目的 探讨丹参酮ⅡA磺酸钠(STS)对过氧化氢(H2O2)诱导后人脐静脉内皮细胞(HUVECs)焦亡的影响及其可能机制。 方法 2021年11月至2022年9月，采用无锡市第九人民医院保存的HUVECs细胞株作为研究对象，实验分为4组：空白对照组(正常条件)、空白＋STS组、H2O2组、H2O2＋STS组。待细胞长至80%融合度时，H2O2组和H2O2＋STS组加入500.00 μmol/L H2O2诱导3 h，去除含500.00 μmol/L H2O2的培养基之后，空白＋STS组和H2O2＋STS组加入5.00 μg/ml STS与HUVECs共培养24 h。通过CCK-8实验检测不同浓度(0、0.05、0.50、5.00、50.00、500.00 μg/ml)的STS对HUVECs增殖的影响，TUNEL染色检测DNA损伤阳性细胞，实时荧光定量PCR(RT-PCR)检测炎症小体核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)的表达来确定H2O2诱导焦亡的最适浓度。检测试剂盒检测H2O2诱导后活性氧自由基(ROS)表达。细胞划痕实验和基质胶体外成管实验探究STS对焦亡状态下HUVECs迁移能力和成管能力的影响。RT-PCR和Western blotting检测NLRP3、Caspase-1、白细胞介素-18、白细胞介素-1β的基因和蛋白表达。不同时点浓度比较采用重复测量方差分析，2组间数据比较采用t检验，多组间数据比较采用单因素方差分析，P<0.05为差异有统计学意义。 结果 50.00 μg/ml以下的STS对HUVECs的增殖无影响，500.00 μmol/L H2O2诱导HUVECs焦亡效果最佳。TUNEL染色显示，与空白对照组相比，H2O2组TUNEL阳性细胞率显著升高，差异有统计学意义(P<0.01)，与H2O2＋STS组TUNEL阳性细胞率的差异无统计学意义(P>0.05)。ROS检测结果显示，与H2O2组比，H2O2＋STS组细胞内ROS明显降低，差异有统计学意义(P<0.01)。细胞划痕和体外成管实验显示，与空白对照组相比，H2O2组细胞迁移率和成管能力明显减弱，差异均有统计学意义(P均<0.01)，与H2O2＋STS组差异均无统计学意义(P均>0.05)。RT-PCR和Western blotting结果显示，H2O2＋STS组与H2O2组比较，细胞焦亡相关因子表达降低，差异有统计学意义(P<0.05)。 结论 STS可通过抑制ROS的过量产生，促进细胞焦亡诱导后HUVECs的细胞迁移和管样形成，减轻H2O2诱导的HUVECs细胞焦亡，从而促进血管生成。 Objective To investigate the effect of sodium tanshinone ⅡA sulfonate (STS) on pyroptosis of human umbilical vein endothelial cells (HUVECs) induced by H2O2 and its possible mechanism. Methods From November 2021 to September 2022, HUVECs were used as the research subjects at Wuxi Ninth People’s Hospital. The experiment was divided into four groups: the blank control group (normal condition), blank + STS group, H2O2 group and H2O2 + STS group. When the cells reached 80% fusion, 500.00 μmol/L of H 2O2 was added to H2O2 group and H2O2 + STS group for 3 hours, and then the medium containing 500.00 μmol/L H 2O2 was removed. After that, the blank+ STS group and the H2O2+ STS group were each supplemented with 5.00 μg/ml of STS and co-cultured with HUVECs for 24 hours. CCK-8 was used to assess the impact of STS at various concentrations (0.00, 0.05, 0.50, 5.00, 50.00, 500.00 μg/ml) on the proliferation of HUVECs. DNA damage-positive cells were detected with TUNEL staining. The expression of NOD-like receptor protein 3 (NLRP3) was detected using real-time PCR (RT-PCR) to investigate the optimal concentration of pyroptosis induced by H 2O2. A detection kit was used to measure the expression of reactive oxygen species (ROS) induced by H2O2. The effect of STS on the migration and tube formation of HUVECs during pyroptosis was examined using a cell scratch test and a matrix gel tube formation test. The expressions of NLRP3, caspase-1, interleukin-18, and interleukin-1β were detected using RT-PCR and Western blotting. Repeated measures ANOVA was used to compare the concentrations at different time points,t-tests were used to compare data between two groups, and one-way ANOVA was used to compare data between multiple groups. P<0.05 was considered statistically significant. Results STS below 50.00 μg/ml had no effect on the proliferation of HUVECs, while 500.00 μmol/L H 2O2 had the most significant effect on inducing pyroptosis in HUVECs. TUNEL staining showed that compared with the control group, the number of TUNEL-positive cells in H2O2 group was significantly increased, and the difference was statistically significant (P<0.01). However, there was no significant difference in the number of TUNEL-positive cells in the H2O2+ STS group (P>0.05). The results of ROS detection showed that compared with the H2O2 group, intracellular ROS levels in the H2O2+ STS group was significantly decreased, and the difference was statistically significant (P<0.01). Cell scratch and tube formationin vitro experiments showed that compared with the control group, cell mobility and tube formation ability were significantly decreased in the H2O2 group (all P<0.01), and there was no statistical significance in the H2O2+ STS group (all P>0.05). RT-PCR and Western blotting results showed that, compared with the H2O2 group, the expression of pyroptosis-related factors in the H2O2+ STS group was significantly decreased (all P<0.05). Conclusion STS can inhibit the excessive production of ROS, promote the cell migration and tubular formation of HUVECs after pyroptosis induction, and alleviate H2O2-induced pyroptosis of HUVECs, thereby promoting angiogenesis.
