丹参酮ⅡA通过半乳糖凝集素-3抑制心肌重塑的机制研究

Mechanism of tanshinoneⅡA inhibiting myocardial remodeling by Galectin-3

目的分析丹参酮ⅡA对缺血/再灌注(I/R)所致心力衰竭(心衰)模型大鼠心肌重构的影响。方法①体内实验:将30只健康雄性SD大鼠按随机数字表法分为假手术组、心衰模型组和丹参酮ⅡA组,每组10只。采用结扎大鼠左冠状动脉(冠脉)待心电监护出现明显ST段抬高30 min后放松结扎线进行再灌注2 h来制备I/R后心衰模型;假手术组于同期开胸,仅丝线绕过左冠脉而不结扎。丹参酮ⅡA组术后3 d开始腹腔注射丹参酮ⅡA 10 mg/kg,连续用药9周;其他两组于同期腹腔注射等量生理盐水。干预9周后观察各组大鼠的行为学表现,检测血流动力学相关指标。处死大鼠取心脏组织,Masson染色后光镜下观察心肌组织纤维化程度;采用酶联免疫吸附试验(ELISA)检测半乳糖凝集素-3(Galectin-3)含量;采用荧光定量反转录-聚合酶链反应(qRT-PCR)检测Ⅰ型胶原、Ⅲ型胶原、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶抑制剂-1(TIMP-1)的mRNA表达;采用蛋白质免疫印迹试验(Western blotting)检测MMP-2和TIMP-1的蛋白表达。②体外实验:提取并分离大鼠的原代心肌成纤维细胞,分为空白对照组、血管紧张素Ⅱ组(给予7~10 mmol/L血管紧张素Ⅱ)和血管紧张素Ⅱ+丹参酮ⅡA组(同时给予7~10 mmol/L血管紧张素Ⅱ+5~10 mmol/L丹参酮ⅡA)。分别于培养24 h和48 h时,采用四甲基偶氮唑蓝(MTT)检测细胞吸光度(A)值并计算细胞增殖率;采用qRT-PCR法检测型胶原、型胶原、MMP-2和TIMP-1的mRNA表达;采用ELISA法检测Galectin-3含量。结果①体内实验:大鼠活动状态、毛发顺帖程度及进食量由好到差依次为假手术组、丹参酮ⅡA组和心衰模型组。与假手术组相比,心衰模型组大鼠心率(HR)明显下降、心功能明显受损,Ⅰ型胶原、Ⅲ型胶原、TIMP-1的mRNA和蛋白表达及Galectin-3含量均明显升高,MMP-2的mRNA和蛋白表达均明显降低。与心衰模型组相比,丹参酮ⅡA组大鼠HR明显升高、心功能明显改善,Ⅰ型胶原、Ⅲ型胶原的mRNA表达均明显降低,TIMP-1的mRNA和蛋白表达及Galectin-3含量均明显降低,MMP-2的mRNA和蛋白表达均明显升高,以制模9周变化最为明显〔Ⅰ型胶原mRNA(2^(-ΔΔCt)):4.70±1.19比10.21±1.62,Ⅲ型胶原mRNA(2^(-ΔΔCt)):3.03±0.46比13.84±1.93,TIMP-1 mRNA(2^(-ΔΔCt)):1.90±0.19比4.55±0.43,TIMP-1/GAPDH:0.33±0.04比0.67±0.05,Galectin-3(ng/L):489.93±79.30比821.72±94.09,MMP-2 mRNA(2^(-ΔΔCt)):0.37±0.07比0.03±0.01,MMP-2/GAPDH:0.69±0.09比0.21±0.04,均P<0.05〕。Masson染色显示,心衰模型组大鼠心肌组织出现明显纤维化,而丹参酮ⅡA干预能够改善大鼠心肌组织的纤维化。②体外实验:与空白对照组相比,血管紧张素组培养24 h和48 h心肌成纤维细胞增殖率、Ⅰ型胶原、Ⅲ型胶原和TIMP-1表达及Galectin-3含量均明显升高,MMP-2表达明显降低。与血管紧张素组相比,血管紧张素+丹参酮ⅡA组心肌成纤维细胞增殖率及Ⅰ型胶原、Ⅲ型胶原和TIMP-1表达及Galectin-3含量均明显降低,MMP-2 mRNA表达明显升高,以培养48 h改变最明显〔细胞增殖率:(57.0±3.7)%比(67.0±2.4)%,Ⅰ型胶原mRNA(2^(-ΔΔCt)):551.43±67.10比871.48±12.25,Ⅲ型胶原mRNA(2^(-ΔΔCt)):233.76±18.73比385.51±31.35,TIMP-1 mRNA(2^(-ΔΔCt)):238.69±17.37比351.84±26.17,Galectin-3(ng/L):283.76±28.73比415.51±31.35,MMP-2 mRNA(2^(-ΔΔCt)):108.54±12.10比51.47±6.25,均P<0.05]。结论丹参酮ⅡA可以改善I/R后心衰大鼠的心功能,抑制心肌纤维化,改善心衰大鼠的心肌重构。

