沉默信息调节因子相关酶1(SIRT1)调控p38MARK信号通路对糖尿病视网膜病变大鼠视网膜神经节细胞的保护作用及机制
|
摘要
目的探讨沉默信息调节因子相关酶1(silment information regulator factor related enzymes 1,SIRT1)对糖尿病视网膜病变大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)的保护作用及其机制。方法取健康清洁级雄性Sprague-Dawley大鼠60只,应用随机数字表法分为正常对照组(正常组)、糖尿病组(病变组)、SIRT1激动剂白藜芦醇治疗组(治疗组)。病变组和治疗组大鼠按60 mg·kg~(-1)单次腹腔注射链脲佐菌素以诱导糖尿病大鼠模型;正常组按60 mg·kg~(-1)腹腔注射枸橼酸钠缓冲液。72 h后取鼠尾静脉血检测血糖,血糖值>16.7 mmol·L~(-1)定为糖尿病大鼠。自造模成功后第2天起治疗组每天每只鼠给予白藜芦醇20 g·kg~(-1)灌胃,正常组和病变组每天每只鼠给予亚甲砜灌胃。8周后进行视网膜免疫组织化学染色,TUNEL法检测视网膜RGCs凋亡,Western blot检测SIRT1、p38 MAPK、Caspase-3蛋白的表达。结果正常组、病变组、治疗组8周后RGCs凋亡指数分别为:(0.848±0.131)%、(19.038±1.327)%、(10.461±1.089)%,三组间差异有统计学意义(F=670.497,P=0.000)。进一步两两比较:正常组RGCs凋亡指数与病变组、治疗组间差异均有统计学意义(均为P=0.000);治疗组与病变组间差异亦有统计学意义(P=0.000)。与正常组(0.132±0.043)相比,病变组(0.060±0.028)和治疗组(0.073±0.026)大鼠视网膜SIRT1蛋白表达降低,总体差异有统计学意义(F=1310.663,P=0.000)。进一步两两比较,病变组和治疗组与正常组间,以及病变组与治疗组间差异均有统计学意义(均为P=0.000)。病变组(1.121±0.082,0.266±0.005)和治疗组(0.574±0.012,0.190±0.060)大鼠视网膜p38MAPK、Caspase-3蛋白表达较正常组(0.402±0.012,0.156±0.006)明显增加,总体差异有统计学意义(F=604.500、1056.709,P=0.000、0.000)。进一步两两比较:p38 MAPK、Caspase-3蛋白表达在正常组与病变组间、正常组与治疗组间以及病变组与治疗组间差异均有统计学意义(均为P=0.000)。结论在糖尿病视网膜病变模型中,SIRT1表达上调,抑制RGCs的凋亡,对糖尿病视网膜病变RGCs起保护作用。其抗凋亡作用机制可能与其抑制p38 MAPK的表达相关。p38 MAPK信号通路是糖尿病视网膜病变中SIRT1介导的神经保护作用的重要通路之一。
Objective To investigate the effect of silment information regulator factor related enzym es 1( SIRT1) on the apoptosis of retinal ganglion cells( RGCs) in rats with diabetic retinopathy and its downstream m olecular m echanism s. M ethods Together 6 0 healthy m ale Sprague-Dawley rats were collected and random ly divided into norm al group,diabetic group,SIRT1 activator-resveratrol treatm ent group( treatm ent group),and diabetic rat m odel was induced by intraperitoneal injection of streptozotocin at 6 0 m g · kg~(-1)in the latter two group rats,while the norm al group was injected with sodium citrate buffer at 6 0 m g · kg~(-1). Then,after 7 2 h,rats with blood glucose > 1 6. 7m m ol· L~(-1)were designated as diabetic rats by blood glucose test. Then each rat in the treatm ent group was treated with SIRT1 activator-resveratrol at 2 0 g · kg~(-1)once a day at the 2 nd day after the success of the m odel,and the norm al group and diabetic group were given m ethylene chloride. Finally,after im m unohistochem ical staining for retina,TUNEL assay was used to evaluate the apoptosis of RGCs,while the expression of SIRT1,p3 8 M AP K and Caspase-3 protein was detected by W estern blot. Results The apoptotic index of RGCs in the norm al group,diabetic group and treatm ent group was( 0. 848 ± 0. 131) %,( 19. 038 ± 1. 327) %,( 10. 461 ± 1. 089) % respectively at 8weeks,and the difference am ong the three groups was statistically significant( F =6 7 0. 4 9 7,P = 0. 0 0 0),while the differences between each two groups were also statistically significant( all P = 0. 0 0 0). Furtherm ore,when com pared with the norm al group( 0. 132 ± 0. 043),the expression of SIRT1 protein in the diabetic group( 0. 060 ± 0.0 2 8) and the treatm ent group( 0. 0 7 3 ± 0. 0 2 6) was significantly decreased,and the overall difference am ong the three groups was statistically significant( F = 1 3 1 0. 6 6 3,P= 0. 0 0 0),while the differences between each two groups were also statistically significant( all P = 0. 