摘要
目的探讨核心蛋白聚糖(DCN)对高糖诱导的心肌细胞H9c2转化生长因子β(TGF-β)/p38丝裂原活化蛋白激酶(p38MAPK)/环磷酸腺苷反应元件结合蛋白(CREB)通路及纤维化的影响。方法将心肌细胞H9c2随机分为5组:正常对照组(使用含5.6mmol/L葡萄糖的无血清培养液培养)、高糖组(使用含33mmol/L葡萄糖的无血清培养液培养)、DCN低浓度组(使用含33mmol/L葡萄糖和0.25μg/mlDCN的无血清培养液培养)、DCN中浓度组(使用含33mmol/L葡萄糖和1.25μg/mlDCN的无血清培养液培养)、DCN高浓度组(用含33mmol/L葡萄糖和2.5μg/mlDCN的无血清培养液培养),MTT法检测心肌细胞H9c2存活率;平板克隆实验检测各组H9c2细胞克隆形成数;流式细胞术检测H9c2细胞周期;划痕实验检测各组H9c2细胞迁移能力;Westernblot法检测H9c2细胞结缔组织生长因子(CTGF)、基质金属蛋白酶9(MMP-9)、纤维连接蛋白(FN)、TGF-β、p38MAPK表达水平以及CREB磷酸化水平。结果高糖组细胞存活率、克隆形成数、S期、G_(2)/M期、MMP-9表达水平显著低于正常对照组,G_(0)/G_(1)期、划痕愈合率、CTGF、FN、TGF-β、p38MAPK及磷酸化CREB表达水平显著高于正常对照组(P<0.05);与高糖组比较,DCN低、中、高浓度组细胞存活率[(90.77±1.13)%、(95.35±2.05)%、(98.70±2.30)%vs(73.23±1.09)%]、克隆形成数[(75.39±8.24)个、(92.28±9.94)个、(112.22±12.42)个vs(54.57±6.19)个]依次上升(P<0.05);高糖组G_(0)/G_(1)期、划痕愈合率、CTGF、FN、TGF-β、p38MAPK及磷酸化CREB水平显著高于DCN低、中、高浓度组,S期、G_(2)/M期、MMP-9表达水平显著低于DCN低、中、高浓度组(P<0.05)。结论DCN能够促进高糖诱导下H9c2细胞增殖,抑制迁移和心肌纤维化,可能与通过阻滞TGF-β/p38MAPK/CREB通路有关。
Objective To investigate the effects of decorin(DCN)on TGF-β/p38MAPK/cAMP/CREB signaling pathway and fibrosis in H9c2 cardiomyocytes induced by high glucose.Methods H9c2 cells were randomly divided into normal control group(cultured with serum-free medium containing 5.6 mmol/L glucose),high glucose group(cultured with serum-free medium containing 33 mmol/L glucose),low concentration group(cultured with serum-free medium containing 33 mmol/L glucose and 0.25μg/ml DCN),medium concentration group(cultured with serum-free medium containing 33 mmol/L glucose and 1.25μg/ml DCN),high concentration group(cultured with serum-free medium containing 33 mmol/L glucose and 2.5μg/ml DCN).MTT assay was used to measure the survival rate of the cells.Plate cloning assay,flow cytometry and scratch-wound assay were employed to observe the number of cell colonies,cell cycle and migration ability in the H9c2 cells.Then,Western blotting was adopted to detect the expression levels of CTGF,MMP-9,FN,TGF-β,p38MAPK and p-CREB in each group of cells.Results The cell survival rate,number of colony formation,cells arrested at S phase and G_(2)/M phase and expression level of MMP-9 were significantly lower,while cells arrested at G_(0)/G_(1)phase,scratch healing rate,and expression levels of CTGF,FN,TGF-β,p38MAPK and p-CREB were obviously higher in the high glucose group than the normal control group(P<0.05).Compared to the high glucose group,the cell survival rate[(90.77±1.13)%,(95.35±2.05)%and(98.70±2.30)%vs(73.23±1.09)%]and number of colonies(75.39±8.24,92.28±9.94 and 112.22±12.42 vs 54.57±6.19)were increased gradually in in the low-,medium-and high-dose DCN groups(P<0.05).The 3 doses of treatment also resulted in lower percentage of cells at G_(0)/G_(1)stage,scratch healing rate,and expression levels of CTGF,FN,TGF-β,p38MAPK and p-CREB,and higher percentages of cells at S stage and G_(2)/M stage and MMP-9 level when compared with the high glucose group(P<0.05).Conclusion DCN can promote the proliferation of H9c2 cells induce
作者
赵茂宇
付静
熊秋璨
钟灵
Zhao Maoyu;Fu Jing;Xiong Qiucan;Zhong Ling(Department of Cardiology,Second Affiliated Hospital of Army Medical University,Chongqing 400037,China)
出处
《中华老年心脑血管病杂志》
CAS
北大核心
2023年第7期751-755,共5页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
