活血化瘀方对糖尿病大鼠视网膜周细胞的保护作用
|
摘要
目的探究自拟活血化瘀方对糖尿病大鼠视网膜周细胞(retinal capillary pericytes,RCPs)的保护作用。方法 40只SD大鼠随机分成4组,其中10只为正常组,另30只腹腔注射链脲佐菌素造糖尿病模型成功后再分为活血化瘀方高(6.75 g·kg~(-1))、低剂量组(3.375 g·kg~(-1))及模型组,每组各10只,灌胃60 d后取视网膜,对RCPs进行免疫组化及Bax、Bak、Bcl-2、caspase-3蛋白表达分析。在体外实验中,将高糖(30 mmol·L~(-1))培养的RCPs用不同浓度的活血化瘀方进行干预,CCK-8检测RCPs增殖,JC-1染色后荧光显微镜观察细胞形态和RCPs凋亡情况,并使用流式细胞仪进行定量分析。结果糖尿病模型大鼠较正常组RCPs凋亡明显,而活血化瘀方可减少RCPs凋亡。与0μg·m L~(-1)剂量增殖效果相比,25～100μg·m L~(-1)活血化瘀方有显著性差异(P<0.01或P<0.05)。在抗高糖诱导凋亡方面,25～100μg·m L~(-1)与0μg·m L~(-1)相比,有显著性差异。结论活血化瘀方可通过主要外膜蛋白/Bcl-2途径抑制糖尿病大鼠RCPs凋亡,且对高糖诱导的RCPs凋亡具有保护作用。
OBJECTIVE To determine the protective effect of Huoxue Huayu prescription on retinal capillary pericytes(RCPs) of diabetic rats. METHODS Forty SD rats were conducted and randomly separated into 4 groups, ten of them were in the normal group, while other 30 rats were divided into Huoxue Huayu prescription high-dose diabetes group(6.75 g·kg~(-1), n=10), low-dose diabetes group(3.375 g·kg~(-1), n=10) and model group(n=10) after successfully established diabetic model by intraperitoneal injection of streptozotocin. Retina was taken after gastric administration for 60 d for RCPs immunohistochemical study, while Western blot assay was adopted to test the Bax, Bak, Bcl-2, caspase-3 protein expression. In vitro assay, RCPs were incubated with high glucose(30 mmol·L-1) and intervened by different concentrations of Huoxue Huayu prescription. CCK-8 assay was employed to measure the relative growth rate. Apoptosis of RCPs was observed with a fluorescence microscope using JC-1 stain, and quantitied by flow cytometry using Annexin V-FITC stain. RESULTS More obvious RCPs apoptosis were observed in diabetic model rats than that in normal group, and the Huoxue Huayu prescription could inhibit the apoptosis of RCPs. Compared with 0 μg·mL~(-1), the proliferation in 25-100 μg·mL~(-1) of Huoxue Huayu prescription increased significantly(P<0.05 or P<0.01). There was a significant difference between 25-100 μg·mL~(-1) and 0 μg·mL~(-1) in anti-apoptotic effect induced by high glucose. CONCLUSION Huoxueguayu prescription can inhibited high glucose induced apoptosis in RCPs, and the preliminary mechanism may be associated with major outer membrane proteins/BCL-2 pathway.
OBJECTIVE To determine the protective effect of Huoxue Huayu prescription on retinal capillary pericytes(RCPs) of diabetic rats. METHODS Forty SD rats were conducted and randomly separated into 4 groups, ten of them were in the normal group, while other 30 rats were divided into Huoxue Huayu prescription high-dose diabetes group(6.75 g·kg~(-1), n=10), low-dose diabetes group(3.375 g·kg~(-1), n=10) and model group(n=10) after successfully established diabetic model by intraperitoneal injection of streptozotocin. Retina was taken after gastric administration for 60 d for RCPs immunohistochemical study, while Western blot assay was adopted to test the Bax, Bak, Bcl-2, caspase-3 protein expression. In vitro assay, RCPs were incubated with high glucose(30 mmol·L-1) and intervened by different concentrations of Huoxue Huayu prescription. CCK-8 assay was employed to measure the relative growth rate. Apoptosis of RCPs was observed with a fluorescence microscope using JC-1 stain, and quantitied by flow cytometry using Annexin V-FITC stain. RESULTS More obvious RCPs apoptosis were observed in diabetic model rats than that in normal group, and the Huoxue Huayu prescription could inhibit the apoptosis of RCPs. Compared with 0 μg·mL~(-1), the proliferation in 25-100 μg·mL~(-1) of Huoxue Huayu prescription increased significantly(P<0.05 or P<0.01). There was a significant difference between 25-100 μg·mL~(-1) and 0 μg·mL~(-1) in anti-apoptotic effect induced by high glucose. CONCLUSION Huoxueguayu prescription can inhibited high glucose induced apoptosis in RCPs, and the preliminary mechanism may be associated with major outer membrane proteins/BCL-2 pathway.
