丝胶对糖尿病大鼠视网膜氧化应激和微炎症状态的改善作用
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摘要
目的探讨丝胶对糖尿病大鼠视网膜氧化应激和微炎症状态的改善作用。方法采用高脂高糖饲料喂养联合链脲佐菌素腹腔注射法建立糖尿病大鼠模型,将24只成模大鼠随机分为丝胶治疗组和糖尿病模型组,每组12只,另取同周龄12只正常大鼠作为正常对照组,成模后丝胶治疗组给予丝胶溶液空腹灌胃、糖尿病模型组及正常对照组给予等体积生理盐水灌胃每天1次,共35 d。药物干预后,检测3组视网膜组织中丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)的含量;采用Western blot法检测视网膜核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素氧合酶1(heme oxygenase 1,HO-1)、核因子-κB(nuclear factor kappa-B,NF-κB)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白的表达,苏木素-伊红染色法观察视网膜形态结构。结果各指标三组间整体比较差异显著(均为P<0.01)。丝胶治疗组大鼠视网膜中MDA含量、NF-κB和TNF-α蛋白表达水平分别为(4.145±0.282)mmol·gprot-1、0.232±0.027和0.761±0.058,较糖尿病模型组的(6.813±0.446)mmol·gprot-1、0.334±0.024、0.994±0.084均显著降低(均为P<0.05)。丝胶治疗组大鼠视网膜中还原型GSH含量、Nrf2和HO-1蛋白表达水平分别为(78.518±4.317)mg·gprot-1、0.591±0.054和0.954±0.091,较糖尿病模型组的(59.890±5.932)mg·gprot-1、0.351±0.044、0.585±0.054均显著升高(均为P<0.05)。糖尿病模型组大鼠视网膜各层细胞排列紊乱,内界膜肿胀,神经节细胞可见空泡、水肿样改变,丝胶治疗组大鼠视网膜各层细胞形态较规则、排列轻度紊乱,病理变化较糖尿病模型组明显减轻。结论丝胶可改善糖尿病视网膜氧化应激和炎症介质的损伤,延缓糖尿病视网膜病变发展。
Objective To investigate the improvement effects of sericin on the retinal oxidative stress and micro-inflammatory state in diabetic rats. Methods A diabetic rat model was established by using high-fat and high-sugar diet and intraperitoneal injection of streptozotocin.Then 24 diabetic rats were randomly divided into sericin treatment group and diabetic model group,with 12 rats for each group,and additional 12 normal rats with the same age were collected as a normal control group. Next,the rats in the sericin treatment group received sericin solution,while the other two groups was given the same amount of normal saline once a day for 35 days.After the agent intervention,the content of malondialdehyde( MDA) and glutathione( GSH) in the retina of all rats were detected by related kits. The expressions of nuclear factor erythroid 2-related factor 2( Nrf2),heme oxygenase 1( HO-1),nuclear factor-κB( NF-κB) and tumor necrosis factor-α( TNF-α) protein were detected by Western blot,and finally,the retinal morphology was examined by hematoxylin-eosin staining in the 3 groups. Results The content of MDA,NF-κB and TNF-α protein expression in the sericin treatment group was( 4. 145 ± 0. 282) mmol·gprot-1,0. 232 ± 0. 027 and 0. 761 ± 0. 058,respectively,which was significantly lower than that in the diabetic model group[( 6. 813 ± 0. 446) mmol·gprot-1,0. 334 ± 0. 024 and 0. 994 ± 0.084]( all P < 0. 05). The content of GSH,Nrf2 and HO-1 protein expression in rat retina in the sericin treatment group was( 78. 518 ± 4. 317) mg·gprot-1,0. 591 ± 0. 054 and 0. 954 ± 0. 091,respectively,which was significantly higher than that in the diabetic model group [( 59. 890 ± 5.932) mg · gprot-1,0. 351 ± 0. 044 and 0. 585 ± 0. 054]( all P < 0. 05). The diabetic model group presented the disorder arrangement of the neurocyte in different levels in the retina,irregular and swollen inner limiting membrane,vacuoles in the ganglion cells,but in the sericin treatment group,the morphology of retinal layers was more regular and mildly disorderly arranged. The pathological damages in the retina were alleviated significantly. Conclusion Sericin can ameliorate oxidative stress and inflammation in diabetic retina,thereby delaying the development of diabetic retinopathy.
