筋脉通含药血清对高糖培养大鼠背根神经节神经元氧化应激及UCP3、IGF-1表达的影响
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摘要
目的研究中药筋脉通含药血清对高糖培养大鼠背根神经节神经元(DRGn)氧化应激以及解耦联蛋白3(UCP3)、胰岛素样生长因子(IGF-1)表达的影响。方法雄性Sprague Dawley(SD)大鼠随机分为正常对照组、筋脉通组及牛磺酸组,连续灌胃筋脉通、牛磺酸或蒸馏水3 d以制备筋脉通含药血清、牛磺酸含药血清和正常对照血清。用于制备细胞悬液的背根神经节取自孕15 d的SD胎鼠。CCK-8比色法确定指标检测时间点。体外培养的DRGn分为高糖组(HG组)、筋脉通组(JMT组)、牛磺酸组(Tau组)及正常对照组(CON组),分别加入相应血清培养24 h后,采用原位末端标记法检测DRGn细胞凋亡,荧光探针法检测DRGn中活性氧(ROS)水平,免疫荧光法+免疫印迹法检测UCP3和IGF-1的蛋白表达,实时荧光定量PCR法检测UCP3和IGF-1的mRNA。结果与CON组相比,HG组DRGn细胞活性显著降低,细胞凋亡率显著增加,ROS水平显著升高(P<0.01),UCP3和IGF-1蛋白及mRNA表达水平显著下降(P<0.01);与HG组比较,JMT组和Tau组的细胞凋亡率、ROS水平显著降低,UCP3和IGF-1蛋白及mRNA表达水平显著升高(P<0.05,P<0.01)。结论筋脉通可上调高糖培养DRGn中UCP3和IGF-1的表达,减轻高糖状态下氧化应激对神经细胞的损伤。
Objective To study the impacts of jinmaitong medicated serum on the oxidative stress and the expressions of UCP3 and IGF-1 in the dorsal root ganglion neurons( DRGn) of the rats cultured with high glucose medium. Methods The male Sprague Dawley( SD) rats were randomized into normal control group,Jinmaitong group and taurine group. The lavage with Jinmaitong,taurine or distilled water was given for3 days to prepare Jinmaitong medicated serum,taurine medicated serum and normal control serum separately.The cell suspension was prepared from DRGn in the SD rat embryo in 15-day gestation. CCK-8 assay was used to determine the time point of indicator detection. The DRGn cultured in vitro was divided into high glucose group( HG group),Jinmaitong group( JMT group),taurine group( Tau group) and normal control group( CON group). The corresponding serums were given accordingly. In 24 h culture,the in situ end labeling method was used to detect DRGn cell apoptosis,the fluorescent probe method to detect the reactive oxygen species( ROS) level in DRGn,the immunofluorescence method and western blot method to detect the protein expressions of UCP3 and IGF-1,and the real-time fluorescent quantitative RCR method to detect mRNA of UCP3 and IGF-1. Results Compared with the CON group,the DRGn cell activity was reduced significantly,the apoptosis rate was increased significantly( P < 0. 01); the protein and mRNA expressions of UCP3 and IGF-1 were reduced significantly( P < 0. 01) in the HG group. Compared with the HG group,the apoptosis rate and ROS level were reduced significantly and the protein and mRNA expressions of UCP3 and IGF-1were increased significantly in the JMT group and the Tau group( P < 0. 05,P < 0. 01). Conclusion Jinmaitong can up-regulates UCP3 and IGF-1 expressions in DRGn cultured with high glucose medium and alleviates the injury of the neural cell caused by the high glucose induced oxidative stress.
Objective To study the impacts of jinmaitong medicated serum on the oxidative stress and the expressions of UCP3 and IGF-1 in the dorsal root ganglion neurons( DRGn) of the rats cultured with high glucose medium. Methods The male Sprague Dawley( SD) rats were randomized into normal control group,Jinmaitong group and taurine group. The lavage with Jinmaitong,taurine or distilled water was given for3 days to prepare Jinmaitong medicated serum,taurine medicated serum and normal control serum separately.The cell suspension was prepared from DRGn in the SD rat embryo in 15-day gestation. CCK-8 assay was used to determine the time point of indicator detection. The DRGn cultured in vitro was divided into high glucose group( HG group),Jinmaitong group( JMT group),taurine group( Tau group) and normal control group( CON group). The corresponding serums were given accordingly. In 24 h culture,the in situ end labeling method was used to detect DRGn cell apoptosis,the fluorescent probe method to detect the reactive oxygen species( ROS) level in DRGn,the immunofluorescence method and western blot method to detect the protein expressions of UCP3 and IGF-1,and the real-time fluorescent quantitative RCR method to detect mRNA of UCP3 and IGF-1. Results Compared with the CON group,the DRGn cell activity was reduced significantly,the apoptosis rate was increased significantly( P < 0. 01); the protein and mRNA expressions of UCP3 and IGF-1 were reduced significantly( P < 0. 01) in the HG group. Compared with the HG group,the apoptosis rate and ROS level were reduced significantly and the protein and mRNA expressions of UCP3 and IGF-1were increased significantly in the JMT group and the Tau group( P < 0. 05,P < 0. 01). Conclusion Jinmaitong can up-regulates UCP3 and IGF-1 expressions in DRGn cultured with high glucose medium and alleviates the injury of the neural cell caused by the high glucose induced oxidative stress.