引文
[1]WANG X A,XU Y S.Train of thought about differentiation of symptoms and signs in treatment of changes in retina caused by diabetes by TCM[J].Jilin J Tradit Chin Med(吉林中医药),2010,30(7):565-566.
[2]刘峥嵘,秦裕辉.中医治疗糖尿病视网膜病变的现代研究概况[J].湖南中医杂志,2015,31(2):156-158.
[3]SUN R,HUI S X.In different periods of diabetic retinopathy in the observation of the curative effect of treatment of Huayumingmu decoction and its influence on VEGF[J].World Chin Med(世界中医药),2016,11(1):75-78.
[4]黄建良,喻干龙.活血化瘀法治疗糖尿病视网膜病变38例[J].湖南中医杂志,2000,16(1):29.
[5]RAO V R,PRESCOTT E,SHELKE N B,et al.Delivery of sar1118 to the retina via ophthalmic drops and its effectiveness in a rat streptozotocin(stz)model of diabetic retinopathy(DR)[J].Invest Ophthalmol Vis Sci,2010,51(10):5198-5204.
[6]WANG J,CHENG X X,ZHU X L,et al.Effect of the union of Yiqi Bushen fang and tripterygium wilfordii polyglycosidium on expression of VEGF and secretion of IL-6 and TNF-αin rats with diabetic nephropathy[J].Chin J Mod Appl Pharm(中国现代应用药学),2016,33(6):711-716.
[7]EJAZ S,CHEKAROVA I,EJAZ A,et al.Importance of pericytes and mechanisms of pericyte loss during diabetes retinopathy[J].Diabetes Obes Metab,2008,10(1):53-63.
[8]MOGENSEN C,BERGNER B,WALLNER S,et al.Isolation and functional characterization of pericytes derived from hamster skeletal muscle[J].Acta Physiol,2011,201(4):413-426.
[9]PFISTER F,FENG Y H F,HOFFMANN S,et al.Pericyte migration:a novel mechanism of pericyte loss in experimental diabetic retinopathy[J].Diabetes,2008,57(9):2495-2502.
[10]REERS M,SMILEY S T,MOTTOLA-HARTSHORN C,et al.Mitochondrial membrane potential monitored by JC-1 dye[J].Method Enzymol,1995(260):406-417.
[11]SMITH S W,CHAND S,SAVAGE C O.Biology of the renal pericyte[J].Nephrol Dial Transpl,2012,27(6):2149-2155.
[12]SABANAYAGAM C.Epidemiology of diabetic retinopathy,diabetic macular edema and vision loss due to diabetes[J].Diabetes Res Clin Pr,2016,120(1):S14.
[13]KLAASSEN I,NOORDEN C J F V,SCHLINGEMANN R O.Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions[J].Prog Retin Eye Res,2013,34(5):19-48.
[14]WANG Y L,HUI Y N,GUO B.Inhibition of proliferation of retinal microvascular endothelial cells by pericytes through down-regulating KDR/FIK-1 in a co-culture system[J].Int JOphthalmol(国际眼科杂志),2006,6(2):255-263.
[15]ARMULIK A,GENOVéG,M?E M,et al.Pericytes regulate the blood-brain barrier[J].Nature,2010,468(7323):557-561.
