AMPK在高糖诱导大鼠肾小球系膜细胞Ⅳ型胶原过度表达中的作用
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摘要
目的：
肾小球系膜基质增生是糖尿病肾病重要的病理表现之一。阻断和逆转上述病理变化是防治糖尿病肾病的重要策略。研究发现,高糖环境下的肾小球上皮细胞AMPK活性明显降低。AMPK是否参与了糖尿病状态下系膜基质增生的过程目前尚不清楚。本研究观察高糖环境下大鼠肾小球系膜细胞AMPK表达和活性的变化,以及这种变化与肾小球系膜基质增生的关系；并引入AMPK激活剂AICAR,观察其是否对高糖环境诱导的肾小球系膜基质增生具有纠正作用。
方法：
1、高糖环境的建立：分别采用DMEM低糖与高糖细胞培养基,糖浓度5.6mmol／L为低糖对照组、糖浓度为22.0mmol／L为高糖实验组。
2、实验分组：对照组(糖浓度5.6mmol／L),高糖组(糖浓度22.0mmol／L),低浓度AICAR组(高糖+0.5mmol／L AICAR),高浓度AICAR组(高糖+1.0mmol／L AICAR)。分组培养24小时后进行下一步实验。
3、AMPK表达及其活性的测定：采用RT-PCR方法测定AMPKα1亚单位mRNA水平；采用Western blot方法测定总AMPKα蛋白水平及磷酸化AMPKα蛋白水平。
4、Ⅳ型胶原蛋白表达的测定：采用Western blot方法测定Ⅳ型胶原蛋白α5亚单位水平。
结果：
1、高糖环境对细胞AMPK表达及活性的影响：RT-PCR显示,在高糖环境下培养系膜细胞24小时后,其AMPKα1亚单位的mRNA水平显著低于对照组(p<0.01)；利用Western blot方法测定的总AMPKα蛋白水平及磷酸化AMPKα蛋白水平也显著低于对照组(p<0.01)。
2、高糖环境对肾小球系膜细胞Ⅳ型胶原蛋白α5(COL4α5)表达水平的影响：高糖培养24小时后,肾小球系膜细胞COL4α5表达水平显著高于对照组(p<0.01)。
3、AICAR对高糖环境下肾小球系膜细胞AMPK表达及其活性的影响：与高糖组相比,AICAR组AMPKα1亚单位的mRNA水平显著升高(p<0.01),总AMPKα蛋白水平及磷酸化AMPKα蛋白表达水平也显著升高(p<0.01)。
4、AICAR对高糖环境中肾小球系膜细胞COL4α5表达的影响：与高糖组比较,AICAR组COL4α5表达水平显著下降(p<0.01)。
结论：
1、高糖环境下肾小球系膜细胞AMPK的表达及活性均受到抑制。
2、高糖环境下肾小球系膜细胞COL4α5的表达水平升高。
3、AMPK激活剂AICAR可以抑制高糖诱导的肾小球系膜细胞COL4α5过度表达,其作用可能与AICAR增强肾小球系膜细胞AMPK的表达及活性有关。
4、高糖诱导的系膜细胞COL4α5过度表达可能与AMPK的表达及活性降低有关。
Objective:
Hyperplasia of glomerular mesangial matrix is one of pathological changes of diabetic nephropathy (DN). One of important methods to prevent and cure DN is to block and reverse this pathological change. Recent studies indicated that activity of AMPK was significantly depressed in glomerular epithelial cells in high glucose. However, whether this mechanism is involved in hyperplasia of glomerular mesangial matrix in diabetes remains unclear. This study was designed to observe the expression and activity of AMPK in high glucose stimulated mesangial cells, and to explore the relationship between this change and hyperplasia of glomerular mesangial matrix. AMPK-activator, AICAR, was used to further explore the protective role of AMPK to glomerular mesangial cells in high glucose environment.
Methods:
1. Establishment of high glucose environment:The DMEM cell-culture mediums with low and high glucose were applied; glucose 5.6mmol/L was used for control group, glucose 22.0mmol/L for high glucose group.
