微小RNA-30b-3p在肝脏缺血再灌注损伤小鼠肝组织中的表达及意义

Significance of microRNA-30b-3p expression in liver tissues of mice with hepatic ischemia-reperfusion injury

目的探讨微小RNA-30b-3p(miR-30b-3p)在肝脏缺血再灌注损伤(IRI)小鼠肝组织中的表达,并阐述可能的机制。方法将40只无特定病原体级雄性健康C57BL/6小鼠随机分为IRI组、假手术组、miRNA阴性对照(miR-NC)组和miR-30b-3p agomir组,每组10只。IRI组、miR-NC组和miR-30b-3p agomir组小鼠建立肝脏IRI模型,假手术组小鼠仅进行开腹及关腹等操作。造模前24 h,miR-NC组和miR-30b-3p agomir组小鼠分别经尾静脉注射10 nmol·L^(-1)的miR-NC或miR-30b-3p agomir 0.3 m L;假手术组和IRI组小鼠术前不进行干预。采用生物化学法检测各组小鼠血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平;苏木精-伊红染色法观察小鼠肝组织病理学改变;荧光脱氧核糖核苷酸末端转移酶介导的缺口末端标记法标记肝细胞凋亡情况;免疫组织化学法检测各组小鼠肝组织中Caspase-3表达与分布;实时荧光定量聚合酶链反应检测小鼠肝组织中miR-30b-3p表达;Western blot法检测小鼠肝组织中Bcl-2、Fas相关死亡域蛋白(FADD)、Bax及Cleave Caspase-3蛋白的表达。结果 IRI组和miR-NC组小鼠血清ALT、AST水平显著高于假手术组(P<0.05),miR-30b-3p agomir组与假手术组小鼠血清ALT、AST水平比较差异无统计学意义(P>0.05)。miR-30b-3p agomir组小鼠血清ALT、AST水平显著低于IRI组(P<0.05);miR-NC组与IRI组小鼠血清ALT、AST水平比较差异无统计学意义(P>0.05)。miR-30b-3p agomir组小鼠血清ALT、AST水平均显著低于miR-NC组(P<0.05)。IRI组、miR-NC组小鼠肝组织中miR-30b-3p相对表达量显著低于假手术组(P<0.05),miR-30b-3p agomir组小鼠肝组织中miR-30b-3p相对表达量显著升高(P<0.05);miR-30b-3p agomir组小鼠肝组织中miR-30b-3p相对表达量显著高于IRI组(P<0.05),miR-NC组与IRI组小鼠肝组织中miR-30b-3p相对表达量比较差异无统计学意义(P>0.05),miR-30b-3p agomir组小鼠肝组织中miR-30b-3p相对表达量显著高于miR-NC组(P<0.05)。IRI组和miR-NC组小鼠肝细胞凋亡率显著高于假手术组(P<0.05),miR-30b-3p agomir组小鼠肝细胞凋亡率与假手术组比较差异无统计学意义(P>0.05);miR-30b-3p agomir组小鼠肝细胞凋亡率显著低于IRI组(P<0.05);miR-NC组与IRI组小鼠肝细胞凋亡率比较差异无统计学意义(P>0.05);miR-30b-3p agomir组小鼠肝细胞凋亡率显著低于miR-NC组(P<0.05)。IRI组和miR-NC组小鼠肝组织中Caspase-3相对表达量显著高于假手术组(P<0.05);miR-30b-3p agomir组与假手术组小鼠肝组织中Caspase-3相对表达量比较差异无统计学意义(P>0.05);miR-30b-3p agomir组小鼠肝组织中Caspase-3相对表达量显著低于IRI组(P<0.05),miR-NC组与IRI组小鼠肝组织中Caspase-3相对表达量比较差异无统计学意义(P>0.05);miR-30b-3p agomir组小鼠肝组织中Caspase-3相对表达量显著低于miR-NC组(P<0.05)。与假手术组比较,IRI组、miR-NC组小鼠肝组织中FADD、Bax、Cleave Caspase-3相对表达量均显著增加(P<0.05),Bcl-2相对表达量显著减少(P<0.05);miR-30b-3p agomir组与假手术组小鼠肝组织中FADD、Bax、Cleave Caspase-3及Bcl-2相对表达量比较差异均无统计学意义(P>0.05);与IRI组比较,miR-30b-3p agomir组小鼠肝组织中FADD、Bax及Cleave Caspase-3相对表达量显著降低(P<0.05),Bcl-2相对表达量显著增加(P<0.05);miR-NC组与IRI组小鼠肝组织中FADD、Bax、Cleave Caspase-3及Bcl-2相对表达量比较差异均无统计学意义(P>0.05);与miR-NC组比较,miR-30b-3p agomir组小鼠肝组织中FADD、Bax及Cleave Caspase-3相对表达量均显著降低(P<0.05),Bcl-2相对表达量显著增加(P<0.05)。结论 miR-30b-3p可减轻小鼠肝脏IRI,其机制可能与抑制FADD/Caspase-3信号通路所介导的肝细胞凋亡有关。

