β-榄香烯影响糖尿病大鼠视网膜中IL-1β、ICAM-1表达分析
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摘要
目的研究β-榄香烯对糖尿病大鼠视网膜病变中白细胞介素-1β(IL-1β)、细胞间黏附分子1(ICAM-1)表达变化的影响,以期为β-榄香烯应用于糖尿病视网膜病变临床防治提供理论依据。方法选取40只成年SD健康大鼠,随机分为正常对照组、糖尿病组、β-榄香烯治疗组(40 mg/kg)和β-榄香烯治疗组(80 mg/kg)。采用链脲佐菌素(STZ)腹腔内注射大鼠给予构建糖尿病模型,对β-榄香烯治疗组大鼠实施β-榄香烯溶液灌胃,持续治疗12周。在给药12周末对各组大鼠进行处死,将大鼠的视网膜组织进行分离,同时收集大鼠房水与血清,使用ELISA法测定IL-1β、ICAM-1蛋白在大鼠视网膜组织、房水与血清中的表达情况;免疫组织化学检测大鼠视网膜组织中IL-1β、ICAM-1蛋白表达水平;蛋白质免疫印迹法检测大鼠视网膜组织中IL-1β、ICAM-1蛋白的表达量。结果 ELISA法检测结果显示,与正常对照组比较,糖尿病组大鼠血清与视网膜中IL-1β、ICAM-1蛋白含量升高;但β-榄香烯治疗组(40 mg/kg)和β-榄香烯治疗组(80 mg/kg)大鼠血清与视网膜中IL-1β、ICAM-1含量低于糖尿病组,其中β-榄香烯治疗组(80 mg/kg)对血清与视网膜IL-1β、ICAM-1蛋白的降低作用较β-榄香烯治疗组(40 mg/kg)作用更强;各组房水中IL-1β、ICAM-1蛋白比较差异无统计学意义。正常对照组视网膜IL-1β蛋白微量表达,β-榄香烯治疗组(40 mg/kg)和β-榄香烯治疗组(80 mg/kg)IL-1β均较糖尿病组表达低;与正常对照组比较,糖尿病组视网膜IL-1β、ICAM-1蛋白表达量升高;β-榄香烯治疗组(40 mg/kg)和β-榄香烯治疗组(80 mg/kg)视网膜IL-1β、ICAM-1蛋白表达量均低于糖尿病组,且β-榄香烯治疗组(80 mg/kg)视网膜IL-1β、ICAM-1蛋白表达量低于β-榄香烯治疗组(40 mg/kg)。结论β-榄香烯可以抑制糖尿病大鼠血清及视网膜组织中的IL-1β、ICAM-1炎性因子的产生,且β-榄香烯剂量越大,抑制效果越明显,提示β-榄香烯可能用于糖尿病视网膜病变的防治。
Objective To study the effect of beta elemene on IL-1 beta and intercellular adhesion molecule 1(ICAM-1) expression levels in rats with diabetic retinopathy, to validate the potential of beta elemene in the prevention and treatment of diabetic retinopathy. Methods Forty adult SD healthy rats were randomly divided into four groups: normal control group, diabetic group, 40 mg/kg beta elemene treatment group, and 80 mg/kg beta elemene treatment group. Diabetes was induced in rats by intraperitoneal injection of streptozotocin(STZ). The rats in the beta elemene treatment group were treated with beta elemene solution for 12 weeks. They were then executed, and their retinal tissue, aqueous humor, and serum were extracted. ELISA was used to determine the IL-1 beta and ICAM-1 protein expression in retina, aqueous humor, and serum of rats. Immunohistochemistry was used to detect the expression levels of IL-1 beta and ICAM-1 proteins in retinal tissues of rats. Western blotting was done to evaluate the expression levels of IL-1 beta and ICAM-1 proteins in the retinal tissue. Results ELISA assay revealed that, compared with the normal control group, the levels of IL-1 beta and ICAM-1 proteins in diabetic rat serum and retina were significantly higher(P<0.05); however, 40 mg/kg treatment group and 80 mg/kg treatment exhibited significantly lower IL-1 beta and ICAM-1 levels in rat serum and retina compared to the diabetic group(P<0.05), among which 80 mg/kg beta elemene treatment group exhibited lower serum and retinal IL-1 beta and ICAM-1 protein levels compared to 40 mg/kg beta elemene treatment group(P<0.05). There was no significant difference in IL-1 beta and ICAM-1 protein levels in aqueous humor among the groups(P>0.05). Immunohistochemistry results showed that compared with the normal control group, retinal IL-1 beta protein levels were higher in the diabetic group; however, 40 mg/kg treatment group and 80 mg/kg treatment group exhibited lower IL-1 beta levels compared with diabetic group; the retinal tissue isolated from normal group exhibited almost no expression of ICAM-1 protein, whereas the diabetic group exhibited high expression of ICAM-1 protein. The 40 mg/kg treatment group and the 80 mg/kg treatment group exhibited lower expression levels of ICAM-1 compared to diabetic group. Western blotting revealed that, compared with the normal control group, diabetes group exhibited significantly higher expression levels of retinal IL-1 beta and ICAM-1 proteins(P<0.05); 40 mg/kg treatment group and 80 mg/kg treatment group exhibited significantly lower retinal IL-1 beta and ICAM-1 protein levels compared to diabetic group(P<0.05), among which 80 mg/kg treatment group exhibited significantly lower retinal IL-1 beta and ICAM-1 protein levels compared to 40 mg/kg treatment group(P<0.05). Conclusion Beta elemene can inhibit the production of IL-1 beta and ICAM-1 inflammatory factors in serum and retinal tissue of diabetic rats, suggesting that beta elemene may be used in the prevention and treatment of diabetic retinopathy.
