目的 观察橙皮素（hesperetin, Hst）对心肌细胞肥大的影响,并探讨相关的机制。方法 通过血管紧张素 Ⅱ（angiotensin II,Ang Ⅱ）诱导H9c2细胞建立体外心肌细胞肥大模型;选择浓度为40~680 μmol/L的Hst进行干预,采用细胞增殖检测试剂盒（cell counting kit-8,CCK-8）检测细胞活力,最终选取三个浓度的Hst进行效应及机制研究。采用小麦胚芽凝集素（wheat germ agglutinin,WGA）染色实验和免疫印迹检测（Western blot）检测Hst对H9c2细胞面积和心肌肥大标志物的变化;免疫印迹检测磷酸化核因子κB p65亚基（p-p65）、κB抑制因子α（ inhibitor of kappa B alpha,IκBα）、NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3, NLRP3）蛋白表达水平;酶联免疫吸附实验（Enzyme linked immunosorbent assay,ELISA）检测肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin -1β,IL-1β)、白细胞介素-6（interleukin -6,IL-6）的表达水平。结果 与对照组相比,浓度为1 μmol/L的Ang Ⅱ诱导H9c2细胞肥大效果最显著（P<0.05）。Ang Ⅱ组NLRP3、p-p65的蛋白表达水平较对照组显著升高,IκBα出现降解,表达降低（P<0.05）;下游炎症因子TNF-α、IL-1β、IL-6的表达水平较对照组显著升高（P<0.05）;与Ang Ⅱ组相比,Hst干预后能够缩小心肌细胞面积（P<0.05）,降低心肌肥大标志物ANP、β-MHC的表达水平（P<0.05）,NLRP3、p-p65的蛋白表达水平下降（P<0.05）,IκBα降解减少,表达增多（P<0.05）,下游炎症因子TNF-α、IL-1β、IL-6的表达水平显著降低（P<0.05）。结论 Hst能显著抑制Ang Ⅱ诱导的心肌细胞肥大,其机制可能通过抑制NLRP3/NF-κB以及炎症因子信号通路发挥作用。
Abstract
Objective To investigate the effect of hesperetin (Hst) on cardiomyocyte hypertrophy and explore the underlying mechanism. Methods An in vitro model of cardiomyocyte hypertrophy was established by treating H9c2 cells with different concentrations of angiotensin II (Ang II). Hst at concentrations ranging from 40 to 680 μmol/L was used for intervention. Cell viability was assessed using the cell counting kit-8 (CCK-8) to ultimately select adequate concentrations for the following experiments. Wheat germ agglutinin (WGA) staining and Western blot were employed to detect changes in cell surface area and expression of myocardial hypertrophy markers in H9c2 cells. Western blot was also used to measure the protein expression levels of phosphorylated RELA (p-p65), inhibitor of kappa B alpha (IκBα), and NOD-like receptor pyrin domain-containing protein 3 (NLRP3). Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Results Compared with the control group, treatment with 1 μmol/L Ang II produced the most significant hypertrophic effect in H9c2 cells (P<0.05). In the Ang II group, the protein expression levels of NLRP3 and p-p65 were significantly increased, while IκBαwas degraded and its expression decreased (P<0.05). The expression levels of downstream inflammatoryfactors TNF-α, IL-1β, and IL-6 were also significantly elevated (P<0.05). Compared with the Ang II group, Hst intervention reduced cardiomyocyte surface area (P<0.05) and decreased the expression levels of hypertrophy markers ANP and β-MHC (P<0.05). The protein expression levels of NLRP3 and p-p65 were downregulated (P<0.05), while IκBα degradation was attenuated and its expression increased (P<0.05). The expression levels of downstream inflammatory factors TNF-α, IL-1βand IL-6 were significantly reduced (P<0.05). Conclusion Hst significantly inhibits Ang II-induced cardiomyocyte hypertrophy, potentially through suppression of the NLRP3/NF-κB signaling pathway and downstream inflammatory cytokine expression.
关键词
橙皮素 /
心肌肥大 /
NLRP3 /
NF-κB /
炎症因子
Key words
hesperetin /
cardiac hypertrophy /
NLRP3 /
NF-κB /
inflammatory factors
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