白香丹胶囊及白芍提取物对过表达5-HTR2C诱导的海马神经元中PKC的影响
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摘要
目的:根据前期对愤怒模型大鼠基因芯片研究,构建大鼠5-HTR2C过表达海马神经元,研究白香丹胶囊及君药白芍提取物对5-HTR2C及PKCα/β2的影响,为阐明白香丹胶囊调整愤怒情绪的作用机制提供科学依据。方法:采用基因工程技术,构建5-HTR2C真核表达载体,应用电转染技术,使5-HTR2C在大鼠原代神经元中过量表达,建立5-HTR2C体外高表达的细胞模型,结合中药血清药理学,制备白香丹胶囊和白芍提取物的含药血清,以氟西汀为阳性对照药,干预转染后的神经元,提取各组蛋白后,Western-blot技术检测5-HTR2C及PKCα/β2中相应基因的蛋白表达。结果:与正常组相比,5-HTR2C转染组中5-HTR2C的蛋白表达显著升高(P <0. 001),PKCα的蛋白表达显著下降(P <0. 001),P-PKCα的蛋白表达显著升高(P <0. 001),PKCβ2的蛋白表达显著下降(P <0. 001),P-PKCβ2的蛋白表达显著升高(P <0. 001);与5-HTR2C过表达组相比,白香丹、白芍提取物及氟西汀干预组中5-HTR2C的蛋白表达均显著下调(P <0. 01,P <0. 01,P <0. 05),PKCα的蛋白表达均显著上调(P <0. 05,P <0. 01,P <0. 01),P-PKCα的蛋白表达显著下降(P <0. 001,P <0. 01,P <0. 05),PKCβ2的蛋白表达均显著上调(P <0. 001,P <0. 01,P <0. 05),PPKCβ2的蛋白表达显著下降(P <0. 001,P <0. 001,P <0. 01)。结论:白香丹胶囊能够调整5-HTR2C高表达引起的PKCα/β2及其磷酸化水平的异常变化,且作用比氟西汀稍强,充分显示了整体调节的优势。但是,作为其中主要组分的白芍提取物无显著的调节作用,推测白香丹胶囊其他组分-香附酮及丹皮酚在愤怒致病治疗中也有一定的作用,需进一步研究。
Objective:On the basis of the early research about gene Microarray of the rat model of anger,we studied the effect of Baixiangdan Capsule and Jun-drug radix paeoniae alba extract on 5-HTR2C and PKCα/β2,by constructing the 5-HTR2C overexpression hippocampus neurons. And to clarify the mechanism of the effect of Baixiangdan Capsule on adjusting anger emotion. Methods:First,we prepared the 5-HTR2C overexpression cell model by importing the 5-HTR2C eukaryotic expression vector into rat hippocampus neurons by using electric transfection technique. Second,we prepared the serum containing the Baixiangdan Capsules and white peony extract. After that,we intervened the transfected neurons by using the serum,and detected protein expression of 5-HTR2C and PKCα/β2 with fluoxetine as positive control. Results:Compared with the normal group,in 5-HTR2C transfected group protein expression of the 5-HTR2C significantly increased( P < 0. 001),PKCα decreased significantly(P < 0. 001),P-PKCα significantly increased(P < 0. 001),PKCβ2 decreased(P < 0. 001) and P-PKCβ2 significantly increased( P < 0. 001). Compared with the 5-HTR2C transfected group,in the Baixiangdan Capsule group,white peony root extract group and fluoxetine group,the 5-HTR2C protein expression was significantly lowered( P < 0. 01,P < 0. 001,P < 0. 05),PKCα significantly upregulated( P < 0. 05,P < 0. 01,P < 0. 01),P-PKCα significantly decreased( P < 0. 001,P < 0. 01,P <0. 05),PKCβ2 significantly raised( P < 0. 001,P < 0. 01),P < 0. 05) and P-PKCβ2 significantly with decreased( P < 0. 001,P < 0. 001,P < 0. 01). Conclusion:Baixiangdan Capsule can adjust the abnormal changes of PKCα/β2 and its phosphorylation level caused by 5-HTR2C high expression,and the effect is slightly stronger than fluoxetine,which shows the advantages of overall regulation. However,as one of the main components,the peony extract had no significant regulatory effect. It is presumably other components-incense of Baixiangdan Capsule,such as cyperone and paeonol,also has a certain role in anger pathogenic treatment. So the above research findings need further research.
