环磷酰胺致小鼠心脏功能损伤超声评价及其与血清外泌体miRNA的相关性

Serum exosomal miRNA change and its correlation with cardiotoxicity analyzed by echocardiography in cytoxan treated mice

目的分析环磷酰胺心脏毒性小鼠模型血清外泌体miRNA的改变,为更准确的评估环磷酰胺导致心脏功能损伤的程度提供新方法。方法选用7~8周龄的C57BL/6小鼠,随机分为对照组和环磷酰胺组2组。环磷酰胺组小鼠给予环磷酰胺100 mg/(kg·周),根据临床化疗方案,连续腹腔注射2周后,休息1周,再连续注射2周,共5周;对照组小鼠腹腔注射等量0.9%氯化钠溶液。第5周进行小鼠心脏超声检查,测量或计算左心室射血分数(LVEF)、短轴缩短率(FS)、左心室等容舒张期时间(IVRT)及Tei指数,同时行血清外泌体候选miRNA的q-PCR检测。采用t检验比较环磷酰胺组与对照组小鼠LVEF、FS、IVRT、Tei指数、血清外泌体miRNA表达量。结果与对照组小鼠比较,环磷酰胺组小鼠LVEF、FS均降低[0.62±0.05 vs 0.72±0.06,(28.00±3.10)%vs(35.10±4.95)%],且差异均有统计学意义(t=3.591,P=0.003;t=3.453,P=0.004);Tei指数、IVRT均升高[0.71±0.17 vs 0.59±0.14,(27.42±8.42)ms vs(23.40±6.70)ms],但差异无统计学意义。与对照组小鼠比较,环磷酰胺组小鼠血清外泌体中mi R-133a-3p、mi R-146a-5p表达增高(1.931±1.460 vs 0.072±0.077,1.895±1.059 vs 0.790±0.296),且差异均有统计学意义(t=4.072,P=0.001;t=3.168,P=0.006);mi R-30c-5p、mi R-99a-5p表达差异无统计学意义(0.870±0.327 vs0.530±0.670,0.404±0.134 vs 0.714±0.404)。结论 C57BL/6小鼠腹腔注射环磷酰胺能够引起心脏功能明显损伤,心脏功能受损小鼠血清外泌体中mi R-133a-3p和mi R-146a-5p表达增高,使其有望成为诊断环磷酰胺心脏功能受损新的血清标志物。

Objective To analyze the serum exosomal miRNA change in mice with cytoxan induced cardiotoxicity assessed by echocardiography,and then to explore its possibility in evaluating the cytoxan related cardiotoxicity.Methods C57BL/6 mice aged at 7-8 weeks were randomly divided into two groups,one with control treatment while the other with cytoxan treatment.Cytoxan treated mice were given cyclophosphamide 100 mg/(kg?week),according to the clinical chemotherapy regimen,continuous intraperitoneal injection for 2 weeks,rest 1 week,and then continuous injection for 2 weeks,a total of 5 weeks;Control mice were injected intraperitoneally with an equal volume of 0.9%sodium chloride solution.Five weeks after initial treatment,mice were subjected to echocardiography for cardiac function analysis,maesuring or calculating left ventricular ejection fraction(LVEF),short-axis fractional shortening(FS),left ventricular isovolumic relaxation time(IVRT)and Tei index.Serum exosome isolation were also performed,exosomal miRNA candidates were analyzed by qPCR.The t test was used to compare LVEF,FS,IVRT,Tei index,and serum exosomal miRNA expression levels between the cytoxan treated group and the control group.Results Compared with the control mice,cytoxan treated mice had decreased LVEF and FS[0.62±0.05 vs 0.72±0.06,(28.00±3.10)%vs(35.10±4.95)%],and the difference was statistically significant(t=3.591,P=0.003;t=3.453,P=0.004);The Tei index and IVRT were increased[0.71±0.17 vs 0.59±0.14,(27.42±8.42)ms vs(23.40±6.70)ms],but the difference was not statistically significant.Compared with the control mice,the expression of miR-133a-3p and miR-146a-5p in serum exosomes increased in the cytoxan treated group(1.931±1.460 vs 0.072±0.077,1.895±1.059 vs 0.790±0.296),and the differences were statistically significant(t=4.072,P=0.001;t=3.168,P=0.006);There was no significant difference in the expression of miR-30c-5p and miR-99a-5p(0.870±0.327 vs 0.530±0.670,0.404±0.134 vs 0.714±0.404).Conclusions Cytoxan treatment has an obvious cardiotoxicity in mice,and miR-133a-3p and miR-146a-5p increase.This study suggests that these exosomal miRNAs might serve as a biomarker for cytoxan related cardiotoxicity.