小鼠内毒素性心肌损伤时Sestrin2与mtDNA-NLRP3炎性小体通路的关系

Relationship between Sestrin2 and mtDNA-NLRP3 inflammasome pathway during endotoxin-induced myocardial injury in mice

目的评价小鼠内毒素性心肌损伤时Sestrin2与线粒体DNA(mtDNA)-NOD样受体相关蛋白3(NLRP3)炎性小体通路的关系。方法清洁级健康雄性ICR小鼠184只,8~12周龄,体质量20~25 g。取小鼠168只,采用随机数字表法分为7组(n=24):正常对照组(N组)、LPS组(L组)、mtDNA组、LPS+mtDNA组(M组)、正常对照+阴性对照腺相关病毒(AAV-NC)组(NC组)、LPS+mtDNA+AAV-NC组(MC组)和LPS+mtDNA+Sestrin2过表达腺相关病毒(AAV-Sestrin2)组(MS组)。另取10只小鼠用于检测AAV-Sestrin2转染效果,其余6只小鼠用于提取mtDNA。采用腹腔注射LPS 10 mg/kg制备内毒素性心肌损伤模型。mtDNA组腹腔注射mtDNA 5 mg/kg;M组腹腔注射LPS后30 min时腹腔注射mtDNA 5 mg/kg;MS组尾静脉注射AAV-Sestrin2150μl,MC组和NC组尾静脉注射等容量AAV-NC。病毒注射后4周,MS和MC组腹腔注射LPS 10 mg/kg,30 min后腹腔注射mtDNA 5 mg/kg。注射LPS后24 h时收集血液标本,采用全自动生化分析仪测定血清CK-MB和LDH活性,ELISA法测定血清cTnI、IL-18和IL-1β浓度,qRT-PCR法检测血清mtDNA表达水平。取血结束后处死小鼠,取心肌组织,采用DHE染色法测定ROS含量,铁离子还原/抗氧化能力法测定总抗氧能力(T-AOC)含量,化学发光法测定ATP含量,Western blot法测定NLRP3、半胱氨酸蛋白酶-1活性亚基p20(caspase-1p20)、凋亡相关点样蛋白(ASC)表达,HE染色观察病理学结果。结果与N组比较,L组和mtDNA组血清CK-MB、LDH、cTnI、IL-1β和IL-18水平升高,mtDNA表达上调,心肌组织ROS含量升高,T-AOC和ATP含量降低,NLRP3、caspase-1p20和ASC表达上调(P<0.05),心肌病理学损伤加重。与L组和mtDNA组比较,M组血清CK-MB、LDH、cTnI、IL-1β、IL-18水平升高,mtDNA表达上调,心肌组织ROS含量升高,T-AOC和ATP含量降低,NLRP3、caspase-1p20和ASC表达上调(P<0.05),心肌病理学损伤加重。与M组比较,MS组血清CK-MB、LDH、cTnI、IL-1β、IL-18水平降低,mtDNA表达下调,心肌组织ROS含量降低,T-AOC和ATP含量升高,NLRP3、caspase-1p20和ASC表达下调(P<0.05),心肌病理学损伤减轻。结论Sestrin2可通过减轻线粒体损伤、抑制氧化应激,保护mtDNA免受氧化损伤,进而抑制mtDNA-NLRP3炎性小体通路,减轻小鼠内毒素性心肌损伤。

Objective To evaluate the relationship between Sestrin2 and mitochondrial DNA(mtDNA)-NOD-like receptor associated protein 3(NLRP3)inflammasome pathway during endotoxin-induced myocardial injury in mice.Methods One hundred and eighty-four clean-grade healthy male ICR mice,aged 8-12 weeks,weighing 20-25 g,were used in this study.One hundred and sixty-eight mice were divided into 7 groups(n=24 each)using the random number table method:normal control group(N group),lipopolysaccaride(LPS)group(L group),mtDNA group,LPS+mtDNA group(M group),normal control+negative control adeno-associated virus(AAV-NC)group(NC group),LPS+mtDNA+AAV-NC group(MC group),and LPS+mtDNA+Sestrin2 overexpression adeno-associated virus(AAV-Sestrin2)group(MSgroup).Another 10 mice were used to detect the transfection effect of AAV-Sestrin2,and the left 6 mice were used for mtDNA extraction.The model of endotoxemia was developed by intraperitoneal injection of LPS 10 mg/kg.mtDNA 5 mg/kg was intraperitoneally injected in mtDNA group,and mtDNA 5 mg/kg was intraperitoneally injected at 30 min after LPS injection in M group.AAV-Sestrin2150μl was injected via the tail vein in MS group,and the equal volume of AAV-NC was injected via the tail vein in MC and NC groups.Four weeks after virus injection,LPS 10 mg/kg was intraperitoneally injected and 30 min later mtDNA 5 mg/kg was intraperitoneally injected in MS and MC groups.Blood samples were collected at 24 h after LPS injection for determination of serum creatine kinase-MB(CK-MB)and lactate dehydrogenase(LDH)activities(by biochemical assay),concentrations of serum cardiac troponin I(cTnI),interleukin-18(IL-18)and interleukin-1beta(IL-1β)(by enzyme-linked immunesorbent assay),and expression of mtDNA(by quantitative real-time polymerase chain reaction).The animals were sacrificed after the end of blood sampling and myocardial tissues were obtained for determination of the contents of reactive oxygen species(ROS),total antioxidant capacity(T-AOC),and adenosine triphosphate(ATP)and expression of NOD-like receptor associated protein 3(NLRP3),active subunit p20 of caspase-1(caspase-1p20)and apoptosis-associated microprotein(ASC)in myocardial tissues(by Western blot)and for microscopic examination of the pathological changes after HE staining(with a light microscope).Results Compared with N group,the levels of CK-MB,LDH,cTnI,IL-1βand IL-18 in serum were significantly increased,the expression of mtDNA was up-regulated,the ROS content in myocardial tissues was increased,the T-AOC and ATP contents in myocardial tissues were decreased,the expression of NLRP3,caspase-1p20 and ASC in the myocardial tissues was up-regulated(P<0.05),and the pathological changes of myocardial tissues were aggravated in L group and mtDNA group.Compared with L group and mtDNA group,the levels of CK-MB,LDH,cTnI,IL-1βand IL-18 in serum were significantly increased,the expression of mtDNA was up-regulated,the ROS content in myocardial tissues was increased,the T-AOC and ATP contents in myocardial tissues were decreased,the expression of NLRP3,caspase-1p20 and ASC in the myocardial tissues was up-regulated(P<0.05),and the pathological changes of myocardial tissues were aggravated in M group.Compared with M group,the levels of CK-MB,LDH,cTnI,IL-1βand IL-18 in serum were significantly decreased,the expression of mtDNA was down-regulated,the ROS content in myocardial tissues was decreased,the T-AOC and ATP contents in myocardial tissues were increased,the expression of NLRP3,caspase-1p20 and ASC in the myocardial tissues was down-regulated(P<0.05),and the pathological changes of myocardial tissues were significantly attenuated in MS group.Conclusions Sestrin2 can reduce endotoxin-induced myocardial injury in mice by alleviating mitochondrial damage,inhibiting oxidative stress,protecting mtDNA from oxidative damage,and then inhibiting mtDNA-NLRP3 inflammasome pathway.