Objective:On the basis of the early research about gene Microarray of the rat model of anger,we studied the effect of Baixiangdan Capsule and Jun-drug radix paeoniae alba extract on 5-HTR2C and PKCα/β2,by constructing the 5-HTR2C overexpression hippocampus neurons. And to clarify the mechanism of the effect of Baixiangdan Capsule on adjusting anger emotion. Methods:First,we prepared the 5-HTR2C overexpression cell model by importing the 5-HTR2C eukaryotic expression vector into rat hippocampus neurons by using electric transfection technique. Second,we prepared the serum containing the Baixiangdan Capsules and white peony extract. After that,we intervened the transfected neurons by using the serum,and detected protein expression of 5-HTR2C and PKCα/β2 with fluoxetine as positive control. Results:Compared with the normal group,in 5-HTR2C transfected group protein expression of the 5-HTR2C significantly increased( P < 0. 001),PKCα decreased significantly(P < 0. 001),P-PKCα significantly increased(P < 0. 001),PKCβ2 decreased(P < 0. 001) and P-PKCβ2 significantly increased( P < 0. 001). Compared with the 5-HTR2C transfected group,in the Baixiangdan Capsule group,white peony root extract group and fluoxetine group,the 5-HTR2C protein expression was significantly lowered( P < 0. 01,P < 0. 001,P < 0. 05),PKCα significantly upregulated( P < 0. 05,P < 0. 01,P < 0. 01),P-PKCα significantly decreased( P < 0. 001,P < 0. 01,P <0. 05),PKCβ2 significantly raised( P < 0. 001,P < 0. 01),P < 0. 05) and P-PKCβ2 significantly with decreased( P < 0. 001,P < 0. 001,P < 0. 01). Conclusion:Baixiangdan Capsule can adjust the abnormal changes of PKCα/β2 and its phosphorylation level caused by 5-HTR2C high expression,and the effect is slightly stronger than fluoxetine,which shows the advantages of overall regulation. However,as one of the main components,the peony extract had no significant regulatory effect. It is presumably other components-incense of Baixiangdan Capsule,such as cyperone and paeonol,also has a certain role in anger pathogenic treatment. So the above research findings need further research.
引文
[1]严灿,徐志伟.肝主疏泄调畅情志功能的中枢神经生物学机制探索[J].中国中西医结合杂志,2005,25(5):459-461.
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[3] HR Melowic,RV Stahelin,NR Blatner,et al. Mechanism of Diacylglyerol-induced membrane targeting and activation of protein kinase Ctheta[J]. J Biol Chem,2007,282(29):2146-2147.
[4]王杰琼,高冬梅,于艳红,等. HPLC法对白香丹胶囊中芍药苷在大鼠体内药时曲线的测定[J].世界科学技术:中医药现代化,2016,18(2):302-306.
[5]邵继红,韩珍,杨艳,等.白芍抗抑郁作用的实验研究[J].宁夏医学杂志,2008,30(6):490.
[6]王杰琼,高冬梅,于艳红,等. 5-HTR2C重组质粒电转染至大鼠海马神经元对细胞活性的影响[J].世界科学技术:中医药现代化,2015(4):794-799.
[7]詹向红,李伟,赵君玫,等.慢性愤怒应激对大鼠衰老进程及其神经内分泌免疫机制的影响[J].中国中医药杂志,2010,25(1):111-113.
[8] Di Matteo V,Marisa C,Di Giulio C,et al. Role of serotonin(2C)receptors in the control of brain dopamiaergle function[J]. Phamacology Biochemistry and Behavior,2002,71(4):727.
[9] Manji HK,Lenox RH,Ziskind. Protein kinase C signaling in the brain:molecular transduction of mood stabilization in the treatment of manic-depressive illness[J]. Biol Psychiatry,1999,46(10):1328-1351.
[10] Manji HK,Mc Namara R,Chen G. Signalling pathways in the brain:cellular transduction of mood stabilisation in the treatment of manic depressive illness[J]. Aust NZJ Psychiatry,1999,33:65-83.
