活血解毒方对糖尿病视网膜病变细胞凋亡及相关细胞因子的影响
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摘要
目的观察中药复方活血解毒方(HXJD)对糖尿病大鼠视网膜细胞凋亡及凋亡相关因子NF-κB和Caspase-3 mRNA和蛋白表达的影响。方法采用链脲佐菌素(65 mg/kg)一次性腹腔注射的方法,建立糖尿病大鼠模型,将50只SD大鼠随机分为模型组和HXJD高剂量组(15.4 g/kg)、中剂量组(7.70 g/kg)、低剂量组(3.85 g/kg)及导升明组(0.167 g/kg),每组10只,另设10只正常对照组。造模成功后,灌胃给药,20周后处死动物。应用TUNEL法检测细胞凋亡程度,免疫组化和蛋白印迹技术检测视网膜全层中NF-κB和Caspase-3的表达;采用Real-time PCR法检测视网膜NF-κB和Caspase-3 mRNA的表达。同时,体外培养大鼠视网膜神经节细胞株(RGC-5),制备HXJD含药血清。利用55.5 mmol/L葡萄糖浓度模拟高糖环境诱导,将细胞分为5组:空白组、高糖组、含药血清低剂量组、含药血清中剂量组、含药血清高剂量组。采用CCK-8和AnnexinⅤ-FITC法检测不同血清干预高糖诱导下RGC细胞24 h增殖活性及细胞凋亡的影响。结果与正常对照组比较,模型组凋亡细胞增加,大鼠视网膜组织NF-κB和Caspase-3蛋白及mRNA的表达明显升高(P<0.05);与模型组比较,活血解毒方各剂量组和导升明组视网膜细胞凋亡的程度、视网膜NF-κB和Caspase-3蛋白和mRNA的表达量均降低(P<0.05)。与空白组比较,高糖组细胞活性降低,细胞凋亡比例增加(P<0.05)。与高糖组比较,含药血清高、中剂量组细胞活性增高(P<0.05),含药血清各剂量组细胞凋亡比例减少(P<0.05,P<0.01)。结论活血解毒方可能通过改善糖尿病大鼠视网膜中细胞凋亡的相关细胞因子NF-κB和Caspase-3的表达,同时调节视网膜神经节细胞活力,改善高糖条件下RGC细胞的凋亡程度。
Objective To observe the effects of Huoxue Jiedu Recipe(HXJD)on the apoptosis and the expression of NF-κB and Caspase-3 in the reina tissue of early diabetic rats. Methods The diabetic rat model was established by using one single intraperitoneal injection of streptozotocin(STZ, 65 mg/kg). Then the model rats were randomly divided into five groups,i.e.. the model group, the low dose HXJD group(3.85 g/kg), the middle dose HXJD group(7.70 g/kg), the high dose HXJD group(15.40 g/kg) and the Western medicine treatment group(Calcium Dobesilate capsule, 0.167 g/kg), ten rats in each group. A normal control group consisting of ten rats was also set up. The nomal group and model group were given normal water and the other rats were medicated by intragastric administrate for 20 weeks. All rats were sacrificed after 20 weeks. Apoptotic level was detected by TUNEL. Immunological histochemistry assay and Western blot were used to observe the protein expression of NF-κB and Caspase-3. The expressive abundance of NF-κB mRNA and Caspase-3 mRNA in rats′ retina were detected by Real-time quantitative PCR. At the same time, rat retinal ganglion cells(RGC-5) were cultured in vitro, the application of serum pharmacology of HXJD was prepared. Meanwhile, 55.5 mmol/l glucose was used to induce the high glucose environment. The cells were divided into five groups, i.e.. the control group, the high glucose group, HXJD low dose containing serum group, HXJD middle dose containing serum group, HXJD high dose containing serum group. The proliferation activity and apoptotic of RGC were detected by CCK8 and Annexin Ⅴ-FITC methods. Results Compared to the normal group, the number of apoptotic cells in model group was increased obviously, and the expression of NF-κB and Caspase-3 protein and mRNA were also increased(P<0.05). Compared with the model group, the apoptotic level and the expression of NF-κB and Caspase-3 in HXJD groups and the Western medicine treatment group were decreased evidently(P<0.05). Compared with the control group,the proliferation activity of RGC cells in the high glucose group decreased, and the apoptosis ratio of RGC cells was increased(P<0.05). Compared with the high glucose group, the proliferation activity of RGC cells in the high and middle dose HXJD containing serum group significantly improved(P<0.05), the apoptosis ratio of RGC cells in each dose HXJD containing serum group was reduced(P<0.05, P<0.01). Conclusion Huoxue Jiedu Recipe can inhibit the expression of NF-κB and Caspase-3 in retina of diabetic rats, and improve the proliferation and the apoptotic level of RGC cells, so it can inhibit apoptosis in retinal cell and protect the tissue in diabetic retinopathy.
