不同波形低强度电磁场对高糖培养的肾小球系膜细胞(HBZY-1细胞株)细胞外基质合成的影响

Effect of low-intensity electromagnetic fields on ECM metabolism of glomerular mesangial cell(HBZY-1) cultured in high glucose condition

目的:研究低强度电磁场对糖尿病肾病模型细胞外基质(ECM)代谢产物的表达以及参与调节ECM的主要信号通路是否有积极的影响,并找到3种波形中的最优参数。方法:实验采用1.6 m T和15 Hz的正弦、方波、脉冲群分别对高糖培养的HBZY-1细胞株进行干预,每天作用8 h,连续3 d。完成后采用MTT法检测细胞活性,ELISA法检测上清液中转化生长因子β1(TGFβ1)、纤维连接蛋白(FN)、基质金属蛋白酶(MMP-2)蛋白含量,Western blot法检测p38MAPK、ERK、Smad3总蛋白及磷酸化蛋白的表达。结果:(1)所有高糖组在1 d后都表现出较强的细胞活性,与正常组比较具有显著差异;与高糖对照组进行比较,方波和脉冲群组在干预2 d开始抑制HBZY-1细胞株的增殖,正弦组始终没有显著差异;脉冲群组细胞活性显著低于方波组。(2)所有高糖组的TGFβ1、FN蛋白表达显著高于正常组,MMP-2则相反;方波和脉冲群组TGFβ1、FN蛋白表达显著低于高糖对照组,而MMP-2蛋白的表达显著高于高糖对照组,正弦组在这些蛋白表达上与高糖对照组无显著差异;脉冲群组在TGFβ1蛋白表达显著低于方波组,而MMP-2蛋白表达则相反,FN蛋白表达则无显著差异。(3)所有高糖组的p-Smad3蛋白表达显著高于正常组;方波和脉冲群组p-Smad3蛋白表达显著低于高糖对照组,而正弦组无显著差异;与方波组相比,脉冲群组的抑制效果更显著。结论:方波和脉冲群能有效地抑制高糖诱导的HBZY-1细胞增殖,一方面通过抑制系膜细胞TGFβ1、FN的合成减少ECM的沉积,另一方面通过促进MMP-2的合成加速ECM降解。此外,方波和脉冲群还通过抑制TGFβ1/Smad3信号通路减少系膜细胞的损伤。脉冲群对系膜细胞的作用效果显著好于方波。

Objective To study on the effects of low-intensity electromagnetic fields on the expression of extracellular matrix（ECM） metabolites of diabetic nephropathy model and the main signal pathways participating in the regulation of ECM, and to find the optimal parameters of three waveforms. Methods The glomerular mesangial cell（HBZY- 1） cultured in high glucose condition was intervened with the sine wave, square wave, and pulsed electromagnetic fields（PEMFs） of 1.6 m T and 15 Hz, with the intervention of 8 h/d for 3 consecutive days. After the stimulation, methyl thiazolyl tetrazolium assay was used to detect cell activity; enzyme-linked immunosorbent assay was used to detect the protein content of transforming growth factor（TGF） beta 1, fibronectin（FN）, matrix metalloproteinase-2（MMP-2） in the supernatant; Western blot was use to detect the expression of p38 MAPK, ERK, Smad3 total protein and phosphorylated protein. Results After 1 d of intervention, the high glucose groups showed stronger cell activity than the normal group, with significant differences.Compared with the high glucose control groups, the square wave group and PEMFs group began to inhibit the proliferation of HBZY-1 from the second day of intervention, and the sine wave group did not showed any significant differences. The cell activity in PEMFs group was significantly lower than that in square wave group. The TGF beta 1 and FN protein expressions in high glucose groups were significantly higher than those in normal group, while the MMP-2 in high glucose groups was lower than that in normal group. The TGF beta 1 and FN protein expressions in square wave group and PEMFs group were significantly lower than those in high glucose control group, but the MMP-2 protein expression in square wave group and PEMFs group was significantly higher than that in high glucose control group. No significant differences were found in the protein expressions between sine wave group and high glucose control group. Compared with square wave group, TGF beta 1 protein expression in PEMFs group was significantly lower, but the MMP- 2 protein expression in PEMFs group was higher. No significant differences were found in FN protein expression between PEMFs group and square wave group. The phosphorylated Smad3（p- Smad3） protein expression in high glucose groups was significantly higher than that in normal group. The p- Smad3 protein expressions in square wave group and PEMFs group were significantly lower than those in high glucose control groups, having no significant differences with sine wave group.Compared with the square wave group, the inhibitory effect of PEMFs group was more significant. Conclusion Square wave and PEMFs can effectively inhibit the proliferation of high glucose induced HBZY-1, decreasing ECM deposition by inhibiting the synthesis of mesangial cell TGF beta 1 and FN, and accelerating the ECM degradation by promoting the MMP- 2 synthesis. Besides, the square wave and PEMFs can reduce the damage of mesangial cells by inhibiting the TGF beta1/Smad3 signal pathway. The effect of PEMFs on mesangial cells was significantly better than square wave.