0 0 0). The expression levels of p3 8 M AP K and Caspase-3 were increased in diabetic group( 1. 1 2 1 ± 0. 0 8 2,0. 2 6 6 ± 0. 0 0 5) and treatm ent group( 0.5 7 4 ± 0. 0 1 2,0. 1 9 0 ± 0. 0 6 0) respectively,and the overall difference and pairwise com-parison in the three groups approached statistically significance( all P = 0. 0 0 0,0.0 0 0). Conclusion Up-regulation of SIRT1,can inhibit the apoptosis of RGCs,and protect RGCs against apoptosis in rat model of diabetic retinopathy,which may be correlated with the down-regulation of p38 MAPK signal pathway.
Objective To investigate the effect of silment information regulator factor related enzym es 1( SIRT1) on the apoptosis of retinal ganglion cells( RGCs) in rats with diabetic retinopathy and its downstream m olecular m echanism s. M ethods Together 6 0 healthy m ale Sprague-Dawley rats were collected and random ly divided into norm al group,diabetic group,SIRT1 activator-resveratrol treatm ent group( treatm ent group),and diabetic rat m odel was induced by intraperitoneal injection of streptozotocin at 6 0 m g · kg~(-1)in the latter two group rats,while the norm al group was injected with sodium citrate buffer at 6 0 m g · kg~(-1). Then,after 7 2 h,rats with blood glucose > 1 6. 7m m ol· L~(-1)were designated as diabetic rats by blood glucose test. Then each rat in the treatm ent group was treated with SIRT1 activator-resveratrol at 2 0 g · kg~(-1)once a day at the 2 nd day after the success of the m odel,and the norm al group and diabetic group were given m ethylene chloride. Finally,after im m unohistochem ical staining for retina,TUNEL assay was used to evaluate the apoptosis of RGCs,while the expression of SIRT1,p3 8 M AP K and Caspase-3 protein was detected by W estern blot. Results The apoptotic index of RGCs in the norm al group,diabetic group and treatm ent group was( 0. 848 ± 0. 131) %,( 19. 038 ± 1. 327) %,( 10. 461 ± 1. 089) % respectively at 8weeks,and the difference am ong the three groups was statistically significant( F =6 7 0. 4 9 7,P = 0. 0 0 0),while the differences between each two groups were also statistically significant( all P = 0. 0 0 0). Furtherm ore,when com pared with the norm al group( 0. 132 ± 0. 043),the expression of SIRT1 protein in the diabetic group( 0. 060 ± 0.0 2 8) and the treatm ent group( 0. 0 7 3 ± 0. 0 2 6) was significantly decreased,and the overall difference am ong the three groups was statistically significant( F = 1 3 1 0. 6 6 3,P= 0. 0 0 0),while the differences between each two groups were also statistically significant( all P = 0. 0 0 0). The expression levels of p3 8 M AP K and Caspase-3 were increased in diabetic group( 1. 1 2 1 ± 0. 0 8 2,0. 2 6 6 ± 0. 0 0 5) and treatm ent group( 0.5 7 4 ± 0. 0 1 2,0. 1 9 0 ± 0. 0 6 0) respectively,and the overall difference and pairwise com-parison in the three groups approached statistically significance( all P = 0. 0 0 0,0.0 0 0). Conclusion Up-regulation of SIRT1,can inhibit the apoptosis of RGCs,and protect RGCs against apoptosis in rat model of diabetic retinopathy,which may be correlated with the down-regulation of p38 MAPK signal pathway.
引文
[1]SIMO R,HERNANDEZ C.Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence[J].Prog Retin Eye Res,2015,48(9):160-180.
