目的:观察圣草次苷对大鼠心肌缺血再灌注损伤(myocardial ischemia-reperfusion injury,MIRI) 的保护作用,并探讨其作用机制。方法:取60 只健康SD 大鼠,随机分为假手术组、模型组以及圣草次苷低、中、高剂量组(8、16、32 mg·kg-1)、和地尔硫组,每组10 只,采用手术结扎法构建心肌缺血再灌注损伤模型;圣草次苷低、中、高剂量组以及地尔硫组于造模前1 周开始灌喂给药,模型组和假手术组给予等量生理盐水灌喂。再灌注结束后,通过苏木精-伊红(hematoxylin-eosin,HE)染色观察大鼠心肌组织病理改变;脱氧核糖核苷酸末端转移酶介导的脱氧三磷酸尿苷缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL)染色检测心肌细胞凋亡情况;采用酶联免疫吸附法检测血清肿瘤坏死因子α(tumor necrosis factor α,TNF-α)、白介素6(interleukin 6,IL-6)、白介素1β(interleukin 1β,IL-1β)、肌钙蛋白(cardiac troponin,cTnI)、肌红蛋白(myohemoglobin,Mb)、肌酸激酶同工酶(creatine kinase MB,CK-MB)含量水平;Western blot 检测心肌组织c-Myc、Bcl-2 相关X 蛋白(Bcl-2 associated X protein,Bax)、B 淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(caspase-3)、半胱氨酸天冬氨酸蛋白酶3 剪切体(Cleaved caspase-3)、JAK 激酶2(Janus kinase 2,JAK2)、磷酸化JAK2(phosphorylated JAK2,p-JAK2)、信号转导与转录激活因子3(signal transducer and activator oftranscription 3,STAT3)、磷酸化STAT3(phosphorylated STAT3,p-STAT3)蛋白表达。结果:与假手术组比较,模型组HR、LVEF、FS、LVSP、LVWT、c-Myc 水平显著降低(P<0.05), cTnI、Mb、CK-MB、TNF-α、IL-6、IL-1β、Bax/Bcl-2、Cleaved caspase-3/caspase-3、p-JAK2/JAK2、p-STAT3/STAT3 水平显著升高(P<0.05)。与模型组比较,圣草次苷 16、32 mg·kg-1 组及地尔硫卓组HR、LVEF、FS、LVSP、LVWT、c-Myc 水平显著升高(P<0.05),cTnI、Mb、CK-MB、TNF-α、IL-6、IL-1β、 Bax/Bcl-2、Cleaved caspase-3/caspase-3、p-JAK2/JAK2、p-STAT3/STAT3 水平显著降低(P<0.05)。结论:圣草次苷可能通过减少促炎细胞因子表达,同时调节JAK2/STAT3 信号通路抑制心肌细胞凋亡,在MIRI 中发挥心肌保护作用。
Objective: To investigate the protective effect of eriocitrin from myocardial ischemia-reperfusion injury (MIRI) in rats,and explore its action mechanism. Methods: Sixty healthy SD rats were collected and randomly divided into sham operation group,model group,low-dose,medium-dose,high-dose eriocitrin groups (erioctrin 8,16,32 mg·kg-1) and diltiazem group,with 10 rats per group. MIRI model was constructed by surgical ligation method. The low-dose,medium-dose,high-dose eriocitrin groups and diltiazem group were given administration at 1 week before modeling,while model group and sham group were given the same volume of normal saline. After reperfusion,pathological changes of myocardial tissues were observed by hematoxylin-eosin (HE) staining. The apoptosis of cardiomyocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The levels of serum tumor necrosis factor α (TNF-α),interleukin 6 (IL-6), interleukin 1β (IL-1β),cardiac troponin I (cTnI),myoglobin (Mb) and creatine kinase MB (CK-MB) were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of c-Myc,Bcl-2 associated X protein (Bax),B-cell lymphoma-2 (Bcl-2),cysteinyl aspartate specific protease-3 (caspase-3),cleaved caspase-3, Janus kinase 2 (JAK2),phosphorylated JAK2 (p-JAK2),signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) proteins in myocardial tissues were detected by Western blot. Results: compared with sham group,levels of HR,LVEF,FS,LVSP,LVWT and c-Myc were significantly decreased (P<0.05), while cTnI,Mb,CK-MB,TNF-α, IL-6, IL-1β, Bax/Bcl-2, cleaved caspase-3/caspase-3, p-JAK2/JAK2 and p-STAT3/STAT3 levels were significantly increased in model group (P<0.05). Compared with model group,levels of HR,LVEF,FS,LVSP,LVWT and c-Myc were significantly increased (P<0.05), while cTnI, Mb, CK-MB,TNF-α, IL-6, IL-1β, Bax/Bcl-2, cleaved caspase-3/caspase-3, p-JAK2/JAK2 and p-STAT3/STAT3 levels were significantly decreased in 16 mg·kg-1 and 32 mg·kg-1 eriocitrin groups,and diltiazem group (P<0.05). Conclusion: Eriocitrin may play a myocardial protection role in MIRI by reducing the expressions of pro-inflammatory cytokines and simultaneously regulating JAK2/STAT3 signaling pathways to inhibit myocardial apoptosis.
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