目的 探讨芍药苷（paeoniflorin, PF）对热应激（heat stress, HS）诱导心肌损伤的保护作用及机制。方法 体内实验：每组随机选取10只小鼠,将小鼠分为对照（PBS-Ctrl）组、热应激（PBS-HS）组、芍药苷预处理（PF-Pre-HS）组。PBS-Ctrl组和PBS-HS组注射200 µl的PBS,PF-Pre-HS组注射200 µl浓度为15 kg/mg的PF,每天1次,连续注射7 d。HS模型的建立：小鼠置于39.5℃、湿度为40%的高温模拟舱中,以首只小鼠死亡为节点,停止热应激,室温恢复6 h取材。以乳酸脱氢酶（lactate dehydrogenase, LDH）、超氧化物歧化酶（superoxide dismutase, SOD）活性检测心肌损伤;二氢乙锭（dihydroethidium,DHE）染色检测心肌ROS水平;蛋白质印迹（Western blotting,WB）检测热休克转录因子 1（heat shock factor 1,HSF1）和热休克蛋白70（heat shock protein,HSP70）表达水平。体外实验：建立H9c2细胞热应激损伤模型（45°C,2 h,细胞铺板密度为4×104/ml）,热应激前用浓度为1.25、2.5、5、10 µmol/L的PF处理细胞24 h,细胞增殖检测试剂盒（cell counting kit-8,CCK-8）测定细胞活力,摸索PF最佳保护浓度。实验设置对照（Ctrl）组、热应激（HS）组以及芍药苷预处理（HS+PF）组。RNA 测序（RNA sequencing,RNA-seq）分析H9c2细胞基因表达情况;检测LDH、SOD和谷胱甘肽（glutathione, GSH）水平;活性氧（reactive oxygen species, ROS）染色评估H9c2细胞的氧化应激水平;四氯四乙基苯并咪唑羧酸碘盐（5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide,JC-1）检测细胞线粒体膜电位（mitochondrial membrane potential,MMP）;流式细胞术检测细胞凋亡水平。结果 体内实验：相较于PBS-HS组,PF-Pre-HS组小鼠热应激下存活率升高;相较于PBS-Ctrl组,PBS-HS组小鼠血清和心肌组织的LDH水平升高（P<0.05）、心肌组织中SOD活性降低（P<0.01）;和PBS-HS组相比,PF-Pre-HS组小鼠血清和心肌组织的LDH水平降低（P<0.05）、心肌组织中SOD活性升高（P<0.05）;与PBS-Ctrl组比较,PBS-HS组心肌组织中HSF1和HSP70的蛋白水平降低（P<0.01）,相较于PBS-HS组,PF-Pre-HS组HSF1、HSP70蛋白表达显著升高（P<0.05）。体外实验：与HS组相比,PF预处理可逆转氧化应激和凋亡相关基因的表达（Rasgrf1、S100a14）;与Ctrl组相比,HS组细胞活力、SOD和GSH活性、线粒体膜电位显著降低（P<0.05）,ROS、LDH水平、细胞凋亡率升高（P<0.05）;而同HS组相比,PF 预处理可显著逆转HS造成的损伤,具体表现为HS+PF 组的细胞活力、SOD 和 GSH 活性以及线粒体膜电位显著升高（P<0.05）,ROS、LDH 水平以及细胞凋亡率显著下降（P<0.05）。结论 PF可以减轻热应激诱导的心肌损伤,其机制与线粒体膜电位和氧化应激调节有关,HSF1/HSP70在其中发挥重要作用。
Abstract
Objective s To investigate the protective effect and mechanism of paeoniflorin (PF) on heat stress (HS)-induced myocardial injury in mice. Methods In vivo experiments: 10 mice were randomly selected and allocated into the control (PBS-Ctrl) group, heat stress (PBS-HS) group, and paeoniflorin pre-treated (PF-Pre-HS) group. The PBS-Ctrl and PBS-HS groups received 200 µl of PBS, while the PF-Pre-HS group was administered 200 µl of PF at a concentration of 15 kg/mg daily for 7 days. The HS model was established by placing mice in a high-temperature simulation chamber at 39.5°C with 40% humidity. The stress was terminated upon the death of the first mouse and a 6 h recovery allowed at room temperature before tissue collection. Myocardial injury was assessed by measuring lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities. Reactive oxygen species (ROS) levels in the myocardium were detected using dihydroethidium (DHE) staining and the expression levels of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70) were examined via Western blotting (WB). In vitro experiments: a HS injury model was established in H9c2 cells (45°C for 2 h, at a seeding density of 4×104 cells/ml), pre-treated with PF at concentrations of 1.25, 2.5, 5, and 10 µmol/L for 24 h to determine the optimal protective concentration using a cell counting kit-8 (CCK-8) assay. Experiments were set up with control (Ctrl), heat stress (HS), and paeoniflorin pre-treatment (HS+PF) groups. RNA sequencing (RNA-seq) was conducted to analyze gene expression in H9c2 cells. LDH, SOD, and glutathione (GSH) levels were measured. Oxidative stress levels in H9c2 cells were assessed using reactive oxygen species (ROS) staining. Mitochondrial membrane potential (MMP) was detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) and apoptotic levels in cells were determined by flow cytometry. Results In vivo experiments: compared to the PBS-HS group, the PF-Pre-HS group exhibited an increased survival rate under HS conditions. Compared to the PBS-Ctrl group, the levels of LDH in both the serum and myocardial tissue of mice in the PBS-HS group were increased (P<0.05), while the activity of SOD in the myocardial tissue was decreased (P<0.01). Conversely, in the PF-Pre-HS group, the levels of LDH in both serum and myocardial tissue were reduced (P<0.05), and the SOD activity in the myocardial tissue was elevated when compared to the PBS-HS group (P<0.05). Protein levels of HSF1 and HSP70 in cardiac tissue were decreased in the PBS-HS group (P<0.01), while they were significantly increased in the PF-Pre-HS group compared to the PBS-HS group (P<0.05). In vitro experiments: compared with the PBS-HS group, pretreatment with PF reversed the expression of genes associated with oxidative stress and apoptosis (Rasgrf1, S100a14). Compared to the Ctrl group, the HS group showed a significant decrease in cell viability, SOD and GSH levels, and MMP (P<0.05), along with elevated levels of ROS, LDH, and apoptosis rate (P<0.05). Conversely, PF pre-treatment significantly ameliorated the HS-induced injury, as evidenced by a marked increase in cell viability, SOD and GSH levels, and MMP in the HS+PF group (P<0.05), accompanied by a significant reduction in ROS, LDH levels, and apoptosis rate (P<0.05). Conclusion PF alleviates HS-induced myocardial injury by modulating MMP and oxidative stress, in which the HSF1/HSP70 pathway plays a significant role.
关键词
细胞培养 /
小鼠 /
H9c2细胞 /
芍药苷 /
热应激 /
心肌损伤 /
热休克因子1（HSF1） /
热休克蛋白70（HSP70）
Key words
cell culture /
mice /
H9c2 cells /
paeoniflorin /
heat stress /
myocardial injury /
heat shock factor1 (HSF1) /
heat shock protein (HSP70)
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