引文
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[13]Vidal-Puig AJ,Grujic D,Zhang CY,et al.Energy metabolism in uncoupling protein 3 gene knockout mice[J].J Biol Chem,2000,275(21):16258-16266.
[14]梁晓春.糖尿病周围神经病变与消渴兼证“筋痹”及其中医治疗[J].中国临床医生,2006,34(5):17-18.
[15]中国中西医结合学会内分泌专业委员会.糖尿病中西医结合诊疗规范(2010)[M].北京:军事医学科学出版社,2010:1-6.
[16]梁晓春,崔丽英,郭赛珊,等.筋脉通治疗糖尿病周围神经病变的临床观察[J].中国中西医结合杂志,1999,19(9):517-519.
[17]孙青,梁晓春,张宏,等.筋脉通含药血清对高糖培养背根神经节神经元氧化应激及细胞凋亡的影响[J].中成药,2013,35(7):1390-1395.
[18]刘伟,梁晓春,孙青,等.筋脉通对糖尿病大鼠背根神经节氧化应激及细胞凋亡的影响[J].中国医学科学院学报,2013,35(6):649-654.
[19]Liu D,Liang X,Zhang H.Effects of High Glucose on Cell Viability and Differentiation in Primary Cultured Schwann Cells:Potential Role of ERK Signaling Pathway[J].Neurochem Res,2016,41(6):1281-1290.
[2]Brownlee M.The pathobiology of diabetic complications:a unifying mechanism[J].Diabetes,2005,54(6):1615-1625.
[3]EdwardsJL,VincentAM,ChengHT,etal.Diabetic neuropathy:mechanisms to management[J].Pharmacol Ther,2008,120(1):1-34.
[4]Leinninger GM,Edward JL,Lipshaw MJ,et al.Mechanisms of disease:mitochondria as new therapeutic targets in diabetic neuropathy[J].Nat Clin Pract Neurol,2006,2(11):620-628.
[5]Migdalis IN,Kalogeropoulou K,Kalantzis L,et al.Insulin-like growth factor-I and IGF-I receptors in diabetic patients with neuropathy[J].Diabet Med,1995,12(9):823-827.
[6]Ju DT,Liao HE,Shibu MA,et al.Nerve Regeneration Potential of Protocatechuic Acid in RSC96 Schwann Cells by Induction of Cellular Proliferation and Migration through IGF-IR-PI3K-Akt Signaling[J].Chin J Physiol,2015,58(6):412-419.
[7]Chang YM,Kuo WH,Lai TY,et al.RSC96 Schwann Cell Proliferation and Survival Induced by Dilong through PI3K/Akt Signaling Mediated by IGF-I[J].Evid Based Complement Alternat Med,2011,2011:216148.
[8]Kurmasheva RT,Houghton PJ.IGF-I mediated survival pathways in normal and malignant cells[J].Biochim Biophys Acta,2006,1766(1):1-22.
[9]Schrauwen P,Mensink M,Schaart G,et al.Reduced skeletal muscle uncoupling protein-3 content in prediabetic subjects and type 2 diabetic patients:restoration by rosiglitazone treatment[J].J Clin Endocrinol Metab,2006,91(4):1520-1525.
[10]Vincent AM,Olzmann JA,Brownlee M,et al.Uncoupling proteins prevent glucose-induced neuronal oxidative stress and programmed cell death[J].Diabetes,2004,53(3):726-734.
[11]Skulachev VP.Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants[J].Q Rev Biophys,1996,29(2):169-202.
[12]Gong DW,Monemdjou S,Gavrilova O,et al.Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3[J].J Biol Chem,2000,275(21):16251-16257.
[13]Vidal-Puig AJ,Grujic D,Zhang CY,et al.Energy metabolism in uncoupling protein 3 gene knockout mice[J].J Biol Chem,2000,275(21):16258-16266.
[14]梁晓春.糖尿病周围神经病变与消渴兼证“筋痹”及其中医治疗[J].中国临床医生,2006,34(5):17-18.
[15]中国中西医结合学会内分泌专业委员会.糖尿病中西医结合诊疗规范(2010)[M].北京:军事医学科学出版社,2010:1-6.
[16]梁晓春,崔丽英,郭赛珊,等.筋脉通治疗糖尿病周围神经病变的临床观察[J].中国中西医结合杂志,1999,19(9):517-519.
[17]孙青,梁晓春,张宏,等.筋脉通含药血清对高糖培养背根神经节神经元氧化应激及细胞凋亡的影响[J].中成药,2013,35(7):1390-1395.
[18]刘伟,梁晓春,孙青,等.筋脉通对糖尿病大鼠背根神经节氧化应激及细胞凋亡的影响[J].中国医学科学院学报,2013,35(6):649-654.
[19]Liu D,Liang X,Zhang H.Effects of High Glucose on Cell Viability and Differentiation in Primary Cultured Schwann Cells:Potential Role of ERK Signaling Pathway[J].Neurochem Res,2016,41(6):1281-1290.