基于水协同转运蛋白KCC1、KCC3、KCC4、NKCC1含量及Na~+ -K~+ -ATP酶活性变化研究湿阻中焦证的水液代谢机制
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摘要
目的:以前期研究的水通道蛋白为平台,通过观察并分析水协同转运蛋白KCC1、KCC3、KCC4、NKCC1含量及Na~+-K~+-ATP酶活性变化,揭示湿阻中焦致水液代谢紊乱及平胃散对其修复作用机制的科学内涵,为中医临床治疗本证型疾病提供实验参考;同时搭建中药复方水通道调节剂的研究平台。方法:模拟"外湿过盛,困阻脾胃、饮食不节,湿从内生、情志不遂,气机受阻、水湿不运三大致湿病因,建立湿阻中焦证模型。用蛋白定量法测定Na~+-K~+-ATP酶活性,用酶联免疫法(ELISA)测定肾脏组织KCC1,KCC3,KCC4,NKCC1的含量。结果:与空白组比较,模型组肾脏KCC1、KCC4含量升高,KCC3、NKCC1、Na~+-K~+-ATP酶活力降低,与模型组比较,自然恢复组肾脏KCC1、KCC3、NKCC1含量降低,Na~+-K~+-ATP酶活力明显升高(P<0.01),KCC4含量有上升趋势,经药物干预后,各给药组KCC1含量均明显降低(P<0.01),平胃散中剂量组KCC3、KCC4含量明显升高(P<0.01),平胃散低剂量组NKCC1含量明显降低(P<0.01);平胃散低剂量组肾脏Na~+-K~+-ATP酶活力明显升高(P<0.01);呋塞米组和平胃散加泽泻组KCC1、KCC3、KCC4、NKCC1含量及Na~+-K~+-ATP酶活性均明显降低,差异有统计学意义(P<0.01);与自然恢复组比较,平胃散中剂量组肾脏KCC1、KCC3、KCC4含量及Na~+-K~+-ATP酶活力有上升趋势,平胃散低剂量组NKCC1含量下降明显(P<0.01),呋塞米组和平胃散加泽泻组KCC1、KCC3、KCC4、NKCC1含量及Na~+-K~+-ATP酶活性均明显降低,差异有统计学意义(P<0.01)。结论:(1)湿阻中焦证模型不仅能够影响体内能量代谢,还能够影响KCC1、KCC3、KCC4、NKCC1含量表达,这可能是湿阻中焦导致水代谢输布障碍的机制之一;(2)平胃散能够调整Na~+-K~+-ATP酶活性和KCC1、KCC3、KCC4、NKCC1含量分布参与能量代谢和水液代谢调控,这可能是平胃散燥湿运脾、行气导滞、散中焦之湿阻治疗湿阻中焦证的分子机理之一;(3)水协同转运蛋白之间可能存在互补代偿机制。
Objective: This experiment,in previous studies of water channel protein as a platform,through observation and analysis of water cotransporter KCC1,KCC3,KCC4 and NKCC1 contents and Na~+-K~+-ATPase activity changes,revealed wet block middle energizer induced water metabolism and Pingwei Power on the repair mechanism of the scientific connotation,for TCM clinical treatment of the syndromes of disease and provide experimental reference. At the same time,we build a research platform of traditional Chinese medicine compound water channel modulators. Methods: We simulated three causes of damp,including excessive internal dampness obstructing spleen and stomach,itnernal dmapness due to improper diet,emotional disorder causing Qi stagnation,water and dampness failing to transport and then established the model of accumulation of dampness in middle Jiao syndrome. Na~+-K~+-ATPase activity was determined with quantitative method for protein and enzyme linked immunosorbent assay( ELISA) determined kidney tissue KCC1,KCC3,KCC4 and NKCC1 contents. Results: Compared with the blank group,model group's KCC1 and KCC4 contents in kidney increased and KCC3,NKCC1 and Na~+-K~+-ATP enzyme activity decreased. Compared with the model group,natural recovery group's KCC1,KCC3 and NKCC1 contents decreased,Na~+-K~+-ATPase increased significantly( P < 0. 01) and KCC4 had a increased trend. After intervention,the contents of each treatment groups' KCC1 were significantly lower( P < 0. 01). Pingwei Powder middle dose group's KCC3 and KCC4 were significantly increased( P< 0. 01) and low dose group's NKCC1 decreased significantly( P < 0. 01). Pingwei Powder low dose group's Na~+-K~+-ATPase activity was significantly increased( P < 0. 01). The furosemide group and Pingwei Powder plus Alisma group's KCC1,KCC3 and KCC4 contents and Na~+-K~+-ATPase activity were significantly decreased and the difference was statistically significant( P <0. 01). Compared with spontaneous recovery group,Pingwei Powder middle dose group' s KCC1,KCC3 and KCC4 contents and Na~+-K~+-ATPase activity had a increased trend. Pingwei Powder low dose group's NKCC1 content decreased significantly( P< 0. 01) and furosemide group and Pingwei Powder plus Alisma group' s KCC1,KCC3,KCC4 and NKCC1 contents and Na~+-K~+-ATPase activity were significantly lower and the difference was statistically significant( P < 0. 01). Conclusion:( 1) Accumulation of dampness in middle Jiao syndrome model can not only affect the energy metabolism in vivo,but also can affect KCC1,KCC3,KCC4 and NKCC1 contents,which may be one of the mechanisms of water metabolism disorder of distribution.( 2) Pingwei Powder can adjust Na~+-K~+-ATPase activity and KCC1,KCC3,KCC4 and NKCC1 contents distribution,involve in energy metabolism and water fluid metabolism,which may be the mechansim of Pingwei Powder can dry dampness and activate spleen,improve Qi movement and relieve stagnation,disperse dampness in the middle.( 3) Water cotransporter may complement compensatory mechanisms.
