Effect of Yiqi Huoxue Lishui Recipe on calcium transport and endoplasmic reticulum stress in cardiomyocytes f rats with heart failure
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摘要:目的
探讨益气活血利水方对心力衰竭(HF)大鼠心肌细胞钙转运、内质网应激凋亡及血管紧张素Ⅱ(AngⅡ)、转化生长因子β1(TGF-β1)表达的影响。
方法将10只健康大鼠设为Sham组，另将45只建模成功大鼠分为HF组、低干预组、中干预组、高干预组和西药组，每组9只。低干预组、中干预组、高干预组分别灌胃2.6、5.2、10.4 g/(kg·d)益气活血利水溶液，西药组灌胃10.0 mg/(kg·d)曲美他嗪, Sham组和HF组分别灌胃等体积0.9%NaCl溶液。采用苏木精-伊红(HE)染色法及电镜分别观察各组大鼠心肌组织病理形态; 采用Bradford染料结合法分析心肌肌质网中钙摄取量; 采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法检测各组大鼠心肌细胞凋亡情况; 采用免疫印迹法检测心肌组织中内质网应激相关蛋白[B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2关联X蛋白(Bax)、半胱天冬酶-3(Caspase3)蛋白]和钙运转相关蛋白[钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、兰尼碱受体2(RyR2)、肌浆网钙离子ATP酶2a(SERCA2a)]表达水平; 采用免疫组织化学法检测心肌组织中TGF-β1、AngⅡ阳性表达率。
结果Sham组、HF组、低干预组、中干预组、高干预组、西药组大鼠心肌肌质网钙摄取速率分别为(19.63±5.63)、(10.25±4.11)、(13.70±5.12)、(14.08±4.80)、(16.91±5.03)、(16.65±5.10) μmol/(mg·min), 多组间比较差异有统计学意义(F=3.946, P=0.004); Sham组、HF组、低干预组、中干预组、高干预组、西药组大鼠心肌细胞凋亡率分别为(7.52±2.01)%、(30.63±6.58)%、(19.14±5.03)%、(18.41±5.21)%、(13.61±3.65)%、(13.17±4.11)%, 多组间比较差异有统计学意义(F=28.900，P < 0.001)。与Sham组比较, HF组大鼠心肌组织Bcl-2、SERCA2a表达均降低, Bax、Caspase3、CaMKⅡ、RyR2、TGF-β1、AngⅡ表达均升高，差异有统计学意义(P < 0.05); 与HF组比较，低干预组、中干预组、高干预组、西药组Bcl-2、SERCA2a表达均升高, Bax、Caspase3、CaMKⅡ、RyR2、TGF-β1、AngⅡ表达均降低，差异有统计学意义(P < 0.05); 低干预组与中干预组上述8项指标比较，差异无统计学意义(P>0.05); 高干预组上述8项指标与中干预组比较，差异有统计学意义(P < 0.05), 但与西药组比较，差异无统计学意义(P>0.05)。
结论益气活血利水方可改善HF大鼠心肌细胞钙转运，通过抑制内质网应激减少心肌细胞凋亡，并呈现浓度依赖性，其机制可能与抑制TGF-β1、AngⅡ表达有关。
Abstract:ObjectiveTo investigate the effects of Yiqi Huoxue Lishui Recipe on calcium transport, endoplasmic reticulum stress apoptosis and the expressions of angiotensin Ⅱ (Ang Ⅱ) and transforming growth factor-β1 (TGF-β1) in cardiomyocytes of heart failure (HF) rats.
MethodsTen healthy rats were set as Sham group, and another 45 successfully modeled rats were divided into HF group, low intervention group, medium intervention group, high intervention group and western medicine group, with 9 rats in each group. The low intervention group, middle intervention group and high intervention group were gavaged with 2.6, 5.2 and 10.4 g/(kg·d) of Yiqi Huoxue Lishui Recipe, the western medicine group was gavaged with 10 mg/(kg·d) trimetazidine, and the Sham and HF groups were gavaged with the same volume of 0.9% NaCl solution, respectively. HE staining and electron microscopy were used to observe the pathological morphology of myocardial tissue. Bradford dye binding method was used to analyze the calcium intake in the myocardial sarcoplasmic reticulum. The apoptosis of cardiomyocytes in each group was detected by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL). Western blotting was used to detect the expressions of endoplasmic reticulum stress-related proteins[B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-3 protein (Caspase3)]and calcium transport-related proteins[calcium/calmodulin-dependent protein kinase Ⅱ (CAMK Ⅱ), ryanodine receptor (RyR2) and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a); the positive expression rates of TGF-β1 and Ang Ⅱ in myocardial tissue were detected by immunohistochemistry.