Objective To explore the effect of tanshinoneⅡA on myocardial remodeling in ischemia/reperfusion(I/R)-induced heart failure of rodent model.Methods①In vivo,30 SD rats were randomly divided into sham operation,heart failure and tanshinoneⅡA treatment group,with 10 rats in each group.The I/R model was established by ligating the left coronary artery until ST segment elevation for 30 minutes,then the ligation was removed for 2 hours as reperfusion.In the sham operation group,the rat chest was opened without artery ligation.Three days after model establishment,tanshinoneⅡA(10 mg/kg)were given intraperitoneal injected in tanshinoneⅡA group for 9 weeks.In the other two groups,normal saline was administrated in the same way.The behavioral manifestations of the rats in each group were observed;hemodynamic indexes were evaluated;Masson staining was performed to observe the degree of myocardial fibrosis;enzyme linked immunosorbent assay(ELISA)was used to detect the content of Galectin-3 in myocardial tissue;quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the expressions of collagenⅢ,collagenⅠ,matrix metalloproteinase 2(MMP-2)and tissue inhibitor of metalloproteinase(TIMP-1).②In vitro,rats primary cardiac fibroblasts were extracted and isolated,and divided into blank control group,angiotensinⅡgroup(7-10 mmol/L angiotensinⅡ)and angiotensinⅡ+tanshinoneⅡA group(7-10 mmol/L angiotensinⅡ+5-10 mmol/L tanshinoneⅡA).At 24 hours and 48 hours of culture,the cell proliferation in each group was detected by methyl thiazolyl tetrazolium(MTT);the expressions of collagenⅢ,collagenⅠ,MMP-2 and TIMP-1 were detected by qRT-PCR;the content of Galectin-3 in cardiac fibroblasts was detected by ELSIA.Results①In vivo,the rats'activity status,hair conformity and food intake were ranked from good to bad in order of sham operation group,tanshinoneⅡA group and heart failure model group.Compared with the sham-operated group,the heart rate(HR)of the rats in the heart failure model group was significantly decreased and the heart function was significantly impaired.The mRNA and protein expression of collagenⅠ,collagenⅢ,TIMP-1 and Galectin-3 content were significantly increased,while the mRNA and protein expression of MMP-2 were significantly decreased.Compared with the heart failure model group,rats in the tanshinoneⅡA group showed significantly higher HR and improved cardiac function,significantly lower mRNA expression of collagenⅠand collagenⅢ,significantly lower mRNA and protein expression of TIMP-1 and Galectin-3,and significantly higher mRNA and protein expression of MMP-2,and the most obvious changes were in the 9th weeks of modeling[collagenⅠmRNA(2^(-ΔΔCt)):4.70±1.19 vs.10.21±1.62,collagenⅢmRNA(2^(-ΔΔCt)):3.03±0.46 vs.13.84±1.93,TIMP-1 mRNA(2^(-ΔΔCt)):1.90±0.19 vs.4.55±0.43,TIMP-1/GAPDH:0.33±0.04 vs.0.67±0.05,Galectin-3(ng/L):489.93±79.30 vs.821.72±94.09,MMP-2 mRNA(2^(-ΔΔCt)):0.37±0.07 vs.0.03±0.01,MMP-2/GAPDH:0.69±0.09 vs.0.21±0.04,all P<0.05].Masson staining showed that myocardial tissue fibrosis was obvious in the heart failure group,and the degree of fibrosis in the tanshinoneⅡA group was reduced.②In vitro,compared with the blank control group,the proliferation rate,collagenⅠ,collagenⅢand TIMP-1 expression and Galectin-3 content of myocardial fibroblasts were significantly increased,and MMP-2 expression was significantly decreased in the angiotensin group at 24 h and 48 h of culture.Compared with the angiotensin group,the proliferation rate of cardiac fibroblasts and the expression of collagenⅠ,collagenⅢand TIMP-1 and the content of Galectin-3 were significantly decreased,and the expression of MMP-2 mRNA was significantly increased in the angiotensin+tanshinoneⅡA group,and the most significant changes were at 48 hours of culture[proliferation rate:(57.0±3.7)%vs.(67.0±2.4)%,collagenⅠmRNA(2^(-ΔΔCt)):551.43±67.10 vs.871.48±12.25,collagenⅢmRNA(2^(-ΔΔCt)):233.76±18.73 vs.385.51±31.35,TIMP-1 mRNA(2^(-ΔΔCt)):238.69±17.37 vs.351.84±26.17,Galectin-3(ng/L):283.76±28.73 vs.415.51±31.35,MMP-2 mRNA(2^(-ΔΔCt)):108.54±12.10 vs.51.47±6.25,all P<0.05].Conclusion TanshinoneⅡA can improve cardiac function,inhibit myocardial fibrosis and improve myocardial remodeling in rats with I/R-induced heart failure.