摘要
目的探讨微RNA-204(miR-204)靶向硫氧还蛋白相互作用蛋白(TXNIP)对脓毒症大鼠急性肺损伤的保护作用。方法选择无特定病原体级雄性Sprague Dawley大鼠48只,随机分为假手术组、模型组、阴性对照(NC)组、miR-204过表达(miR-204 agomir)组,每组12只。模型组、NC组、miR-204 agomir组大鼠通过盲肠结扎穿刺法建立脓毒症模型,假手术组大鼠仅开腹不做盲肠结扎及穿孔;脓毒症模型建立后,NC组、miR-204 agomir组大鼠分别通过尾静脉注射2μL agomir NC和2μL miR-204 agomir,模型组和假手术组大鼠注射等体积的生理盐水。于造模24 h后,断颈处死各组大鼠,取肺组织,采用光学显微镜观察各组大鼠肺组织病理变化,采用实时荧光定量聚合酶链式反应法检测各组大鼠肺组织中miR-204、TXNIP及硫氧还蛋白(Trx)mRNA表达水平;取HEK-293T细胞,采用双荧光素酶报告基因法检测试剂盒检测miR-204和TXNIP 3′UTR非编码区(3′UTR)靶向关系;Western blot法检测各组大鼠肺组织中TXNIP及Trx蛋白表达水平;酶联免疫吸附测定法检测各组大鼠肺组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平。结果假手术组大鼠肺泡形态结构正常;模型组和NC组大鼠肺泡壁增厚,肺组织中出现大量炎症细胞浸润;与模型组和NC组比较,miR-204 agomir组大鼠肺组织炎症细胞浸润减少,肺泡损伤明显改善。模型组和NC组大鼠肺组织miR-204相对表达量显著低于假手术组(P<0.05);模型组与NC组大鼠肺组织中miR-204相对表达量比较差异无统计学意义(P>0.05);miR-204 agomir组大鼠肺组织中miR-204相对表达量显著高于假手术组、模型组和NC组(P<0.05)。模型组和NC组大鼠肺组织TXNIP mRNA和蛋白相对表达量显著高于假手术组(P<0.05);模型组与NC组大鼠肺组织TXNIP mRNA和蛋白相对表达量比较差异无统计学意义(P>0.05);miR-204 agomir组大鼠肺组织TXNIP mRNA和蛋白相对表达量显著低于假手术组、模型组和NC组(P<0.05)。miR-204 agomir+TXNIP 3′UTR-野生型组荧光素酶活性显著低于阴性对照+TXNIP 3′UTR-野生型组(P<0.05);miR-204 agomir+TXNIP 3′UTR-突变型组与阴性对照+TXNIP 3′UTR-突变型组荧光素酶活性比较差异无统计学意义(P>0.05)。模型组、NC组和miR-204 agomir组大鼠肺组织Trx mRNA和蛋白相对表达量显著低于假手术组(P<0.05);miR-204 agomir组大鼠肺组织Trx mRNA和蛋白相对表达量显著高于模型组和NC组(P<0.05);模型组与NC组大鼠肺组织中Trx mRNA和蛋白相对表达量比较差异无统计学意义(P>0.05)。模型组、NC组和miR-204 agomir组大鼠肺组织中TNF-α、IL-6和MDA水平显著高于假手术组,SOD水平显著低于假手术组(P<0.05);miR-204 agomir组大鼠肺组织中TNF-α、IL-6和MDA水平显著低于模型组和NC组,SOD水平显著高于模型组和NC组(P<0.05);模型组与NC组大鼠肺组织中TNF-α、IL-6、MDA和SOD水平比较差异均无统计学意义(P>0.05)。结论miR-204可通过靶向TXNIP抑制脓毒症急性肺损伤大鼠肺组织炎症反应和氧化应激,其机制可能与Trx表达升高有关。
Objective To explore the protective effect of microRNA-204(miR-204)on acute lung injury of rats with sepsis by targeting thioredoxin interacting protein(TXNIP).Methods A total of 48 specific pathogen free grade Sprague Dawley male rats were randomly divided into the sham operation group,model group,negative control(NC)group and miR-204 overexpression(miR-204 agomir)group,with 12 rats in each group.Cecal ligation and puncture was used to establish sepsis model of rats in the model group,NC group and miR-204 agomir group;the rats in the sham operation group were only performed with laparotomy,but without cecal ligation and perforation.After the establishment of sepsis models,the rats in the NC group and miR-204 agomir group were injected 2μL of NC and 2μL of miR-204 agomir by tail vein,respectively;and the rats in the model group and sham operation group were injected with the same amount of normal saline.After 24 hours of modeling,the rats in each group were killed by cutting the neck.The lung tissue of rats in each group was taken,the pathological changes of lung tissue were observed by light microscope,the expressions of miR-204,TXNIP mRNA and thioredoxin(Trx)mRNA were detected by real-time fluorescent quantitative polymerase chain reaction.HEK-293T cells were collected,and the targeting relationship between miR-204 and TXNIP 3′untranslated coding regions(3′UTR)were detected by dual luciferase reporter gene detection kit.The expressions of TXNIP and Trx protein in the lung tissue of rats in each group were detected by Western blot.