汉黄芩素减轻LPS诱导BV-2细胞炎症并保护SH-SY5Y细胞的机制  

Mechanism of Wogonin in Alleviating LPS-Induced Inflammation in BV-2 Cells and Protecting SH-SY5Y Cells

作　　者：孙梦菲 欧阳竞锋[1] 吴春阳 程娇娇 SUN Mengfei;OUYANG Jingfeng;WU Chunyang;CHENG Jiaojiao(Beijing Key Research Laboratory of Traditional Chinese Medicine for Prevention and Treatment of Major Diseases,Experimental Research Center of Chinese Academy of Medical Sciences,Beijing 100700,China;Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100007,China)

机构地区：[1]中国中医科学院医学实验中心中医药防治重大疾病基础研究北京市重点实验室,北京100700 [2]北京中医药大学东直门医院,北京100007

出　　处：《中国实验方剂学杂志》2024年第20期62-69,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程重大攻关项目(CI2021A00602);国家自然科学基金面上项目(81470171)。

摘　　要：目的:探讨汉黄芩素对脂多糖(LPS)诱导的BV-2小胶质细胞条件培养基培养SH-SY5Y细胞的保护机制。方法:将BV-2细胞分为空白组、LPS组、汉黄芩素低、中、高浓度组(4、8、16μmol·L^(-1)),其中LPS组给予1 mg·L^(-1)的LPS,汉黄芩素组分别用相应浓度的汉黄芩素预处理4 h后,给予1 mg·L^(-1)的LPS并使用以上各组的条件培养基培养SH-SY5Y细胞。通过细胞增殖与活性检测(CCK-8)试剂盒检测以上各组BV-2细胞的细胞活力;酶联免疫吸附测定法(ELISA)检测各组BV-2细胞上清液中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量;免疫组织化学法(IHC)检测SH-SY5Y细胞的酪氨酸羟化酶(TH)、α-突触核蛋白(α-Syn)的表达情况;免疫荧光法(IF)检测SH-SY5Y细胞核转录因子-κB p65(NF-κB p65)蛋白核转移和荧光表达强度;蛋白免疫印迹法(Western blot)检测SH-SY5Y细胞Toll样受体4(TLR4)/髓样分化因子88(MyD88)/NF-κB通路相关蛋白表达情况。结果:与空白组比较,LPS组BV-2细胞上清IL-6、TNF-α含量均显著升高(P<0.01);与LPS组比较,汉黄芩素低浓度组BV-2细胞上清IL-6含量降低(P<0.05),汉黄芩素中高浓度组IL-6和TNF-α含量均降低(P<0.05,P<0.01),其中汉黄芩素高浓度组IL-6和TNF-α含量降低最显著(P<0.01),干预效果最佳。与空白组比较,LPS组条件培养基培养的SH-SY5Y细胞α-Syn蛋白表达显著升高、TH蛋白表达显著降低(P<0.01);与LPS组比较,汉黄芩素中高浓度组α-Syn蛋白表达明显降低(P<0.05,P<0.01),汉黄芩素低、中、高浓度组TH蛋白表达明显升高(P<0.05,P<0.01)。与空白组比较,LPS组NF-κB p65蛋白逐渐向核内聚集、荧光表达强度显著增强(P<0.01);与LPS组比较,汉黄芩素各浓度组NF-κB p65蛋白逐渐向核外分散、荧光表达强度逐渐减弱,汉黄芩素高浓度组荧光强度显著减弱(P<0.01)。与空白组比较,LPS组TLR4、磷酸化(p)-NF-κB p65、MyD88蛋白表达明显升高(P<0.05,P<0.01);与LPS组比较,汉黄芩Objective:To examine the protective mechanism of wogonin in SH-SY5Y cells cultured in the conditioned media with lipopolysaccharide(LPS)-induced BV-2 microglia.Method:BV-2 microglia were divided into the blank group,LPS group,low concentration group of wogonin(4μmol∙L-1),medium concentration group of wogonin(8μmol∙L-1),and high concentration group of wogonin(16μmol∙L-1).The LPS group was given 1 mg·L^(-1) LPS,and the other three groups were treated with the corresponding concentration of wogonin for 4 h and then given 1 mg·L^(-1) LPS.The conditioned media from these groups were used to cultivate SH-SY5Y cells.Cell counting kit-8(CCK-8)was used to assess the vitality of BV-2 cells in the above groups.The contents of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in the supernatant of BV-2 cells were determined by enzyme-linked immunosorbent assay(ELISA).The expression of tyrosine hydroxylase(TH)andα-Synuclein(α-Syn)in SH-SY5Y cells was detected by immunohistochemical staining(IHC).The nuclear transfer and fluorescence expression intensity of nuclear transcription factor-κB p65(NF-κB p65)protein in SH-SY5Y cells were detected by immunofluorescence staining(IF).Western blot was used to detect the expression of Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/NF-κB pathwayrelated proteins in SH-SY5Y cells.Result:The levels of IL-6 and TNF-αin the supernatant of BV-2 cells in the LPS group were significantly higher than those in the blank group(P<0.01).Compared with those in the LPS group,the IL-6 content of BV-2 cells in the low concentration group of wogonin was statistically significantly lower(P<0.05),whereas the IL-6 and TNF-αcontents of the medium and high concentration groups of wogonin were statistically lower(P<0.05,P<0.01).The IL-6 and TNF-αcontents in the high concentration group of wogonin decreased most significantly(P<0.01),and the intervention effect was the best.Compared with that in the blank group,the expression ofα-Syn protein in SH-SY5Y cells cultur

关 键 词：帕金森病 汉黄芩素 脂多糖(LPS) Toll样受体4(TLR4)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5R161.1