黄芩素抑制小胶质细胞活化及保护SH-SY5Y神经细胞的机制  

Mechanism of Baicalein in Inhibiting Microglial Activation and Protecting SH-SY5Y Nerve Cells

作　　者：尹梦霞 欧阳竞锋[1] 崔拓拓 刘鑫 孙梦菲 荆志伟[3] YIN Mengxia;OUYANG Jingfeng;CUI Tuotuo;LIU Xin;SUN Mengfei;JING Zhiwei(Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China;Dongfang Hospital,Beijing University of Chinese Medicine,Beijing 100078,China;Institute of Basic Research in Clinical Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,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]北京中医药大学东方医院,北京100078 [3]中国中医科学院中医临床基础医学研究所,北京100700

出　　处：《中国实验方剂学杂志》2023年第10期93-101,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医药管理局全国中医药创新骨干人才培训项目(ZX2020003)。

摘　　要：目的:探讨黄芩素(BAI)对脂多糖(LPS)激活的BV-2细胞条件培养基下SH-SY5Y细胞损伤的干预作用及机制。方法:用LPS 1 mg·L^(-1)激活BV-2细胞建立LPS组条件培养基,同时设置空白组、BAI低、中、高浓度组(4、8、16μmol·L^(-1)),使用各组条件培养基培养SH-SY5Y细胞。通过细胞增殖与活性检测(CCK-8)试剂盒测定干预后各组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细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)含量;采用免疫组化(IHC)观察SH-SY5Y细胞中α-突触核蛋白(α-syn)、酪氨酸羟化酶(TH)蛋白表达情况,免疫荧光(IF)法观察SH-SY5Y细胞中核转录因子-κB p65蛋白(NF-κB p65,以下简称p65)核转移情况,蛋白免疫印迹法(Western blot)检测SH-SY5Y细胞Toll样受体4(TLR4)、p65、磷酸化(p)-p65、髓样分化因子88(My D88)蛋白表达情况。结果:与空白组比较,LPS组中BV-2细胞活力显著降低(P<0.01),细胞上清液中TNF-α、IL-6、IL-1β含量均显著升高(P<0.01);与LPS组比较,BAI低、中、高浓度组细胞活力显著升高(P<0.01),细胞上清液中TNF-α均显著降低(P<0.01),BAI高浓度(16μmol·L^(-1))组细胞上清液中IL-6含量明显降低(P<0.05),BAI中、高浓度组细胞上清液中IL-1β含量显著降低(P<0.01)。条件培养基处理SH-SY5Y细胞的结果显示,与空白组比较,LPS组SH-SY5Y细胞中p65蛋白表达向核内聚集,TH蛋白表达显著下降(P<0.01),α-syn、TLR4、My D88、p-p65蛋白表达均明显升高(P<0.05,P<0.01);与LPS组比较,BAI低、中、高浓度组SH-SY5Y细胞中p65蛋白表达逐渐分散至细胞质中,TH蛋白表达均显著升高(P<0.01),α-syn蛋白表达均显著降低(P<0.01),BAI高浓度组TLR4、My D88、p-p65蛋白表达明显降低(P<0.05,P<0.01),BAI中浓度组p-p65和My D88蛋白表达均明显降低(P<0.05),BAI低浓度组My D88蛋白表达明显降低(P<0.05)。各组p65蛋白表达差异无统计学意义。结论:BAI�Objective:To investigate the effect of baicalein(BAI)on SH-SY5Y cell injury in lipopolysaccharide(LPS)-activated BV-2 cells conditioned medium and its mechanism.Method:The BV-2 cells were activated with 1 mg∙L-1 of LPS to establish the conditioned medium of the LPS group,and a blank group and groups of BAI with low,medium,and high concentrations(4,8,16μmol∙L-1)were established.SH-SY5Y cells were cultured with the conditioned medium of each group.The cell viability of BV-2 cells in each group after the intervention was determined by cell counting kit-8(CCK-8).The content of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the supernatant of BV-2 cells in each group was determined by enzyme-linked immunosorbent assay(ELISA).The protein expression ofα-synuclein(α-syn)and tyrosine hydroxylase(TH)in SH-SY5Y cells was observed by immunohistochemical(IHC)staining,and the nuclear transfer of nuclear factor kappa-B p65 protein(NF-κB p65,p65)in SH-SY5Y cells was observed by immunofluorescence(IF).The protein expression of Toll-like receptor 4(TLR4),p65,phosphorylated p65(p-p65),and Myeloid differentiation factor 88(MyD88)in SH-SY5Y cells was observed by Western blot.Result:As compared with the blank group,the viability of BV-2 cells in the LPS group was significantly decreased(P<0.01),and the content of TNF-α,IL-6,and IL-1βin the cell supernatant was significantly increased(P<0.01).As compared with the LPS group,the cell viability was significantly increased in groups of BAI with low,medium,and high concentrations(P<0.01),and TNF-αin the cell supernatant was significantly decreased(P<0.01).The content of IL-6 in the cell supernatant was decreased in the BAI group with high concentration(P<0.05),and the content of IL-1βin the cell supernatant was significantly decreased in the BAI groups with medium and high concentrations(P<0.01).The results of conditioned medium cultured SH-SY5Y cells showed that as compared with the blank group,the protein expression of p65 in the LPS group e

关 键 词：帕金森病 脂多糖(LPS) 黄芩素(BAI) Toll样受体4(TLR4) 

分 类 号：R2-0[医药卫生—中医学] R22R33R289R742