[2]中华医学会糖尿病学分会.中国2型糖尿病防治指南(2013年版)[J].中华内分泌代谢杂志,2014,30(10):893-942.CHINESE DIABETES SOCIETY.China guideline for type 2 diabetes mellitus(2013 Edition)[J].Chin J Endocrinol Metab,2014,30(10):893-942.
[3]王月欣,陈松.糖尿病视网膜病变神经损伤的发病机制和保护防治研究进展[J].中华眼底病杂志,2014,30(2):209-211.WANG YX,CHEN S.Research advance of nerve lesions in the pathogenesis of diabetic retinopathy and treatment of nerve protection[J].Chin J Ocul Fundus Dis,2014,30(2):209-211.
[4]HERNANDEZ C,DLMONTE M,SIMO R,CASINI G.Neuroprotection as a therapeutic target for diabetic retinopathy[J].J Diabetes Res,2016,2016(24):9508541.
[5]SIMO R,HERNANDEZ C.Neurodegeneration is an early event in diabetic retinopathy:therapeutic implications[J].Br J Ophthalmol,2012,96(10):1285-1290.
[6]HERNANDEZ C,GARCIA-RAMIREZ M,CORRALIZA L,FERNADEZ-CARNEADO J,FARRERA-SINFREU J,PONSATI B,et al.Topical administration of somatostatin prevents retinal neurodegeneration in experimental diabetes[J].Diabetes,2013,62(7):2569-2578.
[7]刘姝林,陈有信.SIRT1在糖尿病视网膜病变发病机制中的作用[J].国际眼科纵览,2013,37(6):374-378.LIU SL,CHEN YX.The role of SIRT1 in the pathogenesis of diabetic retinopathy[J].Int Rev Ophthalmol,2013,37(6):374-378.
[8]彭惠,洪苏玲,李平华.p38丝裂原活化蛋白激酶信号通路在糖尿病性视网膜病变中的作用研究进展[J].眼视光学杂志,2007,9(2):139-141.PENG H,HONG SL,LI PH.Recent progress in research on the p38MAPK cell signal pathway in diabetic retinopathy[J].Chin J Opt Ophthalmol,2007,9(2):139-141.
[9]李东洁,吴迪,张旭乡.糖尿病视网膜神经节细胞损伤的研究进展[J].国际眼科杂志,2016,16(4):670-672.LI DJ,WU D,ZHANG XX.Research advance of diabetic retinal ganglion cell lesions[J].Int Eye Sci,2016,16(4):670-672.
[10]于常红,韩彦弢,曹玉,安明,时肖,杨军廷,等.左卡尼汀对糖尿病大鼠视网膜神经节细胞保护作用的实验研究[J].中国药理学通报,2013,29(11):1502-1505.YU CH,HAN YT,CAO Y,AN M,SHI X,YANG JT,et al.Protective effect of L carnitine on retinal ganglion cells in the diabetic rat[J].Chin Pharmacol Bulletin,2013,(11):1502-1505.
[11]KIM D,NGUYEN MD,DOBBIN MM,FISCHER A,SANANBENESI F,RODGERS JT,et al.SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s disease and amyotrophic lateral sclerosis[J].EMBO J,2007,26(13):3169-3179.
[12]诸葛淳淳,徐国彤,徐晶莹.去乙酰化酶Sirt1在视神经视网膜疾病中的神经保护作用[J].国际眼科纵览,2012,36(4):222-227.ZHU-GE CC,XU GT,XU JY.The neuroprotective role of Sirt1 in retinal and optic nerve diseases[J].Int Rev Ophthalmol,2012,36(4):222-227.
[13]BORRA MT,SMITH BC,DENU JM.Mechanism of human SIRT1activation by resveratrol[J].J Biol Chem,2005,280(17):17187-17195.
[14]CAO D,WANG M,QIU X,LIU D,JIANG H,YANG N,et al.Structural basis for allosteric,substrate-dependent stimulation of SIRT1 activity by resveratrol[J].Genes Dev,2015,29(12):1316-1325.
[15]LI YH,ZHUO YH,LU L,CHEN LY,HUANG XH,ZHANG JL,et al.Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes[J].Chin Med J(Engl),2008,121(24):2566-2571.
