摘要
目的评价肝组织微小RNA-146a(miR-146a)表达与大鼠缺血/再灌注(I/R)肝损伤的关系.方法按随机数字表法将144只SD大鼠分为对照组(N组)、假手术组(S组)及I/R组,各组再分为4个亚组(n=12),分别在实验前1 h静脉给予220μL生理盐水(A组)、20μL miR-146a模拟剂+200μL生理盐水(B组)、20μL miR-146a模拟剂+200μL超声微泡造影剂(C组)、20μL miR-146a抑制剂+200μL超声微泡造影剂(D组).通过制备载miR-146a的超声微泡造影剂,运用超声靶向破坏微泡,于实验前及实验后24 h检测血浆丙氨酸转氨酶(ALT)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量,用反转录-聚合酶链反应(RT-PCR)检测肝组织miR-146a表达,用蛋白质免疫印迹试验(Western Blot)检测肝组织Toll样受体4(TLR4)、白细胞介素-1受体相关激酶1(IRAK-1)、IL-6、TNF-α蛋白表达,光镜下观察肝组织细胞损伤情况.结果实验前各组以及实验后24 h N组和S组血浆ALT、IL-6、TNF-α含量差异均无统计学意义,病理观察也无明显肝组织细胞损伤.实验后24 h,与S组比较,I/R A组和D组肝组织miR-146a表达显著下降(miR-146a/U6 snRNA:0.51±0.13、0.22±0.09比1.01±0.02,均P<0.01),血浆ALT、IL-6、TNF-α含量及肝组织TLR4、IRAK-1、IL-6、TNF-α表达量显著升高〔ALT(U/L):103.23±26.64比44.16±18.55,176.46±7.26比49.74±6.83;IL-6(μg/L):64.28±16.19比17.68±7.54,88.49±3.23比15.58±2.38;TNF-α(μg/L):31.28±2.57比5.58±3.35,59.12±8.74比5.27±1.37;TLR4/GAPDH:2.43±0.36、3.23±0.71比0.96±0.24;IRAK-1/GAPDH:2.34±0.52、3.14±0.63比0.76±0.21;IL-6/GAPDH:1.02±0.22、1.11±0.16比0.98±0.37;TNF-α/GAPDH:2.05±0.48、2.86±0.27比0.59±0.16;均P<0.01〕,且以I/R D组升高更为显著(均P<0.01);病理显示肝组织细胞损伤明显;而I/R B组和C组转染miR-146a模拟剂后肝组织miR-146a表达量显著升高(miR-146a/U6 nsRNA:1.56±0.31、2.40±0.53比1.01±0.02,均P<0.01),肝组织TLR4、IRAK-1、IL-6、TNF-α的表达量显著下降(TLR4/GAPDH:0.77±0.18、0.65±0.27比0.96±0.24,IRAK-1/GAPDH:0.61±0.14、0.47±0.20比0.76±0.21,IL-6/GAPDH:0.80±0.13、0.54±0.22比0.98±0.37,TNF-α/GAPDH:0.41±0.14、0.16±0.03比0.59±0.16,均P<0.01),且以C组结合超声微泡后表达更为显著(P<0.01),病理显示肝组织细胞损伤也更为轻微,B组和C组血浆ALT、IL-6及TNF-α含量也与S组差异无统计学意义.结论超声微泡能高效转染miR-146a模拟剂和抑制剂于肝组织细胞;miR-146a可能负调控TLR信号通路介导的I/R肝损伤.
