(P>0.05)。(2)RT-PCR检测显示,中、低剂量中药组的Bcl-2 mRNA相对表达量较模型组增多(P<0.01),高剂量中药组较模型组,无统计学意义(P>0.05);中、低剂量中药组Bax mRNA相对表达量均较模型组减少(P<0.05),而高剂量组较模型组无统计学意义(P>0.05);中剂量中药组的Caspase-3 mRNA相对表达量较模型组减少(P<0.01),而高、低剂量组与模型组比较无统计学意义(P>0.05)。结论益精杞菊地黄颗粒剂对慢性高眼压大鼠RGCs凋亡有保护作用,其机理可能与Bcl-2蛋白的表达上调,Bax和Caspase-3蛋白的表达下调有关,中剂量中药组的作用比较明显。
OBJECTIVE To observe the effects of Yijing Qiju Dihuang granule on the expression of Bcl-2, Bax and Caspase-3 in the retina of rats with chronic high intraocular pressure METHODS Sixty male Sprague-Dawley rats were divided into 5 groups randomly: blank group,model group,high dose Chinese medicine group, medium dose group and low dose group in scheme of 12 rats per group. In addition to the blank group, rats in the other 4 groups were made into chronic ocular hypertension model. All groups had been intervened for one month. We used the immunohistochemical staining to detect the protein expression of Bcl-2, bax and Caspase-3 of all rats RGCs cells.And the relative expression of Bcl-2 mRNA, Bax mRNA, Caspase-3 mRNA of rats were detected by RT-PCR. RESULTS(1) Compared with the normal group, there were significant differences in the model group and medication group(P<0.01); Compared with the model group, medium dose group showed higher expression of Bcl-2(P<0.01) and lower expression of Bax and Caspase-3(P<0.01); No statistical significance was found between model group and high, low dose group in terms of three indexes(P>0.05).(2) The RT-PCR showed that the relative expressions of Bcl-2 mRNA in medium and low dose group were higher than in the model group(P<0.01). No statistical significance showed between the high dose group and the model group(P>0.05). The Bax mRNA and Caspase-3 mRNA in medium dose group were lower than the model group(P<0.05, P<0.01). The Bax mRNA of low dose group were a bit lower than the model group(P <0.05). While the result of high dose group and model group showed no statistical difference(P>0.05). The Caspase-3 mRNA of high and low dose group showed no statistical significance in contrast to it of the model group(P>0.05).CONCLUSIONS The Yijing Qiju Dihuang granule possessed protective effects on rats with chronic high intraocular pressure and its mechanism might link to up-regulated expression of Bcl-2 and the down-regulated expression of Bax and Caspase-3. And medium dose of the formula showed the most favorable effect.
OBJECTIVE To observe the effects of Yijing Qiju Dihuang granule on the expression of Bcl-2, Bax and Caspase-3 in the retina of rats with chronic high intraocular pressure METHODS Sixty male Sprague-Dawley rats were divided into 5 groups randomly: blank group,model group,high dose Chinese medicine group, medium dose group and low dose group in scheme of 12 rats per group. In addition to the blank group, rats in the other 4 groups were made into chronic ocular hypertension model. All groups had been intervened for one month. We used the immunohistochemical staining to detect the protein expression of Bcl-2, bax and Caspase-3 of all rats RGCs cells.And the relative expression of Bcl-2 mRNA, Bax mRNA, Caspase-3 mRNA of rats were detected by RT-PCR. RESULTS(1) Compared with the normal group, there were significant differences in the model group and medication group(P<0.01); Compared with the model group, medium dose group showed higher expression of Bcl-2(P<0.01) and lower expression of Bax and Caspase-3(P<0.01); No statistical significance was found between model group and high, low dose group in terms of three indexes(P>0.05).(2) The RT-PCR showed that the relative expressions of Bcl-2 mRNA in medium and low dose group were higher than in the model group(P<0.01). No statistical significance showed between the high dose group and the model group(P>0.05). The Bax mRNA and Caspase-3 mRNA in medium dose group were lower than the model group(P<0.05, P<0.01). The Bax mRNA of low dose group were a bit lower than the model group(P <0.05). While the result of high dose group and model group showed no statistical difference(P>0.05). The Caspase-3 mRNA of high and low dose group showed no statistical significance in contrast to it of the model group(P>0.05).CONCLUSIONS The Yijing Qiju Dihuang granule possessed protective effects on rats with chronic high intraocular pressure and its mechanism might link to up-regulated expression of Bcl-2 and the down-regulated expression of Bax and Caspase-3. And medium dose of the formula showed the most favorable effect.