Objective To study the effect of beta elemene on IL-1 beta and intercellular adhesion molecule 1(ICAM-1) expression levels in rats with diabetic retinopathy, to validate the potential of beta elemene in the prevention and treatment of diabetic retinopathy. Methods Forty adult SD healthy rats were randomly divided into four groups: normal control group, diabetic group, 40 mg/kg beta elemene treatment group, and 80 mg/kg beta elemene treatment group. Diabetes was induced in rats by intraperitoneal injection of streptozotocin(STZ). The rats in the beta elemene treatment group were treated with beta elemene solution for 12 weeks. They were then executed, and their retinal tissue, aqueous humor, and serum were extracted. ELISA was used to determine the IL-1 beta and ICAM-1 protein expression in retina, aqueous humor, and serum of rats. Immunohistochemistry was used to detect the expression levels of IL-1 beta and ICAM-1 proteins in retinal tissues of rats. Western blotting was done to evaluate the expression levels of IL-1 beta and ICAM-1 proteins in the retinal tissue. Results ELISA assay revealed that, compared with the normal control group, the levels of IL-1 beta and ICAM-1 proteins in diabetic rat serum and retina were significantly higher(P<0.05); however, 40 mg/kg treatment group and 80 mg/kg treatment exhibited significantly lower IL-1 beta and ICAM-1 levels in rat serum and retina compared to the diabetic group(P<0.05), among which 80 mg/kg beta elemene treatment group exhibited lower serum and retinal IL-1 beta and ICAM-1 protein levels compared to 40 mg/kg beta elemene treatment group(P<0.05). There was no significant difference in IL-1 beta and ICAM-1 protein levels in aqueous humor among the groups(P>0.05). Immunohistochemistry results showed that compared with the normal control group, retinal IL-1 beta protein levels were higher in the diabetic group; however, 40 mg/kg treatment group and 80 mg/kg treatment group exhibited lower IL-1 beta levels compared with diabetic group; the retinal tissue isolated from normal group exhibited almost no expression of ICAM-1 protein, whereas the diabetic group exhibited high expression of ICAM-1 protein. The 40 mg/kg treatment group and the 80 mg/kg treatment group exhibited lower expression levels of ICAM-1 compared to diabetic group. Western blotting revealed that, compared with the normal control group, diabetes group exhibited significantly higher expression levels of retinal IL-1 beta and ICAM-1 proteins(P<0.05); 40 mg/kg treatment group and 80 mg/kg treatment group exhibited significantly lower retinal IL-1 beta and ICAM-1 protein levels compared to diabetic group(P<0.05), among which 80 mg/kg treatment group exhibited significantly lower retinal IL-1 beta and ICAM-1 protein levels compared to 40 mg/kg treatment group(P<0.05). Conclusion Beta elemene can inhibit the production of IL-1 beta and ICAM-1 inflammatory factors in serum and retinal tissue of diabetic rats, suggesting that beta elemene may be used in the prevention and treatment of diabetic retinopathy.
