SIRT1/NLRP3信号通路在七氟烷后处理减轻HT22细胞氧糖剥夺-复氧复糖损伤中的作用

Role of SIRT1/NLRP3 signaling pathway in sevoflurane postconditioning-induced attenuation of oxygen-glucose deprivation and restoration injury in HT22 cells

目的评价沉默信息因子1(SIRT1)/NOD样受体蛋白3(NLRP3)信号通路在七氟烷后处理减轻小鼠海马神经元(HT22细胞)氧糖剥夺-复氧复糖(OGD/R)损伤中的作用。方法培养HT22细胞,以5×10^(4)个/ml的密度接种于培养板或培养皿,采用随机数字表法分为4组(n=24):空白对照组(Control组)、氧糖剥夺-复氧复糖组(OGD/R组)、七氟烷后处理组(SPC组)和SIRT1小干扰RNA组(si-SIRT1组)。Control组将细胞置于37℃常氧培养箱中培养,OGD/R组将细胞培养基更换为无糖无血清培养基,置于37℃培养箱(95%N2+5%CO_(2))中培养4 h,随后将无糖无血清培养基更换为原细胞培养基,置于37℃常氧培养箱中继续培养24 h制备氧糖剥夺-复氧复糖损伤模型,SPC组在细胞氧糖剥夺4 h后将无糖无血清培养基更换为原细胞培养基,将细胞放入缺氧小室中,在复氧即刻向小室内充入2%七氟烷,之后将小室置于37℃常氧培养箱中培养1 h,最后将细胞从缺氧小室中取出,置于常氧培养箱中继续培养23 h进行七氟烷后处理,si-SIRT1组于实验前24 h时加入SIRT1小干扰RNA 150 pmol孵育,其余操作同SPC组。采用MTT法检测细胞存活情况,TUNEL染色检测细胞凋亡情况,采用PCR法检测SIRT1、NLRP3、IL-1β和IL-18的mRNA表达,采用Western blot法检测SIRT1、NLRP3、IL-1β和IL-18的表达。结果与Control组比较,OGD/R组细胞存活率降低,细胞凋亡率升高,SIRT1及其mRNA表达下调,NLRP3、IL-1β和IL-18及其mRNA表达上调(P<0.05);与OGD/R组比较,SPC组细胞存活率升高,细胞凋亡率降低,SIRT1及其mRNA表达上调,NLRP3、IL-1β和IL-18及其mRNA表达下调(P<0.05);与SPC组比较,si-SIRT1组细胞存活率降低,细胞凋亡率升高,SIRT1及其mRNA表达下调,NLRP3、IL-1β和IL-18及其mRNA表达上调(P<0.05)。结论SIRT1/NLRP3信号通路激活参与了七氟烷后处理减轻HT22细胞OGD/R损伤的过程。

Objective To evaluate the role of silent information regulator-1(SIRT1)/nucleotide-binding domain(NOD)-like receptor protein-3(NLRP3)signaling pathway in sevoflurane postconditioning-induced attenuation of oxygen-glucose deprivation and restoration(OGD/R)injury in mouse hippocampal neuronal cell line(HT22)cells.Methods The HT22 cells were seeded in a culture plate(96-well plate,100μl/well;6-well plate,2 ml/well)at the density of 5×10^(4) cells/ml or in a culture dish(6 cm in diameter)and then divided into 4 groups(n=24 each)using a random number table method:control group(Control group),OGD/R group,sevoflurane postconditioning group(SPC group),and SIRT1 small interfering RNA group(si-SIRT 1 group).In Control group,cells were cultured at 37℃in normal culture atmosphere.In OGD/R group,the culture medium was replaced with glucose-free serum-free culture medium,and cells were exposed to 95%N2+5%CO_(2)for 4 h in an incubator at 37℃,and then the glucose-free serum-free culture medium was replaced with the primary culture medium,and cells were cultured for 24 h at 37℃in normal culture atmosphere.In SPC group,the glucose-free serum-free culture medium was replaced with the primary cell culture medium after 4-h oxygen and glucose deprivation,the cells were put into the hypoxia incubator chamber which was filled with 2%sevoflurane immediately after start of reoxygenation,then the chamber was placed in an incubator and the cells were cultured for 1 h at 37℃in normal culture atmosphere,and finally the cells were removed from the chamber and cultured for 23 h at 37℃in normal culture atmosphere.In si-SIRT1 group,SIRT1 small interfering RNA 150 pmol was added at 24 h before surgery,cells were then incubated,and the other procedures were the same as those previously described in group SPC.The cell survival rate was determined using MTT assay.TUNEL assay was used to detect cell apoptosis,and the apoptosis rate was calculated.The expression of SIRT1,NLRP3,IL-1βand IL-18 mRNA was determined using polymerase chain reaction.The expression of SIRT1,NLRP3,interleukin-1beta(IL-1β)and IL-18 was detected using Western blot.Results Compared with Control group,the cell survival rate was significantly decreased,the apoptosis rate was increased,the expression of SIRT1 protein and mRNA was down-regulated,and the expression of NLRP3,IL-1βand IL-18 protein and mRNA was up-regulated in OGD/R group(P<0.05).Compared with OGD/R group,the cell survival rate was significantly increased,the apoptosis rate was decreased,the expression of SIRT1 protein and mRNA was up-regulated,and the expression of NLRP3,IL-1βand IL-18 protein and mRNA was down-regulated in SPC group(P<0.05).Compared with SPC group,the cell survival rate was significantly decreased,the apoptosis rate was increased,the expression of SIRT1 protein and mRNA was down-regulated,and the expression of NLRP3,IL-1βand IL-18 protein and mRNA was up-regulated in si-SIRT1 group(P<0.05).Conclusions Activation of SIRT1-NLRP3 signaling pathway is involved in sevoflurane postconditioning-induced attenuation of OGD/R injury in HT22 cells.