秋葵总黄酮通过抑制炎症反应对糖尿病小鼠的心脏保护作用

Flavonoids derived from abelmoschus esculentus attenuates diabetic cardiomyopathy in rats via inhibition of inflammation

目的 秋葵中提取的总黄酮(TFA)具有抗氧化、抑制纤维化等作用,心肌细胞在长期高糖刺激下出现过度的氧化应激,产生并释放大量的炎症因子,导致糖尿病心肌病(DCM)。文章将探讨TFA对DCM的影响及其相关机制。方法 将4周龄C57BL/6雄性小鼠分成4组:对照组、TFA组、糖尿病心肌病组(STZ组)及糖尿病心肌病+TFA组(STZ+TFA组)。利用腹腔注射链脲佐菌素(STZ)100 mg/kg构建DCM模型,STZ+TFA组在STZ注射后每日喂食300 mg/kg TFA。通过血清生化检测、心脏超声及心脏活检评估各组小鼠的生理状况,并应用Western Blot与qPCR法分别比较激活核因子κB(NF-κB)及炎症因子的mRNA表达水平。同时,将H9C2株心肌细胞分为6组:低糖对照组、低糖TFA组、高糖组(HG组)、高糖+TFA组(HG+TFA组)、高糖+p65过表达组(HG+pCNDA-p65组)及高糖+TFA+p65过表达组(HG+TFA+pCNDA-p65组),利用正常的DMEM培养基内加D-葡萄糖,配置成含葡萄糖浓度为33 mmol/L的高糖培养基,pCDNA-p65转染H9C2株心肌细胞过表达p65。通过细胞免疫荧光实验对比细胞NF-κB入核水平,应用qPCR法检测细胞内IL-1、IL-6、TNF-α水平。结果 16周时STZ+TFA组心脏质量/体重(4.73±0.29)较STZ组(5.65±0.35)明显降低(P<0.05),血糖有下降趋势,但差异无统计学意义(P>0.05),对照组、TFA组、STZ组和STZ+TFA组心率差异无统计学意义(P>0.05);心超结果显示TFA明显改善糖尿病心肌病小鼠的左室扩大以及左室的收缩及舒张功能(P<0.05);HE染色与Masson染色显示TFA抑制糖尿病心肌病小鼠的心肌重构并降低心肌纤维化水平;并且给予TFA后,小鼠组织中的NF-κB、IL-1、IL-6、TNF-α水平明显下调(P<0.01)。在细胞水平,TFA预处理的心肌细胞予高糖环境刺激后,NF-κB入核水平减少并且细胞内IL-1、IL-6、TNF-α水平下调(P<0.01);过表达心肌细胞内的NF-κB后,TFA对于由高糖引起的高水平IL-1、IL-6、TNF-α不起作用。结论 TFA通过抑制NF-κB入核下调IL-1、IL-6、TNF-α水平,改善糖尿病心肌病。

Objective The total flavonoids from astragalus(TFA) extracted from okra have antioxidant and anti-fibrotic effects. The cardiomyocytes suffer from excessive oxidative stress under long-term high glucose stimulation, resulting in the production and release of a large number of inflammatory factors, leading to diabetic cardiomyopathy(DCM). This paper will discuss the effects of TFA on DCM and its related mechanisms. Methods 4-week-old C57 BL/6 male mice were divided into 4 groups: control group, TFA group, diabetic cardiomyopathy group(STZ group) and diabetic cardiomyopathy +TFA group(STZ+TFA group). DCM model was established by intraperitoneal injection of streptozotocin(STZ) 100 mg/kg, and the STZ+TFA group was fed 300 mg/kg TFA daily after STZ injection.Serum biochemical assay, cardiac ultrasound and cardiac biopsy were used to evaluate the physiological status of each group.The expression of NF-κB and mRNA of inflammatory factors were compared by Western Blot and qPCR, respectively.Meanwhile, H9 C2 strains of cardiomyocytes were divided into 6 groups: low glucose group, low glucose TFA group, high glucose group(HG group), high glucose +TFA group(HG+TFA group), high glucose +p65 overexpression group(HG+pCNDA p65 group) and high glucose+TFA+ p65 overexpression group(HG+TFA+pCNDA p65 group) D-glucose was added into DMEM medium to prepare a high glucose medium containing 33 mmol/L glucose, pCNDA-p65 was transfected into H9 C2 cells to overexpress p65. The nuclear translocation of NF-κB were compared by immunofluorescence assay, and the levels of IL-1, IL-6 and TNF-α were detected by qPCR. Results At 16 weeks, the heart weight/body weight of STZ+TFA group(4.73±0.29) was significantly lower than that of STZ group(5.65±0.35)(P<0.05), and blood glucose showed a downward trend, but the difference was not statistically significant(P>0.05). There was no significant difference in heart rate between control group, TFA group, STZ group and STZ+TFA group(P>0.05).The results of echocardiography showed that TFA significantly improved left ventricular enlargement, systolic and diastolic function of left ventricle in diabetic cardiomyopathy mice(P<0.05). HE staining and Masson staining showed that TFA inhibited myocardial remodeling and reduced myocardial fibrosis in diabetic cardiomyopathy mice. The levels of NF-κB, IL-1, IL-6 and TNF-α in mice were decreased significantly after TFA treatment(P<0.01).As for the cellular level, the levels of NF-κB and IL-1, IL-6 and TNF-α in TFA pretreated cardiomyocytes were decreased after high glucose stimulation(P<0.01). After overexpression of NF-κB in myocardium, TFA had no effect on high levels of IL-1, IL-6 and TNF-α induced by high glucose. Conclusion TFA can reduce the levels of IL-1, IL-6 and TNF-α by inhibiting nuclear translocation of nuclear translocation of NF-κB and improving diabetic cardiomyopathy.