Objective To evaluate the relationship between the expression of rnicroRNA-146a(miR-146a)in liver tissue and the inflammatory hepatic injury induced by ischemia/reperfusion(I/R)in rats.Methods One hundred and forty-four Sprague-Dawley(SD)rats were randomly divided into three groups:control(group N),sham operation(group S)and group I/K.Each group was subdivided into four subgroups(n=12),and different substances were respectively injected intravenously to rats in different subgroups at 1 hour before the experiment:220μL physiological saline(group A),20μL miR-146a mimic+200 ph physiological saline(group B).20μL miR-146a mimic+200μL ultrasound microblibble contrast agent(group C)and 20 pL miR-146a inhibitor+200μL ultrasound microbubble contrast agent(group D).Before the experiment and after experiment for 24 hours,the plasma concentrations of alanine ami not ra nsferase(ALT),interleukin-6(IL-6)and tumor ne crosis factor-α(TNF-α)were detected,the reverse transcription-polvmerase chain reaction(RT-PCR)was used to measure the expression of miR-146a in liver tissue,and Western Blot was applied to detect protein expressions of Toll-like receptor 4(TLR4),IL-1 receptor associated kinase 1(IRAK-1),IL-6 and TNF-α.and the pathological hepatic cell injury was observed.Results Before the experiment and 24 hours after experiment in various subgroups of N and S groups,there were no statistical significant differences in the plasma concentrations of ALT,IL-6 and TNF-α,and the expression of miR-146a level and the protein expressicns of TLR4.IRAK-1.IL-6 and TNF-αin liver tissues:the pathological examination also did not show any obvious hepatic cell injury.After the experiment for 24 hours:compared to the group S.the liver tissue miR-146a expression was significantly decreased in the subgroups A and D of group I/R(miR-146a/LI6nsRNA:0.51±0.13,0.22±0.09\s.1.01+0.02,both P<0.01),and the plasma concentrations of ALT,IL-6 and TNF-αand the protein expressions of TLR4.IRAK-1,IL-6 and TNF-αin liver tissues were significantly increased[ALT(U/L):103.23±26.64 vs.44.16±18.55.176.46±7.26 vs.49.74±6.83.IL-6(μg/L):64.28±i6.19 vs.17.68±7.54.88.49±3.23 vs.15.58±2.38;TNF-α(μg/L):31.28±2.57 vs.5.58±3.35,59.12±8.74 vs.5.27±1.37:TLR4/GAPDH:2.43±0.36,3.23±0.71 vs.0.96±0.24.IRAK-l/GAPDH:2.34±0.52,3.14土0.63 vs.0.76±0.21,IL-6/GAPDH:1.01±0.22,1.11±0.16 vs.0.98±0.37,TNF-α/GAPDH:2.05±0.48、2.86±0.27 vs.0.59±0.16.all P<0.01],moreover,the hepatic pathological lesions were obvious:the liver tissue expression of miR-146a was significantly increased after being transfected with miR-146a mimic in subgroups B and C of group I/K(miR-146a/U6nsRNA:1.56±0.31,2.40±0.53 vs.1.01±0.02,both P<0.01).especially in group C combined with ultrasound microhubble(P<0.01).However,the protein expressions of TLR4.IRAK-1.IL-6 and TNF-αin liver tissues were significantly decreased(TLR4/GAPDH:0.77±0.18、0.65±0.27 vs.0.96±0.24.IRAK-1/GAPDH:0.61±0.14.0.47±0.20 vs.0.76±0.21.IL-6/GAPDH:0.80±0.13,0.54±0.22 vs.0.98±0.37,TNF-α/GAPDH:0.41±0.14,0.16±0.03 vs.0.59±0.16:all P<0.01),and the expressions were more significant in the group C combined with ultrasound microbubbles(P<0.01),and the hepatic pathological damage was mild,however,the plasma concentrations of ALT,IL-6 and TNF-αwere of no statistical significant differences.Conclusion Ultrasound mic robubble can efficiently transfect miR-146a mimic and inhibitor into the liver tissue,and miR-146a may negatively regulate the I/R inflanimaton liver injury mediated by TLB signaling pathway.
作者
晏春根
朱冬芳
夏国园
Yan Chungen;Zhu Dongfang;Xia Guoyuan(Department of Gastroenterology,Affiliated Hospital of Shaoxing University,Shaoxing 312000,Zhejiang,China;Department of Lab Medicine,Affiliated Hospital of Shaoxing University,Shaoxing 312000,Zhejiang,China;Department of Ultrasound Diagnosis,Affiliated Hospital of Shaoxing University,Shaoxing 312000,Zhejiang,China)
出处
《中国中西医结合急救杂志》
CAS
CSCD
北大核心
2019年第1期21-25,共5页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
浙江省自然科学基金(LY15H180008)
浙江省医药卫生科研项目(2019333712)
浙江省绍兴市科技计划项目(2015014001).
