天麻素与薯蓣皂苷元配伍对大鼠缺氧损伤脑微血管内皮细胞的保护作用

Protective Effects of Gastrodin and Diosgenin on Hypoxia Injury of Rats Brain Microvascular Endothelial Cells

目的探讨天麻素(Gas)、薯蓣皂苷元(Dio)及其组合物(GDC)对大鼠缺氧损伤脑微血管内皮细胞(BMECs)的保护作用和对脑微血管新生的影响及其作用机制。方法将BMECs分为5组:正常对照组,模型对照组,Gas组(1、2、4、8、16、32μmol·L^(-1)),Dio组(0.25、0.5、1、2、4、8μmol·L^(-1)),GDC组(Gas和Dio浓度分别为1和0.25、2和0.5、4和1、8和2、16和4、32和8μmol·L^(-1))。分组给药12 h后,对模型对照组、Gas组、Dio组和GDC组通过氧糖剥夺法建立BMECs缺氧模型。检测乳酸脱氢酶(LDH)漏出率、白细胞介素1β(IL-1β)含量、超氧化物歧化酶(SOD)活力、丙二醛(MDA)浓度;观察Hoechst 33258、FITC-Annexin V/PI染色,采用Transwell小室实验、细胞划痕实验和小管形成实验,考察Gas、Dio及GDC对缺氧损伤BMECs的保护作用及促进血管新生作用。进一步利用网络药理学和分子对接方法研究GDC抗脑缺血的作用机制。结果Gas、Dio以2、0.5μmol·L^(-1)组合时,可显著提高缺氧损伤BMECs细胞活力(P<0.05),促进细胞增殖;与模型对照组比较,Gas组LDH漏出率降低(P<0.05);Gas组、Dio组与GDC组IL-1β含量均显著下降(均P<0.05),Gas组、GDC组SOD活力升高(P<0.05),Gas组、Dio组以及GDC组凋亡小体减少。GDC可促进BMECs缺氧模型的增殖、侵袭、迁移以及小管形成,表明其在细胞水平上一定程度促进血管新生。网络药理学研究发现,Gas与半胱天冬酶3(CASP3)、过氧化物酶体增生激活受体γ(PPARG)、凝血因子Ⅱ(F2)、基质金属蛋白酶-9(MMP-9)、丝氨酸苏氨酸蛋白激酶(Akt)1蛋白具有良好的结合活性,Dio与内皮一氧化氮合成酶(NOS3)、KDR蛋白具有良好的结合活性,Gas、Dio可能主要通过血管内皮生长因子(VEGF)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、磷酯酰肌醇3-激酶(PI3K)-Akt信号通路以及缺氧诱导因子-1(HIF-1)信号通路调控脑缺血后血管新生。结论Gas、Dio及GDC具有抗缺氧损伤作用并可促进缺氧损伤后血管新生,作用机制可能与其作用于Akt1、NOS3、VEGFR2等蛋白,调控VEGF/Akt/MAPK等信号通路促进血管新生有关。

Objective To investigate the protective effects of gastrodin(Gas),diosgenin(Dio)and their composition(GDC)on the hypoxia injury of rat cerebral microvascular endothelial cells(BMECs),and to study their mechanism of angiogenesis after cerebral microvascular hypoxia injury.Methods BMECs were divided into five kinds of groups:normal control,model control,Gas groups(1,2,4,8,16,32μmo·L^(-1)),Dio groups(0.25,0.5,1,2,4,8μmo·L^(-1)),and GDC groups(the concentrations of Gas and Dio were 1 and 0.25,2 and 0.5,4 and 1,8 and 2,16 and 4,32 and 8μmo·L^(-1),respectively).After 12 hours of treatment,BMECs were subjected to hypoxia-induced by oxygen-glucose deprivation in the control,Gas,Dio,and GDC groups.The LDH leakage rate,IL-1βcontent,SOD viability,MDA concentration,Hoechst 33258,FITC-Annexin V/PI staining.Transwell chamber assay,scratch wound healing assay,and tube formation assay were employed,and investigated the protective effects of Gas,Dio,and GDC on BMECs in promoting angiogenesis and further using network pharmacology and molecular docking methods to focus on angiogenesis aspects to investigate the mechanism of GDC against cerebral ischemia.Results When Gas and Dio are combined in 2 and 0.5μmo·L^(-1),it can significantly increase the cell viability of BMECs(P<0.05),Promote cell proliferation;Compared with the model control group,LDH leakage rate was decreased in the Gas group(P<0.05);IL-1βcontent was decreased significantly in Gas,Dio,and GDC groups(P<0.05),SOD vitality was increased in Gas and GDC groups(P<0.05),Apoptotic bodies were reduced in the Gas,Dio groups and in the GDC group.GDC can promote proliferation,invasion,migration and tubule formation in the hypoxia model of BMECs,indicating a degree of promoting angiogenesis effect at the cellular level.Network pharmacology found good binding activity between Gas and Caspase-3(CASP3),peroxisome proliferative activated receptor,gamma(PPARG),F2,matrix metalloproteinase-9(MMP-9),and serine-threonine kinase(Akt)1,Dio to nitric oxide synthase 3(NOS3)and KDR proteins,and Gas and Dio may regulate angiogenesis after cerebral ischemia mainly through VEGF signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway and hypoxia inducible factor-1(HIF-1)signaling pathway.Conclusions Gas,Dio,and GDC have protective effects against hypoxic injury and can promote angiogenesis after hypoxia injury.Their mechanism of action may be related to herb action on Akt 1,NOS 3,VEGFR2 and other proteins,as well as the regulation of signaling pathways such as VEGF/Akt/MAPK to promote angiogenesis.