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心房颤动(atrial fibrillation,AF)简称房颤,主要表现为心脏迅速而又不规则地跳动,是临床上最常见的一种心律失常,其发病率及致死率居高不下,危害性堪比恶性肿瘤,严重影响病人的生命质量[1]。心房纤维化的特征在于心肌成纤维细胞异常增殖所致的心肌细胞外基质(extracellular matrix, ECM)的异常积累[2],它作为AF发生的触发器,可引起心房电传导异常,更易形成折返,有利于AF的发作和维持,是AF发生、发展的病理基础[3]。二十二碳六烯酸(docosahexaenoic acid, DHA)属于ω-3多不饱和脂肪酸家族中的重要成员,在膳食鱼类中广泛存在。研究[4]表明,增加多不饱和脂肪酸的摄入可有效降低冠心病和心血管疾病事件发生的风险。最新研究[5]也证实,多不饱和脂肪酸可发挥抗AF的作用。丝裂原激活蛋白激酶磷酸酶(mitogen activates protein kinase phosphatase, MKP-1)是一种苏氨酸/酪氨酸磷酸酯酶,可调控丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPKs)及P38磷酸化状态。正常情况下,MAPKs和MKP-1处于动态平衡状态,这种平衡状态在决定细胞生存或凋亡方面起重要作用[6]。另有研究[7]发现, MAPK通路可介导成纤维细胞的增殖和迁移。而DHA是否通过影响P38 MAPK通路发挥抗AF的作用尚未见报道。本研究观察DHA对大鼠心房纤维化的影响,并探讨该作用是否与调控P38 MAPK通路介导的胶原表达有关。
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大鼠尾静脉注射乙酰胆碱-氯化钙混合液1~2 s后即出现典型的AF心电图(见图 1)。
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CON组和DHA组尾静脉注射0.9%氯化钠溶液,给药前后心电图无明显变化,未见典型AF心电图。AF组和DHA+AF组通过观察心电图,结果显示随着实验时间的增加,AF大鼠的AF持续时间逐渐延长,实验第4、10、17天,与AF组相比,DHA+AF组大鼠AF持续时间明显缩短(P<0.05~P<0.01)(见表 1)。
分组 第3天 第4天 第10天 第17天 AF组 6.20±1.6 11.8±3.1 28.8±2.4 41.0±2.7 DHA+AF组 7.40±1.1 9.20±1.8 16.6±2.5 17.6±1.7 t 1.95 2.29 11.13 23.19 P >0.05 < 0.05 < 0.01 < 0.01 表 1 AF组与DHA+AF组大鼠AF持续时间比较(x±s;ni=10)
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与CON组大鼠相比,AF组ERP明显缩短,DHA组及DHA+AF ERP组延长(P<0.01);与AF组相比,DHA+AF组大鼠ERP明显延长(P<0.01)(见表 2)。
分组 ERP/ms F P MS组内 CON组 80.0±2.6## 424.99 < 0.01 6.055 AF组 60.3 ±1.5** DHA组 95.7±1.5**## DHA+AF组 83.0±3.6**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 2 各组大鼠心房肌ERP比较(x±s;ni=12)
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与CON组相比,AF组大鼠心房肌细胞APD50和APD90明显缩短(P<0.01),DHA组与DHA+AF组延长(P<0.01);与AF组相比,DHA+AF组大鼠心房肌细胞APD50和APD90延长(P<0.01)(见表 3、4)。
分组 APD50 F P MS组内 CON组 0.99±0.08##▲▲ 86.74 < 0.01 0.006 AF组 1.22 ±0.10**## DHA组 0.88±0.04**▲▲ DHA+AF组 1.08±0.07**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 3 DHA对大鼠心房肌细胞APD50的影响(x±s;ni=24)
分组 APD90 F P MS组内 CON组 0.99±0.07##▲▲ 129.50 < 0.01 0.006 AF组 0.81±0.06**##▲▲ DHA组 1.20 ±0.08**## DHA+AF组 1.15±0.09**## q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 4 DHA对大鼠心房肌细胞APD90的影响(x±s;ni=24)
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与CON组相比,AF组Ⅰ型、Ⅲ型胶原表达明显增高(P<0.01),DHA组Ⅰ型、Ⅲ型胶原表达明显降低(P<0.01);与AF组相比,DHA、DHA+AF组Ⅰ型、Ⅲ型胶原表达均明显降低(P<0.01)(见表 5、6)。
分组 Ⅰ型胶原水平/(μg/L) F P MS组内 CON组 40.6±8.8##▲▲ 85.58 < 0.01 31.133 AF组 63.3±4.8**▲▲ DHA组 27.2±3.8**## DHA+AF组 43.9±3.1##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 5 DHA对大鼠心房肌Ⅰ型胶原表达的影响(x±s;ni=12)
分组 Ⅲ型胶原水平(μg/L) F P MS组内 CON组 4.89±0.85## 12.58 < 0.01 1.841 AF组 6.97±0.91**▲▲ DHA组 3.66 ±2.17## DHA+AF组 4.67±1.05## q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 6 DHA对大鼠心房肌Ⅲ型胶原表达的影响(x±s;ni=12)
