氢对脂多糖-尼日利亚菌素诱导巨噬细胞焦亡的影响及lncRNA NEAT1在其中的作用

Effect of hydrogen on lipopolysaccharide and nigericin-induced pyroptosis in macrophages and the role of lncRNA NEAT1

目的评价氢对脂多糖(LPS)-尼日利亚菌素诱导巨噬细胞焦亡的影响及长链非编码RNA(lncRNA)核富集转录体1(NEAT1)在其中的作用。方法体外培养人单核巨噬细胞系THP-1,采用随机数字表法分为4组(n=25):对照组(C组)、LPS-尼日利亚菌素组(LN组)、富氢液培养基+LPS-尼日利亚菌素组(H+LN组)和慢病毒转染+富氢液培养基+LPS-尼日利亚菌素组(LV+H+LN组)。C组细胞使用普通培养基正常培养24 h;LN组加入终浓度为100 ng/ml的LPS和10 μmol/L的尼日利亚菌素孵育24 h;H+LN组将普通培养基更换为0.6 mmol/L的富氢液培养基,随即加入终浓度为100 ng/ml的LPS和10 μmol/L的尼日利亚菌素孵育24 h;LV+H+LN组使用经慢病毒转染致NEAT1稳定过表达的THP-1细胞,并将普通培养基更换为0.6 mmol/L的富氢液培养基,随即加入终浓度为100 ng/ml的LPS和10 μmol/L的尼日利亚菌素孵育24 h。采用CCK-8法检测细胞活力,比色法检测LDH释放量,ELISA法检测培养基IL-1β和IL-18浓度,流式细胞术检测细胞焦亡率,Western blot法检测NOD样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(caspase-1)和削皮素D(GSDMD)的表达,qRT-PCR法检测NEAT1基因的表达。结果与C组比较,LN组细胞活力降低,LDH释放量、IL-1β和IL-18浓度、细胞焦亡率、NEAT1基因、NLRP3、ASC、caspase-1和GSDMD表达水平升高(P<0.05);与LN组比较,H+LN组细胞活力升高,LDH释放量、IL-1β和IL-18浓度、细胞焦亡率、NEAT1基因、NLRP3、ASC、caspase-1和GSDMD表达水平降低(P<0.05);与H+LN组比较,LV+H+LN组细胞活力降低,LDH释放量、NEAT1基因、IL-1β和IL-18浓度、细胞焦亡率、NLRP3、ASC、caspase-1和GSDMD表达水平升高(P<0.05)。结论氢可减轻LPS-尼日利亚菌素诱导的巨噬细胞焦亡,其机制与下调lncRNA NEAT1表达有关。

Objective To evaluate the effect of hydrogen on lipopolysaccharide(LPS)and nigericin-induced pyroptosis in macrophages and the role of long non-coding RNA(lncRNA)nuclear-enriched abundant transcript 1(NEAT1).Methods Human monocyte-derived macrophages THP-1 cells were cultured in vitro and divided into 4 groups(n=25 each)using a random number table method:control group(group C),LPS and nigericin group(group LN),hydrogen-rich medium+LPS and nigericin group(group H+LN),and lentiviral transfection+hydrogen-rich medium+LPS-nigericin group(group LV+H+LN).THP-1 cells were cultured in the common culture medium for 24 h in group C.LPS at a final concentration of 100 ng/ml and nigericin 10μmol/L were added to the culture medium,and the cells were incubated for 24 h in group LN.In group H+LN,the culture medium was replaced with 0.6 mmol/L hydrogen-rich medium,then LPS at a final concentration of 100 ng/ml and nigericin 10μmol/L were immediately added,and the cells were incubated for 24 h.In group LV+H+LN,THP-1 cells over-expressing NEAT1 stably after being transfected with lentivirus were used,then LPS at a final concentration of 100 ng/ml and nigericin 10μmol/L were immediately added,and the cells were incubated for 24 h.Cell viability was detected by CCK-8 assay.Lactic dehydrogenase(LDH)release was assessed by colorimetric method.The amount of LDH released was measured by colorimetry.The concentrations of interleukin-1beta(IL-1β)and IL-18 in culture medium were measured by enzyme-linked immunosorbent assay.The pyroptotic rate was detected by flow cytometry.The expression of nucleotide-binding oligomerization domain-like receptor-containing pyrin domain 3(NLRP3),apoptosis-associated speck-like protein(ASC),caspase-1 and gasdermin D(GSDMD)was detected by Western blot.The expression of NEAT1 gene was determined by quantitative real-time polymerase chain reaction.Results Compared with group C,the cell viability was significantly decreased,the amount of LDH released,concentrations of IL-1βand IL-18,and pyroptotic rate were increased,and the expression of NEAT1 gene,NLRP3,ASC,caspase-1 and GSDMD was up-regulated in group LN(P<0.05).Compared with group LN,the cell viability was significantly increased,the amount of LDH released,concentrations of IL-1βand IL-18,and pyroptotic rate were decreased,and the expression of NEAT1 gene,NLRP3,ASC,caspase-1 and GSDMD was down-regulated in group H+LN(P<0.05).Compared with group H+LN,the cell viability was significantly decreased,the amount of LDH released,concentrations of IL-1βand IL-18,and pyroptotic rate were increased,and the expression of NEAT1 gene,NLRP3,ASC,caspase-1 and GSDMD was up-regulated in group LV+H+LN(P<0.05).Conclusions Hydrogen can ameliorate LPS and nigericin-induced pyroptosis in macrophages,and the mechanism may be associated with down-regulating the expression of lncRNA NEAT1.