葛花总黄酮对阿霉素所致中毒性心肌炎的保护作用研究

Protective effect of flos puerariae flavonoid on adriamycin-induced toxic myocarditis

目的探讨葛花总黄酮对阿霉素中毒性心肌炎的保护作用及其机制。方法健康昆明种小鼠96只,雄雌不拘,分为6组,每组16只。正常对照组(control):每天经口灌胃(ig)生理盐水(NS)20 mL/kg,并于实验第2天起,腹腔注射(ip)NS 10 mL/kg,隔天1次,共7次;阿霉素模型对照组(model):每天ig 20 mL/kg NS,同时于实验第2天起,ip阿霉素3 mg/kg,隔天1次,共7次;阿霉素+葛花总黄酮小剂量组(50 mg/kg);阿霉素+葛花总黄酮中剂量组(100 mg/kg);阿霉素+葛花总黄酮大剂量组(200 mg/kg);维生素E阳性对照组(Vit E):造模同阿霉素模型对照组,同时每天ig Vit E 0.04 g/kg。阿霉素+葛花总黄酮各组的造模方法同阿霉素模型对照组,同时每天分别ig葛花总黄酮相应剂量,各组实验连续进行15 d。采用HE染色检测心肌组织结构变化,采用免疫组织法检测心肌组织凋亡相关蛋白Bax和Bcl-2表达情况,并进行组织学检查,观察心肌结构变化。结果与正常对照组比较,阿霉素(3 mg/kg,ip,7次)可致小鼠血清肌酸激酶(CK)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)和iNOS活力均显著升高(P<0.01),同时使心肌丙二醛(MDA)含量明显上升(P<0.01),而超氧化物歧化酶(SOD)活力则显著下降(P<0.01)。另外,小鼠心肌细胞凋亡数明显升高,细胞凋亡率达(40.5±5.2)%,凋亡相关蛋白Bax和Bcl-2含量均明显升高,与正常对照组比较差异有显著性(P<0.01),但Bcl-2/Bax值降低。葛花总黄酮(50、100、200 mg/kg,15 d)和Vit E组剂量依赖性的逆转阿霉素所致的上述改变,表现为降低小鼠血清CK、GOT、LDH和iNOS活力,降低心肌MDA含量和增加SOD活力,降低促凋亡基因Bax蛋白表达,使Bcl-2/Bax值增加,尤其以高剂量组作用最为明显。光镜和电镜结果对心肌形态学的观察也证实了葛花总黄酮对阿霉素中毒性心肌炎保护作用。结论阿霉素可以诱导心肌细胞凋亡,引起实验小鼠中毒性心肌炎。葛花总黄酮能拮抗阿霉素所致的心肌损伤,其作用机制与增强心肌SOD活力和抗心肌脂质过氧化、降低凋亡基因Bax蛋白表达等有关。

Objective To investigate the protective effect of flos puerariae flavonoid on adriamycin (ADR)-induced toxic myocarditis and its mechanisms from morphological,biochemical and molecular levels.Methods 96 healthy Kunming male mice were randomly divided into 6 groups:normal control group,ADR model control group,ADR+low dose of flos puerariae flavonoid group(50 mg/kg),ADR +middle dose of flos puerariae flavonoid group(100 mg/kg),ADR +high dose of flos puerariae flavonoid group(200 mg/kg),and Vit E positive control group(40 mg/kg),16 in each group.The drugs were orally administered for consecutive 15 d and the model of toxic myocarditis was induced by intraperitoneal injection of ADR(3 mg/kg)in mice from day 2,one time every other day,for 7 times Colorimetry was used to measure the changes of marker enzymes about myocardial injury and inducible nitric oxide synthase(iNOS)activity in serum and tissue;immunohistochemical method was adopted to detecte the expression of myocardial apoptosis related proteins Bax and bcl-2;HE staining was conducted to observe the pathological changes of cardiac structure.Results Compared with normal control group,ADR(3 mg/kg,ip,7 times)induced the elevation of serum creatine kinase (CK),lactate dehydrogenase (LDH),aspartate transaminase(GOT)and iNOS activity increased significantly in mice(P<0.01).Meanwhile myocardial superoxide dismutase(SOD)activity decreased, and the malondialdehyde(MDA)content increased(P<0.01).Myocardial cell apoptosis in mice increased significantly,and the apoptosis rate was(40.5 ±&nbsp;5.2)%;the expressions of Bax and Bcl-2 were significantly increased(P<0.01),However,the Bcl-2/Bax ratio decreased.The flos puerariae flavonoid (50,100,200 mg/kg,ig,15 d)and Vit E positive control group could reverse the changes induced by ADR,decrease serum CK,LDH,GOT and iNOS activities,increased myocardial SOD activity,lower MDA content and the expression of bax protein,and elevated Bcl-2/Bax ratio,in a dose-dependent manner.Light microscopy confirmed that flos puerariae flavonoid significantly alleviated the changes of myocardial microstructure.Conclusion ADR could induce myocardial cell apoptosis and lead toxic myocarditis in experimental mice.The flos puerariae flavonoid has protective effect on ADR-induced myocardial injury and the mechanism may be related to elevating myocardial SOD activity and anti-lipid peroxidation,inhibiting the expression of Bax protein and adriamycin-induced cardiomyocyte apoptosis.