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各组P38表达无明显变化,与CON组相比,DHA组P-P38表达明显下降,MKP-1表达明显增加(P<0.01);AF组P-P38表达明显增高,MKP-1表达明显降低(P<0.01);与AF组相比,DHA+AF组P-P38表达明显下降,MKP-1表达明显增加(P<0.01)(见表 7、8)。
分组 P-P38/P38 F P MS组内 CON组 0.60±0.05## 3.03 < 0.05 0.054 AF组 0.82±0.04**▲▲ DHA组 0.56 ±0.46## DHA+AF组 0.71±0.03**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 7 DHA对大鼠心房肌组织P-P38和P38蛋白表达的影响(x±s;ni=12)
项目分组 MKP-1/β-actin F P MS组内 CON组 0.70±0.06##▲▲ 125.90 < 0.01 0.003 AF组 0.46±0.04**▲▲ DHA组 0.87 ±0.06**## DHA+AF组 0.75±0.05##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 8 DHA对大鼠心房肌组织MKP-1蛋白表达的影响(x±s;ni=12)
二十二碳六烯酸介导MAPK途径抗房颤的作用及机制研究
Study on the effects and mechanism of MAPK pathway mediated by docosahexaenoic acid against atrial fibrillation
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摘要:
目的探讨二十二碳六烯酸(DHA)通过影响P38 MAPK途径调控胶原表达发挥抗房颤作用可能的机制。 方法将80只对乙酰胆碱(66 μg/mL)-氯化钙(50 mg/mL)混合液敏感的SD大鼠随机分为对照组(CON组)、对照DHA处理组(DHA组)、房颤组(AF组)和房颤DHA处理组(DHA+AF组),每组20只复制大鼠房颤模型。BL-420F描记心电图、连续双刺激法测定心房有效不应期(effective refractory period,ERP)、全细胞膜片钳技术记录心房肌细胞动作电位时程(action potential duration,APD)变化,酶免疫吸附法(ELISA)检测大鼠心房肌组织Ⅰ型、Ⅲ型胶原表达情况,蛋白印迹检测P38、P-P38及MKP-1表达情况。 结果大鼠房颤持续时间随实验时间增加而逐渐延长,而DHA可以缩短房颤持续时间(P < 0.05~P < 0.01)。AF组大鼠心房ERP、APD明显缩短、DHA可明显延长大鼠心房ERP、APD(P < 0.01);各组P38表达无明显变化,与CON组相比,AF组大鼠P-P38表达水平明显升高,MKP-1表达明显下降(P < 0.01);与AF组相比,DHA+AF组P-P38表达水平明显降低,MKP-1表达明显增加(P < 0.01)。ELISA结果证实,DHA可明显下调大鼠心房肌组织Ⅰ型、Ⅲ型胶原表达(P < 0.01)。 结论DHA可能通过调控P38 MAPK信号转导通路介导胶原表达发挥抗房颤作用。 Abstract:ObjectiveTo investigate the possible mechanism of docosahexaenoic acid(DHA) regulating collagen expression by affecting the P38 MAPK pathway to play an anti-atrial fibrillation. MethodsEighty SD rats sensitive to acetylcholine(66 μg/mL)-calcium chloride(50 mg/mL) mixture were randomly divided into the control group(CON group), control DHA treatment group(DHA group), atrial fibrillation group(AF group) and atrial fibrillation DHA treatment group(DHA+AF group)(20 rats in each group model of atrial fibrillation).The BL-420F tractive electrocardiogram and continuous double stimulation method were used to detect the atrial effective refractory period(ERP), the whole cell patch clamp technique was used to record the atrial muscle cell action potential duration(APD) change, the enzyme immunosorbent assay(ELISA) was used to detect the typeⅠ, Ⅲ collagen expression in atrial muscle tissue of rats, and the expression levels of P38 and P-PP38 and MKP 1 protein were detected using Western blotting. ResultsThe duration of atrial fibrillation in rats prolonged with the increasing of experimental time, while DHA could shorten the