[16]ADAMIEC-MROCZEK J,ZZJAC-PYTRUS H,MISIUK-HOJLO M.Caspase-dependent apoptosis of retinal ganglion cells during the development of diabetic retinopathy[J].Adv Clin Exp Med,2015,24(3):531-535.
[17]苟文军,吕红彬,杨旭,李恒,刘灵琳,李利文,等.罗格列酮对糖尿病大鼠视网膜神经节细胞凋亡的影响[J].眼科新进展,2016,36(9):818-821.GOU WJ,LV HB,YANG X,LI H,LIU LL,LI LW,et al.Effects of rosiglitazone on retinal ganglion cells apoptosis in rats with diabetes mellitus[J].Rec Adv Ophthalmol,2016,36(9):818-821.
[18]李德冠,樊飞跃,孟爱民.p38 MAPK通路在造血系统调节中的作用[J].中国药理学通报,2011,27(1):4-6.LI DG,FAN FY,MENG AM.p38 MAPK signaling pathways in the regulation of hematopoietic system[J].Chin Pharmacol Bull,2011,27(1):4-6.
[19]章必成,张巍.TNF,p38 MAPK与细胞凋亡[J].国外医学(生理、病理科学与临床分册),2001,21(6):464-466.ZHANG BC,ZHANG W.p38 MAPK and cell apoptosis[J].Foreign Med Sci(Sec Pathophysiol Clin Med),2001,21(6):464-466.
[20]李永浩,吕林,陈凌燕,黄新华,张静琳,李石毅,等.P38丝裂素活化蛋白激酶信号通路阻断对糖尿病鼠早期血视网膜屏障和视网膜节细胞的保护作用[J].中华眼底病杂志,2010,26(2):139-142.LI TH,LV L,CHEN LY,HUANG XH,ZHANG JL,LI SY,et al.Protective effect of blocking the signal path of p38 mitogen activated protein kinase on blood retinal barrier and retinal ganglion cells in early diabetic rats[J].Chin J Ocul Fundus Dis,2010,26(2):139-142.
[21]KUME S,KITADA M,KANASAKI K,MAEGAWA H,KOYA D.Anti-aging molecule,Sirt1:a novel therapeutic target for diabetic nephropathy[J].Arch Pharm Res,2013,36(2):230-236.
[2]中华医学会糖尿病学分会.中国2型糖尿病防治指南(2013年版)[J].中华内分泌代谢杂志,2014,30(10):893-942.CHINESE DIABETES SOCIETY.China guideline for type 2 diabetes mellitus(2013 Edition)[J].Chin J Endocrinol Metab,2014,30(10):893-942.
[3]王月欣,陈松.糖尿病视网膜病变神经损伤的发病机制和保护防治研究进展[J].中华眼底病杂志,2014,30(2):209-211.WANG YX,CHEN S.Research advance of nerve lesions in the pathogenesis of diabetic retinopathy and treatment of nerve protection[J].Chin J Ocul Fundus Dis,2014,30(2):209-211.
[4]HERNANDEZ C,DLMONTE M,SIMO R,CASINI G.Neuroprotection as a therapeutic target for diabetic retinopathy[J].J Diabetes Res,2016,2016(24):9508541.
[5]SIMO R,HERNANDEZ C.Neurodegeneration is an early event in diabetic retinopathy:therapeutic implications[J].Br J Ophthalmol,2012,96(10):1285-1290.
[6]HERNANDEZ C,GARCIA-RAMIREZ M,CORRALIZA L,FERNADEZ-CARNEADO J,FARRERA-SINFREU J,PONSATI B,et al.Topical administration of somatostatin prevents retinal neurodegeneration in experimental diabetes[J].Diabetes,2013,62(7):2569-2578.
[7]刘姝林,陈有信.SIRT1在糖尿病视网膜病变发病机制中的作用[J].国际眼科纵览,2013,37(6):374-378.LIU SL,CHEN YX.The role of SIRT1 in the pathogenesis of diabetic retinopathy[J].Int Rev Ophthalmol,2013,37(6):374-378.
[8]彭惠,洪苏玲,李平华.p38丝裂原活化蛋白激酶信号通路在糖尿病性视网膜病变中的作用研究进展[J].眼视光学杂志,2007,9(2):139-141.PENG H,HONG SL,LI PH.Recent progress in research on the p38MAPK cell signal pathway in diabetic retinopathy[J].Chin J Opt Ophthalmol,2007,9(2):139-141.
