PAG双向调节H_(2)S诱导GES-1细胞免疫反应  

PAG Induces immune responses of GES-1 cells by bidirectional regulation of H_(2)S

作　　者：陈刚 马春杰 林悦铭 伦志强 林秀英 王玉 夏春辉[2] Chen Gang;Ma Chunjie;Lin Yueming;Lun Zhiqiang;Lin Xiuying;Wang Yu;Xia Chunhui(Research Institute of Medicine and Pharmacy,Qiqihar Medical University,Qiqihar 161006,China;Pharmacy Department,Qiqihar Medical University,Qiqihar 161006,China;Basic Medicine Department,Qiqihar Medical University,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院医药科学研究院分子生物学研究室,黑龙江齐齐哈尔161006 [2]齐齐哈尔医学院药学院化学教研室,黑龙江齐齐哈尔161006 [3]齐齐哈尔医学院基础医学院细胞生物学教研室,黑龙江齐齐哈尔161006

出　　处：《国际免疫学杂志》2023年第4期378-385,共8页International Journal of Immunology

基　　金：齐齐哈尔市科技计划联合引导项目(LHYD-2021018)。

摘　　要：目的探讨硫化氢(hydrogen sulfide,H_(2)S)诱导胃黏膜上皮细胞-1(gastric mucosal epithelial-1,GES-1)的免疫反应机制。方法分别设立对照组、炔丙基甘氨酸(propargylglycine,PAG)浓度组(PAG分别为100μmol/L,PAG 500μmol/L,PAG 1000μmol/L)和NaHS(NaHS 400μmol/L)作用组(NaHS组,NaHS-PAG 100μmol/L组,NaHS-PAG 500μmol/L组,NaHS-PAG 1000μmol/L组)。在不同浓度H_(2)S合成酶胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)抑制剂PAG(100μmol/L,500μmol/L,1000μmol/L)作用下,通过添加400μmol/L外源性NaHS作用于胃黏膜细胞GES-1进行研究。Calcein/PI细胞活性与细胞毒性实验检测NaHS对GES-1细胞膜渗透性的影响;酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测细胞内的CSE的活性及H_(2)S、白细胞介素(interleukin,IL)-1β和IL-18含量;DCF(DCFH-DA)/Hoechst33342双标染色检测细胞内活性氧(reactive oxygen species,ROS)水平。结果Calcein/PI细胞活性与细胞毒性实验结果显示,PAG和NaHS不破坏GES-1细胞膜完整性,PAG 500μmol/L、PAG 1000μmol/L及NaHS 400μmol/L抑制细胞增殖,抑制率分别为(48.11±2.29)%、(60.87±2.42)%、(59.83±2.3)%。100μmol/LPAG阻碍了NaHS的增殖抑制作用,抑制率为(47.80±2.65)%,PAG 500μmol/L和PAG1000μmol/L促进了NaHS的增殖抑制作用,抑制率分别为(65.16±1.02)%、(70.90%±1.30)%;ELISA结果显示,NaHS作用GES-1细胞后,与对照组相比,细胞内CSE的活性明显增高[U/mL:(23.33±0.77)比(6.55±1.84)]、H_(2)S含量[μmol/L:(13.27±0.83)比(1.39±0.29)],炎性因子IL-1β水平增加[μg/L:(3.48±0.26)比(0.77±0.11)],IL-18水平降低[ng/L:(1.15±0.36)比(3.19±0.90)],差异均有统计学意义(P值分别为0.00,0.00,0.00,0.02),镜下观察显示ROS含量增加。PAG 100μmol/L弱化NaHS的作用,差异均具有统计学意义(P值均<0.05),而PAG 500μmol/L和PAG 1000μmol/L增强NaHS的作用,差异均具有统计学意义(P值均<0.05)。此外,IL-18表达与IL-1β的表达呈负相关(r=-0.49,P<0.05)。结论PAG双向调节Objective To explore the mechanism of hydrogen sulfide(H_(2)S)-induced immune response in gastric mucosal cells(GES-1).Methods Control group,propargylglycine(PAG)concentration group(PAG 100μmol/L,PAG 500μmol/L,PAG 1000μmol/L)and NaHS(HaHS 400μmol/L)action group(NaHS group,NAHS-PAG 100μmol/L,NaHS-PAG 500μmol/L group,NaHS-PAG 1000μmol/L group)were set up,respectively.In the presence of different concentrations(100μmol/L,500μmol/L,1000μmol/L)of the cystathionine-γ-lyase(CSE)inhibitor PAG,the GES-1 cells was treated with NaHS(400μmol/L),the effect of NaHS or/and PAG on the membrane permeability of GES-1 cells were determined by the Calcein/PI cell activity and cytotoxicity assay,the activity of CSE and H_(2)S,interleukin(IL)-1βand IL-18 content were determined by enzyme-linked immunosorbent assay(ELISA),and intracellular reactive oxygen species(ROS)were detected by DCF(DCFH-DA)/Hoechst33342 double standard staining.Results The Calcein/PI cell activity and cytotoxicity assay showed that PAG and NaHS did not disrupt GES-1 membrane integrity,PAG 500μmol/L,PAG 1000μmol/L,and NaHS 400μmol/L inhibited cell proliferation with(48.11±2.29)%,(60.87±2.42)%,and(59.83±2.3)%,respectively.The PAG 100μmol/L prevented the inhibitory proliferation of NaHS with the inhibition rate of(47.80±2.65)%,while PAG 500μmol/L and PAG 1000μmol/L promoted the inhibitory proliferation of NaHS,with(65.16±1.02)%and(70.90±1.30)%,respectively.After GES-1 treatment with cells NaHS,the activity of CSE[U/mL:(23.33±0.77)vs(6.55±1.84)],content of H_(2)S[μmol/L:(13.27±0.83)vs(1.39±0.29)],and inflammatory factors IL-1β[μg/L:(3.48±0.26)vs(0.77±0.11)]were increased,while IL-18[ng/L:(1.15±0.36)vs(3.19±0.90)]were reduced(P values were 0.00,0.00,0.00 and 0.02,respectively).Increased ROS level was observed under the microscope.The PAG 100μmol/L weakened the effect of NaHS(all P<0.05).However,the PAG 500μmol/L and PAG 1000μmol/L aggravate the effects of NaHS,respectively(all P<0.05).In addition,IL-18 expression was negatively c

关 键 词：硫化氢 炔丙基甘氨酸 白细胞介素-1Β 白细胞介素-18 胃黏膜细胞 

分 类 号：R392[医药卫生—免疫学]