毛蕊异黄酮对异丙肾上腺素诱导心肌肥厚的影响

Effects of calycosin on isoprenaline-induced cardiac hypertrophy

目的 探讨毛蕊异黄酮（CA）对异丙肾上腺素（ISO）诱导心肌肥厚的影响.方法 C57小鼠30只随机分为对照组（n=10）、ISO组（n=10）、CA＋ISO组（n=10）.ISO组小鼠腹腔注射ISO（50 mg·kg-1·d-1）,CA＋ISO组小鼠腹腔注射ISO（50 mg·kg-1·d-1）的同时进行CA（50 mg·kg-1·d-1）灌胃处理,对照组小鼠腹腔注射生理盐水（50 ml·kg-1·d-1）.处理14 d后测量体质量（BM）后处死小鼠,收集心脏标本,测量心质量（HM）、胫骨长（TL）和心肌细胞横截面积.实时定量PCR检测小鼠心脏组织心房钠尿肽（ANP）、β-肌球蛋白重链（β-MHC）mRNA表达,免疫印迹法进行心肌肥厚相关分子通路检测.将原代培养的大鼠心肌细胞分为对照组、ISO组和ISO＋CA组：对照组加入PBS,ISO组给予ISO（10μmol/L）处理,ISO＋CA组同时加入ISO（10μmol/L）和不同浓度的CA（1、10、50、100μmol/L）进行处理.48 h后收集细胞,实时定量PCR检测ANP mRNA表达,免疫荧光染色进行心肌细胞面积测量.结果 与对照组比较,ISO处理后小鼠心脏体积增大,HM/BM、HM/TL、心肌细胞横截面积均增加（均P〈0.05）;与ISO组比较,CA处理可降低小鼠心脏体积,减少HM/BM、HM/TL和心肌细胞横截面积（均P〈0.05）.实时定量PCR显示,与对照组比较,ISO处理后小鼠心肌肥厚标志物ANP和β-MHC mRNA表达上调,而CA干预能显著抑制ISO诱导的小鼠ANP和β-MHC mRNA表达升高（均P〈0.05）.免疫印迹显示,ISO处理后小鼠心脏组织蛋白激酶B（AKT）、糖原合成酶激酶3β（GSK3β）和细胞外信号调节激酶（ERK）的磷酸化水平明显上调,而CA干预后上述信号通路的活化受到明显抑制（均P〈0.05）.ISO处理48 h后,原代培养的大鼠心肌细胞面积明显增大、ANP mRNA表达升高（均P〈0.05）;与ISO组比较,10、50和100μmol/L CA处理可显著降低心肌细胞ANP mRNA表达,100μmol/L CA处理可显著减少心肌细胞面积（均P〈0.05）.结论 CA可抑制ISO诱导的心肌肥厚.

Objective To explore the effects of calycosin （CA） on isoprenaline （ISO）-induced cardiac hypertrophy. Methods Thirty C57 mice were randomly divided into the control group（n=10）,ISO group（n=10）,and CA＋ISO group（n=10）. The mice in ISO group received intraperitoneal injection with ISO（50 mg＆#183;kg-1＆#183;d-1）,those in CA＋ISO group received intraperitoneal injection with ISO（50 mg＆#183;kg-1＆#183;d-1） and intragastric gavage with CA （50 mg ＆#183; kg-1 ＆#183; d-1）,and those in control group received intraperitoneal injection with normal saline（50 ml ＆#183; kg-1 ＆#183; d-1）. At 14 days after the treatment,the mice were measured for body mass（BM）,sacrificed and harvested for cardiac specimens. The heart mass（HM）,tibial length（TL） and myocardial cell cross-sectional area were measured. Real-time quantitative PCR was used to determine the mRNA expression of atrial natriuretic peptide（ANP） and β-myosin heavy chain（β-MHC）. Immunoblotting was used to investigate the molecular pathway related to cardiac hypertrophy. Primary cultured rat cardiac myocytes were divided into the control group,ISO group and ISO＋CA group. The control group was added with PBS,ISO group treated with ISO（10μmol/L）,and ISO＋CA group added with ISO （10μmol/L）and different concentrations of CA（1,10,50,100μmol/L）. After 48 h,the myocardial cells were collected and determined for ANP mRNA expression by real-time quantitative PCR,and the area of myocardial cell was measured by immunofluorescence staining. Results Compared with the control group, the cardiac volume,HM/BM and HM/TL ratios,and cross-sectional area of myocardial cells were increased in those of the ISO group（all P〈0.05）. Compared with the ISO group,CA treatment was shown to reduce the cardiac volume,the ratios of HM/BM and HM/TL,and the cross-sectional area of myocardial cells（all P〈0.05）. Real-time quantitative PCR showed that the expressions of ANP andβ-MHC mRNA were up-regulated in ISO-treated mice as compared with the control group,whereas CA treatment significantly inhibited the ISO-induced up-regulation of ANP andβ-MHC mRNA expressions（all P〈0.05）. Immunoblotting showed that the phosphorylation levels of protein kinase B（AKT）,glycogen synthase kinase 3β（GSK3β）and extracellular signal-regulated kinase（ERK）were significantly upregulated in the cardiac tissues of ISO-treated mice,and the activation of these signal pathways was significantly inhibited by CA （all P〈0.05）. At 48 h after ISO treatment,the primary cultured rat cardiac myocytes showed significantly increased area and up-regulated level of ANP mRNA expression（both P〈0.05）. Compared with the ISO group,CA treatment at concentration of 10,50 and 100 μmol/L significantly reduced the ANP mRNA expressions in the cardiac myocytes;moreover, the myocyte area was significantly reduced with 100 μmol/L CA treatment （all P〈0.05）. Conclusion CA may inhibit ISO-induced cardiac hypertrophy.