Effect of exocarpium on TGF-β1/Smad signal pathway in diabetic cardiomyopathy rat myocardial cells
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摘要:
目的 观察化橘红对糖尿病心肌病（DCM）大鼠心肌细胞TGF-β1/Smad信号通路的干预作用。 方法 （1）构建DCM大鼠模型，将40只大鼠用链脉佐菌素处理后，15只血糖浓度<16.7 mmol/L的大鼠纳入空白对照组，剩余的25只血糖浓度>16.7 mmol/L的大鼠随机分为DCM组（n=11）与化橘红组（n=14）。化橘红组大鼠给予化橘红400 mg/(kg·d)灌胃，DCM组和对照组给予等量生理盐水灌胃。测量各组大鼠不同时间点血糖（1、8、16、22周）；测量心功能相关指标；光镜下观察心肌形态学改变，电镜下心肌纤维化定量分析；测定心肌胶原总量；免疫组化测定心肌胶原；Western印迹检测心肌细胞TGF-β1、Smad、MMP-9、MMP-2、TIMP-1等蛋白含量；RT-PCR检测TGF-β1、MMP-9、MMP-2、TIMP-1等蛋白的mRNA表达。（2）构建心肌成纤维细胞增殖模型，随机分为两组，化橘红预处理组为血清培养基加0.5 mmol/L化橘红，而对照组为等量血清培养基。检测两组一氧化氮变化、TGF-β1蛋白、c-fos蛋白、p27蛋白，iNOS蛋白、Cyclin D蛋白表达情况。 结果 化橘红组的大鼠各时间点血糖、心肌纤维化、心肌胶原总量及各型胶原较DCM组显著下降，心收缩、舒张功能明显改善（P<0.05），TGF-β1、Smad、MMP-9、MMP-2、TIMP-1蛋白的表达明显下调（P<0.05），相关蛋白的mRNA表达也明显抑制（P<0.05）。化橘红预处理组一氧化氮、TGF-β1蛋白、c-fos蛋白、p27蛋白，iNOS蛋白、Cyclin D蛋白表达情况较对照组相比明显下降（P<0.05）。 结论 化橘红对DCM中TGF-β1/Smad通路起到了抑制作用，从而调控其下游的MMPs/TIMPs等通路，改善DCM的心肌纤维化进程，抑制心肌肥厚，减少心肌的损伤。 Abstract:Objective To observe the effect of exocarpium on TGF-β1/Smad signal pathway in diabetic cardiomyopathy (DCM) rat myocardial cells. Methods 1) To construct the model of DCM rats, 40 rats were treated with streptozotocin, 15 rats with blood glucose <16.7 mmol/L were in blank control group. The remaining 25 rats with blood glucose >16.7 mmol/L were randomly divided into diabetic cardiomyopathy group (DCM group, n=11) and exocarpium group (n=14). Rats of exocarpium group were given exocarpium [400 mg/(kg·d)], DCM group and control group were given normal saline. Different time of blood glucose (1, 8, 16, 22 weeks) were measured.Heart function index measurement, myocardial morphological changes were observed under light microscope.Quantitative analysis of myocardial fibrosis was performed by electron microscopy. Myocardial collagen amount, myocardial collagen content was determined by immunohistochemistry. Western blot detection of myocardial protein of TGF-β1, Smad, MMP-9, MMP-2, TIMP-1 and RT-PCR detection of mRNA of TGF-β1, MMP-9, MMP-2, TIMP-1.2) To construct the myocardial fibroblast proliferation model.All were randomly divided into two groups, there is serum medium with 0.5 mmol L-1 exocarpium in exocarpium pretreatment group, while the control group with serum medium. To detect two groups of NO changes, TGF-β1 protein, c-fos protein, p27 protein, iNOS protein, the expression of Cyclin D protein. Results Compared with DCM group, each time point of blood glucose, myocardial fibrosis, myocardial collagen and the total collagen were significantly