Objective To observe the effects of Huoxue Jiedu Recipe(HXJD)on the apoptosis and the expression of NF-κB and Caspase-3 in the reina tissue of early diabetic rats. Methods The diabetic rat model was established by using one single intraperitoneal injection of streptozotocin(STZ, 65 mg/kg). Then the model rats were randomly divided into five groups,i.e.. the model group, the low dose HXJD group(3.85 g/kg), the middle dose HXJD group(7.70 g/kg), the high dose HXJD group(15.40 g/kg) and the Western medicine treatment group(Calcium Dobesilate capsule, 0.167 g/kg), ten rats in each group. A normal control group consisting of ten rats was also set up. The nomal group and model group were given normal water and the other rats were medicated by intragastric administrate for 20 weeks. All rats were sacrificed after 20 weeks. Apoptotic level was detected by TUNEL. Immunological histochemistry assay and Western blot were used to observe the protein expression of NF-κB and Caspase-3. The expressive abundance of NF-κB mRNA and Caspase-3 mRNA in rats′ retina were detected by Real-time quantitative PCR. At the same time, rat retinal ganglion cells(RGC-5) were cultured in vitro, the application of serum pharmacology of HXJD was prepared. Meanwhile, 55.5 mmol/l glucose was used to induce the high glucose environment. The cells were divided into five groups, i.e.. the control group, the high glucose group, HXJD low dose containing serum group, HXJD middle dose containing serum group, HXJD high dose containing serum group. The proliferation activity and apoptotic of RGC were detected by CCK8 and Annexin Ⅴ-FITC methods. Results Compared to the normal group, the number of apoptotic cells in model group was increased obviously, and the expression of NF-κB and Caspase-3 protein and mRNA were also increased(P<0.05). Compared with the model group, the apoptotic level and the expression of NF-κB and Caspase-3 in HXJD groups and the Western medicine treatment group were decreased evidently(P<0.05). Compared with the control group,the proliferation activity of RGC cells in the high glucose group decreased, and the apoptosis ratio of RGC cells was increased(P<0.05). Compared with the high glucose group, the proliferation activity of RGC cells in the high and middle dose HXJD containing serum group significantly improved(P<0.05), the apoptosis ratio of RGC cells in each dose HXJD containing serum group was reduced(P<0.05, P<0.01). Conclusion Huoxue Jiedu Recipe can inhibit the expression of NF-κB and Caspase-3 in retina of diabetic rats, and improve the proliferation and the apoptotic level of RGC cells, so it can inhibit apoptosis in retinal cell and protect the tissue in diabetic retinopathy.
引文
[1] Chen MS, Kao CS, Chang CJ, et al. Prevalence and risk factors of diabetic retinopathy among noninsulin-dependent diabetic subjects[J]. Am J Ophthalmol, 1992, 114(6): 723-730.
[2] Kempen JH, O'Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States[J]. Arch Ophthalmol, 2004, 122(4): 552-563.
