O-GlcNAc修饰调控NLRP3炎症小体对H9c2大鼠心肌细胞缺氧复氧损伤的影响

Effect of NLRP3 inflammasome O-GlcNAcylation during hypoxia-reoxygenation injury in H9c2 cardiomyocytes

目的:体外探讨O-连接的N-乙酰葡萄糖胺(O-linked N-acetylglucosamine,O-GlcNAc)修饰调控核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体对H9c2大鼠心肌细胞缺氧复氧(hypoxia/reoxygenation,H/R)损伤的影响及可能的机制。方法:对数生长期的H9c2大鼠心肌细胞感染O-GlcNAc转移酶(O-GlcNAc transferase,OGT)过表达腺病毒(Ad-OGT)以提高细胞内OGlcNAc修饰水平,通过缺氧6 h再恢复常氧12 h建立细胞H/R模型。将细胞随机分为空载腺病毒(Ad-Null)预处理常氧对照细胞组、Ad-Null预处理H/R细胞组、Ad-OGT预处理常氧对照细胞组和Ad-OGT预处理H/R细胞组。通过RT-qPCR检测炎症细胞因子白细胞介素1β(interleukin-1β,IL-1β)和IL-18的mRNA水平;TUNEL染色检测细胞凋亡;Western blot测定NLRP3炎症小体组成蛋白[NLRP3、含caspase募集结构域的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a caspase recruitment domain,ASC)和caspase-1]及其效应产物表达;蛋白质免疫共沉淀法检测NLRP3炎症小体组成蛋白相互作用及修饰状态。LPS诱导NLRP3炎症小体表达,再次验证OGlcNAc修饰的调节作用。结果:H/R导致细胞内O-GlcNAc修饰水平显著降低,过表达OGT可显著对抗H/R损伤造成的O-GlcNAc修饰水平下降(P<0.05)。与Ad-Null预处理H/R细胞组相比,Ad-OGT预处理H/R细胞组NLRP3炎症小体活化后效应产物IL-1β和IL-18 mRNA转录被抑制、相应蛋白表达水平降低、细胞凋亡现象减轻(P<0.05);虽然NLRP3炎症小体的3种组成蛋白(NLRP3、ASC和caspase-1)表达水平并无显著下调(P>0.05),但相互结合能力减弱(P<0.05),同时检测到NLRP3的O-GlcNAc修饰程度显著增强(P<0.05)。通过刺激物LPS诱导NLRP3炎症小体表达激活,再次验证了NLRP3的O-GlcNAc修饰状态对炎症小体组分相互结合的影响。结论:NLRP3发生OGlcNAc修饰后可通过阻碍NLRP3炎症小体的组装活化缓解H/R对体外培养H9c2大鼠心肌细胞的损伤。

AIM:To investigate the effect of nucleotide-binding oligomerization domain-like receptor protein3(NLRP3)inflammasome regulated by O-linked N-acetylglucosamine(O-GlcNAc)modification(O-GlcNAcylation)on hypoxia-reoxygenation(H/R)injury in H9c2 rat cardiomyocytes and its mechanisms in vitro.METHODS:The H9c2 cardiomyocytes in logarithmic phase of growth were infected with O-GlcNAc transferase(OGT)over-expressing adenovirus(Ad-OGT)to increase intracellular global O-GlcNAcylation level.The cells were incubated under hypoxic conditions for 6 h and then restored to normoxia for 12 h to establish H/R model.Null-load adenovirus(Ad-Null)and normoxia as control(Ctrl)were also set.The cells were randomly divided into Ctrl group and H/R group with Ad-Null or Ad-OGT pretreatment.The transcription of inflammatory cytokines interleukin-1β(IL-1β)and IL-18 was detected by RT-qPCR.Apoptosis was detected by TUNEL staining.The expression of NLRP3 inflammasome components NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)and caspase-1,and its downstream products were detected by Western blot.The interaction and modification status of NLRP3 inflammasome components were detected by protein immunoprecipitation.The regulatory role of O-GlcNAcylation on NLRP3 inflammasome definitely induced by lipopolysaccharide(LPS)was verified once again.RESULTS:The H/R injury led to a significant decrease in cell global OGlcNAcylation level(P<0.05).Over-expression of OGT protein notably counteracted the deceased O-GlcNAcylation level caused by H/R injury.Compared with Ad-Null+H/R group,the transcription of IL-1βand IL-18 induced by NLRP3 inflammasome activation were inhibited in Ad-OGT+H/R group,the corresponding protein expression levels were reduced,and the apoptosis were mitigated(P<0.05).However,the expression of the 3 proteins constituting the NLRP3 inflammasome(NLRP3,ASC and caspase-1)had no obvious reduction(P>0.05).Further analysis showed that the binding ability of these 3 proteins was significantly weakened while the O-GlcNAcylation level of NLRP3 protein was increased(P<0.05).We verified once again the inhibitory effect of O-GlcNAcylation on NLRP3 inflammasome assembly and activation using LPS.CONCLUSION:O-GlcNAcylation of NLRP3 alleviates the inflammatory damage of H9c2 rat cardiomyocytes in vitro during H/R injury via hindering the assembly and activation of NLRP3 inflammasome.