cAMP 对高血压鼠血管平滑肌细胞增殖的影响

Effect of cAMP on proliferation of vascular smooth muscle cells in hypertensive rats

摘要:

目的 探讨3'，5'- 环化腺苷一磷酸（cAMP）对高血压鼠血管平滑肌细胞增殖的影响，并进一步证 明分子机制。方法 复制高血压鼠模型，提取血管平滑肌细胞。噻唑蓝比色（MTT）法检测db-cAMP 对正常 鼠和高血压鼠在对照组、db-cAMP 组和腺苷酸环化酶激活剂Forskolin（FSK）组血管平滑肌细胞活性的影响。 Western blot 法检测不同浓度的db-cAMP 对正常鼠和高血压鼠血管平滑肌细胞中抑制性蛋白α 亚基2（Giα2） 和抑制性蛋白α 亚基3（Giα3）蛋白表达以及对表皮生长因子受体（EGFR）蛋白、细胞外调节蛋白激酶 （ERK1/2）蛋白和原癌基因（c-Src）蛋白磷酸化水平的影响。流式细胞仪检测不同浓度的db-cAMP 对正 常鼠和高血压鼠血管平滑肌细胞内活性氧（ROS）生成的影响。结果 在正常鼠中，与对照组的细胞活性水平 相比，db-cAMP 组和FSK 组的细胞活性水平差异无统计学意义（P >0.05）。在高血压鼠中，与对照组的细胞活 性水平比较，db-cAMP 组和FSK 组的细胞活性水平差异有统计学意义（P <0.05），db-cAMP 组和FSK 组均下 降；与正常鼠不添加db-cAMP 比较，高血压鼠不添加db-cAMP 的血管平滑肌细胞中Giα2 和Giα3 的蛋白表 达增加，超氧阴离子O2 - 生成，EGFR 蛋白、ERK1/2 蛋白和c-Src 蛋白的磷酸化水平均增加。与正常鼠不 添加db-cAMP 比较，正常鼠添加0.5 mmol/L db-cAMP 的血管平滑肌细胞中上述参数部分差异有统计学意 义（P <0.05）。与高血压鼠不添加db-cAMP 相比，在高血压鼠血管平滑肌细胞中分别加入0.1、0.3、0.5 和 1 mmol/L db-cAMP 后上述参数均降低，差异有统计学意义（P <0.05）。结论 初步认为db-cAMP 可能通过 抑制细胞中Giα 蛋白的表达、氧化应激水平及下游信号通路，从而抑制高血压鼠血管平滑肌细胞增殖。

Abstract:

Objective To discuss the effect of 3’, 5’-cyclic adenosine monophosphate (cAMP) on the proliferation of vascular smooth muscle cells in hypertensive rats and further demonstrate the molecular mechanism. Methods The hypertensive rat models were constructed and the vascular smooth muscle cells were extracted. MTT method was used to detect the effect of db-cAMP on the activity of vascular smooth muscle cells in the control group, the db-cAMP group and the Forskolin (cAMP activator) group. Western blot was used to detect the effect of db-cAMP with different concentrations on the levels of inhibitory protein α subunit 2 (Giα2) and inhibitory protein α subunit 3 (Giα3) and the phosphorylation levels of epidermal growth factor receptor (EGFR) protein, ERK1/2 protein and c-Src protein of vascular smooth muscle cells in the normal and hypertensive rats. Flow cytometry was used to detect the effect of db-cAMP with different concentrations on the formation of reactive oxygen species (ROS) in the vascular smooth muscle cells of the normal and hypertensive rats. Results In the normal rats, the cell viability in the dbcAMP group and the Forskolin group was not significantly different from that in the control group (P > 0.05). In the hypertensive rats, the cell viability in the db-cAMP group and the Forskolin group was significantly lower than that in the control group (P < 0.05). Compared with the normal rats without adding db-cAMP, the protein expression levels of Giα2 and Giα3, the generation of superoxide anion O2 -, the phosphorylation levels of EGFR, ERK1/2 and c-Src proteins were significantly increased in the vascular smooth muscle cells of the hypertensive rats without adding dbcAMP (P < 0.05). Compared with the normal rats without adding db-cAMP, some of the above parameters in the vascular smooth muscle cells were statistically different in the normal rats adding 0.5 mmol/L db-cAMP (P < 0.05). Compared with the hypertensive rats without adding db-cAMP, the above parameters were reduced in the vascular smooth muscle cells of the hypertensive rats adding 0.1, 0.3, 0.5 and 1 mmol/L db-cAMP (P < 0.05). Conclusions It is suggested that db-cAMP may inhibit the proliferation of vascular smooth muscle cells in hypertensive rats by inhibiting the expression level of Giα protein, oxidative stress and downstream signaling pathway.