Objective: This experiment,in previous studies of water channel protein as a platform,through observation and analysis of water cotransporter KCC1,KCC3,KCC4 and NKCC1 contents and Na~+-K~+-ATPase activity changes,revealed wet block middle energizer induced water metabolism and Pingwei Power on the repair mechanism of the scientific connotation,for TCM clinical treatment of the syndromes of disease and provide experimental reference. At the same time,we build a research platform of traditional Chinese medicine compound water channel modulators. Methods: We simulated three causes of damp,including excessive internal dampness obstructing spleen and stomach,itnernal dmapness due to improper diet,emotional disorder causing Qi stagnation,water and dampness failing to transport and then established the model of accumulation of dampness in middle Jiao syndrome. Na~+-K~+-ATPase activity was determined with quantitative method for protein and enzyme linked immunosorbent assay( ELISA) determined kidney tissue KCC1,KCC3,KCC4 and NKCC1 contents. Results: Compared with the blank group,model group's KCC1 and KCC4 contents in kidney increased and KCC3,NKCC1 and Na~+-K~+-ATP enzyme activity decreased. Compared with the model group,natural recovery group's KCC1,KCC3 and NKCC1 contents decreased,Na~+-K~+-ATPase increased significantly( P < 0. 01) and KCC4 had a increased trend. After intervention,the contents of each treatment groups' KCC1 were significantly lower( P < 0. 01). Pingwei Powder middle dose group's KCC3 and KCC4 were significantly increased( P< 0. 01) and low dose group's NKCC1 decreased significantly( P < 0. 01). Pingwei Powder low dose group's Na~+-K~+-ATPase activity was significantly increased( P < 0. 01). The furosemide group and Pingwei Powder plus Alisma group's KCC1,KCC3 and KCC4 contents and Na~+-K~+-ATPase activity were significantly decreased and the difference was statistically significant( P <0. 01). Compared with spontaneous recovery group,Pingwei Powder middle dose group' s KCC1,KCC3 and KCC4 contents and Na~+-K~+-ATPase activity had a increased trend. Pingwei Powder low dose group's NKCC1 content decreased significantly( P< 0. 01) and furosemide group and Pingwei Powder plus Alisma group' s KCC1,KCC3,KCC4 and NKCC1 contents and Na~+-K~+-ATPase activity were significantly lower and the difference was statistically significant( P < 0. 01). Conclusion:( 1) Accumulation of dampness in middle Jiao syndrome model can not only affect the energy metabolism in vivo,but also can affect KCC1,KCC3,KCC4 and NKCC1 contents,which may be one of the mechanisms of water metabolism disorder of distribution.( 2) Pingwei Powder can adjust Na~+-K~+-ATPase activity and KCC1,KCC3,KCC4 and NKCC1 contents distribution,involve in energy metabolism and water fluid metabolism,which may be the mechansim of Pingwei Powder can dry dampness and activate spleen,improve Qi movement and relieve stagnation,disperse dampness in the middle.( 3) Water cotransporter may complement compensatory mechanisms.
引文
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[10]Pearson MM,Lu J,Mount DB,et al.Localization of the K+-cl contranspoters,KCC3 in the central and peripheral nervous systems[J].Neuroscience,2013,103:481-491.
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[2]彭晋,王良,黄秀深,等.湿阻中焦证模型胃肠水通道蛋白1的病理特征性表达分布谱以及平胃散干预的研究[J].辽宁中医杂志,2011,38(12):2469-2472,2514.
[3]王良,邓利平,黄秀深.湿阻中焦证模型胃肠水通道蛋白9(AQP9)的病理特征性表达分布谱以及平胃散干预的研究[J].时珍国医国药,2012,23(3):543-545.
[4]张晓丹,黄秀深,杨成,等.湿阻中焦证模型大鼠胃肠水通道蛋白3(AQP3)的病理特征性表达分布谱以及平胃散干预的研究[J].辽宁中医杂志,2012,39(12):2500-2503.
[5]陈继兰.平胃散对湿阻中焦证大鼠水糖协同转运蛋白影响的研究[D].成都:成都中医药大学,2014.
[6]杨成.平胃散调控湿阻中焦证模型致胃肠水通道蛋白异常表达及细胞信号转导的影响[D].成都:成都中医药大学,2012.
[7]徐叔云,卞如濂,陈修.药理实验方法学[J].中国药理学通报,1992(1):19.
[8]李连成,路志正.湿阻的流行病学调查[J].中医杂志,1992,33(6):44-45.
[9]王克穷,张立华,刘伟.湿阻脾胃证的流行病学调查及相关因素的初步研究[J].中国中医基础医学杂志,1996,2(2):41-44.
[10]Pearson MM,Lu J,Mount DB,et al.Localization of the K+-cl contranspoters,KCC3 in the central and peripheral nervous systems[J].Neuroscience,2013,103:481-491.
[11]Macalay N,Zeuthen.Twanter transport between CNS compartments:controbitions aqaporions and contransporters[J].J Neurosci,2009,530:367-378.