2. The groups:Control group(glucose 5.6 mmol/L), high glucose group (glucose 22.0 mmol/L), low AICAR group (22.0mmol/L glucose+0.5 mmol/L AICAR), high AICAR group (22.0mmol/L glucose+1.0 mmol/L AICAR). GMCs were incubated in these four groups for 24 hours, and then correlative changes were detected.
3. Detection of the expression and activity of AMPK:AMPKα1 mRNA level was observed by RT-PCR and protein levels of AMPKa, p-AMPKa by Western blot.
4. Detection of the expression of COL4a5:The protein level of COL4a5 was observed by Western blot.
Results:
1. Effect of high glucose on the expression and activity of AMPK in mesangial cells:RT-PCR showed that, compared with control group, high glucose increased AMPKal mRNA synthesis of GMCs distinctly (P＜0.01); and western blot showed that, compared with control group, high glucose increased total AMPKa and p-AMPKa protein synthesis of GMCs obviously (P＜0.01).
2. Effect of high glucose on the expression of COL4a5:Compared with control group, high glucose enhanced COL4a5 protein level distinctly (P＜0.01).
3. Effect of AICAR on the expression and activity of AMPK in high glucose:Compared with high glucose group, AICAR group enhanced AMPKal mRNA level (P＜0.01) and protein levels of AMPKa (P<0.01), p-AMPKa (P<0.01).
4. Effect of AICAR on the expression of COL4a5 in high glucose: Compared with high glucose group, AICAR enhanced protein level of COL4a5 distinctly (P＜0.01).
Conclusions:
1. High glucose can suppress the expression and activity of AMPKa.
2. High glucose can enhance the expression of COL4a5.
3. AMPK activator, AICAR, can suppress high glucose induced over-expression of COL4a5, which may correlate with the enhanced expression and activity of AMPKa by AICAR.
4. High glucose induced over-expression of COL4a5 may be concerned with the impaired expression and activity of AMPKa in the environment.
肾小球系膜基质增生是糖尿病肾病重要的病理表现之一。阻断和逆转上述病理变化是防治糖尿病肾病的重要策略。研究发现,高糖环境下的肾小球上皮细胞AMPK活性明显降低。AMPK是否参与了糖尿病状态下系膜基质增生的过程目前尚不清楚。本研究观察高糖环境下大鼠肾小球系膜细胞AMPK表达和活性的变化,以及这种变化与肾小球系膜基质增生的关系；并引入AMPK激活剂AICAR,观察其是否对高糖环境诱导的肾小球系膜基质增生具有纠正作用。
方法：
1、高糖环境的建立：分别采用DMEM低糖与高糖细胞培养基,糖浓度5.6mmol／L为低糖对照组、糖浓度为22.0mmol／L为高糖实验组。
2、实验分组：对照组(糖浓度5.6mmol／L),高糖组(糖浓度22.0mmol／L),低浓度AICAR组(高糖+0.5mmol／L AICAR),高浓度AICAR组(高糖+1.0mmol／L AICAR)。分组培养24小时后进行下一步实验。
3、AMPK表达及其活性的测定：采用RT-PCR方法测定AMPKα1亚单位mRNA水平；采用Western blot方法测定总AMPKα蛋白水平及磷酸化AMPKα蛋白水平。
4、Ⅳ型胶原蛋白表达的测定：采用Western blot方法测定Ⅳ型胶原蛋白α5亚单位水平。
结果：
1、高糖环境对细胞AMPK表达及活性的影响：RT-PCR显示,在高糖环境下培养系膜细胞24小时后,其AMPKα1亚单位的mRNA水平显著低于对照组(p<0.01)；利用Western blot方法测定的总AMPKα蛋白水平及磷酸化AMPKα蛋白水平也显著低于对照组(p<0.01)。
2、高糖环境对肾小球系膜细胞Ⅳ型胶原蛋白α5(COL4α5)表达水平的影响：高糖培养24小时后,肾小球系膜细胞COL4α5表达水平显著高于对照组(p<0.01)。
3、AICAR对高糖环境下肾小球系膜细胞AMPK表达及其活性的影响：与高糖组相比,AICAR组AMPKα1亚单位的mRNA水平显著升高(p<0.01),总AMPKα蛋白水平及磷酸化AMPKα蛋白表达水平也显著升高(p<0.01)。
4、AICAR对高糖环境中肾小球系膜细胞COL4α5表达的影响：与高糖组比较,AICAR组COL4α5表达水平显著下降(p<0.01)。
结论：
1、高糖环境下肾小球系膜细胞AMPK的表达及活性均受到抑制。
2、高糖环境下肾小球系膜细胞COL4α5的表达水平升高。
3、AMPK激活剂AICAR可以抑制高糖诱导的肾小球系膜细胞COL4α5过度表达,其作用可能与AICAR增强肾小球系膜细胞AMPK的表达及活性有关。
