白皮杉醇通过抑制HMGB1/TLR4/NF-κB信号减轻多柔比星诱导的H9c2心肌细胞损伤

Through inhibition of HMGB1/TLR4/NF-κB signaling, piceatannol attenuates the damage caused to H9c2 cardiomyocytes by doxorubicin

关键词:  白皮杉醇  多柔比星  H9c2心肌细胞  高迁移率族蛋白B1/Toll样受体4/核因子-κB通路  

Key words:  piceatannol  doxorubicin  H9c2 cells  HMGB1/TLR4/NF-κB

摘要:

目的 探讨白皮杉醇(Piceatannol,PIC)对多柔比星(Doxorubicin,DOX)引起的H9c2心肌细胞损伤的保护作用及机制。方法 分组培养H9c2大鼠心肌细胞后,加入终浓度为1 μmol.L-1的多柔比星和梯度浓度的白皮杉醇处理24小时,应用MTT法检测细胞存活率并筛选出最佳联合给药浓度。采用Hoechst 33342染色及流式细胞术检测H9c2心肌细胞的凋亡情况。采用二氯荧光黄双乙酸盐(DCFH-DA)染色荧光显微镜照像检测活性氧(ROS)水平。蛋白免疫印迹(Western Blot)法测定细胞凋亡蛋白BCL2、BAX、CLEAVED CASPASE-3蛋白表达水平,并使用TLR4激动剂脂多糖(lipopolysaccharide,LPS)进一步验证白皮杉醇与HMGB1/TLR4/NF-κB信号通路的关系。结果 MTT表明DOX可以导致H9c2心肌细胞活性明显降低,而白皮杉醇可抑制此过程,与正常对照组比较,AnnexinV-FITC/PI 染色和Heochest 33342染色表明白皮杉醇可以降低DOX诱导的H9c2心肌细胞的凋亡率,ROS染色结果示,白皮杉醇对DOX干预后细胞ROS水平的升高有明显降低作用,Western blot结果表明白皮杉醇可以一定程度上促进DOX干预后抗凋亡蛋白BCL-2表达抑制促凋亡蛋白(BAX、CLEAVEDCASPASE-3)的表达,进一步研究发现白皮杉醇能够抑制多柔比星引起的H9c2心肌细胞内TLR4蛋白水平的升高,而TLR4激动剂LPS逆转了这一作用。结论 白皮杉醇可能通过抑制HMGB1/TLR4/NF-κB通路改善多柔比星诱导的H9c2心肌细胞损伤。

Abstract:

ABSTRACT: OBJECTIVE The objective of this study is to examine the potential protective effect and underlying mechanism of Piceatannol (PIC) against Doxorubicin (DOX)-induced injury in H9c2 cardiomyocytes. METHODS H9c2 cardiomyocytes were cultured in groups and subsequently treated with doxorubicin at a final concentration of 1 μmol·L-1 and Piceatannol at various levels. The cells were incubated for 24 hours, and the MTT method was employed to assess the cell survival rate and determine the most effective co-administration concentration. Hoechst 33342 staining and flow cytometry were utilized to detect apoptosis in H9c2 cardiomyocytes. Reactive oxygen species (ROS) levels were assessed using DCFH-DA staining and fluorescence microscopy. Protein immunoblotting (Western Blot) was employed to determine the expression levels of apoptotic proteins BCL-2, BAX, and CLEAVED CASPASE-3. The relationship between piceatannol and the HMGB1/TLR4/NF-κB signaling pathway was further confirmed by using the TLR4 agonist lipopolysaccharide (LPS). RESULTS DOX treatment resulted in a significant decrease in H9c2 cardiomyocyte activity, which was inhibited by piceatannol. AnnexinV-FITC/PI staining and Hoechst 33342 staining demonstrated that piceatannol reduced the apoptotic rate of DOX-induced H9c2 cardiomyocytes compared to the normal control group. ROS staining results showed that piceatannol significantly decreased cellular ROS levels compared to the model control group. Western blot analysis revealed that piceatannol increased the expression level of the anti-apoptotic protein BCL-2 and decreased the expression levels of the pro-apoptotic proteins (BAX, CLEAVED CASPASE-3). Furthermore, it was found that piceatannol inhibited the increase of TLR4 protein level in H9c2 cardiomyocytes induced by doxorubicin, and this effect was reversed by the TLR4 agonist LPS. CONCLUSION Piceatannol may ameliorate doxorubicin-induced H9c2 cardiomyocyte injury by inhibiting the HMGB1/TLR4/NF-κB pathway.