[3] Nakajima M, Cooney MJ, Tu AH, et al. Normalization of retinal vascular permeability in experimental diabetes with genistein[J]. Invest Ophthalmol Vis Sci, 2001, 42(9): 2110-2114.
[4] Malone JI, Morrison AD, Pavan PR, et al. Prevalence and significance of retinopathy in subjects with type 1 diabetes of less than 5 years' duration screened for the diabetes control and complications trial[J]. Diabetes Care, 2001, 24(3): 522-526.
[5] Fletcher EL, Phipps JA, Ward MM, et al. Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy[J]. Curr Pharm Des, 2007, 13(26): 2699-2712.
[6] Hu L, Yang H, Ai M, et al. Inhibition of TLR4 alleviates the inflammation and apoptosis of retinal ganglion cells in high glucose[J]. Graefes Arch Clin Exp Ophthalmol, 2017, 255(11): 2199-2210.
[7] 姚青, 韩静, 黄黎明, 等. 活血解毒方对糖尿病早期大鼠视网膜功能的影响[J]. 中国中西医结合杂志, 2012, 32(9): 1271-1274.
[8] 姚青, 韩静, 余俊达, 等. 活血解毒方对糖尿病大鼠视网膜病变的影响[J]. 中国中西医结合杂志, 2012, 32(3): 362-366.
[9] 赵卫红, 寿好长, 闫福岭主编. 细胞凋亡[M]. 河南: 河南医科大学出版社, 1997: 4.
[10] Fletcher EL, Phipps JA, Ward MM. Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy[J]. Curr Pharm Des, 2007, 13(26): 699-712.
[11] 何晶. 三七的药理作用及研究进展[J]. 天津药学, 2004, 16(5): 58-60.
[12] 邹晨辉, 申竹芳. 黄连生物碱抗糖尿病机制的研究进展[J]. 中草药, 2004, 35(11): 2-5.
[13] 李玉杰, 龚慕辛, 来媛媛, 等. 鬼箭羽不同提取部位对糖尿病大鼠药理作用的研究[J]. 北京中医药大学学报, 2010, 33(3): 179-182.
[14] 姚青, 韩静, 黄黎明, 王伟.活血解毒方对糖尿病大鼠视网膜病变内皮素1表达的影响[J]. 中国实验方剂学杂志, 2012, 18(20): 169-172.
[15] Barber AJ, Gardner TW, Abcouwer SF. The significance of vascular and neural apoptosis to the pathology of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2011, 52(2): 1156-1163.
[16] Kim D, Mecham RP, Trackman PC, et al. Downregulation of Lysyl oxidase protects retinal endothelial cells from high glucose-induced apoptosis[J]. Invest Ophthalmol Vis Sci, 2017, 58(5): 2725-2731.
[17] 汪东生, 杨金奎. 糖尿病视网膜病变发病情况调查与发病机制研究的新进展[J]. 中华眼科医学杂志(电子版), 2016, 12(6): 273-282.
[18] Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences [J]. Cell, 1986, 46(5): 705-716.
[19] Mincheva-Tasheva S, Soler RM. NF-κB signaling pathways: role in nervous system physiology and pathology[J]. Neuroscientist, 2013, 19(2): 175-194.
[20] Shen H, Rong H. Pterostilbene impact on retinal endothelial cells under high glucose environment[J]. Int J Clin Exp Pathol, 2015, 8(10): 12589-12594.
[21] Adamiec-Mroczek J, Zajac-Pytrus H, Misiuk-Hoj?o M. Caspase-dependent apoptosis of retinal ganglion cells during the development of diabetic retinopathy[J]. Adv Clin Exp Med, 2015, 24(3): 531-535.
[22] 于晓, 牛膺筠, 王颖立. EPO对糖尿病大鼠视网膜Caspases-3、NF-κB和TNF-α表达的影响[J]. 眼科研究, 2008, 26(10): 743-746.
[2] Kempen JH, O'Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States[J]. Arch Ophthalmol, 2004, 122(4): 552-563.
