表皮生长因子对缺氧缺糖模型大鼠骨骼肌细胞氧化损伤的保护作用
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摘要
目的:构建体外骨骼肌L6细胞缺氧缺糖(OGD)模型,在体外水平上探讨表皮生长因子(EGF)对压疮深部组织损伤(DTI)的保护机制。方法:将对数生长期大鼠骨骼肌L6成肌细胞分为5组,即正常对照组、OGD组、5μg·L-1 EGF+OGD组、10μg·L-1 EGF+OGD组和20μg·L-1 EGF+OGD组。MTT法检测各组细胞生存率,流式细胞术检测各组细胞凋亡率,DCFH-DA法检测各组L6成肌骨骼肌细胞中活性氧(ROS)水平,Rhodamine 123检测线粒体膜电势,Western blotting法检测各组骨骼肌细胞中Bcl-2和Bax蛋白表达。结果:与正常对照组比较,OGD组OGD 24h时骨骼肌细胞生存率明显降低(P<0.01),细胞凋亡率明显升高(P<0.01),ROS水平升高(P<0.01),线粒体膜电势下降(P<0.01),Bcl-2/Bax比值明显下降(P<0.01)。与OGD组比较,不同浓度EGF组细胞存活率明显升高,细胞凋亡率降低,其中10和20μg·L-1 EGF组差异有统计学意义(P<0.05或P<0.01);不同浓度EGF组骨骼肌细胞中ROS水平呈浓度依赖性降低,线粒体膜电势明显增加,10和20μg·L-1 EGF组差异有统计学意义(P<0.05或P<0.01);Bcl-2/Bax比值明显下降,并具有浓度依赖性,其中10和20μg·L-1 EGF组差异有统计学意义(P<0.05或P<0.01)。结论:EGF通过降低细胞内ROS水平保护线粒体功能,改善OGD诱导的大鼠骨骼肌细胞损伤,并具有浓度依赖性。
Objective:To set up the rat skeletal muscle L6 cell models of oxygen-glucose deprivation(OGD)in vitro,and to investigate the protective effect of EGF in deep tissue injury(DTI)of pressure sores.Methods:The rat skeletal muscle cells in the logarithmic phase were divided into normal control group,OGD group,5μg·L-1 EGF+OGD group,10μg·L-1 EGF+ OGD group and 20μg·L-1 EGF+ OGD group.The survival rates of skeletal muscle cells in various groups were measured by MTT assay;the cell apoptotic rates in various groups were detected by flow cytometry;the reactive oxygen species(ROS)levels were detected by DCFH-DA;Rhodamine 123 was used to detect the mitochondrial membrane potential;the expressions of Bax and Bcl-2 proteins were determined by Western blotting method.Results:Compared with normal control group,the survival rates of skeletal muscle cells in OGD group after 24 h OGD was significantly decreased(P<0.05);the apoptotic rate was markedly increased(P <0.01);the ROS level was increased(P <0.01);the mitochondrial membrane potential was decreased(P<0.01);the ratio of Bcl-2/Bax was significantly decreased(P<0.01).Compared with OGD group,the survival rates of skeletal muscle cells in different concentrations of EGF groups were increased and the apoptotic rates were decreased,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P<0.01);the ROS levels in skeletal muscle cells in different concentrations of EGF groups were decreased and the mitochondrial membrane potential were increased,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P<0.01);the Bcl-2/Bax ratios were significantly decreased in a concentration-dependent manner,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P <0.01).Conclusion:EGF can improve the skeletal muscle cell injury induced by OGD in a concentration-dependent manner via decreasing the ROS levels and protecting the cell mitochondrial function.
Objective:To set up the rat skeletal muscle L6 cell models of oxygen-glucose deprivation(OGD)in vitro,and to investigate the protective effect of EGF in deep tissue injury(DTI)of pressure sores.Methods:The rat skeletal muscle cells in the logarithmic phase were divided into normal control group,OGD group,5μg·L-1 EGF+OGD group,10μg·L-1 EGF+ OGD group and 20μg·L-1 EGF+ OGD group.The survival rates of skeletal muscle cells in various groups were measured by MTT assay;the cell apoptotic rates in various groups were detected by flow cytometry;the reactive oxygen species(ROS)levels were detected by DCFH-DA;Rhodamine 123 was used to detect the mitochondrial membrane potential;the expressions of Bax and Bcl-2 proteins were determined by Western blotting method.Results:Compared with normal control group,the survival rates of skeletal muscle cells in OGD group after 24 h OGD was significantly decreased(P<0.05);the apoptotic rate was markedly increased(P <0.01);the ROS level was increased(P <0.01);the mitochondrial membrane potential was decreased(P<0.01);the ratio of Bcl-2/Bax was significantly decreased(P<0.01).Compared with OGD group,the survival rates of skeletal muscle cells in different concentrations of EGF groups were increased and the apoptotic rates were decreased,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P<0.01);the ROS levels in skeletal muscle cells in different concentrations of EGF groups were decreased and the mitochondrial membrane potential were increased,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P<0.01);the Bcl-2/Bax ratios were significantly decreased in a concentration-dependent manner,especially in 10 and 20μg·L-1 EGF groups(P<0.05 or P <0.01).Conclusion:EGF can improve the skeletal muscle cell injury induced by OGD in a concentration-dependent manner via decreasing the ROS levels and protecting the cell mitochondrial function.
