丙氨酰谷氨酰胺对心肌缺血模型大鼠心肌纤维化及缝隙连接蛋白43重构的影响及其机制

摘要

目的：研究丙氨酰谷氨酰胺（Ala-Gln）诱导热休克蛋白70（HSP70）高表达对心肌缺血模型大鼠心肌纤维化及缝隙连接蛋白43（Cx43）重构的影响及其机制。　　方法：32只SPF级雄性SD大鼠（200±20）g，按随机数字表法随机等分为4组：对照组、盐酸异丙肾上腺素（ISO）[5mg/（kg·d）]组（模型组）、ISO[5mg/（kg·d）]+Ala-Gln [0.75mg/（kg·d）]组（干预组）、ISO[5mg/（kg·d）]+槲皮素[100mg/（kg·d）]+Ala-Gln [0.75mg/（kg·d）]+二甲基亚矾（DMSO）组（槲皮素组）。每组每天1次给予相应试剂，ISO连续给药7d后停止，余处理继续至第4周时处死大鼠。伊红素（HE）、Masson染色法观察心肌纤维化情况并计算胶原容积分数；免疫组织化学法观察心肌组织中HSP70、磷酸化细胞外信号调节激酶1/2（p-ERK1/2）及Cx43分布，并通过软件进行半定量分析。　　结果：伊红素、Masson染色结果显示对照组无明显心肌纤维化，模型组和槲皮素组出现明显的纤维化，而干预组较模型组和槲皮素组心肌纤维化程度均减弱（P均<0.01），干预组与对照组的心肌纤维化程度无明显差异（P>0.05）。对照组、模型组、槲皮素组中HSP70的含量差异无统计学意义（P>0.05），而干预组中HSP70的含量较上述各组明显升高,且差异有统计学意义（P均<0.01）。心肌组织中p-ERK1/2的含量干预组较模型组、槲皮素组均降低，差异有统计学意义（P均<0.01）。Cx43含量在对照组与干预组中差异无统计学意义且呈线性规律分布，干预组较模型组和槲皮素组均增加，差异有统计学意义（P均<0.01），分布无规律性，且侧面分布相对增多。　　结论：Ala-Gln诱导HSP70高表达可减轻ISO诱发的心肌缺血模型大鼠心肌纤维化程度和Cx43的重构，其机制可能与HSP70下调p-ERK1/2的表达，抑制细胞外信号调节激酶通路激活有关。%Objective: To investigate the effects of Ala-Gln induced heat shock protein 70 (HSP70) expression on myocardial ifbrosis and connexin43 (Cx43) remodeling in experimental rats’ model. Methods: A total of 32 SPF male SD rats were randomly divided into 4 groups:①Control group,②Model group, the rats received isoprenaline (ISO) 5m/(kg·d),③Intervention group, the rats received ISO+Ala-Gln 0.75mg/(kg·d),④Quercetin group, the rats received ISO+quercetin 100mg/(kg·d)+Ala-Gln+DMSO. n=8 in each group. All animals were treated for 7 days and killed in 4 weeks for relevant examinations. HE and Masson staining was conducted to observe myocardial ifbrosis, then calculate collagen volume fraction; immunohistochemistry was applied to measure myocardial HSP70 expression, extracellular signal regulated kinase (p-ERK1/2) and Cx43 distribution, then conduct semi-quantitative analysis by speciifc soft ware. Results: HE and Masson staining indicated that Control group had no obvious myocardial ifbrosis, Model group and Quercetin group had obvious ifbrosis, and Intervention group showed less ifbrosis than the other 2 groups, all P<0.01. The fibrosis level was similar between Intervention group and Control group, P>0.05. The myocardial HSP70 expression was similar among Control, Model and Quercetin groups, P>0.05, while HSP70 expression was signiifcantly higher in Intervention group than the other 3 groups, all P<0.01. The myocardial p-ERK1/2 level was lower in Intervention group than Model and Quercetin groups, all P<0.01. The myocardial Cx43 level was similar between Control group and Intervention group with linear distribution, while it was higher in Intervention group than Model and Quercetin groups with disordered distribution, all P<0.01. Conclusion: Ala-Gln inducing the higher expression of HSP70, which may reduce myocardial ifbrosis in experimental rats, it could be because of HSP70 down regulating p-ERK1/2 expression and inhibiting ERK signaling pathway.