引文
[1] QUIGLEY HA, NICKELLS RW, KERRIGAN LA, et al. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis[J]. Invest Ophthalmol Vis Sci,1995,36(5):774-786.
[2] GARCIA-VALENZUELA E, SHAREEF S, WALSH J, et al. Programmed cell death of retinal ganglion cells during experimental glaucoma[J]. Exp Eye Res,1995,61(1):33-44.
[3] KERRIGAN LA, ZACK DJ, QUIGLEY HA, et al. TUNEL-positive ganglion cells in human primary open-angle glaucoma[J]. Arch Ophthalmol,1997,115(8):1031-1035.
[4] OKISAKA S, MURAKAMI A, MIZUKAWA A,et al. Apoptosis in retinal ganglion cell decrease in human glaucomatous eyes[J].Jpn J Ophthalmol,1997,41(2):84-88.
[5]张兆康,倘孟莹,滕月,等.益精杞菊地黄颗粒对慢性高眼压大鼠视网膜神经节细胞凋亡的影响[J].中国中医眼科杂志,2018,28(6):363-368.
[6] SHAEREF S R,GAERIA-VALENZUELA E,SALIEMO A,et al.Chronic Ocular Hypertension following episcleral venous occlusion in rats[J].Exp Eye Res,1995,61(3):379-382.
[7] SAWADA A,NEUFELD A H. Confirmation of the rat model of chronic, moderately elevated intraocular pressure[J].Exp Eye Res,1999,69(5):525-531.
[8] LEE D,SHIM MS,KIM KY,et al. Coenzyme Q10 inhibits glutamate excitotoxicity and oxidative stress-mediated mitochondrial alteration in a mouse model of glaucoma[J].Invest Ophthalmol Vis Sci,2014,55:993-1005.
[9] NETLAND PA,DREYER EB. Calcium channel blockers in the management of low tension and open-angle glaucoma[J]. Am J Ophthalmol,1993,115:608-613.
[10] HUSAIN S,ABDUL Y,SINGH S,et al.Regulation of nitric oxide production byδ-Opioid receptors during glaucomatous injury[J].PLos One,2014,9(10):e110397.
[2] GARCIA-VALENZUELA E, SHAREEF S, WALSH J, et al. Programmed cell death of retinal ganglion cells during experimental glaucoma[J]. Exp Eye Res,1995,61(1):33-44.
[3] KERRIGAN LA, ZACK DJ, QUIGLEY HA, et al. TUNEL-positive ganglion cells in human primary open-angle glaucoma[J]. Arch Ophthalmol,1997,115(8):1031-1035.
[4] OKISAKA S, MURAKAMI A, MIZUKAWA A,et al. Apoptosis in retinal ganglion cell decrease in human glaucomatous eyes[J].Jpn J Ophthalmol,1997,41(2):84-88.
[5]张兆康,倘孟莹,滕月,等.益精杞菊地黄颗粒对慢性高眼压大鼠视网膜神经节细胞凋亡的影响[J].中国中医眼科杂志,2018,28(6):363-368.
[6] SHAEREF S R,GAERIA-VALENZUELA E,SALIEMO A,et al.Chronic Ocular Hypertension following episcleral venous occlusion in rats[J].Exp Eye Res,1995,61(3):379-382.
[7] SAWADA A,NEUFELD A H. Confirmation of the rat model of chronic, moderately elevated intraocular pressure[J].Exp Eye Res,1999,69(5):525-531.
[8] LEE D,SHIM MS,KIM KY,et al. Coenzyme Q10 inhibits glutamate excitotoxicity and oxidative stress-mediated mitochondrial alteration in a mouse model of glaucoma[J].Invest Ophthalmol Vis Sci,2014,55:993-1005.
[9] NETLAND PA,DREYER EB. Calcium channel blockers in the management of low tension and open-angle glaucoma[J]. Am J Ophthalmol,1993,115:608-613.
[10] HUSAIN S,ABDUL Y,SINGH S,et al.Regulation of nitric oxide production byδ-Opioid receptors during glaucomatous injury[J].PLos One,2014,9(10):e110397.