miR-135b-5p抑制脓毒症引起的小鼠急性肺损伤(ALI)的作用及机制  

The role of miR-135b-5p in inhibiting mice acute lung injury(ALI)induced by sepsis and its mechanism

作　　者：臧宾宾[1] 李华[1] 杨颖 谢航 徐晓婷 ZANG Bin-bin;LI Hua;YANG Ying;XIE Hang;XU Xiao-ting(Department of Critical Care Medicine,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450002;Department of Liver,Gallbladder,Spleen and Stomach,Zhengzhou Hospital of Traditional Chinese Medicine,Zhengzhou 450007,China)

机构地区：[1]河南省中医院重症医学科,郑州450002 [2]郑州市中医院肝胆脾胃科,河南郑州450007

出　　处：《中国应用生理学杂志》2022年第4期366-372,共7页Chinese Journal of Applied Physiology

基　　金：河南省高校重点科研项目(21A320009)。

摘　　要：目的:探究miR-135b-5p在小鼠脓毒症(sepsis)引起的急性肺损伤(ALI)模型中的表达水平及其对小鼠肺部炎症反应和细胞焦亡的影响。方法:将C57BL/6小鼠随机分为6组,每组8只,通过盲肠结扎穿刺法(CLP)手术构建CLP诱导的脓毒症小鼠模型:腹腔注射0.1 mg/kg的巴比妥麻醉,腹部纵向切开暴露盲肠,结扎盲肠并用注射器针头进行穿孔,挤出部分肠道内容物后缝合伤口。假手术组(Sham组)开腹后不做任何处理缝合伤口,无CLP手术处理。治疗组分为CLP+NC mimic组,CLP+miR-135b-5p mimic组,CLP+NC mimic+empty vector组,CLP+消皮素D(GSDMD)组,CLP+miR-135b-5p mimic+GSDMD组。治疗组小鼠在CLP手术前一周皮下注射200μl溶解于生理盐水的NC mimic(200 nmol/L),miR-135b-5p mimic(200 nmol/L),empty vector(100 nmol/L),GSDMD vector(100 nmol/L),每天注射1次,连续一周。术后24 h采用二氧化碳窒息法实施安乐死。采用qRT-PCR检测小鼠肺组织样本中miR-135b-5p和GSDMD mRNA的表达水平;苏木精-伊红(HE)染色检测小鼠肺组织形态和损伤状态;采用5 ml生理盐水冲洗小鼠右肺3次,每次持续约3~5 min,收集肺泡灌洗液(BALF),酶联免疫吸附实验(ELISA)检测小鼠肺泡灌洗液(BALF)中GSDMD、白介素1β(IL-1β)和白介素18(IL-18)的表达水平;蛋白免疫印迹法检测小鼠肺组织内含NLR家族PYRIN域蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶1(caspase 1)以及切割后的N-端GSDMD端蛋白结构域(cleaved-GSDMD-N)的表达水平。双荧光素酶报告基因检测系统验证miR-135b-5p与GSDMD的靶向结合关系。结果:与对照组相比,CLP组小鼠肺组织中有大量的炎症细胞浸润,肺泡损伤,细胞间质水肿及肺泡塌陷等病理特征,小鼠肺组织内细胞焦亡相关蛋白(NLRP3,caspase-1和GSDMD)的表达水平明显增加(P<0.01),但miR-135b-5p的表达水平明显下调(P<0.01);与CLP组相比,超表达miR-135b-5p能够明显抑制CLP诱导的小鼠肺组织内细胞焦亡(P<0.01),靶向抑制GSDMD的�Objective:To investigate the expression status of miR-135 b-5 p in the sepsis induced acute lung injury(ALI)mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues.Methods:The cecal ligation puncture(CLP)method was employed to construct sepsis-induced ALI mice models.The C57 BL/6 mice were randomly divided into 6 groups with 8 mice in each group.The sepsis mouse models were constructed by performing CLP surgery:mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital,the abdomen was cut longitudinally to expose the cecum,the cecum was ligated and perforated with syringe needle,the wound was sutured after extruding part of the intestinal contents.The sham operation group(Sham group)did not undergo any treatment and suture wounds after laparotomy,and no CLP operation was performed.The treatment groups were divided into CLP+NC mimic group,CLP+miR-135 b-5 p mimic group,CLP+NC mimic+Empty vector group,CLP+GSDMD group,and CLP+miR-135 b-5 p mimic+GSDMD group.One week before CLP surgery,mice in the treatment group were injected subcutaneously with 200μL NC mimic(200 nmol/L),miR-135 b-5 p mimic(200 nmol/L),Empty vector(100 nmol/L),GSDMD Vector(100 nmol/L),and miR-135 b-5 p mimic(200 nmol/L),once a day for a week.The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation.The qRT-PCR was utilized to determine miR-135 b-5 p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues.The mice right lung tissues were flushed with 5 ml saline for 3 times,and each flush lasted for 3~5 min to collect the BALF,and the levels of GSDMD,IL-1βand IL-18 in BALF were determined by ELISA.The protein levels of NLRP3,caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis;Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135 b-5 p and GSDMD.Results:Compared with the control group mice,there were a large number of inflammatory cell

关 键 词：脓毒症 小鼠 急性肺损伤 miR-135b-5p 膜穿孔蛋白D 焦亡 

分 类 号：R392[医药卫生—免疫学]