右美托咪定通过Akt/mTOR自噬通路介导NLRP3炎症小体失活而减轻脓毒症大鼠肠损伤

Dexmedetomidine mediates inactivation of NLRP3 inflammasome through Akt/mTOR autophagy pathway to reduce intestinal injury in septic rats

目的:探讨右美托咪定(DEX)是否通过蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)自噬通路介导核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体失活从而减轻脓毒症大鼠肠损伤。方法:随机数字法对50只大鼠分组:假手术组、模型组、DEX组(0、3和6 h腹腔注射10μg/kg DEX)、DEX+NVP-BEZ235组(在DEX组基础上腹腔注射30 mg/kg NVP-BEZ235)及DEX+3-甲基腺嘌呤(3-MA)组(在DEX组基础上腹腔注射15 mg/kg 3-MA),每组10只。除假手术组外,其余各组盲肠结扎穿孔术(CLP)建立脓毒症大鼠模型,建模后给药。HE染色观察肠道组织形态,对结肠标本进行结肠大体形态损伤指数(CMDI)评分;ELISA检测血清中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平;免疫组化检测肠道组织中自噬相关蛋白beclin-1和LC3-II蛋白水平;Western blot检测肠道组织中Akt、p-Akt、mTOR、p-mTOR、NLRP3、含caspase募集结构域的凋亡相关斑点样蛋白(ASC)、caspase-1及其前体pro-caspase-1的蛋白水平。结果:模型组大鼠肠道组织完整性被破坏,细胞脱落和炎症浸润现象明显;DEX组大鼠肠道组织完整,仅出现部分细胞脱落和炎症浸润现象;DEX+NVP-BEZ235组和DEX+3-MA组大鼠肠道完整性被破坏,细胞脱落和炎症浸润明显。与假手术组相比,模型组大鼠CMDI评分,血清中IL-1β和TNF-α水平,肠道组织中beclin-1和LC3-II蛋白阳性率,p-Akt/Akt、p-mTOR/mTOR、NLRP3、ASC和caspase-1/pro-caspase-1蛋白水平升高(P<0.05);与模型组相比,DEX组大鼠CMDI评分,血清中IL-1β和TNF-α水平,肠道组织中NLRP3、ASC和caspase-1/pro-caspase-1蛋白水平降低(P<0.05),肠道组织中beclin-1和LC3-II蛋白阳性率及p-Akt/Akt和pmTOR/mTOR蛋白水平升高(P<0.05);与DEX组相比,DEX+NVP-BEZ235组和DEX+3-MA组血清中IL-1β和TNF-α水平及肠道组织中NLRP3、ASC和caspase-1/pro-caspase-1蛋白水平升高(P<0.05),肠道组织中beclin-1和LC3-II蛋白阳性率及p-Akt/Akt和p-mTOR/mTOR蛋白水平降低(P<0.05)。结论:DEX能够通过激活Akt/mTOR通路促进自噬而介导NLRP3炎症小体失活,从而减轻脓毒症大鼠肠损伤。

AIM:To investigate whether dexmedetomidine(DEX)reduces intestinal injury in septic rats by mediating the inactivation of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome through protein kinase B(PKB/Akt)/mammalian target of rapamycin(mTOR)autophagy pathway.METHODS:A total of 50 rats were randomly divided into 5 groups:sham operation group,model group,DEX group(intraperitoneal injection of 10μg/kg DEX at 0,3 and 6 h),DEX+NVP-BEZ235 group(intraperitoneal injection of 30 mg/kg NVP-BEZ235 on the basis of DEX group)and DEX+3-methyladenine(3-MA)group(intraperitoneal injection of 15 mg/kg 3-MA on the basis of DEX group),with 10 rats in each group.Except for sham operation group,cecal ligation and puncture(CLP)were performed to establish a rat model of sepsis in the other groups,and the rats were administered after modeling.Hematoxylineosin(HE)staining was used to observe the intestinal tissue morphology,and colon macroscopic damage index(CMDI)was evaluated.Enzyme-linked immunosorbent assay(ELISA)was used to detect the serum levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α).Immunohistochemistry was used to detect the autophagy-related proteins beclin-1 and LC3-II in intestinal tissues.Western blot was used to detect the protein levels of Akt,p-Akt,mTOR,pmTOR,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),caspase-1 and its precursor pro-caspase-1 in intestinal tissues.RESULTS:The integrity of the intestinal tissue in model group was destroyed,and the phenomenon of cell shedding and inflammatory infiltration was obvious.The intestinal tissues in DEX group were intact,with only some cell shedding and inflammatory infiltration.The integrity of the intestinal tissue in DEX+NVP-BEZ235 and DEX+3-MA groups was destroyed,and the cell shedding and inflammation was obvious.Compared with sham operation group,the CMDI,the levels of serum IL-1βand TNF-α,the positive rates of beclin-1 and LC3-II in intestinal tissues,and the protein levels of p-Akt/Akt,p-mTOR/mTOR,NLRP3,ASC and caspase-1/pro-caspase-1 increased in model group(P<0.05).Compared with model group,the CMDI,the serum levels of IL-1β and TNF-α,and the protein levels of NLRP3,ASC and caspase-1/pro-caspase-1 in intestinal tissues reduced in DEX group(P<0.05),while the positive rates of beclin-1 and LC3-II in intestinal tissues,and the protein levels of p-Akt/Akt and p-mTOR/mTOR increased(P<0.05).Compared with DEX group,the levels of serum IL-1β and TNF-α,and the protein levels of NLRP3,ASC and caspase-1/pro-caspase-1 in intestinal tissues increased in DEX+NVP-BEZ235 and DEX+3-MA groups(P<0.05),while the positive rates of beclin-1 and LC3-II in intestinal tissues,and the protein levels of p-Akt/Akt and pmTOR/mTOR reduced(P<0.05).CONCLUSION:Dexmedetomidine activates the Akt/mTOR pathway to promote autophagy,thereby promoting the inactivation of NLRP3 inflammasome and reducing intestinal injury in septic rats.