目的:脓毒症心肌损伤可增加患者的死亡率,这是脓毒症的重要并发症。心肌细胞凋亡在脓毒症心肌损伤中起关键作用。本文探讨了虾青素(ATX)抑制脂多糖(LPS)诱导的心肌细胞凋亡的可能机制。
方法:H9C2细胞实验分三个部分进行。在第一部分,我们设置了三组:对照组,LPS组(10µg/ml),脓毒症心肌损伤模型,和LPS+ATX(5,10,30µM);在第二部分,我们设置了四组:对照组,LPS组,LPS+PTP1B-IN-1,一种蛋白酪氨酸磷酸酶1B(PTP1B)抑制剂,和LPS+PTP1B-IN-1+ATX;在第三部分,我们设置了四组:对照组,LPS组,LPS+茴香霉素,c-Jun氨基末端激酶(JNK)激活剂,和LPS+茴香霉素+ATX。我们使用细胞计数试剂盒-8(CCK-8)测定评估H9C2细胞活力。我们使用流式细胞术分析观察细胞凋亡。我们使用JC-1染色测试了线粒体膜电位(ΔkW)。为了识别ATX的分子靶标,通过SwissTargetPrediction数据库预测虾青素靶标。我们使用微型热电泳(MST)验证了ATX及其靶标的结合亲和力。我们使用免疫荧光染色研究了p-JNK的表达。最后,Westernblot用于评估PTP1B,JNK,p-JNK和线粒体凋亡相关蛋白表达。
结果:LPS以时间依赖性方式抑制H9C2细胞活力,ATX处理以浓度依赖性方式提高LPS给药后H9C2细胞活力。ATX抑制LPS诱导的H9C2细胞凋亡和线粒体膜电位丧失。正如SwissTargetPrediction数据库所预测的那样,PTP1B是ATX的潜在靶点,MST进一步验证了ATX与PTP1B之间的相互作用。ATX减弱了LPS诱导的PTP1B和p-JNK的蛋白表达,不管PTP1B抑制。免疫荧光染色和免疫印迹均显示ATX抑制LPS诱导的H9C2细胞中p-JNK的表达,不管茴香霉素给药。此外,通过添加茴香霉素来过度表达JNK,ATX抑制LPS诱导的细胞凋亡,通过JNK信号传导H9C2细胞中线粒体膜电位的丧失和线粒体凋亡相关蛋白的上调。
结论:ATX通过PTP1B/JNK途径抑制LPS诱导的H9C2细胞线粒体凋亡,PTP1B是ATX的靶点。
OBJECTIVE: Myocardial injury induced by sepsis can increase the patient\'s mortality, which is an important complication of sepsis. Myocardial apoptosis plays a key role in septic myocardial injury. Here we explored the potential mechanism of astaxanthin (ATX) inhibiting myocardial apoptosis induced by lipopolysaccharide (LPS) in vitro.
METHODS: The H9C2 cell experiment was conducted in three parts. In the first part, we set up three groups: control group, LPS group (10 µg/ml), a model of septic myocardial injury, and LPS + ATX (5, 10, 30 µM); In the second part, we set up four groups: control group, LPS group, LPS + PTP1B-IN-1, a protein tyrosine phosphatase 1B (PTP1B) inhibitor, and LPS + PTP1B-IN-1 + ATX; In the third part, we set up four groups: control group, LPS group, LPS + Anisomycin, a c-Jun N-terminal kinase (JNK) activator, and LPS + Anisomycin + ATX. We assessed H9C2 cell viability using the Cell Counting Kit-8 (CCK-8) assay. We observed cell apoptosis using flow cytometry analysis. We tested the mitochondrial membrane potential (ΔΨm) using JC-1 staining. To identify the molecular targets of ATX, Astaxanthin targets were predicted through the SwissTargetPrediction database. We verified the binding affinity of ATX and its targets using microscale thermophoresis (MST). We investigated the p-JNK expression using immunofluorescence staining. Finally, Western blot was used to evaluate PTP1B, JNK, p-JNK and the mitochondrial apoptosis-associated protein expression.
RESULTS: LPS inhibited H9C2 cell viability in a time-dependent manner and ATX treatment enhances H9C2 cell viability in a concentration dependent manner after LPS administration. ATX inhibited the LPS-induced apoptosis and loss of mitochondrial membrane potential in H9C2 cells. As predicted by the SwissTargetPrediction database, PTP1B was a potential target of ATX, and the interaction between ATX and PTP1B was further verified by MST. ATX attenuated the LPS-induced protein expression of PTP1B and p-JNK, regardless of PTP1B inhibition. Both immunofluorescence staining and Western blotting showed that ATX suppressed the LPS-induced p-JNK expression in H9C2 cells, regardless of Anisomycin administration. In addition, by adding Anisomycin to overexpress JNK, ATX inhibited the LPS-induced apoptosis, loss of mitochondrial membrane potential and upregulation of mitochondrial apoptosis-associated proteins in H9C2 cells via JNK signaling.
CONCLUSIONS: ATX inhibited LPS-induced mitochondrial apoptosis of H9C2 cells by PTP1B/JNK pathway and PTP1B was the target of ATX.