柚皮素对脂多糖诱导的H9c2心肌细胞炎症和凋亡的作用机制研究

Effect and mechanism of naringenin on lipopolysaccharide-induced inflammation and apoptosis in H9c2 cardiomyocytes

目的探讨柚皮素对脂多糖(LPS)诱导的H9c2心肌细胞的炎症和凋亡的作用机制。方法将大鼠H9c2心肌细胞随机分为4组:空白对照组、模型组和低、高2个浓度实验组。用10μg·mL-1 LPS处理模型组和实验组的细胞造成细胞损伤,空白对照组用正常含血清培养液处理细胞。低、高2个浓度实验组分别加入5,20μmol·mL-1柚皮素处理12 h。用实时荧光定量-PCR法检测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子α(TNF-α)基因的表达水平,缺口末端标记法检测心肌细胞凋亡情况(阳性细胞数与总细胞数比值),蛋白质印迹法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和核转录因子-κB (NF-κB)蛋白表达水平。结果空白对照组、模型组和低、高2个浓度实验组的IL-1β基因相对表达量分别为1.00±0.18,1.57±0.17,1.55±0.26和1.15±0.13;IL-6基因相对表达量分别为1.00±0.16,1.75±0.21,1.71±0.16和1.32±0.20;TNF-α基因相对表达量分别为1.00±0.15,2.68±0.17,2.65±0.23和1.51±0.18;心肌细胞凋亡率分别为(2.87±0.81)%,(13.82±1.34)%,(13.78±1.82)%和(7.32±0.64)%。上述指标:模型组与空白对照组比较,或高浓度实验组与模型组比较,差异均有统计学意义(均P<0.05)。蛋白结果的趋势与基因一致。结论柚皮素通过调控NF-κB通路改善脂多糖诱导的H9c2心肌细胞的炎症反应和细胞凋亡。

Objective To investigate the effect and mechanism of naringenin(NAR) on the inflammation and apoptosis of H9 c2 cardiomyocytes induced by lipopolysaccharide(LPS). Methods Rat H9 c2 cardiomyocytes were randomly divided into four groups:blank control group, model group, experimental-L group and experimental-H group. The cells in the blank control group were treated with normal serum-containing medium, the cells in the model group and the experimental groups were treated with 10 μmol· mL-1 LPS, and the cells in the experimental groups were added with 5 and 20 μmol· mL-1NAR for 12 h. Gene expression levels of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were determined by real time quantitative-PCR.The apoptotic cardiomyocytes were detected by TdT-mediated dUTP nick-end labeling assay(positive cell number vs total cell number),and expressions of the B-cell lymphoma-2(Bcl-2),Bcl 2-associated X(Bax),and nuclear factor kappa B(NF-κB) proteins were detected by Western blot. Results The relative expression levels of IL-1β gene in the blank control group,model group,experimental-L group and experimental-H group were 1. 00 ± 0. 18,1. 57 ± 0. 17,1. 55 ± 0. 26 and1. 15 ± 0. 13;the relative expression levels of IL-6 gene in the 4 groups were 1. 00 ± 0. 16,1. 75 ± 0. 21,1. 71 ± 0. 16 and 1. 32 ± 0. 20;the relative expression levels of TNF-α in the 4 groups were 1. 00 ± 0. 15,2. 68 ± 0. 17,2. 65 ± 0. 23 and 1. 51 ± 0. 18;apoptosis rate of myocardial cells in the 4 groups were(2. 87 ± 0. 81) %,(13. 82 ± 1. 34) %,(13. 78 ± 1. 82) % and(7. 32 ± 0. 64) %. Comparison between model group and blank control group,or comparison between experimental-H group and model group,the difference of the factors were significant(all P < 0. 05). The trend of protein results is consistent with that of gene. Conclusion Naringin regulates the NF-κB pathway to improve the inflammatory response and apoptosis of H9c2 myocardial cells induced by LPS.