Objective To investigate the improvement effects of sericin on the retinal oxidative stress and micro-inflammatory state in diabetic rats. Methods A diabetic rat model was established by using high-fat and high-sugar diet and intraperitoneal injection of streptozotocin.Then 24 diabetic rats were randomly divided into sericin treatment group and diabetic model group,with 12 rats for each group,and additional 12 normal rats with the same age were collected as a normal control group. Next,the rats in the sericin treatment group received sericin solution,while the other two groups was given the same amount of normal saline once a day for 35 days.After the agent intervention,the content of malondialdehyde( MDA) and glutathione( GSH) in the retina of all rats were detected by related kits. The expressions of nuclear factor erythroid 2-related factor 2( Nrf2),heme oxygenase 1( HO-1),nuclear factor-κB( NF-κB) and tumor necrosis factor-α( TNF-α) protein were detected by Western blot,and finally,the retinal morphology was examined by hematoxylin-eosin staining in the 3 groups. Results The content of MDA,NF-κB and TNF-α protein expression in the sericin treatment group was( 4. 145 ± 0. 282) mmol·gprot-1,0. 232 ± 0. 027 and 0. 761 ± 0. 058,respectively,which was significantly lower than that in the diabetic model group[( 6. 813 ± 0. 446) mmol·gprot-1,0. 334 ± 0. 024 and 0. 994 ± 0.084]( all P < 0. 05). The content of GSH,Nrf2 and HO-1 protein expression in rat retina in the sericin treatment group was( 78. 518 ± 4. 317) mg·gprot-1,0. 591 ± 0. 054 and 0. 954 ± 0. 091,respectively,which was significantly higher than that in the diabetic model group [( 59. 890 ± 5.932) mg · gprot-1,0. 351 ± 0. 044 and 0. 585 ± 0. 054]( all P < 0. 05). The diabetic model group presented the disorder arrangement of the neurocyte in different levels in the retina,irregular and swollen inner limiting membrane,vacuoles in the ganglion cells,but in the sericin treatment group,the morphology of retinal layers was more regular and mildly disorderly arranged. The pathological damages in the retina were alleviated significantly. Conclusion Sericin can ameliorate oxidative stress and inflammation in diabetic retina,thereby delaying the development of diabetic retinopathy.
引文
[1]SONG SJ,WONG TY.Current concepts in diabetic retinopathy[J].Diabetes Metab J,2014,38(6):416-425.
[2]TIAN M,LV HB.Research advances in oxidative stress and diabetic retinopathy[J].Rec Adv Ophthalmol,2015,35(7):697-700.田敏,吕红彬.氧化应激与糖尿病视网膜病变的研究进展[J].眼科新进展,2015,35(7):697-700.
[3]CARPI-SANTOS R,FERREIRA MJ,PEREIRA NETTO AD,GIESTAL-DE-ARAUJO E,VENTURA AL,COSSENZA M,et al.Early changes in system and glutathione in the retina of diabetic rats[J].Exp Eye Res,2016,146(1):35-42.
[4]LIU D,PERKINS JT,PETRIELLO MC,HENNIG B.Exposure to coplanar PCBs induces endothelial cell inflammation through epigenetic regulation of NF-kappa B subunit p65[J].Toxicol Appl Pharmacol,2015,289(3):457-465.
[5]LI C,MIAO X,LI FS,WANG SD,LIU Q,ET A.Oxidative stress-related mechanisms and antioxidant therapy in diabetic retinopathy[J/OL].Oxid Med Cell Longev,2017,2017:9702820.
[6]GONG X,RUBIN LP.Role of macular xanthophylls in prevention of common neovascular retinopathies:retinopathy of prematurity and diabetic retinopathy[J].Arch Biochem Biophys,2015,572(1):40-48.
[7]SHARMA S,SAXENA S,SRIVASTAV K,SHUKLA RK,MISHRA N.Nitric oxide and oxidative stress is associated with severity of diabetic retinopathy and retinal structural alterations[J].Clin Exp Ophthalmol,2015,43(5):429-436.