[3] Nakajima M, Cooney MJ, Tu AH, et al. Normalization of retinal vascular permeability in experimental diabetes with genistein[J]. Invest Ophthalmol Vis Sci, 2001, 42(9): 2110-2114.
[4] Malone JI, Morrison AD, Pavan PR, et al. Prevalence and significance of retinopathy in subjects with type 1 diabetes of less than 5 years' duration screened for the diabetes control and complications trial[J]. Diabetes Care, 2001, 24(3): 522-526.
[5] Fletcher EL, Phipps JA, Ward MM, et al. Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy[J]. Curr Pharm Des, 2007, 13(26): 2699-2712.
[6] Hu L, Yang H, Ai M, et al. Inhibition of TLR4 alleviates the inflammation and apoptosis of retinal ganglion cells in high glucose[J]. Graefes Arch Clin Exp Ophthalmol, 2017, 255(11): 2199-2210.
[7] 姚青, 韩静, 黄黎明, 等. 活血解毒方对糖尿病早期大鼠视网膜功能的影响[J]. 中国中西医结合杂志, 2012, 32(9): 1271-1274.
[8] 姚青, 韩静, 余俊达, 等. 活血解毒方对糖尿病大鼠视网膜病变的影响[J]. 中国中西医结合杂志, 2012, 32(3): 362-366.
[9] 赵卫红, 寿好长, 闫福岭主编. 细胞凋亡[M]. 河南: 河南医科大学出版社, 1997: 4.
[10] Fletcher EL, Phipps JA, Ward MM. Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy[J]. Curr Pharm Des, 2007, 13(26): 699-712.
[11] 何晶. 三七的药理作用及研究进展[J]. 天津药学, 2004, 16(5): 58-60.
[12] 邹晨辉, 申竹芳. 黄连生物碱抗糖尿病机制的研究进展[J]. 中草药, 2004, 35(11): 2-5.
[13] 李玉杰, 龚慕辛, 来媛媛, 等. 鬼箭羽不同提取部位对糖尿病大鼠药理作用的研究[J]. 北京中医药大学学报, 2010, 33(3): 179-182.
[14] 姚青, 韩静, 黄黎明, 王伟.活血解毒方对糖尿病大鼠视网膜病变内皮素1表达的影响[J]. 中国实验方剂学杂志, 2012, 18(20): 169-172.
[15] Barber AJ, Gardner TW, Abcouwer SF. The significance of vascular and neural apoptosis to the pathology of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2011, 52(2): 1156-1163.
[16] Kim D, Mecham RP, Trackman PC, et al. Downregulation of Lysyl oxidase protects retinal endothelial cells from high glucose-induced apoptosis[J]. Invest Ophthalmol Vis Sci, 2017, 58(5): 2725-2731.
[17] 汪东生, 杨金奎. 糖尿病视网膜病变发病情况调查与发病机制研究的新进展[J]. 中华眼科医学杂志(电子版), 2016, 12(6): 273-282.
[18] Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences [J]. Cell, 1986, 46(5): 705-716.
[19] Mincheva-Tasheva S, Soler RM. NF-κB signaling pathways: role in nervous system physiology and pathology[J]. Neuroscientist, 2013, 19(2): 175-194.
[20] Shen H, Rong H. Pterostilbene impact on retinal endothelial cells under high glucose environment[J]. Int J Clin Exp Pathol, 2015, 8(10): 12589-12594.
[21] Adamiec-Mroczek J, Zajac-Pytrus H, Misiuk-Hoj?o M. Caspase-dependent apoptosis of retinal ganglion cells during the development of diabetic retinopathy[J]. Adv Clin Exp Med, 2015, 24(3): 531-535.
[22] 于晓, 牛膺筠, 王颖立. EPO对糖尿病大鼠视网膜Caspases-3、NF-κB和TNF-α表达的影响[J]. 眼科研究, 2008, 26(10): 743-746.