目的:探究核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,Nr4a1)Nr4a1激动剂胞孢子酮B(cytosporone B,Csn-B)对小鼠噪声暴露后听力损失的治疗作用。方法:采用双氧水刺激HEI-OC1毛细胞系的方法构建氧化应激细胞...目的:探究核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,Nr4a1)Nr4a1激动剂胞孢子酮B(cytosporone B,Csn-B)对小鼠噪声暴露后听力损失的治疗作用。方法:采用双氧水刺激HEI-OC1毛细胞系的方法构建氧化应激细胞模型;通过实时荧光定量PCR(quantitative real-time PCR,q PCR)检测细胞中Nr4a1的mRNA表达水平;分别通过细胞计数试剂盒(cell counting kit-8,CCK8)及流式细胞术的方法检测细胞活力和细胞凋亡水平以评估Csn-B预处理后经双氧水刺激的细胞状态。构建小鼠噪声性听力损失模型,运用qPCR和免疫荧光技术检测噪声暴露后Nr4a1在小鼠耳蜗中的表达;通过检测听性脑干反应(auditory brainstem response,ABR)评估噪声暴露后以及Csn-B连续治疗13 d后小鼠听力情况。结果:双氧水刺激后HEI-OC1毛细胞中Nr4a1表达上升,细胞活力显著下降,凋亡水平显著升高;Csn-B预处理HEI-OC1毛细胞经双氧水刺激,细胞活力显著高于对照组而凋亡水平则显著低于对照组。在体研究结果显示,噪声暴露后小鼠听力显著降低,Nr4a1在小鼠耳蜗中的表达水平显著升高。噪声暴露后经Csn-B治疗小鼠听力得到改善,主要表现为Click-ABR以及Tone Burst-ABR(4000、8000Hz处)阈值下降。结论:Nr4a1激动剂Csn-B增强内耳毛细胞对氧化应激损伤的抵御能力,部分改善噪声暴露后的小鼠听力。展开更多
目的探究核受体亚家族4 A组成员1(nuclear receptor subfamily 4 group A member 1,NR4A1)在缓解顺铂对近端肾小管上皮细胞毒性的作用及其分子机制。方法通过"Tabula-muris"单细胞转录组测序数据库分析肾脏组织各细胞亚群NR4A...目的探究核受体亚家族4 A组成员1(nuclear receptor subfamily 4 group A member 1,NR4A1)在缓解顺铂对近端肾小管上皮细胞毒性的作用及其分子机制。方法通过"Tabula-muris"单细胞转录组测序数据库分析肾脏组织各细胞亚群NR4A1基因的表达。在近端肾小管上皮细胞HK-2细胞系及原代细胞中,通过慢病毒感染以过表达NR4A1基因。采用细胞增殖与毒性检测试剂盒(cell counting kit-8,CCK-8)检测顺铂的细胞毒性。细胞用碘化丙啶(propidium iodide,PI)单染后通过流式细胞仪检测细胞的死亡比例。通过实时荧光定量PCR和Western印迹法检测细胞中NR4A1和核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,NRF2)基因的表达。通过检测丙二醛(malondialdehyde,MDA)和氧化型谷胱甘肽(oxidized glutathione,GSSG)以及脂质活性氧(reactive oxygen species,ROS)的含量分析细胞铁死亡程度。结果单细胞转录组数据分析的结果表明NR4A1基因表达在肾脏组织的近端肾小管上皮细胞亚群中最低。50μmol/L和100μmol/L顺铂能够显著诱导近端肾小管上皮细胞MDA、GSSG和脂质ROS含量的上升(均P<0.01),且顺铂浓度越高诱导MDA、GSSG和脂质ROS增加越多。相比对照HK-2细胞,过表达NR4A1的HK-2细胞脂质ROS含量及铁离子含量显著较低(均P<0.01),过表达NR4A1抑制了顺铂对近端肾小管上皮细胞的毒性及其诱导的铁死亡。分子机制研究发现,在近端肾小管上皮细胞中,过表达NR4A1上调了抗铁死亡基因NRF2的表达(P<0.01)。进一步单细胞转录组数据分析的结果表明,与NR4A1在肾脏组织细胞亚群的表达状态相似,NRF2表达在近端肾小管上皮细胞中也最低。结论顺铂能够诱导近端肾小管上皮细胞发生铁死亡,且浓度越高越明显。NR4A1通过上调近端肾小管上皮细胞NRF2的表达抑制顺铂诱导的细胞铁死亡,从而缓解顺铂对细胞的毒性。展开更多
核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,NR4A1)是由多种应激源诱导的早期转录因子,参与细胞生理活动与病理进程,尤其是在炎症相关的肺部疾病中发挥关键的调节作用,且其活性表达受转录后修饰的调节。文...核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,NR4A1)是由多种应激源诱导的早期转录因子,参与细胞生理活动与病理进程,尤其是在炎症相关的肺部疾病中发挥关键的调节作用,且其活性表达受转录后修饰的调节。文章探讨了NR4A1在哮喘、急性肺损伤(acute lung injury,ALI)、肺动脉高压(pulmonary arterial hypertension,PAH)和慢性阻塞性肺疾病(chronic obstructive pulmonary diseases,COPD)中的调控作用,并对其小泛素相关修饰物(small ubiquitin‑like modifier,SUMO)修饰、磷酸化、乙酰化转录后修饰调节机制进行综述,为进一步明确NR4A1在肺实质性疾病中转录后修饰的调控机制及寻找临床肺实质相关疾病的防治靶点提供理论依据。展开更多
Background and objective:Acute liver failure(ALF)is a type of disease with high mortality and rapid progression with no specific treatment methods currently available.Glucocorticoids exert beneficial clinical effects ...Background and objective:Acute liver failure(ALF)is a type of disease with high mortality and rapid progression with no specific treatment methods currently available.Glucocorticoids exert beneficial clinical effects on therapy for ALF.However,the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine.The purpose of this study was to investigate the specific immunological mechanism of dexamethasone(Dex)on treatment of ALF induced by lipopolysaccharide(LPS)/D-galactosamine(D-Ga IN)in mice.Methods:Male C57 BL/6 mice were given LPS and D-Ga IN by intraperitoneal injection to establish an animal model of ALF.Dex was administrated to these mice and its therapeutic