基于趋化因子网络研究黄芩素延缓D-半乳糖诱导BV-2细胞衰老作用及机制  

Baicalein retards D-galactose-induced senescence of BV-2 cells through regulation of chemokine network

作　　者：伏蕾 秦雪梅[1,2,3] 高丽 FU Lei;QIN Xuemei;GAO Li(Modern Research Center for Traditional Chinese Medicine,Shanxi University,Taiyuan 030006,China;Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Shanxi University,Taiyuan 030006,China;Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi Province,Taiyuan 030006,China)

机构地区：[1]山西大学中医药现代研究中心,山西太原030006 [2]山西大学化学生物学与分子工程教育部重点实验室,山西太原030006 [3]地产中药功效物质研究与利用山西省重点实验室,山西太原030006

出　　处：《中草药》2024年第21期7300-7312,共13页Chinese Traditional and Herbal Drugs

基　　金：山西省基础研究计划自然科学研究面上项目(202203021211292)。

摘　　要：目的 探讨黄芩素延缓D-半乳糖(D-galactose,D-gal)诱导BV-2小胶质细胞衰老的分子机制。方法 采用200 mmol/L D-gal诱导BV-2细胞衰老模型,通过测定细胞活力、细胞形态、细胞周期、衰老相关β-半乳糖苷酶(senescence-associated-β-galactosidase,SA-β-gal)、细胞周期蛋白依赖性激酶抑制剂1A(cyclin-dependentkinase inhibitor1A,p21)、磷酸化组蛋白H2AX(phospho-histone H2AX,γH2AX)的表达,评价黄芩素延缓小胶质细胞衰老的作用。通过文献检索小胶质细胞衰老相关差异基因,构建基因网络,运用生信分析预测小胶质细胞衰老相关的关键基因及信号通路,采用q RT-PCR技术验证黄芩素对小胶质细胞衰老关键基因的调节作用,并进一步研究黄芩素对趋化因子(C-X-C基元)配体10(C-X-C motif chemokine 10,CXCL10)诱导BV-2细胞衰老的作用。结果 给予50μmol/L黄芩素24 h可通过提高细胞活力(P<0.001)、改善细胞形态、显著降低SA-β-gal蓝染细胞数量(P<0.01、0.001)、下调p21和γH2AX蛋白表达(P<0.05)、减少G0/G1期细胞周期停滞(P<0.01)来延缓小胶质细胞衰老。生信分析结果表明,小胶质细胞衰老前后趋化因子通路显著富集。黄芩素可显著下调趋化因子及其相关基因趋化因子(CC基元)配体2(C-C motif chemokine 2,CCL2)、CCL4、趋化因子(C-X-C基元)受体4(C-X-C chemokine receptor type 4,CXCR4)、趋化因子(C-C基元)受体5(C-C chemokine receptor type 5,CCR5)、CXCL10、Toll样受体2(Toll-like receptor 2,TLR2)、TLR4、肿瘤坏死因子(tumour necrotizing factor,TNF)、白细胞介素-1β(interleukin-1β,IL-1β)的m RNA水平(P<0.05、0.01、0.001),显著下调CXCL10的表达(P<0.05),显著延缓CXCL10引起的细胞衰老(P<0.001)。结论 黄芩素可能通过调节趋化因子及相关基因的表达,延缓D-gal诱导的BV-2细胞衰老。Objective To investigate the molecular mechanism of baicalein in delaying senescence induced by D-galactose(D-gal)in BV-2 microglia.Methods The senescence of BV-2 cell was induced by 200 mmol/L D-gal,and the effects of baicalein in delaying cell senescence were evaluated by measuring cell viability,cell morphology,expression of senescence marker senescence-associated-β-galactosidase(SA-β-gal),cell cycle protein-dependent kinase inhibitor 1A(cyclin-dependent kinase inhibitor 1A,p21)and phospho-histone H2AX(γH2AX),and cell cycle.Differential genes related to microglia senescence were searched by literature,and the gene network was constructed.Key genes and pathways related to microglia senescence were predicted using bioinformatics analysis,and baicalein regulatory effects on key genes related to microglia senescence were verified using qRT-PCR.The effects of baicalein on chemokine C-X-C motif chemokine 10(CXCL10)induced BV-2 cells were investigated.Results The results showed that 50μmol/L baicalein for 24h could delay microglia senescence by increasing cell viability(P<0.001),improving cell morphology,significantly decreasing the number of SA-β-gal blue-stained cells(P<0.01,0.001),down-regulating the expression of p21 andγH2AX proteins(P<0.05),and decreasing cell cycle arrest in the G0/G1 phase(P<0.01).Bioinformatics analysis showed that the chemokine pathway was significantly enriched in microglia senescence.Baicalein significantly down-regulated the mRNA levels of chemokines and their related genes C-C motif chemokine 2(CCL2),CCL4,C-X-C chemokine receptor type 4(CXCR4),C-C chemokine receptor type 5(CCR5),C-X-C motif chemokine 10(CXCL10),toll-like receptor 2(TLR2),TLR4,tumour necrotizing factor(TNF),interleukin-1β(IL-1β)mRNA levels(P<0.05,0.01,0.001),significantly down-regulated the level of CXCL10(P<0.05)and reduced CXCL10-induced cell senescence in BV-2 cells(P<0.001).Conclusion Baicalein may delay senescence induced by D-gal in BV-2 cells by regulating the expression of chemokines and related genes.

关 键 词：黄芩素 小胶质细胞 衰老 趋化因子网络 CXCL10 

分 类 号：R285.5[医药卫生—中药学]