ResultsThe calcium uptake rates of rats in the Sham group, HF group, low intervention group, middle intervention group, high intervention group and western medicine group were (19.63±5.63) μmol/(min·mg), (10.25±4.11) μmol/(min·mg), (13.70±5.12) μmol/(min·mg), (14.08±4.80) μmol/(min·mg), (16.91±5.03) μmol/(min·mg), (16.65±5.10) μmol/(min·mg), respectively, there were differences among different groups (F=3.946, P=0.004). The apoptosis rates of cardiomyocytes in the Sham group, HF group, low intervention group, medium intervention group, high intervention group and western medicine group were (7.52±2.01)%, (30.63±6.58)%, (19.14±5.03)%, (18.41±5.21)%, (13.61±3.65)% and (13.17±4.11)%, respectively, there were differences among different groups (F=28.900, P < 0.001); compared with the Sham group, Bcl-2 and SERCA2a levels in the HF group in myocardial tissue of rats were decreased, and Bax, Caspase3, CaMK Ⅱ, RyR2, TGF-β1 and Ang Ⅱ expression were increased (P < 0.05). Compared with the HF group, expressions of Bcl-2 and SERCA2a were increased in the low, medium and high intervention groups and western medicine group, Bax, Caspase3, CaMK Ⅱ, RyR2, TGF-β1 and Ang Ⅱ were all decreased (P < 0.05). There were no significant differences in the low intervention group in above eight indicators compared with the medium intervention group (P>0.05). Compared with the medium intervention group, the high intervention group showed significant differences in above indexes (P < 0.05), but there was no significant differences compared with the western medicine group in above indexes (P>0.05).
ConclusionYiqi Huoxue Lishui Formula can improve calcium transport in heart failure rats, and reduce myocardial apoptosis by inhibiting endoplasmic reticulum stress in a concentration-dependent manner. The mechanism may be related to the inhibition of TGF-β1 and Ang Ⅱ expression.
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图 3 各组大鼠心肌肌质网钙摄取速率比较
与Sham组比较, *P < 0.05; 与HF组比较, #P < 0.05; 与低干预组比较, △P < 0.05; 与中干预组比较, ▲P < 0.05。
表 1 各组大鼠内质网应激相关蛋白Bcl-2、Bax、Caspase3表达量比较(x±s)
组别 n Bcl-2 Bax Caspase3 Sham组 10 1.25±0.11 0.52±0.04 0.25±0.05 HF组 9 0.65±0.08* 1.54±0.17* 0.96±0.07* 低干预组 9 0.87±0.10*# 1.14±0.11*# 0.72±0.10*# 中干预组 9 0.90±0.06*# 1.12±0.08*# 0.68±0.14*# 高干预组 9 1.10±0.09*#△▲ 0.90±0.06*#△▲ 0.44±0.06*#△▲ 西药组 9 1.07±0.11*#△▲ 0.92±0.08*#△▲ 0.42±0.05*#△▲ F — 47.300 111.100 86.310 P — < 0.001 < 0.001 < 0.001 Bcl-2: B细胞淋巴瘤/白血病-2; Bax: Bcl-2关联X蛋白; Caspase3: 半胱天冬酶-3。与Sham组比较, *P < 0.05;
与HF组比较, #P < 0.05; 与低干预组比较, △P < 0.05; 与中干预组比较, ▲P < 0.05。
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表 2 各组大鼠钙转运蛋白CaMKⅡ、RyR2、SERCA2a表达量比较(x±s)
组别 n CaMKⅡ RyR2 SERCA2a Sham组 10 0.16±0.05 0.34±0.07 1.08±0.07 HF组 9 0.97±0.15* 1.12±0.12* 0.47±0.04* 低干预组 9 0.60±0.07*# 0.84±0.05*# 0.62±0.07*# 中干预组 9 0.58±0.08*# 0.80±0.06*# 0.65±0.04*# 高干预组 9 0.30±0.04*#△▲ 0.51±0.10*#△▲ 0.89±0.11*#△▲ 西药组 9 0.29±0.03*#△▲ 0.49±0.13*#△▲ 0.90±0.08*#△▲ F — 125.900 88.970 90.940 P — < 0.001 < 0.001 < 0.001 CaMKⅡ: 钙/钙调蛋白依赖性蛋白激酶Ⅱ; RyR2: 兰尼碱受体2; SERCA2a: 肌浆网钙离子ATP酶2a。
与Sham组比较, *P < 0.05; 与HF组比较, #P < 0.05; 与低干预组比较, △P < 0.05; 与中干预组比较, ▲P < 0.05。
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表 3 各组大鼠心肌组织TGF-β1、AngⅡ阳性表达率比较(x±s)
% 组别 n TGF-β1 AngⅡ Sham组 10 6.12±1.25 4.20±0.54 HF组 9 25.47±4.17* 15.56±3.41* 低干预组 9 17.33±2.54*# 12.25±2.14*# 中干预组 9 16.95±3.02*# 11.47±2.05*# 高干预组 9 11.24±2.20*#△▲ 8.55±1.28*#△▲ 西药组 9 10.55±2.31*#△▲ 9.02±1.17*#△▲ F — 58.590 36.470 P — < 0.001 < 0.001 AngⅡ: 血管紧张素Ⅱ; TGF-β1: 转化生长因子β1。
与Sham组比较, *P < 0.05; 与HF组比较, #P < 0.05;
与低干预组比较, △P < 0.05; 与中干预组比较, ▲P < 0.05。
下载: 导出CSV
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