熊果酸调控外泌体介导的miR-155-5p保护脓毒症大鼠心肌组织的作用研究

Effect of Ursolic Acid Regulating Exosome Mediated miR-155-5p on Myocardial Protection in Septic Rats

目的研究熊果酸通过外泌体介导微小核糖核酸-155-5p(micro ribonucleic acid-155-5p,miR-155-5p)对脓毒症大鼠心肌的保护作用。方法从60只SD雄鼠中随机挑出50只为建模大鼠,通过腹腔注射内毒素(lipopolysaccharide,LPS)法建立脓毒症大鼠模型。余下10只为正常对照组。将建模成功大鼠随机分为5组,熊果酸高、中、低剂量组、GW4869组、模型组。熊果酸高、中、低剂量组腹腔注射5 mg/kg LPS+50%二甲基亚砜(dimethylsurfoxide,DMSO)后立即尾静脉注射熊果酸120、80、40 mg/kg+50%DMSO混合液;GW4869组腹腔注射5 mg/kg LPS+50%DMSO后立即尾静脉注射鞘磷脂酶抑制剂GW48692.5μg/g+50%DMSO混合液;模型组大鼠腹腔注射5 mg/kg LPS+50%DMSO后,尾静脉注射等体积生理盐水;正常对照组仅腹腔注射等体积生理盐水后,尾静脉注射等体积生理盐水。另将各组外泌体与原代培养乳鼠心肌细胞共培养,设置未加外泌体仅加入等体积PBS的心肌细胞为PBS对照组;通过脂质体转染技术将miR-155-5p minic转染至大鼠心肌细胞,将转染miR-155-5p心肌细胞与动物实验中各组外泌体共培养24 h,另设置未转染miR-155-5p minic且不加外泌体仅加入等体积PBS的心肌细胞为PBS对照组,仅转染miR-155-5p minic且不加外泌体的心肌细胞为miRNA-155-5p minic转染组和GW4869+熊果酸+miR-155-5p minic组(其中熊果酸分别设置低、中、高剂量)。结果模型组和GW4869组大鼠心肌细胞出现明显空泡样变、核肿胀,组织水肿明显,可见明显炎性浸润,熊果酸3剂量组大鼠心肌组织病变均出现好转;各组均从外周血中分离出外泌体;模型组和熊果酸3剂量组外泌体中miR-155-5p表达水平均高于正常对照组(P<0.05),GW4869组外泌体中miR155-5p表达水平低于正常对照组(P<0.05);经培养鉴定证实所取细胞为乳鼠心肌细胞;模型组、GW4869组、熊果酸3剂量组白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)水平均高于磷酸盐缓冲液(phosphate buffered saline,PBS)对照组和正常对照组(P<0.05),熊果酸3剂量组上述指标水平均低于模型组和GW4869组(P<0.05);miR-155-5p minic转染乳鼠心肌细胞后miR-155-5p minic转染组细胞中miR-155-5p表达水平升高(P<0.05)。将各组外泌体与miR-155-5p minic转染后细胞共孵育,GW4869组细胞培养基中IL-1β、IL-6浓度均高于其余各组(P<0.05),模型组+miR-155-5p minic均高于miR-155-5p minic转染组(P<0.05),GW4869+熊果酸高剂量+miR-155-5p minic组与miR-155-5p minic转染组差异均无统计学意义(P>0.05)。结论熊果酸可改善脓毒症大鼠心肌病变,且可通过抑制外泌体中miR-155-5p表达水平,抑制心肌细胞炎症反应。

Objective To study the protective effect of ursolic acid on myocardium in septic rats through exosomes mediated micro ribonucleic acid-155-5p(miR-155-5p).Methods 50 rats were randomly selected from 60 male SD rats to be modeled,and the sepsis rat model was established by intraperitoneal injection of lipopolysaccharide(LPS).The remaining 10 were counted as normal control group.Model rats were randomly divided into 4 groups,ursolic acid high,medium and low dose groups,and model group,GW4869 group.Ursolic acid high,medium and low dose groups were injected intraperitoneally with 5 mg/kg LPS+50%dimethylsurfoxide(DMSO)and then tail vein injection of ursolic acid 120,80,40 mg/kg+50%DMSO mixture immediately.The model group was intraperitoneal injection of 5 mg/kg LPS+50%DMSO,and then tail vein injection of an equal volume of normal saline immediately.The GW4869 group as intraperitoneal injection of 5 mg/kg LPS+50%DMSO,and then tail vein injection of sphingomyelinase inhibitor GW48692.5μg/g+50%DMSO mixture immediately.The normal control group was intraperitoneal injection of same volume normal saline and then tail vein injection of an equal volume of normal saline.In addition,the exosomes of each group were co cultured with primary cultured neonatal rat cardiomyocytes,and the cardiomyocytes without exosomes and only with equal volume PBS were set as PBS control group,miR-155-5p minic was transfected into rat cardiomyocytes by liposome transfection technology.The transfected miR-155-5p cardiomyocytes were co cultured with the exosomes of each group in the animal experiment for 24 hours.In addition,cardiomyocytes without miR-155-5p minic and without exosomes were set as PBS control group,miR-155-5p minic without exosomes were the miR-155-5p minic transfection group and GW4869+ursolic acid+miR-155-5p minic group(low,medium and high doses of ursolic acid were set respectively).Results The cardiomyocytes of the model group and GW4869 group showed obvious vacuolar degeneration,nuclear swelling,obvious tissue edema,and obvious inflammatory infiltration.The myocardial tissue lesions of rats in the ursolic acid 3 dose groups were improved.Exosomes were isolated from peripheral blood in each group.The expression levels of miR-155-5p in exosomes of model group and ursolic acid 3-dose group were higher than those of normal control group(P<0.05),and the expression level of miR-155-5p in exosomes of GW4869 group was lower than those of normal control group(P<0.05).The cultured cells were identified as neonatal rat cardiomyocytes.The levels of interleukin-1β(IL-1β)and interleukin-6(IL-6)in the model group,GW4869 group,ursolic acid 3 dose groups were higher than those of the phosphate buffered saline(PBS)group and the normal control group(P<0.05),and the above indexes in ursolic acid 3 dose group were lower than those in model group and GW4869 group(P<0.05).After miR-155-5p minic was transfected into neonatal rat cardiomyocytes,the expression level of miR-155-5p in the miR-155-5p minic cells transfected group was significantly higher than that in the PBS control group(P<0.05).The exosomes of each group were incubated with the cells transfected with miR-155-5p minic,the concentrations of IL-1βand IL-6in the cell culture medium in GW4869 group were significantly higher than those of the other groups(P<0.05),of which the GW4869 group were higher than those except the PBS control group(P<0.05),and those in the model group+miR-155-5p minic were higher than those in the miR-155-5p minic group(P<0.05),and there was no significant difference between GW4869+ursolic acid high dose+mir-155-5p minic group and miR-155-5p minic group(P>0.05).Conclusion Ursolic acid can improve cardiomyopathy in septic rats,and can inhibit the inflammatory response of cardiomyocytes by inhibiting the expression level of miR-155-5p in exosomes.