College of Traditional Chinese Medicine, Jinan University, Guangzhou, China.
Guangdong Pharmaceutical University, Guangzhou, China.
Oxid Med Cell Longev. 2021 Nov 24;2021:8578452. doi: 10.1155/2021/8578452. eCollection 2021.
We explored the role of ROS in cold-induced vasoconstriction and corresponding mechanism.
Three experiments were performed. First, we measured blood flow in human hands before and after cold exposure. Second, 24 mice were randomly divided into 3 groups: 8 mice received saline injection, 8 received subcutaneous Tempol injection, and 8 received intrathecal Tempol injection. After 30 min, we determined blood flow in the skin before and after cold exposure. Finally, we used Tempol, CCG-1423, and Go 6983 to pretreat HAVSMCs and HUVECs for 24 h. Then, cells in the corresponding groups were exposed to cold (6 h, 4°C). After cold exposure, the cytoskeleton was stained. Intracellular Ca and ROS levels were measured by flow cytometry and fluorescence microscopy. We measured protein expression via Western blotting.
In the first experiment, after cold exposure, maximum skin blood flow decreased to 118.4 ± 50.97 flux units. Then, Tempol or normal saline pretreatment did not change skin blood flow. Unlike intrathecal Tempol injection, subcutaneous Tempol injection increased skin blood flow after cold exposure. Finally, cold exposure for 6 h shrank the cells, making them narrower, and increased intracellular Ca and ROS levels in HUVECs and HAVSMCs. Tempol reduced cell shrinkage and decreased intracellular Ca levels. In addition, Tempol decreased intracellular ROS levels. Cold exposure increased RhoA, Rock1, p-MLC-2, ET-1, iNOS, and p-PKC expression and decreased eNOS expression. Tempol or CCG-1423 pretreatment decreased RhoA, Rock1, and p-MLC-2 levels in HAVSMCs. Furthermore, Tempol or Go 6983 pretreatment decreased ET-1, iNOS, and p-PKC expression and increased eNOS expression in HUVECs.
ROS mediate the vasoconstrictor response within the cold-induced vascular response, and ROS in blood vessel tissues rather than nerve fibers are involved in vasoconstriction via the ROS/RhoA/ROCK1 and ROS/PKC/ET-1 pathways in VSMCs and endothelial cells.
探讨 ROS 在冷诱导血管收缩中的作用及相应机制。
进行了三项实验。首先，我们在冷暴露前后测量了人手的血流量。其次，将 24 只小鼠随机分为 3 组：8 只接受生理盐水注射，8 只接受皮下 Tempol 注射，8 只接受鞘内 Tempol 注射。30 分钟后，我们测定了冷暴露前后皮肤的血流量。最后，我们用 Tempol、CCG-1423 和 Go 6983 预处理 HAVSMCs 和 HUVECs 24 小时。然后，将相应组的细胞暴露于冷（6 小时，4°C）。冷暴露后，对细胞骨架进行染色。通过流式细胞术和荧光显微镜测量细胞内 Ca 和 ROS 水平。通过 Western blot 测定蛋白表达。
在第一项实验中，冷暴露后，最大皮肤血流降至 118.4±50.97 通量单位。然后，Tempol 或生理盐水预处理并不改变皮肤血流。与鞘内 Tempol 注射不同，皮下 Tempol 注射增加了冷暴露后的皮肤血流。最后，冷暴露 6 小时会使细胞收缩，使细胞变窄，并增加 HUVECs 和 HAVSMCs 中的细胞内 Ca 和 ROS 水平。Tempol 减少了细胞收缩并降低了细胞内 Ca 水平。此外，Tempol 降低了细胞内 ROS 水平。冷暴露增加了 RhoA、Rock1、p-MLC-2、ET-1、iNOS 和 p-PKC 的表达，降低了 eNOS 的表达。Tempol 或 CCG-1423 预处理降低了 HAVSMCs 中的 RhoA、Rock1 和 p-MLC-2 水平。此外，Tempol 或 Go 6983 预处理降低了 HUVECs 中 ET-1、iNOS 和 p-PKC 的表达，增加了 eNOS 的表达。
ROS 介导了冷诱导血管反应中的血管收缩反应，而血管组织中的 ROS 而不是神经纤维通过 ROS/RhoA/ROCK1 和 ROS/PKC/ET-1 途径参与了 VSMCs 和内皮细胞的血管收缩。
