活血荣络方调控生物钟蛋白Bmal1改善bEnd.3细胞糖氧剥夺/复氧损伤后血管新生的机制研究

Mechanism of Huoxue Rongluo Recipe on regulating circadian clock protein Bmal1 to improve angiogenesis in bEnd.3 cells after oxygen-glucose deprivation/reperfusion injury

目的 基于阴阳理论及活血荣络方(Huoxue Rongluo Recipe,HXRL)对脑梗死后血管新生的影响,探讨HXRL调控生物钟蛋白脑和肌肉芳烃受体核转位蛋白1(brain and muscle Arnt-like 1,Bmal1)促内皮细胞血管新生的作用机制。方法采用Western blotting检测小鼠脑微血管内皮细胞株(bEnd.3)受到糖氧剥夺/复氧(oxygen-glucose deprivation/reperfusion,OGD/R)损伤后各时间点生物钟蛋白Bmal1和Clock的表达;利用染色质免疫共沉淀-测序分析(chromatin immunoprecipitation sequencing,ChIP-seq)明确Bmal1在全基因组中发挥的作用,以及对bEnd.3细胞生物进程、细胞组成及分子功能的影响。采用CCK-8法检测HXRL含药血清最佳干预浓度,敲降bEnd.3细胞Bmal1基因,设置对照组、模型组、HXRL含药血清组、si-Bmal1组和si-Bmal1+HXRL含药血清组,通过划痕、迁移、成管等实验检测细胞迁移及血管形成能力;采用免疫荧光检测各组血管内皮生长因子(vascular endothelial growth factor,VEGF)和神经源性基因Notch同源蛋白1(neurogenic locus notch homolog protein 1,Notch1)表达;采用Western blotting检测VEGF、基质金属蛋白酶2(matrix metalloproteinases 2,MMP2)、Notch1蛋白胞内结构域(notch1 intracellular domain,NICD)和δ样蛋白4(delta-like protein 4,DLL4)蛋白表达。结果 b End.3细胞受到OGD/R损伤后,Bmal1及Clock蛋白表达逐渐升高,造模8、12、16、20 h后具有显著差异(P<0.05、0.001)。ChIP-seq提示Bmal1在内皮细胞中参与细胞发育、分化、增殖等进程,靶向调节VEGF、Notch1启动子区域。CCK-8实验结果显示,bEnd.3细胞受到OGD/R损伤后,10%的HXRL含药血清可有效改善细胞活力(P<0.001)。划痕、迁移及成管实验证实HXRL含药血清可有效改善bEnd.3细胞OGD/R损伤后的迁移能力及血管生成能力(P<0.05、0.01、0.001),但在Bmal1敲降株中,该促进作用被抑制(P<0.05、0.01、0.001)。免疫荧光及Western blotting结果显示,HXRL含药血清可进一步升高OGD/R损伤的bEnd.3细胞中VEGF、MMP2、NICD、DLL4、Bmal1及Clock蛋白表达(P<0.05、0.01、0.001),在Bmal1敲降株中,HXRL含药血清对VEGF、MMP2、NICD及DLL4的促表达作用被抑制(P<0.05、0.01、0.001)。结论 HXRL可有效促进内皮细胞经OGD/R损伤后的血管生成能力,其作用机制与调控生物钟Bmal1蛋白密切相关。

Objective To explore the mechanism of Huoxue Rongluo Recipe(活血荣络方,HXRL)on promoting endothelial cells angiogenesis via regulating circadian protein brain and muscle arnt-like 1(Bmal1)based on the theory of yin-yang and the effect of HXRL on angiogenesis after cerebral infarction.Methods Western blotting was used to detect the expressions of circadian proteins Bmal1 and Clock at each time point after bEnd.3 cells were injured by oxygen-glucose deprivation/reperfusion(OGD/R).Chromatin immunoprecipitation sequencing(ChIP-seq)was used to determine the role of Bmal1 in genome wide,and its effects on bEnd.3 cell biological process,cell component and molecular function.The optimal intervention concentration of HXRL containing serum was detected by CCK-8 method.Bmal1 gene was knocked down in bEnd.3 cells and divided into control group,model group,HXRL containing serum group,si-Bmal1 and si-Bmal1+HXRL containing serum group.The expressions of vascular endothelial growth factor(VEGF)and neurogenic locus notch homolog protein 1(Notch1)were detected by immunofluorescence.Western blotting was used to detect VEGF,matrix metalloproteinases 2(MMP2),Notch1 intracellular domain(NICD)and delta-like protein 4(DLL4)protein expressions.Results After bEnd.3 cells were injured by OGD/R,the expressions of Bmal1 and Clock proteins were gradually increased,and there were significant differences at 8,12,16 and 20 h points after modeling(P<0.05,0.001).ChIP-seq suggests that Bmal1 was involved in cell development,differentiation,proliferation,and other processes in endothelial cells,and targeted the transcriptional start site region of VEGF and Notch1.The results of CCK-8 experiment showed that after bEnd.3 cells were injured by OGD/R,10%HXRL-containing serum could effectively improve cell viability(P<0.001).Scratch,migration and tube formation experiments confirmed that HXRL-containing serum could effectively improve the migration ability and angiogenesis ability of bEnd.3 cells after OGD/R injury(P<0.05,0.01,0.001),but in Bmal1 knockdown strains,this promotion was suppressed(P<0.05,0.01,0.001).The results of immunofluorescence and Western blotting showed that HXRL-containing serum could further increase the protein expressions of VEGF,MMP2,NICD,DLL4,Bmal1 and Clock in bEnd.3 cells damaged by OGD/R(P<0.05,0.01,0.001).In Bmal1 knockdown strain,the promoting effect of HXRL-containing serum on the expressions of VEGF,MMP2,NICD and DLL4 was inhibited(P<0.05,0.01,0.001).Conclusion HXRL can effectively promote the angiogenesis of endothelial cells after OGD/R injury,and its mechanism is closely related to the regulation of circadian clock Bmal1 protein.