[11] Hahn CG,Umapathy,Wang HY,et al. Lithium and valproic acid treatments reduce PKC act ivat ion and receptor-G protein coupling in platelets of bipolar manic patients[J]. J Psychiatr Res,2005,39(4):355-363.
[12]许晶,刘晶.行为缺损抑郁大鼠脑环磷酸腺苷含量及蛋白激酶C表达的变化[J].中华精神科杂志,2002,35(3):173-176.
[13] Kim SH,Kim MK,Yu HS,et al. Electroconvulsive seizure increases phosphor-ylation of PKC substrates,including GAP-43,MARCKS,and neurogranin,in rat brain[J]. Prog Neuro psychopharmacol Biol Psychiatry,2010,34(1):115-121.
[14]秦至臻,戚欣,李静.蛋白激酶C分子的成熟与活化[J].现代生物医学进展,2011,11(15):2992-2995.
[15] Fuortes MG,Faria LC,Merlin LR. Impact of protein kinase C activation on epileptiform activity in the hippocampal slice[J]. Epilepsy Res,2008,82(1):38-45.
[16]孙丽.经前期综合征肝气逆证大鼠模型血清及不同脑区孕酮及氨基酸检测分析[D].济南:山东中医药大学,2008.
[2]王杰琼,张惠云.愤怒、郁怒反应模型大鼠海马5-HTR2C的基因表达[J].中国药理学通报,2011,27(3):325-328.
[3] HR Melowic,RV Stahelin,NR Blatner,et al. Mechanism of Diacylglyerol-induced membrane targeting and activation of protein kinase Ctheta[J]. J Biol Chem,2007,282(29):2146-2147.
[4]王杰琼,高冬梅,于艳红,等. HPLC法对白香丹胶囊中芍药苷在大鼠体内药时曲线的测定[J].世界科学技术:中医药现代化,2016,18(2):302-306.
[5]邵继红,韩珍,杨艳,等.白芍抗抑郁作用的实验研究[J].宁夏医学杂志,2008,30(6):490.
[6]王杰琼,高冬梅,于艳红,等. 5-HTR2C重组质粒电转染至大鼠海马神经元对细胞活性的影响[J].世界科学技术:中医药现代化,2015(4):794-799.
[7]詹向红,李伟,赵君玫,等.慢性愤怒应激对大鼠衰老进程及其神经内分泌免疫机制的影响[J].中国中医药杂志,2010,25(1):111-113.
[8] Di Matteo V,Marisa C,Di Giulio C,et al. Role of serotonin(2C)receptors in the control of brain dopamiaergle function[J]. Phamacology Biochemistry and Behavior,2002,71(4):727.
[9] Manji HK,Lenox RH,Ziskind. Protein kinase C signaling in the brain:molecular transduction of mood stabilization in the treatment of manic-depressive illness[J]. Biol Psychiatry,1999,46(10):1328-1351.
[10] Manji HK,Mc Namara R,Chen G. Signalling pathways in the brain:cellular transduction of mood stabilisation in the treatment of manic depressive illness[J]. Aust NZJ Psychiatry,1999,33:65-83.
[11] Hahn CG,Umapathy,Wang HY,et al. Lithium and valproic acid treatments reduce PKC act ivat ion and receptor-G protein coupling in platelets of bipolar manic patients[J]. J Psychiatr Res,2005,39(4):355-363.
[12]许晶,刘晶.行为缺损抑郁大鼠脑环磷酸腺苷含量及蛋白激酶C表达的变化[J].中华精神科杂志,2002,35(3):173-176.
[13] Kim SH,Kim MK,Yu HS,et al. Electroconvulsive seizure increases phosphor-ylation of PKC substrates,including GAP-43,MARCKS,and neurogranin,in rat brain[J]. Prog Neuro psychopharmacol Biol Psychiatry,2010,34(1):115-121.
[14]秦至臻,戚欣,李静.蛋白激酶C分子的成熟与活化[J].现代生物医学进展,2011,11(15):2992-2995.
[15] Fuortes MG,Faria LC,Merlin LR. Impact of protein kinase C activation on epileptiform activity in the hippocampal slice[J]. Epilepsy Res,2008,82(1):38-45.
[16]孙丽.经前期综合征肝气逆证大鼠模型血清及不同脑区孕酮及氨基酸检测分析[D].济南:山东中医药大学,2008.