[9]李东洁,吴迪,张旭乡.糖尿病视网膜神经节细胞损伤的研究进展[J].国际眼科杂志,2016,16(4):670-672.LI DJ,WU D,ZHANG XX.Research advance of diabetic retinal ganglion cell lesions[J].Int Eye Sci,2016,16(4):670-672.
[10]于常红,韩彦弢,曹玉,安明,时肖,杨军廷,等.左卡尼汀对糖尿病大鼠视网膜神经节细胞保护作用的实验研究[J].中国药理学通报,2013,29(11):1502-1505.YU CH,HAN YT,CAO Y,AN M,SHI X,YANG JT,et al.Protective effect of L carnitine on retinal ganglion cells in the diabetic rat[J].Chin Pharmacol Bulletin,2013,(11):1502-1505.
[11]KIM D,NGUYEN MD,DOBBIN MM,FISCHER A,SANANBENESI F,RODGERS JT,et al.SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s disease and amyotrophic lateral sclerosis[J].EMBO J,2007,26(13):3169-3179.
[12]诸葛淳淳,徐国彤,徐晶莹.去乙酰化酶Sirt1在视神经视网膜疾病中的神经保护作用[J].国际眼科纵览,2012,36(4):222-227.ZHU-GE CC,XU GT,XU JY.The neuroprotective role of Sirt1 in retinal and optic nerve diseases[J].Int Rev Ophthalmol,2012,36(4):222-227.
[13]BORRA MT,SMITH BC,DENU JM.Mechanism of human SIRT1activation by resveratrol[J].J Biol Chem,2005,280(17):17187-17195.
[14]CAO D,WANG M,QIU X,LIU D,JIANG H,YANG N,et al.Structural basis for allosteric,substrate-dependent stimulation of SIRT1 activity by resveratrol[J].Genes Dev,2015,29(12):1316-1325.
[15]LI YH,ZHUO YH,LU L,CHEN LY,HUANG XH,ZHANG JL,et al.Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes[J].Chin Med J(Engl),2008,121(24):2566-2571.
[16]ADAMIEC-MROCZEK J,ZZJAC-PYTRUS H,MISIUK-HOJLO M.Caspase-dependent apoptosis of retinal ganglion cells during the development of diabetic retinopathy[J].Adv Clin Exp Med,2015,24(3):531-535.
[17]苟文军,吕红彬,杨旭,李恒,刘灵琳,李利文,等.罗格列酮对糖尿病大鼠视网膜神经节细胞凋亡的影响[J].眼科新进展,2016,36(9):818-821.GOU WJ,LV HB,YANG X,LI H,LIU LL,LI LW,et al.Effects of rosiglitazone on retinal ganglion cells apoptosis in rats with diabetes mellitus[J].Rec Adv Ophthalmol,2016,36(9):818-821.
[18]李德冠,樊飞跃,孟爱民.p38 MAPK通路在造血系统调节中的作用[J].中国药理学通报,2011,27(1):4-6.LI DG,FAN FY,MENG AM.p38 MAPK signaling pathways in the regulation of hematopoietic system[J].Chin Pharmacol Bull,2011,27(1):4-6.
[19]章必成,张巍.TNF,p38 MAPK与细胞凋亡[J].国外医学(生理、病理科学与临床分册),2001,21(6):464-466.ZHANG BC,ZHANG W.p38 MAPK and cell apoptosis[J].Foreign Med Sci(Sec Pathophysiol Clin Med),2001,21(6):464-466.
[20]李永浩,吕林,陈凌燕,黄新华,张静琳,李石毅,等.P38丝裂素活化蛋白激酶信号通路阻断对糖尿病鼠早期血视网膜屏障和视网膜节细胞的保护作用[J].中华眼底病杂志,2010,26(2):139-142.LI TH,LV L,CHEN LY,HUANG XH,ZHANG JL,LI SY,et al.Protective effect of blocking the signal path of p38 mitogen activated protein kinase on blood retinal barrier and retinal ganglion cells in early diabetic rats[J].Chin J Ocul Fundus Dis,2010,26(2):139-142.
[21]KUME S,KITADA M,KANASAKI K,MAEGAWA H,KOYA D.Anti-aging molecule,Sirt1:a novel therapeutic target for diabetic nephropathy[J].Arch Pharm Res,2013,36(2):230-236.