[8]KUMAR N,KAR A.Pyrroloquinoline quinone ameliorates oxidative stress and lipid peroxidation in the brain of streptozotocin-induced diabetic mice[J].Can J Physiol Pharmacol,2015,93(1):71-79.
[9]YI EH,XU F,LI P.(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one alleviates lipoteichoic acid-induced photoreceptor cell damage[J].Cutan Ocul Toxicol,2017,24:1-17.
[10]TIAN M,ZHANG SY,HAN PY,LI JY,LV HB.t BHQ activates Nrf2 signaling pathways to enhance retinal protection in type 2 diabetic rats[J].Rec Adv Ophthalmol,2017,37(3):220-224.田敏,张思远,韩佩晏,李晶艳,吕红彬.叔丁基对苯二酚激活Nrf2信号通路增强对2型糖尿病大鼠视网膜的保护作用[J].眼科新进展,2017,37(3):220-224.
[11]TAN SM,DE HAAN JB.Combating oxidative stress in diabetic complications with Nrf2 activators:how much is too much[J]?Redox Rep,2014,19(3):107-117.
[12]XU Z,WEI Y,GONG J,CHO H,PARK JK,SUNG ER,et al.NRF2 plays a protective role in diabetic retinopathy in mice[J].Diabetologia,2014,57(1):204-213.
[13]TANG J,KEM TS.Inflammation in diabetic retinopathy[J].Prog Retin Eve Res,2011,30(5):343-358.
[14]TOMIC M,LJUBIC S,KASTELAN S.The role of inflammation and endothelial dysfunction in the pathogenesis of diabetic retinopathy[J].Coll Antropol,2013,37(1):51-57.
[15]WANG X,WANG C,XING HY,LIU M,ZHANG Z.Effects of Tongxinluo capsule on retina through IKKβ/IKBα/NF-κB pathway in diabetic mouse[J].Rec Adv Ophthalmol,2014,34(11):1005-1008.王杏,王超,邢邯英,刘敏,张哲.通心络胶囊对糖尿病小鼠视网膜IKKβ/IKBα/NF-κB通路的作用[J].眼科新进展,2014,34(11):1005-1008.
[16]REN G,SUN A,DENG C,ZHANG J,WU X.The anti-inflammatory effect and potential mechanism of cardamonin in DSS-induced colitis[J].Am J Physiol Gastrointest Liver Physiol,2015,309(7):517-527.
[17]JIN HY,LIU K,XU X.The relationship between inflammation and anti-inflammatory drugs and early diabetic retinopathy[J].Chin J Ocul Fundus Dis,2008,24(4):312-315.金慧昳,刘堃,许迅.炎症、抗炎药物与早期糖尿病视网膜病变的关系[J].中华眼底病杂志,2008,24(4):312-315.
[18]JIANG T,CHANG Q,CAI J,FAN J,ZHANG X.Protective effects of melatonin on retinal inflammation and oxidative stress in experimental diabetic retinopathy[J].Oxid Med Cell Longev,2016,2016:3528274.
[19]WISNIEWSKA-KRUK J,HOEBEN KA,VOGELS IM,GAILLARD PJ,VAN NOORDEN CJ,et al.A novel co-culture model of the bloodretinal barrier based on primary retinal endothelial cells,pericytes and astrocytes[J].Exp Eye Res,2012,90(S249):181-190.
[20]DASH R,ACHARYA C,BINDU PC,KUNDU SC.Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts[J].BMB Rep,2008,41(3):236-241.
[2]TIAN M,LV HB.Research advances in oxidative stress and diabetic retinopathy[J].Rec Adv Ophthalmol,2015,35(7):697-700.田敏,吕红彬.氧化应激与糖尿病视网膜病变的研究进展[J].眼科新进展,2015,35(7):697-700.
[3]CARPI-SANTOS R,FERREIRA MJ,PEREIRA NETTO AD,GIESTAL-DE-ARAUJO E,VENTURA AL,COSSENZA M,et al.Early changes in system and glutathione in the retina of diabetic rats[J].Exp Eye Res,2016,146(1):35-42.
[4]LIU D,PERKINS JT,PETRIELLO MC,HENNIG B.Exposure to coplanar PCBs induces endothelial cell inflammation through epigenetic regulation of NF-kappa B subunit p65[J].Toxicol Appl Pharmacol,2015,289(3):457-465.
