柚皮苷对高糖诱导大鼠H9c2心肌细胞损伤影响

温以杰; 吴丹; 李厚忠; 孟娜娜; 李玲玉; 初彦辉; 金秀东; 张羽飞

doi:10.11847/zgggws1122166

摘要:

目的探讨柚皮苷对高糖引起的大鼠H9c2心肌细胞损伤作用影响及机制。

方法体外培养H9c2心肌细胞，用35 mmol/L葡萄糖处理建立细胞损伤模型。将细胞随机分为5组，对照组（不加任何干预）、模型组（35 mmol/L葡萄糖培养48 h）、柚皮苷低、中、高剂量组（5、10、20 μmol/L柚皮苷预先孵育4 h，再用35 mmol/L葡萄糖培养48 h。噻唑蓝（MTT）法检测各组细胞活力，原位末端标记法（TUNEL）检测细胞凋亡率；Western blot法检测细胞内质网应激（ERS）标志蛋白葡萄糖调节蛋白78（glucose regulated protein 78，GRP78）、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（C/EBP homologous protein，CHOP）、半胱氨酸天冬氨酸蛋白酶12（cysteinyl aspartate specific proteinase 12，caspase 12）及细胞炎症因子白细胞介素 – 6（interleukin-6，IL-6）、真核细胞转录因子（nuclear factor kappa-B，NF-κB）蛋白表达。

结果与对照组比较，模型组H9c2细胞存活率（70.36 ± 6.31）%明显下降（P < 0.01），凋亡率（41.25 ± 7.44）%升高（P < 0.05），细胞中IL-6、NF-κB、GRP78、CHOP和Caspase12蛋白表达水平分别为（0.34 ± 0.03）、（1.04 ± 0.10）、（0.93 ± 0.65）、（0.52 ± 0.02）、（0.39 ± 0.02）均明显升高（P < 0.01）；与模型组比较，20 μmol/L柚皮苷组H9c2细胞存活率（92.33 ± 5.64）%明显提高、细胞凋亡率（25.0 ± 4.82）%明显减少（P < 0.05），H9c2细胞内GRP78、CHOP、Caspase12表达水平分别为（0.57 ± 0.04）、（0.31 ± 0.04）、（0.23 ± 0.02）明显降低（P < 0.05），炎性因子IL-6、NF-κB表达水平分别为（0.23 ± 0.03）、（0.76 ± 0.06）明显下调（P < 0.05）；呈一定剂量效应关系。

结论柚皮苷可明显减轻高糖诱导的H9c2细胞损伤、降低凋亡率，其机制可能与抑制细胞内质网应激水平和炎症因子表达有关。

Abstract:

Objective To investigate the effect of naringin (Nar) on rat H9c2 cardiomyocyte injury induced by high glucose (HG) and its mechanism.

Methods H9c2 cardiomyocytes were cultured in vitro and treated with glucose to establish the cell injury model. Then the cells were randomly divided into 5 groups: a normal control (without any intervention), a model (with 35 mmol·L^–1 glucose treatment for 48 hours), and the low, moderate, and high dose Nar dose groups (with pretreatment of Nar at doses of 5, 10, 20 μmol·L^–1 for 4 hours before 35 mmol·L^–1 glucose treatment for 48 hours). The viability of the each H9c2 cardiomyocyte group was detected with 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The cell apoptosis was detected with terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. The expressions of inflammatory factor interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), endoplasmic reticulum stress (ERS)-related glucose regulated protein 78kD (GRP 78), CCAAT/enhancer-binding proteins (C/EBP)-homologous protein (CHOP) and cysteinyl aspartate specific proteinase 12 (caspase 12) were detected with Western blot.

Results Compared with the control cells, the H9c2 cells of model group showed significantly decreased survival rate (70.36 ± 6.31%) (P < 0.01) but increased apoptotic rate (41.25 ± 7.44%) and expressions of IL-6 (0.34 ± 0.03), NF-κB (1.04 ± 0.10), GRP 78 (0.93 ± 0.65), CHOP (0.52 ± 0.02), and caspase 12 (0.39 ± 0.02) (all P < 0.01). In comparison to the cells of model group, the H9c2 cells with high dose Nar pretreatment demonstrated significantly increased survival rate (92.33 ± 5.64%, P < 0.05) but decreased apoptotic rate (25.0 ± 4.82%), intracellular expressions of GRP 78 (0.57 ± 0.04), CHOP (0.31 ± 0.04) and caspase 12 (0.39 ± 0.02), and down-regulated expressions of IL-6 (0.23 ± 0.03) and NF-κB (0.76 ± 0.06) in dose response manners (P < 0.05 for all)

Conclusion Naringin could significantly alleviate apoptosis induced by high glucose in H9c2 cells and the effect may be related to the inhibited expression of ERS and inflammatory factor NF-κB and IL-6.

柚皮苷对高糖诱导大鼠H9c2心肌细胞损伤影响

Effects of naringin on high glucose induced damage in rat H9c2 cardiomyocytes