Sodium tanshinone ⅡA sulfonate alleviated pyroptosis of human umbilical vein endothelial cells induced by H 2O 2
Objective To investigate the effect of sodium tanshinone ⅡA sulfonate (STS) on pyroptosis of human umbilical vein endothelial cells (HUVECs) induced by H2O2 and its possible mechanism. Methods From November 2021 to September 2022, HUVECs were used as the research subjects at Wuxi Ninth People’s Hospital. The experiment was divided into four groups: the blank control group (normal condition), blank + STS group, H2O2 group and H2O2 + STS group. When the cells reached 80% fusion, 500.00 μmol/L of H 2O2 was added to H2O2 group and H2O2 + STS group for 3 hours, and then the medium containing 500.00 μmol/L H 2O2 was removed. After that, the blank+ STS group and the H2O2+ STS group were each supplemented with 5.00 μg/ml of STS and co-cultured with HUVECs for 24 hours. CCK-8 was used to assess the impact of STS at various concentrations (0.00, 0.05, 0.50, 5.00, 50.00, 500.00 μg/ml) on the proliferation of HUVECs. DNA damage-positive cells were detected with TUNEL staining. The expression of NOD-like receptor protein 3 (NLRP3) was detected using real-time PCR (RT-PCR) to investigate the optimal concentration of pyroptosis induced by H 2O2. A detection kit was used to measure the expression of reactive oxygen species (ROS) induced by H2O2. The effect of STS on the migration and tube formation of HUVECs during pyroptosis was examined using a cell scratch test and a matrix gel tube formation test. The expressions of NLRP3, caspase-1, interleukin-18, and interleukin-1β were detected using RT-PCR and Western blotting. Repeated measures ANOVA was used to compare the concentrations at different time points,t-tests were used to compare data between two groups, and one-way ANOVA was used to compare data between multiple groups. P<0.05 was considered statistically significant. Results STS below 50.00 μg/ml had no effect on the proliferation of HUVECs, while 500.00 μmol/L H 2O2 had the most significant effect on inducing pyroptosis in HUVECs. TUNEL staining showed that compared with the control group, the number of TUNEL-positive cells in H2O2 group was significantly increased, and the difference was statistically significant (P<0.01). However, there was no significant difference in the number of TUNEL-positive cells in the H2O2+ STS group (P>0.05). The results of ROS detection showed that compared with the H2O2 group, intracellular ROS levels in the H2O2+ STS group was significantly decreased, and the difference was statistically significant (P<0.01). Cell scratch and tube formationin vitro experiments showed that compared with the control group, cell mobility and tube formation ability were significantly decreased in the H2O2 group (all P<0.01), and there was no statistical significance in the H2O2+ STS group (all P>0.05). RT-PCR and Western blotting results showed that, compared with the H2O2 group, the expression of pyroptosis-related factors in the H2O2+ STS group was significantly decreased (all P<0.05). Conclusion STS can inhibit the excessive production of ROS, promote the cell migration and tubular formation of HUVECs after pyroptosis induction, and alleviate H2O2-induced pyroptosis of HUVECs, thereby promoting angiogenesis.
Human umbilical vein endothelial cellPyroptosisSodium tanshinone ⅡA sulfonateAngiogenesis
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