[5]LI C,MIAO X,LI FS,WANG SD,LIU Q,ET A.Oxidative stress-related mechanisms and antioxidant therapy in diabetic retinopathy[J/OL].Oxid Med Cell Longev,2017,2017:9702820.
[6]GONG X,RUBIN LP.Role of macular xanthophylls in prevention of common neovascular retinopathies:retinopathy of prematurity and diabetic retinopathy[J].Arch Biochem Biophys,2015,572(1):40-48.
[7]SHARMA S,SAXENA S,SRIVASTAV K,SHUKLA RK,MISHRA N.Nitric oxide and oxidative stress is associated with severity of diabetic retinopathy and retinal structural alterations[J].Clin Exp Ophthalmol,2015,43(5):429-436.
[8]KUMAR N,KAR A.Pyrroloquinoline quinone ameliorates oxidative stress and lipid peroxidation in the brain of streptozotocin-induced diabetic mice[J].Can J Physiol Pharmacol,2015,93(1):71-79.
[9]YI EH,XU F,LI P.(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one alleviates lipoteichoic acid-induced photoreceptor cell damage[J].Cutan Ocul Toxicol,2017,24:1-17.
[10]TIAN M,ZHANG SY,HAN PY,LI JY,LV HB.t BHQ activates Nrf2 signaling pathways to enhance retinal protection in type 2 diabetic rats[J].Rec Adv Ophthalmol,2017,37(3):220-224.田敏,张思远,韩佩晏,李晶艳,吕红彬.叔丁基对苯二酚激活Nrf2信号通路增强对2型糖尿病大鼠视网膜的保护作用[J].眼科新进展,2017,37(3):220-224.
[11]TAN SM,DE HAAN JB.Combating oxidative stress in diabetic complications with Nrf2 activators:how much is too much[J]?Redox Rep,2014,19(3):107-117.
[12]XU Z,WEI Y,GONG J,CHO H,PARK JK,SUNG ER,et al.NRF2 plays a protective role in diabetic retinopathy in mice[J].Diabetologia,2014,57(1):204-213.
[13]TANG J,KEM TS.Inflammation in diabetic retinopathy[J].Prog Retin Eve Res,2011,30(5):343-358.
[14]TOMIC M,LJUBIC S,KASTELAN S.The role of inflammation and endothelial dysfunction in the pathogenesis of diabetic retinopathy[J].Coll Antropol,2013,37(1):51-57.
[15]WANG X,WANG C,XING HY,LIU M,ZHANG Z.Effects of Tongxinluo capsule on retina through IKKβ/IKBα/NF-κB pathway in diabetic mouse[J].Rec Adv Ophthalmol,2014,34(11):1005-1008.王杏,王超,邢邯英,刘敏,张哲.通心络胶囊对糖尿病小鼠视网膜IKKβ/IKBα/NF-κB通路的作用[J].眼科新进展,2014,34(11):1005-1008.
[16]REN G,SUN A,DENG C,ZHANG J,WU X.The anti-inflammatory effect and potential mechanism of cardamonin in DSS-induced colitis[J].Am J Physiol Gastrointest Liver Physiol,2015,309(7):517-527.
[17]JIN HY,LIU K,XU X.The relationship between inflammation and anti-inflammatory drugs and early diabetic retinopathy[J].Chin J Ocul Fundus Dis,2008,24(4):312-315.金慧昳,刘堃,许迅.炎症、抗炎药物与早期糖尿病视网膜病变的关系[J].中华眼底病杂志,2008,24(4):312-315.
[18]JIANG T,CHANG Q,CAI J,FAN J,ZHANG X.Protective effects of melatonin on retinal inflammation and oxidative stress in experimental diabetic retinopathy[J].Oxid Med Cell Longev,2016,2016:3528274.
[19]WISNIEWSKA-KRUK J,HOEBEN KA,VOGELS IM,GAILLARD PJ,VAN NOORDEN CJ,et al.A novel co-culture model of the bloodretinal barrier based on primary retinal endothelial cells,pericytes and astrocytes[J].Exp Eye Res,2012,90(S249):181-190.
[20]DASH R,ACHARYA C,BINDU PC,KUNDU SC.Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts[J].BMB Rep,2008,41(3):236-241.