decreased and systolic, diastolic function improved significantly In exocarpium groups (P<0.05). TGF-β1, Smad, MMP-9, MMP-2, TIMP-1 protein expression were significantly reduced in exocarpium groups (P<0.05). Protein mRNA expression was significantly inhibited in exocarpium groups (P<0.05). Compared with control group, NO, TGF-β1 protein, c-fos protein, p27 protein, iNOS protein, Cyclin D protein decreased significantly in pretreatment group (P<0.05). Conclusion Exocarpium on TGF-β1/Smad pathway in diabetic cardiomyopathy performs an effect of inhibitory. It regulates downstream MMPs/TIMPs pathway, improve myocardial fibrosis, inhibit myocardial hypertrophy and reduce myocardial injury. -
Key words:
- exocarpium /
- diabetic cardiomyopathy /
- TGF- β1 /
- smad
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表 1 化橘红干预后对血糖和心功能的影响（
$\, \overline{{x}}{{±}}{{s}}$ ）组别 n 血糖（mmol/L） 左室收缩压（mmHg） 左室舒张末压（mmHg） 左室腔内压力变化率最大值（±dp/dt max） 1周 8周 16周 22周 化橘红组 14 5.53±0.59 6.61±1.07 8.83±1.24 10.75±2.16 13.82±3.18 6.28±2.04 4.29±1.39 DCM组 11 8.72±1.37 10.28±2.36 14.47±4.19 19.28±6.03 9.52±2.04 9.19±3.23 2.37±0.68 t –1.532 –2.416 –3.313 –3.912 2.621 –2.128 1.372 P 0.041 0.023 0.019 0.008 0.017 0.032 0.048 
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表 2 3组大鼠心肌各型胶原含量与心肌纤维化情况（
$\, \overline{{x}}{{±}}{{s}}$ ）组别 n Ⅰ型胶原含量(*103) Ⅰ/Ⅲ型胶原比值 心肌羟脯氨酸含量（μg/mg） 心肌胶原总含量（μg/mg） 心肌间质纤维化（%） 化橘红组 14 104.52±43.23* 1.14±0.22* 0.57±0.24* 4.38±0.79* 19.37±9.16* DCM组 11 126.27±53.71# 1.52±0.31# 0.87±0.39# 6.93±1.75# 37.26±16.03# NC组 15 83.63±32.16 0.84±0.22 0.36±0.17 3.52±0.25 2.21±0.35 *P<0.05vs DCM组; #P<0.05vs NC组.
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表 3 化橘红干预对信号通路的影响（
$\, \overline{{x}}{{±}}{{s}}$ ，%）组别 n TGF-β1 mRNA TGF-β1 MMP-9 MMP-2 TIMP-1 Smad2 Smad7 c-fos c-fos mRNA 化橘红组 14 46.38±22.71 34.29±10.37 25.38±8.16 25.94±15.48 23.73±7.04 26.34±9.19 21.47±7.12 25.78±11.48 34.79±16.38 DCM组 11 71.23±39.19 63.12±21.47 49.29±17.37 53.28±31.47 45.81±15.10 51.28±19.16 43.19±18.38 44.39±20.19 53.28±23.29 t –8.192 –6.729 –5.381 3.192 –5.102 –5.729 –4.982 –5.720 –6.182 P 0.000 0.002 0.008 0.031 0.013 0.011 0.019 0.015 0.006
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表 4 化橘红对CFb增殖的影响（
$\, \overline{{x}}{{±}}{{s}}$ ，%）组别 n TGF-β1 iNOS 一氧化氮 p27 Cyclin D 对照组 14 31.28±16.26 21.84±11.74 12.28±6.81 3.14±1.05 24.12±9.64 化橘红预处理组 11 49.19±23.83 30.17±15.12 20.16±8.18 5.92±2.13 37.29±16.12 t –5.917 –3.117 –2.921 –1.883 –4.039 P 0.008 0.021 0.035 0.042 0.009
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