右美托咪定调控lncRNA HOTAIR改善脓毒症小鼠肺损伤作用机制初探

Preliminary study on the mechanism of dexmedetomidine regulating lncRNA HOTAIR to improve lung injury in septic mice

目的探究右美托咪定(DEX)改善脓毒症小鼠肺损伤的分子作用机制。方法将雄性C57BL/6小鼠随机(随机数字法)分为空白组(NC)、假手术组(Sham)、盲肠结扎穿孔组(CLP)和Dex治疗组(CLP+DEX),每组36只。CLP组小鼠在CLP术前15 min腹腔注射1 mL无菌生理盐水,CLP+DEX组小鼠在CLP术前15 min腹腔注射50μg/kg DEX。记录小鼠CLP术后24 h内的存活率,在CLP术后0、3、6、12、24 h时处死小鼠,采集小鼠肺脏,利用qPCR检测小鼠肺脏中细胞因子(IL-6、IL-1β、TNF-α)表达水平及lncRNA-HOTAIR表达水平。体外培养RAW264.7细胞,利用LPS(100 ng/mL)和DEX(1μmol/L DEX)建立了供在脓毒症细胞上研究Dex作用机制的细胞模型,利用qPCR检测脓毒症细胞模型经LPS处理后细胞因子(IL-6、IL-1β、TNF-α)及lncRNA-HOTAIR表达情况。此外,通过敲低或者过表达HOTAIR方式进行了体内外探索HOTAIR对脓毒症的作用。结果CLP+Dex组小鼠CLP术后24 h内的存活率高于CLP组,且在术后的6、12、24 h时小鼠肺脏细胞中IL-6、IL-1β、TNF-α的水平均低于CLP组(P<0.05)。此外,分析lncRNA HOTAIR的结果表明,经Dex治疗后,小鼠肺脏的lncRNA HOTAIR的表达水平均降低,尤其在Dex治疗6 h、12 h和24 h,HOTAIR表达水平分别下降1.1倍(P<0.05)、4.0倍(P<0.01)和3.8倍(P<0.01)。此外,相较于对照组,敲低HOTAIR后可以显著降低脓毒症小鼠的IL-1β、IL-6和TNF-α水平(P<0.05),过表达HOTAIR显著增加了脓毒症小鼠的IL-1β、IL-6和TNF-α水平(P<0.01)。结论DEX能减少脓毒症小鼠肺脏中炎性因子的产生,提高脓毒症小鼠存活率,其机制可能与抑制HOTAIR的表达水平有关。

Objective To investigate the mechanism of dexmetomidine(DEX)in improving lung injury in septic mice.Methods Male C57BL/6 mice were randomly assigned to the blank group(NC),sham operation group(sham),cecal ligation and puncture group(CLP),and Dex treatment group(CLP+DEX),36 mice per group.Mice in the CLP group were intraperitoneally injected with 1 mL sterile saline 15 min before CLP,and mice in the CLP+DEX group were intraperitoneally injected with 50μg/kg DEX 15 min before CLP.The survival rate was recorded within 24 h after CLP.The mice were sacrifi ced at 0,3,6,12,and 24 h after CLP,and lung tissues were collected.The expression levels of cytokines(IL-6,IL-1β,TNF-α)and lncRNAHOTAIR in the lung of mice were detected by qPCR.RAW264.7 cell were cultured in vitro,LPS(100 ng/mL)and DEX(1μmol/L)were used to establish a cell model for studying the mechanism of Dex,and the expression of cytokines(IL-6,IL-1β,TNF-α)and lncRNA-HOTAIR in RAW264.7 cell model were detected by qPCR.In addition,the effect of lncRNA-HOTAIR on sepsis was explored in vivo and in vitro by knockdown or overexpression of HOTAIR.Results The survival rate of the CLP+DEX group was higher than that of the CLP group within 24 h after surgery,and the levels of IL-6,IL-1β,and TNF-αin the lungs were signifi cantly lower than those in the CLP group at 6,12,and 24 h after surgery(P<0.05).In addition,the level of lncRNA HOTAIR showed that the expression level of lncRNA HOTAIR in the lungs of mice were decreased after Dex treatment,and were decreased 1.1 times(P<0.05),4.0 times(P<0.01)and 3.8 times(P<0.01)at 6,12,and 24 h,respectively.Compared with the NC group,knockdown of HOTAIR signifi cantly decreased the levels of IL-1β,IL-6,and TNF-αin septic mice(P<0.05),and overexpression of HOTAIR signifi cantly increased the levels of IL-1β,IL-6,and TNF-αin septic mice(P<0.01).Conclusions DEX can reduce the production of infl ammatory factors in the lungs of septic mice and improve the survival rate of septic mice.The mechanism may be related to the inhibition of HOTAIR expression.