引文
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[8]Shen X,Xu GZ.Role of IL-1βon the glutamine synthetase in retinal müller cells under high glucose conditions[J].Current Eye Research,2009,34(9):727-736.doi:10.1080/02713680903030875.
[9]Nentwich MM,Ulbig MW.Diabetic retinopathy-ocular complications of diabetes mellitus[J].World J Diabetes,2015,6(3):489-499.doi:10.4239/w jd.v6.i3.489.
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[11]韩青.糖尿病视网膜病变患者外周血IL-1β和TNF-α的检测及临床意义[J].中国实验诊断学,2016,20(4):583-586.HAN Qing.Expression and clinical significance of serum IL-1βand TNF-αin patients w ith diabetic retinopathy[J].Chin J Lab Diagn,2016,20(4):583-586.
[12]徐家窈,冼文光,刘俐娜.血清VEGF及ICAM-1水平与糖尿病视网膜病变患者微血管损伤的关系[J].中国老年学杂志,2017,37(8):2000-2002.doi:10.3969/j.issn.1005-9202.2017.08.080.
[13]Veldhuis WB,Derksen JW,Floris S,et al.Interferonbeta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat[J].JCereb Blood Flow M etab,2003,23(9):1029-1039.doi:10.1097/01.WCB.0000080703.47016.B6.
[14]郭建忠,魏尧,谷如雷,等.β-榄香烯诱导血管内皮细胞凋亡的研究[J].中国药物与临床,2016,16(8):1127-1129.doi:10.11655/zgyw ylc2016.08.011.
[15]Zhang X,Li ZZ,Liu DF,et al.Effects of probucol on hepatic tumor necrosis factor-α,interleukin-6 and adiponectin receptor-2 expression in diabetic rats[J].Journal of Gastroenterology and Hepatology,2009,24(6):1058-1063.doi:10.1111/j.1440-1746.2008.05719.x.
[16]胡建廷,王汝霞,高聆.西洛他唑对糖尿病大鼠视网膜ICAM-1和VCAM-1的影响及其机制[J].中国新药杂志,2011,20(1):60-63.HU Jianting,WANG Ruxia,GAO Ling.Cilostazol decreases the expression of ICAM-1 and VCAM-1 in the retina via up-regulating PPAR-γin diabetic rats[J].Chinese Journal of New Drugs,2011,20(1):60-63.
[2]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.
[3]Mc Laughlin F,Hayes BP,Horgan CM,et al.Tumor necrosis factor(TNF)-alpha and interleukin(IL)-1beta dow n-regulate intercellular adhesion molecule(ICAM)-2expression on the endothelium[J].Cell Adhes Commun,1998,6(5):381-400.
[4]Lee SJ,Drabik K,Van Wagoner NJ,et al.ICAM-1-induced expression of proinflammatory cytokines in astrocytes:involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathw ays[J].JImmunol,2000,165(8):4658-4666.
