摘要
目的观察根皮素对白细胞介素(IL)-1β诱导的Graves眼病(GO)患者眼眶成纤维细胞(OFs)中炎症反应和氧化应激的抑制作用及其机制。方法收集2019年1月至2020年12月于河南省立眼科医院确诊为非活动期GO并行眼眶减压术的患者6例6眼的眼眶脂肪及结缔组织。采用组织块培养法分离原代OFs并传代,细胞免疫荧光法鉴定OFs。将细胞分为对照组、IL-1β诱导组以及不同浓度根皮素处理组。采用细胞计数试剂盒8(CCK-8)法检测25、50、75、100和200μmol/L根皮素处理24和48 h后OFs的活性。采用IL-1β诱导OFs模拟GO体外炎症环境。采用荧光探针H2DCF-DA法检测正常对照组、IL-1β诱导组、50μmol/L根皮素组和100μmol/L根皮素组细胞内活性氧簇(ROS)水平。采用酶联免疫吸附法(ELISA)检测正常对照组、IL-1β诱导组以及25、50、75、100μmol/L根皮素组细胞培养上清液中促炎细胞因子IL-6、IL-8和MCP-1质量浓度。采用Western blot法检测正常对照组、IL-1β诱导组及100μmol/L根皮素组细胞内血红素加氧酶1(HO-1)、核因子NF-E2相关因子Nrf2和MAPK信号通路蛋白P38、细胞外调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)及其磷酸化蛋白表达。结果原代分离培养的OFs表达vimentin,证明为间充质来源,desimin、S-100、cytokeratin-18表达阴性,鉴定为成纤维细胞。CCK-8结果显示,25、50、75和100μmol/L根皮素组细胞处理后24和48 h吸光度(A)值与对照组比较,差异均无统计学意义(均P>0.05)。50μmol/L根皮素组和100μmol/L根皮素组细胞内ROS水平分别为21.95±1.71和10.01±1.03,明显低于IL-1β诱导组的39.27±4.01,差异均有统计学意义(均P<0.01)。ELISA结果显示,25μmol/L、50μmol/L、75μmol/L和100μmol/L根皮素组细胞培养上清液中IL-6质量浓度分别为(4544.25±572.98)、(1000.25±133.96)、(724.25±98.63)和(519.50±118.02)pg/ml,均显著低于IL-1β诱导组的(7581.75±565.93)pg/ml,差异均有统计学意义(均P<0.01);50μmol/L、75μmol/L和100μmol/L根皮素组细胞培养上清液中IL-8质量浓度分别为(3679.50±676.76)、(2143.75±616.20)和(1174.75±284.18)pg/ml,显著低于IL-1β诱导组的(8411.00±939.67)pg/ml,差异均有统计学意义(均P<0.01);50μmol/L、75μmol/L和100μmol/L根皮素组细胞培养上清液中MCP-1质量浓度分别为(3783.25±610.24)、(1565.75±457.89)和(745.75±227.01)pg/ml,显著低于IL-1β诱导组的(5533.00±602.87)pg/ml,差异均有统计学意义(均P<0.01)。100μmol/L根皮素组HO-1、Nrf2蛋白相对表达量明显高于IL-1β诱导组,p-P38、p-ERK、p-JNK蛋白相对表达量明显低于IL-1β诱导组,差异均有统计学意义(均P<0.01)。结论根皮素可降低IL-1β诱导的GO患者OFs细胞内氧化应激水平,抑制促炎细胞因子产生,其作用机制与Nrf2/HO-1的激活及MAPK信号通路的抑制相关。
Objective To investigate the inhibitory effect of phloretin on inflammation and oxidative stress in interleukin(IL)-1βinduced orbital fibroblasts(OFs)from Graves orbitopathy(GO)patients and its mechanism.Methods The orbital fat and connective tissue from 6 eyes of 6 patients diagnosed as inactive GO who underwent orbital decompression in Henan Eye Hospital from January 2019 to December 2020 were collected.Primary OFs were isolated and passaged by explant culture and were identified by cell immunofluorescence assay.OFs were divided into control group,IL-1βinduced group,and groups of various phloretin concentrations(25,50,75,100 and 200μmol/L).The viability of OFs after 24-and 48-hour treatment of the various phloretin concentrations was determined by cell counting kit-8(CCK-8).OFs were induced by IL-1βto simulate an inflammatory environment of GO in vitro.Intracellular reactive oxygen species(ROS)levels of the normal control group,IL-1βinduced group,50μmol/L phloretin group and 100μmol/L phloretin group were detected by fluorescent probe(H2DCF-DA).The concentrations of pro-inflammatory cytokines IL-6,IL-8 and monocyte chemoattractant protein-1(MCP-1)in cell culture supernatant of the normal control group,IL-1βinduced group and phloretin treated groups(25,50,75,and 100μmol/L)were examined by enzyme-linked immunosorbent assay(ELISA).The expressions of heme oxygenase-1(HO-1),nuclear factor erythroid 