| 摘要: |
| [目的] 探究连翘苷（Phi）调控鞘氨醇激酶1（SphK1）/1-磷酸鞘氨醇（S1P）/1-磷酸鞘氨醇受体1（S1PR1）通路对H2O2诱导的心肌细胞损伤的影响。[方法] 采用H2O2诱导H9c2心肌细胞氧化损伤模型，并经过不同浓度Phi分别处理H9c2细胞和H2O2诱导的H9c2细胞，CCK-8检测各组细胞活力，筛选Phi处理浓度。将H2O2诱导的H9c2细胞随机分为H2O2组、连翘苷低剂量组（L-Phi组）、连翘苷中剂量组（M-Phi组）、连翘苷高剂量组（H-Phi组）、连翘苷高剂量+pcDNA-NC组（H-Phi+NC组）以及连翘苷高剂量+pcDNA-SphK1组（H-Phi+SphK1组）。5-乙炔基-2’-脱氧尿嘧啶核苷（EDU）检测各组细胞增殖能力；流式细胞术检测各组细胞凋亡；酶联免疫吸附测定（ELISA）试剂盒检测各组细胞乳酸脱氢酶（LDH）释放量以及氧化应激相关因子水平；2’，7’-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光检测线粒体活性氧（ROS）水平；JC-1法检测线粒体膜电位（MMP）变化；蛋白免疫印迹法（Western blot）检测各组细胞增殖、凋亡及SphK1/S1P/S1PR1通路相关蛋白表达。[结果] Phi对H9c2细胞活力无影响（P>0.05）；随着Phi浓度升高，H2O2诱导的H9c2心肌细胞活力升高（P<0.05），以5、10、20 μmol/L分别处理L-Phi组、M-Phi组、H-Phi组细胞。与NC组相比，H2O2组细胞EDU阳性细胞率、SOD、过氧化氢酶（CAT）、MMP、细胞周期蛋白D1（CyclinD1）表达降低，凋亡率、LDH、MDA、ROS荧光强度、Bax、活化的半胱氨酸-天冬氨酸蛋白酶9（Cleaved Caspase-9）、SphK1、S1P、S1PR1表达升高（P<0.05）；与H2O2组相比，L-Phi组、M-Phi组、H-Phi组细胞EDU阳性细胞率、SOD、过氧化氢酶（CAT）、MMP、CyclinD1表达升高，凋亡率、LDH、丙二醛（MDA）、ROS荧光强度、Bax、Cleaved Caspase-9、SphK1、S1P、S1PR1表达降低（P<0.05）；与H-Phi组、H-Phi+NC组相比，H-Phi+SphK1组细胞EDU阳性细胞率、SOD、CAT、MMP、CyclinD1表达降低，凋亡率、LDH、MDA、ROS荧光强度、Bax、Cleaved Caspase-9、SphK1、S1P、S1PR1表达升高（P<0.05）。[结论] Phi能够减轻H2O2诱导的心肌细胞损伤，可能与抑制SphK1/S1P/S1PR1通路激活有关。 |
| 关键词: 连翘苷 SphK1/S1P/S1PR1通路 H2O2 心肌细胞损伤 |
| DOI：10.11656/j.issn.1672-1519.2026.07.10 |
| 分类号:R285.5 |
| 基金项目:河北省中医药管理局资助项目（2020386）。 |
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| Effect of phillyrin on H2O2-induced cardiomyocyte damage by regulating the SphK1/S1P/S1PR1 pathway |
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WANG Yuqian, ZHANG Yan, ZHANG Weili
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Department of Cardiology Ⅰ, Tangshan Hospital of Traditional Chinese Medicine, Tangshan 063000, China
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| Abstract: |
| [Objective] To investigate the effect of phillyrin（Phi）regulating the sphingosine kinase 1（SphK1）/sphingosine 1 phosphate （S1P）/sphingosine 1 phosphate receptor 1（S1PR1）pathway on H2O2-induced cardiomyocyte damage. [Methods] The oxidative damage model of H9c2 cardiomyocytes induced by H2O2 was adopted. H9c2 cells and H2O2-induced H9c2 cells were treated with different concentrations of Phi respectively. The viability of cells in each group was detected by CCK-8，and the treatment concentration of Phi was screened. The H 2O2-induced H9c2 cells were randomly divided into H2O2 group，low-dose phillyrin group（L-Phi group）， medium-dose phillyrin group（M-Phi group），high-dose phillyrin group（H-Phi group），high-dose phillyrin+pcDNA-NC group（H-Phi+ NC group），and high-dose phillyrin+pcDNA-SphK1 group（H-Phi+SphK1 group）. The proliferation ability of cells in each group was detected by EDU. Apoptosis of cells in each group was detected by flow cytometry. ELISA kits were used to detect the release of lactate dehydrogenase（LDH）and the levels of oxidative stress-related factors. The DCFH-DA fluorescence method was used to detect the level of mitochondrial reactive oxygen species（ROS）. The change in mitochondrial membrane potential（MMP）was detected by JC-1 method. In addition，Western blot was used to detect the proliferation，apoptosis of cells in each group and the expression of proteins related to the SphK1/S1P/S1PR1 pathway. [Results] Phi had no effect on the viability of H9c2 cells（P>0.05）. With the increase of Phi concentration，the viability of H9c2 cardiomyocytes induced by H2O2 increased（P<0.05）. Cells in the L-PHI group，M-Phi group and H -Phi group were treated with 5，10 and 20 μmol/L respectively. Compared with the NC group，the H2O2 group had lower rate of EDU positive cells，expressions of SOD，catalase（CAT），MMP，and CyclinD1，and higher apoptosis rate，LDH，MDA，ROS fluorescence intensity，expressions of Bax，Cleaved Caspase-9，SphK1，S1P，and S1PR1（P<0.05）. Compared with the H2O2 group，the L-Phi group， M-Phi group and H-Phi group had higher rate of EDU positive cells，expressions of SOD，CAT，MMP，and CyclinD1，and lower apoptosis rate，LDH，MDA，ROS fluorescence intensity，expressions of Bax，Cleaved Caspase-9，SphK1，S1P，and S1PR1（P<0.05）. Compared with the H-Phi group and H-Phi+NC group，the H-Phi+SphK1 group had lower rate of EDU positive cells，expressions of SOD，CAT，MMP，and CyclinD1，and higher apoptosis rate，LDH，MDA，ROS fluorescence intensity，expressions of Bax，Cleaved Caspase-9，SphK1，S1P，and S1PR1（P<0.05）. [Conclusion] Phi can alleviate H2O2-induced cardiomyocyte damage，which may be related to the inhibition of the activation of the SphK1/S1P/S1PR1 pathway. |
| Key words: phillyrin SphK1/S1P/S1PR1 pathway H2O2 cardiomyocyte damage |