effect was observed.Hematoxylin and eosin(H&E)staining was used to determine liver pathology.Multicolor flow cytometry,cytometric bead array(CBA)method,and next-generation sequencing were performed to detect changes of messenger RNA(m RNA)in immune cells,cytokines,and Kupffer cells,respectively.Results:A mouse model of ALF can be constructed successfully using LPS/D-Ga IN,which causes a cytokine storm in early disease progression.Innate immune cells change markedly with progression of liver failure.Earlier use of Dex,at 0 h rather than 1 h,could significantly improve the progression of ALF induced by LPS/D-Ga IN in mice.Numbers of innate immune cells,especially Kupffer cells and neutrophils,increased significantly in the Dex-treated group.In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor(Gr).Sequencing of Kupffer cells revealed that Dex could increase m RNA transcription level of nuclear receptor subfamily 4 group A member 1(Nr4 a1),and that this effect disappeared after Gr inhibition.Conclusions:In LPS/D-Ga IN-induced ALF mice,early administration of Dex improved ALF by increasing the numbers of innate immune cells,especially Kupffer cells and neutrophils.Gr-dependent Nr4 a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.展开更多
基金Project supported by the Zhejiang Provincial Natural Science Foundation of China(No.LEZ20H260001)the National Natural Science Foundation of China(No.81700552)。
文摘Background and objective:Acute liver failure(ALF)is a type of disease with high mortality and rapid progression with no specific treatment methods currently available.Glucocorticoids exert beneficial clinical effects on therapy for ALF.However,the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine.The purpose of this study was to investigate the specific immunological mechanism of dexamethasone(Dex)on treatment of ALF induced by lipopolysaccharide(LPS)/D-galactosamine(D-Ga IN)in mice.Methods:Male C57 BL/6 mice were given LPS and D-Ga IN by intraperitoneal injection to establish an animal model of ALF.Dex was administrated to these mice and its therapeutic effect was observed.Hematoxylin and eosin(H&E)staining was used to determine liver pathology.Multicolor flow cytometry,cytometric bead array(CBA)method,and next-generation sequencing were performed to detect changes of messenger RNA(m RNA)in immune cells,cytokines,and Kupffer cells,respectively.Results:A mouse model of ALF can be constructed successfully using LPS/D-Ga IN,which causes a cytokine storm in early disease progression.Innate immune cells change markedly with progression of liver failure.Earlier use of Dex,at 0 h rather than 1 h,could significantly improve the progression of ALF induced by LPS/D-Ga IN in mice.Numbers of innate immune cells,especially Kupffer cells and neutrophils,increased significantly in the Dex-treated group.In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor(Gr).Sequencing of Kupffer cells revealed that Dex could increase m RNA transcription level of nuclear receptor subfamily 4 group A member 1(Nr4 a1),and that this effect disappeared after Gr inhibition.Conclusions:In LPS/D-Ga IN-induced ALF mice,early administration of Dex improved ALF by increasing the numbers of innate immune cells,especially Kupffer cells and neutrophils.Gr-dependent Nr4 a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.