4、高糖诱导的系膜细胞COL4α5过度表达可能与AMPK的表达及活性降低有关。
Objective:
Hyperplasia of glomerular mesangial matrix is one of pathological changes of diabetic nephropathy (DN). One of important methods to prevent and cure DN is to block and reverse this pathological change. Recent studies indicated that activity of AMPK was significantly depressed in glomerular epithelial cells in high glucose. However, whether this mechanism is involved in hyperplasia of glomerular mesangial matrix in diabetes remains unclear. This study was designed to observe the expression and activity of AMPK in high glucose stimulated mesangial cells, and to explore the relationship between this change and hyperplasia of glomerular mesangial matrix. AMPK-activator, AICAR, was used to further explore the protective role of AMPK to glomerular mesangial cells in high glucose environment.
Methods:
1. Establishment of high glucose environment:The DMEM cell-culture mediums with low and high glucose were applied; glucose 5.6mmol/L was used for control group, glucose 22.0mmol/L for high glucose group.
2. The groups:Control group(glucose 5.6 mmol/L), high glucose group (glucose 22.0 mmol/L), low AICAR group (22.0mmol/L glucose+0.5 mmol/L AICAR), high AICAR group (22.0mmol/L glucose+1.0 mmol/L AICAR). GMCs were incubated in these four groups for 24 hours, and then correlative changes were detected.
3. Detection of the expression and activity of AMPK:AMPKα1 mRNA level was observed by RT-PCR and protein levels of AMPKa, p-AMPKa by Western blot.
4. Detection of the expression of COL4a5:The protein level of COL4a5 was observed by Western blot.
Results:
1. Effect of high glucose on the expression and activity of AMPK in mesangial cells:RT-PCR showed that, compared with control group, high glucose increased AMPKal mRNA synthesis of GMCs distinctly (P＜0.01); and western blot showed that, compared with control group, high glucose increased total AMPKa and p-AMPKa protein synthesis of GMCs obviously (P＜0.01).
2. Effect of high glucose on the expression of COL4a5:Compared with control group, high glucose enhanced COL4a5 protein level distinctly (P＜0.01).
3. Effect of AICAR on the expression and activity of AMPK in high glucose:Compared with high glucose group, AICAR group enhanced AMPKal mRNA level (P＜0.01) and protein levels of AMPKa (P<0.01), p-AMPKa (P<0.01).
4. Effect of AICAR on the expression of COL4a5 in high glucose: Compared with high glucose group, AICAR enhanced protein level of COL4a5 distinctly (P＜0.01).
Conclusions:
1. High glucose can suppress the expression and activity of AMPKa.
2. High glucose can enhance the expression of COL4a5.
3. AMPK activator, AICAR, can suppress high glucose induced over-expression of COL4a5, which may correlate with the enhanced expression and activity of AMPKa by AICAR.
4. High glucose induced over-expression of COL4a5 may be concerned with the impaired expression and activity of AMPKa in the environment.
引文
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