[16]WILLIMOTT S,MERRAM T,WAGNER S D.Apoptosis induces Bcl-XS and cleaved Bcl-XL in chronic lymphocytic leukaemia[J].Biochem Biophys Res Commun,2011,405(3):480-485.
[17]WANG J,BEANCHEMIN M,BERTRAND R.Bcl-x Lphosphorylation at Set49 by polo kinase 3 during cell cycle progression and checkpoints[J].Cell Signat,2011,23(12):2030-2038.
[18]GU X,YAO Y.Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-xl subcellular distribution[J].Apoptosis,2011,16(8):846-855.
[2]刘峥嵘,秦裕辉.中医治疗糖尿病视网膜病变的现代研究概况[J].湖南中医杂志,2015,31(2):156-158.
[3]SUN R,HUI S X.In different periods of diabetic retinopathy in the observation of the curative effect of treatment of Huayumingmu decoction and its influence on VEGF[J].World Chin Med(世界中医药),2016,11(1):75-78.
[4]黄建良,喻干龙.活血化瘀法治疗糖尿病视网膜病变38例[J].湖南中医杂志,2000,16(1):29.
[5]RAO V R,PRESCOTT E,SHELKE N B,et al.Delivery of sar1118 to the retina via ophthalmic drops and its effectiveness in a rat streptozotocin(stz)model of diabetic retinopathy(DR)[J].Invest Ophthalmol Vis Sci,2010,51(10):5198-5204.
[6]WANG J,CHENG X X,ZHU X L,et al.Effect of the union of Yiqi Bushen fang and tripterygium wilfordii polyglycosidium on expression of VEGF and secretion of IL-6 and TNF-αin rats with diabetic nephropathy[J].Chin J Mod Appl Pharm(中国现代应用药学),2016,33(6):711-716.
[7]EJAZ S,CHEKAROVA I,EJAZ A,et al.Importance of pericytes and mechanisms of pericyte loss during diabetes retinopathy[J].Diabetes Obes Metab,2008,10(1):53-63.
[8]MOGENSEN C,BERGNER B,WALLNER S,et al.Isolation and functional characterization of pericytes derived from hamster skeletal muscle[J].Acta Physiol,2011,201(4):413-426.
[9]PFISTER F,FENG Y H F,HOFFMANN S,et al.Pericyte migration:a novel mechanism of pericyte loss in experimental diabetic retinopathy[J].Diabetes,2008,57(9):2495-2502.
[10]REERS M,SMILEY S T,MOTTOLA-HARTSHORN C,et al.Mitochondrial membrane potential monitored by JC-1 dye[J].Method Enzymol,1995(260):406-417.
[11]SMITH S W,CHAND S,SAVAGE C O.Biology of the renal pericyte[J].Nephrol Dial Transpl,2012,27(6):2149-2155.
[12]SABANAYAGAM C.Epidemiology of diabetic retinopathy,diabetic macular edema and vision loss due to diabetes[J].Diabetes Res Clin Pr,2016,120(1):S14.
[13]KLAASSEN I,NOORDEN C J F V,SCHLINGEMANN R O.Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions[J].Prog Retin Eye Res,2013,34(5):19-48.
[14]WANG Y L,HUI Y N,GUO B.Inhibition of proliferation of retinal microvascular endothelial cells by pericytes through down-regulating KDR/FIK-1 in a co-culture system[J].Int JOphthalmol(国际眼科杂志),2006,6(2):255-263.
[15]ARMULIK A,GENOVéG,M?E M,et al.Pericytes regulate the blood-brain barrier[J].Nature,2010,468(7323):557-561.
[16]WILLIMOTT S,MERRAM T,WAGNER S D.Apoptosis induces Bcl-XS and cleaved Bcl-XL in chronic lymphocytic leukaemia[J].Biochem Biophys Res Commun,2011,405(3):480-485.
[17]WANG J,BEANCHEMIN M,BERTRAND R.Bcl-x Lphosphorylation at Set49 by polo kinase 3 during cell cycle progression and checkpoints[J].Cell Signat,2011,23(12):2030-2038.
[18]GU X,YAO Y.Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-xl subcellular distribution[J].Apoptosis,2011,16(8):846-855.