duration of atrial fibrillation(P < 0.05 to P < 0.01).The ERP and APD significantly shortened, while DHA could significantly prolong the atrial ERP and APD in AF group(P < 0.01).There was no significant change in P38 protein among each group.Compared with the CON group, the expression level of P-P38 significantly increased, while the expression level of MKP-1 decreased in AF group(P < 0.01). Compared with the AF group, the expression level of P-P38 significantly decreased, while the expression level of MKP-1 increased in the DHA+AF group(P < 0.01).The results of ELISA showed that DHA could obviously down-regulate the typeⅠand Ⅲ collagen expression(P < 0.01). ConclusionsDHA may play an anti-atrial fibrillation role by regulating P38 MAPK signal transduction pathway mediating collagen expression. -
Key words:
- atrial fibrillation /
- atrial fibrosis /
- docosahexaenoic acid /
- extracellular matrix /
- MAPK pathway
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表 1 AF组与DHA+AF组大鼠AF持续时间比较(x±s;ni=10)
分组 第3天 第4天 第10天 第17天 AF组 6.20±1.6 11.8±3.1 28.8±2.4 41.0±2.7 DHA+AF组 7.40±1.1 9.20±1.8 16.6±2.5 17.6±1.7 t 1.95 2.29 11.13 23.19 P >0.05 < 0.05 < 0.01 < 0.01 表 2 各组大鼠心房肌ERP比较(x±s;ni=12)
分组 ERP/ms F P MS组内 CON组 80.0±2.6## 424.99 < 0.01 6.055 AF组 60.3 ±1.5** DHA组 95.7±1.5**## DHA+AF组 83.0±3.6**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 3 DHA对大鼠心房肌细胞APD50的影响(x±s;ni=24)
分组 APD50 F P MS组内 CON组 0.99±0.08##▲▲ 86.74 < 0.01 0.006 AF组 1.22 ±0.10**## DHA组 0.88±0.04**▲▲ DHA+AF组 1.08±0.07**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 4 DHA对大鼠心房肌细胞APD90的影响(x±s;ni=24)
分组 APD90 F P MS组内 CON组 0.99±0.07##▲▲ 129.50 < 0.01 0.006 AF组 0.81±0.06**##▲▲ DHA组 1.20 ±0.08**## DHA+AF组 1.15±0.09**## q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 5 DHA对大鼠心房肌Ⅰ型胶原表达的影响(x±s;ni=12)
分组 Ⅰ型胶原水平/(μg/L) F P MS组内 CON组 40.6±8.8##▲▲ 85.58 < 0.01 31.133 AF组 63.3±4.8**▲▲ DHA组 27.2±3.8**## DHA+AF组 43.9±3.1##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 6 DHA对大鼠心房肌Ⅲ型胶原表达的影响(x±s;ni=12)
分组 Ⅲ型胶原水平(μg/L) F P MS组内 CON组 4.89±0.85## 12.58 < 0.01 1.841 AF组 6.97±0.91**▲▲ DHA组 3.66 ±2.17## DHA+AF组 4.67±1.05## q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 7 DHA对大鼠心房肌组织P-P38和P38蛋白表达的影响(x±s;ni=12)
分组 P-P38/P38 F P MS组内 CON组 0.60±0.05## 3.03 < 0.05 0.054 AF组 0.82±0.04**▲▲ DHA组 0.56 ±0.46## DHA+AF组 0.71±0.03**##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 表 8 DHA对大鼠心房肌组织MKP-1蛋白表达的影响(x±s;ni=12)
项目分组 MKP-1/β-actin F P MS组内 CON组 0.70±0.06##▲▲ 125.90 < 0.01 0.003 AF组 0.46±0.04**▲▲ DHA组 0.87 ±0.06**## DHA+AF组 0.75±0.05##▲▲ q检验:与CON组比较**P<0.01;与AF组比较##P<0.01;与DHA组比较▲▲P<0.01 -
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