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),malondialdehyde(MDA)and superoxide dismutase(SOD)in the lung tissue of rats in each group were detected by enzyme linked immunosorbent assay.Results In the sham operation group,the morphology and structure of alveoli of rats were normal.In the model group and NC group,the alveolar wall thickened and a large amount of inflammatory infiltration appeared in lung tissue of rats.Compared with the model group and NC group,the inflammatory infiltration in lung tissue of rats in the miR-204 agomir group decreased and the alveolar injury was significantly improved.The relative expression of miR-204 in lung tissue of rats in the model group and NC group was significantly lower than that in the sham operation group(P<0.05),there was no significant difference in the relative expression of miR-204 in lung tissue of rats between model group and NC group(P>0.05),the relative expression of miR-204 in lung tissue of rats in the miR-204 agomir group was significantly higher than that in the sham operation group,model group and NC group(P<0.05).The relative expressions of TXNIP mRNA and protein in lung tissue of rats in the model group and NC group were significantly higher than those in the sham operation group(P<0.05),there was no significant difference in the relative expressions of TXNIP mRNA and protein in lung tissue of rats between the model group and NC group(P>0.05),the relative expressions of TXNIP mRNA and protein in lung tissue of rats in the miR-204 agomir group were significantly lower than those in the sham operation group,model group and NC group(P<0.05).The luciferase activity in the miR-204 mimics+TXNIP 3′UTR-wild type(WT)group was significantly lower than that in the negative control+TXNIP 3′UTR-WT group(P<0.05),there was no significant difference in luciferase activity between the miR-204 mimic+TXNIP 3′UTR-mutant(MUT)group and negative control+TXNIP 3′UTR-MUT group(P>0.05).The relative expressions of Trx mRNA and protein in lung tissue of rats in the model group,NC group and mir-204 agomir group were significantly lower than those in the sham operation group(P<0.05);the relative expressions of Trx mRNA and protein in lung tissue of rats in the miR-204 agomir group were significantly higher than those in the model group and NC group(P<0.05),there was no significant difference in the relative expression of Trx mRNA and protein in lung tissue of rats between the model group and NC group(P>0.05).The levels of TNF-α,IL-6 and MDA in lung tissue of rats in the model group,agomir NC group and miR-204 agomir group were significantly higher than those in the sham operation group(P<0.05),and the level of SOD was significantly lower than that in the sham operation group(P<0.05);the levels of TNF-α,IL-6 and MDA in lung tissue of rats in the miR-204 agomir group were significantly lower than those in the model group and NC group(P<0.05),and the level of SOD was significantly higher than that in the model group and NC group(P<0.05);there was no significant difference in the levels of TNF-α,IL-6,MDA and SOD in lung tissue of rats between the model group and NC group(P>0.05).Conclusion miR-204 through targeting TXNIP can inhibit inflammatory and oxidative stress in lung tissue of rats with acute lung injury induced by sepsis,the mechanism may be related to the increase of Trx expression.
作者
王光权
李洪
潘在轩
WANG Guangquan;LI Hong;PAN Zaixuan(Department of Critical Care Medicine,Qionghai People′s Hospital,Qionghai 571400,Hainan Province,China;Department of Central Laboratory,Qionghai People′s Hospital,Qionghai 571400,Hainan Province,China;Department of Pathology Laboratory,Qionghai People′s Hospital,Qionghai 571400,Hainan Province,China)
出处
《新乡医学院学报》
CAS
2021年第7期612-618,共7页
Journal of Xinxiang Medical University