引文
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[2]Truong B,Grigson E,Patel M,et al.Pressure ulcer prevention in the hospital setting using silicone foam dressings[J].Cureus,2016,8(8):e730.
[3]Zhao R,Liang H,Clarke E,et al.Inflammation in chronic wounds[J].Int J Mol Sci,2016,17(12):2085.
[4]Preston A,Rao A,Strauss R,et al.Deep tissue pressure injury:A clinical review[J].Am J Nurs,2017,117(5):50-57.
[5]Carmona-Cuenca I,Herrera B,Ventura JJ,et al.EGF blocks NADPH oxidase activation by TGF-beta in fetal rat hepatocytes,impairing oxidative stress,and cell death[J].J Cell Physiol,2006,207(2):322-330.
[6]Seeger MA,Paller AS.The roles of growth factors in keratinocyte migration[J].Adv Wound Care(New Rochelle),2015,4(4):213-224.
[7]Berlanga-Acosta J,Fernández-Montequín J,Valdés-Pérez C,et al.Diabetic foot ulcers and epidermal growth factor:revisiting the local delivery route for a successful outcome[J].Biomed Res Int,2017,2017:2923759.
[8]Alderden J,Rondinelli J,Pepper G,et al.Risk factors for pressure injuries among critical care patients:A systematic review[J].Int J Nurs Stud,2017,71:97-114.
[9]Jiang LP,Tu Q,Wang Y,et al.Ischemia-reperfusion injury-induced histological changes affecting early stage pressure ulcer development in a rat model[J].Ostomy/Wound Manag,2011,57(2):55-60.
[10]Sundin BM,Hussein MA,Glasofer S,et al.The role of allopurinol and deferoxamine in preventing pressure ulcers in pigs[J].Plast Reconstr Surg,2000,105(4):1408-1421.
[11]Hong Y,Zhang B,Yu L,et al.Cell membrane integrity and revascularization:The possible functional mechanism of ischemic preconditioning for skeletal muscle protection against ischemic-reperfusion injury[J].Acta Histochem,2017,119(3):309-314.
[12]刘燕,张连元,张娜,等.BQ123对大鼠肢体缺血再灌注后骨骼肌损伤和细胞凋亡的影响[J].吉林大学学报:医学版,2010,36(1):131-134.
[13]Black JM.Moving toward consensus on deep tissue injury and pressure ulcer staging[J].Adv Skin Wound Care,2005,18(8):415-416.
[14]Liang L,Zhang Z.Gambogic acid inhibits malignant melanoma cell proliferation through mitochondrial p66shc/ROS-p53/Bax-mediated apoptosis[J].Cell Physiol Biochem,2016,38(4),1618-1630.
[15]Liu X,Zhu X,Chen M,et al.Resveratrol protects PC12cells against OGD/R-induced apoptosis via the mitochondrialmediated signaling pathway[J].Acta Biochim Biophys Sin(Shanghai),2016,48(4):342-353.
[16]Jaiswal N,Maurya CK,Arha D,et al.Fructose induces mitochondrial dysfunction and triggers apoptosis in skeletal muscle cells by provoking oxidative stress[J].Apoptosis,2015,20(7):930-947.
[17]Yuzefovych LV,Solodushko VA,Wilson GL,et al.Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress,mitochondrial dysfunction,apoptosis,and impaired insulin signaling in rat L6 skeletal muscle cells[J].Endocrinology,2012,153(1):92-100.
[18]Zhang H,Nan W,Wang S,et al.Epidermal Growth Factor Promotes Proliferation and Migration of Follicular Outer Root Sheath Cells via Wnt/β-Catenin Signaling[J].Cell Physiol Biochem,2016,39(1):360-370.
[19]Shen C,Sun L,Zhu N,et al.Kindlin-1 contributes to EGF-induced re-epithelialization in skin wound healing[J].Int J Mol Med,2017,39(4):949-959.
[20]Klingensmith NJ,Yoseph BP,Liang Z,et al.Epidermal growth factor improves intestinal integrity and survival in murine sepsis following chronic alcohol ingestion[J].Shock,2017,47(2):184-192.