Objective To investigate the expression of microRNA-30 b-3 p（ miR-30 b-3 p） in liver tissues of mice with hepatic ischemia-reperfusion injury（ IRI）,and to explain the possible mechanism. Methods A total of 40 C57 BL/6 male mice of specific pathogen free were randomly divided into IRI group,sham operation group,miRNA negative control group（ miR-NC group） and miR-30 b-3 p agomir group,with 10 mice in each group. The liver IRI model of mice was established in the IRI group,miR-NC group and miR-30 b-3 p agomir group; the mice in the sham operation group were only performed open and close sergery. The mice in the miR-NC group and miR-30 b-3 p agomir group were given 0. 3 ml miR-30 b-3 p agomir（ 10 nmol·L-1） or miR-NC（ 10 nmol·L-1） respectively by tail intravenous injection in 24 h before modeling; the mice in the sham group and IRI group did not give intervention. The levels of serum alanine aminotransferase（ ALT） and aspartate aminotransferase（ AST） of mice in each group were detected. The pathological changes of liver tissues were observed by hematoxylin and eosin staining. The apoptosis of hepatocytes was observed by fluorescence deoxyribonucleotide terminal transferase mediated nick end labeling assay. The expression and distribution of Caspase-3 in liver tissues were detected by immunohistochemistry. The expression of miR-30 b-3 p in liver tissues was detected by the real time fluorescence quantitative polymerase chain reaction. The expression of Bcl-2,Fas-associated with death domain protein（ FADD）,Bax and Cleave Caspase-3 protein in liver tissues was detected by Western blot. Results The levels of serum ALT and AST in the IRI group and miR-NC group were significantly higher than those in the sham operation group（ P 〈 0. 05）. There was no significant difference in the levels of serum ALT and AST between the miR-30 b-3 p agomir group and the sham operation group（ P 〉 0. 05）. The levels of serum ALT and AST in the miR-30 b-3 p agomir group were significantly lower than those in the IRI group（ P 〈 0. 05）. There was no significant difference in the levels of serum ALT and AST between the miR-NC group and the IRI group（ P 〉 0. 05）. The levels of serum ALT and AST in the miR-30 b-3 p agomir group were significantly lower than those in the miR-NC group（ P 〈0. 05）. The relative expression of miR-30 b-3 p in the liver tissues of mice in the IRI group and the miR-NC group was significantly lower than that in the sham operation group（ P 〈 0. 05）. The relative expression of miR-30 b-3 p in the liver tissues of mice in the miR-30 b-3 p agomir group was significantly higher than that in the sham operation group（ P 〈 0. 05）. The relative expression of miR-30 b-3 p in the liver tissues of mice in the miR-30 b-3 p agomir group was significantly higher than that in the IRI group（ P 〈 0. 05）. There was no significant difference in the relative expression of miR-30 b-3 p in the liver tissues of mice between the miR-NC group and the IRI group（ P 〉 0. 05）. The relative expression of miR-30 b-3 p in the liver tissues of mice in the miR-30 b-3 p agomir group was significantly higher than that in the miR-NC group（ P 〈 0. 05）. The hepatocyte apoptosis rate of mice in the IRI group and miR-NC group was significantly higher than that in the sham operation group（ P 〈 0. 05）.There was no significant difference in the hepatocyte apoptosis rate of mice between miR-30 b-3 p agomir group and the sham operation group（ P 〉 0. 05）. The hepatocyte apoptosis rate of mice in the miR-30 b-3 p agomir group was significantly lower than that in the IRI group（ P 〈 0. 05）. There was no significant difference in the hepatocyte apoptosis rate of mice between the miRNC group and the IRI group（ P 〉 0. 05）. The hepatocyte apoptosis rate of mice in the miR-30 b-3 p agomir group was significantly lower than that in the miR-NC group（ P 〈 0. 05）. The expression of Caspase-3 in the liver tissues of mice in the IRI group and miR-NC group was significantly higher than that in the sham operation group（ P 〈 0. 05）. There was no significant difference in the expression of Caspase-3 in the liver tissues of mice between the miR-30 b-3 p agomir group and the sham operation group（ P 〉 0. 05）. The expression of Caspase-3 in the liver tissues of mice in the miR-30 b-3 p agomir group was significantly lower than that in the IRI group（ P 〈 0. 05）. There was no significant difference in the expression of Caspase-3 in the liver tissues of mice between the miR-NC group and the IRI group（ P 〉 0. 05）. The expression of Caspase-3 in the liver tissues of mice in the miR-30 b-3 p agomir group was significantly lower than that in the miR-NC group（ P 〈 0. 05）. Compared with the sham operation group,the expression of FADD,Bax and Cleave Caspase-3 in the liver tissues of mice increased significantly（ P 〈 0. 05）; and Bcl-2 expression decreased significantly in the IRI group and miR-NC group（ P 〈 0. 05）. There was no significantly difference in the expression of FADD,Bax,Cleave Caspase-3 and Bcl-2 in the liver tissues of mice between miR-30 b-3 p agomir group and the sham operation group（ P 〉 0. 05）. Compared with the IRI group,the expression of FADD,Bax and Cleave Caspase-3 in the liver tissues of mice decreased significantly（ P 〈 0. 05）; and Bcl-2 expression increased significantly in the miR-30 b-3 p agomir group（ P 〈 0. 05）. There was no significantly difference in the expression of FADD,Bax,Cleave Caspase-3 and Bcl-2 in the liver tissues of mice between miR-NC group and the IRI group（ P 〉 0. 05）. Compared with the miR-NC group,the expression of FADD,Bax and Cleave Caspase-3 in the liver tissues of mice decreased significantly（ P 〈0. 05）; and Bcl-2 expression increased significantly in the miR-30 b-3 p agomir group（ P 〈 0. 05）. Conclusion MiR-30 b-3 p can reduce the IRI of liver in mice,and its mechanism may be related to the inhibition of FADD/Caspase-3 signal mediated hepatocyte apoptosis.