棕榈酰转移酶修饰的NOD2在小鼠心肺复苏后脑损伤中的作用

Role of palmitoyltransferase modified NOD2 in brain injury after cardiopulmonary resuscitation in mice

目的探讨棕榈酰转移酶(ZDHHC5)和核苷酸结合寡聚化结构域2(NOD2)在小鼠心肺复苏(CPR)后脑损伤中的作用。方法24只C57BL/6J雄性小鼠分为空白组、对照组、ZDHHC5-si组和NOD2-si组,每组6只。空白组无需任何处理,其余3组进行CPR造模。CPR造模前ZDHHC5-si组和NOD2-si组小鼠分别通过尾静脉注射ZDHHC5 siRNA和NOD2 siRNA。改良神经功能缺损量表(mNSS)评估各组造模后24 h、48 h和72 h的神经功能。72 h后采集血液标本和脑组织。实时荧光定量PCR(qPCR)检测脑组织ZDHHC5和NOD2 mRNA表达。酶联免疫吸附试验(ELISA)检测血浆白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)和IL-6;比色法及硫代巴比妥酸(TBA)法分别检测脑组织丙二醛(MDA)和髓过氧化物酶(MPO)。Western blot检测脑组织Cleaved Caspase-3、ZDHHC5及NOD2的蛋白表达。光镜下观察脑组织苏木素伊红(HE)染色病理切片。荧光显微镜下观察脑组织TUNEL染色病理切片。结果与空白组比较,其他3组mNSS评分,IL-1β、TNF-α、IL-6、MDA和MPO的表达水平,Cleaved Caspase-3、ZDHHC5和NOD2的蛋白表达量升高(P<0.05),脑组织损伤和细胞凋亡加重。与对照组比较,ZDHHC5-si组和NOD2-si组上述指标降低(P<0.05),脑组织损伤和细胞凋亡减轻。与NOD2-si组比较,ZDHHC5-si组上述指标降低(P<0.05),脑组织损伤和细胞凋亡进一步减轻。结论在小鼠CPR模型中,NOD2受ZDHHC5的调控后可产生棕榈酰化修饰的NOD2,进而促使炎性因子释放并引起神经细胞凋亡,损伤脑组织并影响神经功能。

Objective To investigate the role of nucleotide-binding oligomerization domain 2(NOD2)modified by palmitoyltransferase(ZDHHC5)in brain injury after cardiopulmonary resuscitation(CPR)in mice.Methods Twenty-four male C57BL/6J mice were divided into the blank group,the control group,the ZDHHC5-si group and the NOD2-si group,with 6 mice in each group.Except for the blank group without any treatment,CPR modeling was performed in the other three groups.At 24 h before CPR,mice in the ZDHHC5-si group and the NOD2-si group were injected with ZDHHC5 siRNA and NOD2 siRNA via tail vein,respectively.The modified neurological deficit scale(mNSS)was used to evaluate the neurological function at 24 h,48 h and 72 h in each group.Blood samples and brain tissue were collected 72 h after modeling.Real-time fluorescent quantitative PCR(qPCR)was used to detect ZDHHC5 and NOD2 in brain tissue.The protein expression levels of IL-1β,TNF-αand IL-6 in plasma were detected by enzyme-linked immunosorbent assay(ELISA).Colorimetric method and thiobarbituric acid(TBA)method were used to detect protein expression levels of MDA and MPO in brain tissue,respectively.Western blot assay was used to detect expression levels of Cleaved Caspase-3,ZDHHC5 and NOD2 in brain tissue.HE pathological sections of brain tissue were observed under light microscope.The pathological sections of brain tissue were observed by TUNEL under fluorescence microscope.Results Compared with the blank group,the mNSS score,the expression levels of IL-1β,TNF-α,IL-6,MDA and MPO,and the protein expression levels of Cleaved Caspase-3,ZDHHC5 and NOD2 were significantly increased(P<0.05),and brain tissue damage and cell apoptosis were aggravated in the other three groups.Compared with the control group,the above indicators were significantly decreased in the ZDHHC5-si group and the NOD2-si group(P<0.05),and brain tissue damage and cell apoptosis were significantly attenuated.Compared with the NOD2-si group,the above parameters were significantly decreased(P<0.05),and brain tissue damage and cell apoptosis were further attenuated in the ZDHHC5-si group.Conclusion In the mouse CPR model,NOD2 can produce palmitoylated NOD2 after regulated by ZDHHC5,which further promotes the release of inflammatory factors and causes neuronal apoptosis,ultimately damaging brain tissue and affecting neurological function.