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.
Key words
cell culture /
mice /
H9c2 cells /
paeoniflorin /
heat stress /
myocardial injury /
heat shock factor1 (HSF1) /
heat shock protein (HSP70)
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
References
[1] Wang Y, Yang C, Elsheikh NAH, et al. HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress[J]. Aging (Albany NY), 2019, 11: 5535-5547.
[2] Slimen IB, Najar T, Ghram A, et al. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review[J]. Int J Hyperthermia, 2014, 30: 513-523.
[3] Ahmad G, Agarwal A, Esteves SC, et al. Ascorbic acid reduces redox potential in human spermatozoa subjected to heat-induced oxidative stress[J]. Andrologia, 2017, 49:e12773.
[4] Yu T, Robotham JL, Yoon Y.Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology[J]. Proc Natl Acad Sci USA, 2006, 103: 2653-2658.
[5] Wang M, Wang RY, Xie XH, et al. Araloside C protects H9c2 cardiomyoblasts against oxidative stress via the modulation of mitochondrial function[J].Biomed Pharmacother, 2019, 117: 109143.
[6] Chen Y, Wu G, Li M, et al. LDHA-mediated metabolic reprogramming promoted cardiomyocyte proliferation by alleviating ROS and inducing M2 macrophage polarization[J]. Redox Biol, 2022, 56: 102446.
[7] Ren S, Wang Y, Zhang Y, et al. Paeoniflorin alleviates Ang II-induced cardiac hypertrophy in H9c2 cells by regulating oxidative stress and Nrf2 signaling pathway[J]. Biomed Pharmacother, 2023, 165: 115253.
[8] 乐娜, 赵丹萍, 张睿, 等. 白芍柔肝解郁有效成分芍药苷抗应激作用机制[J]. 中华中医药杂志, 2021, 12: 7020-7028.
[9] Li L, Wang H, Zhao S, et al. Paeoniflorin ameliorates lipopolysaccharide-induced acute liver injury by inhibiting oxidative stress and inflammation via SIRT1/FOXO1a/SOD2 signaling in rats[J]. Phytother Res, 2022, 36: 2558-2571.
[10] Li X, Sun C, Zhang J, et al. Protective effects of paeoniflorin on cardiovascular diseases: a pharmacological and mechanistic overview[J]. Front Pharmacol,2023, 14: 1122969.
[11] Zhai J, Guo Y.Paeoniflorin attenuates cardiac dysfunction in endotoxemic mice via the inhibition of nuclear factor-κB[J]. Biomed Pharmacother, 2016, 80: 200-206.
[12] 王亚茹, 袁满, 张丽, 等. 槲皮素抗氧化作用及相关机制研究进展[J]. 营养学报, 2022, 44: 204-208.
[13] Liu B, Piao X, Niu W, et al. Kuijieyuan decoction improved intestinal barrier injury of ulcerative colitis by affecting TLR4-dependent PI3K/AKT/NF-κB oxidative and inflammatory signaling and gut microbiota[J]. Front Pharmacol, 2020, 11: 1036.
[14] 王俐, 蔡美琴, 刘秀玲, 等. α-硫辛酸对高糖和H2O2致ECV304细胞线粒体氧化损伤的保护作用[J]. 营养学报, 2010,32: 234-238.
[15] Wang X, Yuan B, Dong W, et al. Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway[J]. Heart Vessels, 2015, 30: 396-405.
[16] Yang H, Jing H, Han X, et al. Synergistic anticancer strategy of sonodynamic therapy combined with PI-103 against hepatocellular carcinoma[J]. Drug Des Devel Ther, 2021, 15: 531-542.
[17] Dressler C, Beuthan J, Mueller G, et al. Fluorescence imaging of heat-stress induced mitochondrial long-term depolarization in breast cancer cells[J]. J Fluoresc, 2006, 16: 689-695.
[18] Hehir MP, Morrison JJ.Paeoniflorin, a novel heat-shock protein inducing compound, and human myometrial contractility in vitro[J]. J Obstet Gynaecol Res, 2016, 42: 302-306.
[19] Bei M, Wang Q, Yu W, et al. Effects of heat stress on ovarian development and the expression of HSP genes in mice[J]. J Therm Biol, 2020, 89: 102532.
[20] Zhou Y, Wang Y, Xu F, et al. Small HSPs play an important role in crosstalk between HSF-HSP and ROS pathways in heat stress response through transcriptomic analysis in lilies[J]. BMC Plant Biol, 2022, 22: 202.