2-related factor(Nrf2)proteins,as well as P38,extracelluar regulated protein kinase(ERK)and c-Jun N-terminal kinase(JNK)proteins as well as their phosphorylated proteins in the MAPK signal pathway of the normal control group,IL-1βinduced group and 100μmol/L phloretin group,were detected by Western blot.The purpose and methods of the study were explained to the patients and their family members.Written informed consent was obtained.The study protocol was approved by the Ethics Committee of Henan Provincial People's Hospital(No.HNEECKY-2020[07]).Results For cultured OFs,the mesenchymal origin was confirmed by positive expression of vimentin and fibroblasts were identified by negative expression of desmin,S-100 and cytokeratin-18.CCK-8 showed that there was no significant difference in absorbance value after 24-and 48-hour treatment between 25μmol/L,50μmol/L,75μmol/L and 100μmol/L phloretin groups and control group(all at P>0.05).The ROS levels of 50μmol/L and 100μmol/L phloretin groups were 21.95±1.71 and 10.01±1.03,respectively,which were significantly lower than 39.27±4.01 of IL-1βinduced group(both at P<0.01).ELISA showed that IL-6 concentrations in 25μmol/L,50μmol/L,75μmol/L and 100μmol/L phloretin groups were(4544.25±572.98),(1000.25±133.96),(724.25±98.63),(519.50±118.02)pg/ml,respectively,which were all significantly lower than(7581.75±565.93)pg/ml in IL-1βinduced group(all at P<0.01).IL-8 concentrations in 50μmol/L,75μmol/L and 100μmol/L phloretin groups were(3679.50±676.76),(2143.75±616.20),(1174.75±284.18)pg/ml,respectively,which were all significantly lower than(8411.00±939.67)pg/ml in IL-1βinduced group(all at P<0.01).The concentrations of MCP-1 in 50μmol/L,75μmol/L and 100μmol/L phloretin groups were(3783.25±610.24),(1565.75±457.89),(745.75±227.01)pg/ml,respectively,which were all significantly lower than(5533.00±602.87)pg/ml in IL-1βinduced group(all at P<0.01).The relative expression levels of HO-1 and Nrf2 were significantly higher and the relative expression levels of p-P38,p-ERK,and p-JNK were significantly lower in 100μmol/L phloretin group than IL-1βinduced group(all at P<0.01).Conclusions Phloretin reduces the oxidative stress level of IL-1βinduced OFs from GO patients and inhibits the production of pro-inflammatory cytokines.The mechanism is related to the activation of Nrf2/HO-1 and the inhibition of the MAPK signal pathway.
作者
靳玮
张黎
李谦
倪瑶
柴昌
Jin Wei;Zhang Li;Li Qian;Ni Yao;Chai Chang(Department of Ophthalmology,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,People's Hospital of Zhengzhou University,Zhengzhou 450003,China;State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-Sen University,Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science,Guangdong Provincial Clinical Research Center for Ocular Diseases,Guangzhou 510060,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2023年第3期233-240,共8页
Chinese Journal Of Experimental Ophthalmology
基金
河南省自然科学基金项目(202300410406)。