[5]Yang CR,Shih KS,Liou JP,et al.Denbinobin upregulates miR-146a expression and attenuates IL-1β-induced upregulation of ICAM-1 and VCAM-1 expressions in osteoarthritis fibroblast-like synoviocytes[J].J Mol Med,2014,92(11):1147-1158.doi:10.1007/s00109-014-1192-8.
[6]李雪,周赟,陈蕾.β-榄香烯主要抗肿瘤机制及在非肿瘤性疾病中的研究进展[J].安徽医药,2015(8):1429-1432.doi:10.3969/j.issn.1009-6469.2015.08.001.LI Xue,ZHOU Yun,CHEN Lei.Research progress on the main anti-tumor mechanisms ofβ-elemene and its applications in non-oncologic diseases[J].Anhui M ed Pharmaceut J,2015(8):1429-1432.doi:10.3969/j.issn.1009-6469.2015.08.001.
[7]周赟,刘宁宁,万超,等.β-榄香烯对糖尿病大鼠视网膜中肿瘤坏死因子-α表达的影响[J].眼科新进展,2015,35(12):1129-1131.doi:10.13389/j.cnki.rao.2015.0309.ZHOU Yun,LIU Ning-Ning,WAN Chao,et al.Effects ofβ-elemene on expression of TNF-αin retina of diabetic rats[J].Rec Adv Ophthalmol,2015,35(12):1129-1131.doi:10.13389/j.cnki.rao.2015.0309.
[8]Shen X,Xu GZ.Role of IL-1βon the glutamine synthetase in retinal müller cells under high glucose conditions[J].Current Eye Research,2009,34(9):727-736.doi:10.1080/02713680903030875.
[9]Nentwich MM,Ulbig MW.Diabetic retinopathy-ocular complications of diabetes mellitus[J].World J Diabetes,2015,6(3):489-499.doi:10.4239/w jd.v6.i3.489.
[10]Matsunaga DR,Yi JJ,De Koo LO,et al.Optical coherence tomography angiography of diabetic retinopathy in human subjects[J].Ophthalmic Surg Lasers Imaging Retina,2015,46(8):796-805.doi:10.3928/23258160-20150909-03.
[11]韩青.糖尿病视网膜病变患者外周血IL-1β和TNF-α的检测及临床意义[J].中国实验诊断学,2016,20(4):583-586.HAN Qing.Expression and clinical significance of serum IL-1βand TNF-αin patients w ith diabetic retinopathy[J].Chin J Lab Diagn,2016,20(4):583-586.
[12]徐家窈,冼文光,刘俐娜.血清VEGF及ICAM-1水平与糖尿病视网膜病变患者微血管损伤的关系[J].中国老年学杂志,2017,37(8):2000-2002.doi:10.3969/j.issn.1005-9202.2017.08.080.
[13]Veldhuis WB,Derksen JW,Floris S,et al.Interferonbeta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat[J].JCereb Blood Flow M etab,2003,23(9):1029-1039.doi:10.1097/01.WCB.0000080703.47016.B6.
[14]郭建忠,魏尧,谷如雷,等.β-榄香烯诱导血管内皮细胞凋亡的研究[J].中国药物与临床,2016,16(8):1127-1129.doi:10.11655/zgyw ylc2016.08.011.
[15]Zhang X,Li ZZ,Liu DF,et al.Effects of probucol on hepatic tumor necrosis factor-α,interleukin-6 and adiponectin receptor-2 expression in diabetic rats[J].Journal of Gastroenterology and Hepatology,2009,24(6):1058-1063.doi:10.1111/j.1440-1746.2008.05719.x.
[16]胡建廷,王汝霞,高聆.西洛他唑对糖尿病大鼠视网膜ICAM-1和VCAM-1的影响及其机制[J].中国新药杂志,2011,20(1):60-63.HU Jianting,WANG Ruxia,GAO Ling.Cilostazol decreases the expression of ICAM-1 and VCAM-1 in the retina via up-regulating PPAR-γin diabetic rats[J].Chinese Journal of New Drugs,2011,20(1):60-63.