| Objective:To identify the active components of essential oil from Ligusicum Chuanxiong Hort.（EVCX）in protecting vascular endothelial cells（EVCs）and further explore the potential mechanism on protected effect of Ligustilide（LIG）.Methods:Firstly,the active components of EVCX were screened with MTT test by the assessment of protecting HUVECs injury induced by Oxygen-glucose deprivation-reperfusion（OGD-R）.Secondly,the corresponding genes and proteins expressions of PI3K-Akt signal pathway were detected with the employment of high content screening（HCS）,western blot,etc,in order to confirm whether the above signal pathway involved the protection of LIG on OGD-R-caused injured HUVECs.Finally,angiogenesis disorder model of zebrafish was established and applied to assess the effect of LIG on angiogenesis.The detailed methods were as follows.1.Screening active components of EVCX on protecting VECs.OGD-R injury model on HUVEC was established by receiving glucose-free DMEM culture mediums with appropriate concentration of Na2S2O4 for 12 h,and then culturing in normal manner for additional 4 h.The protective effect of 1-h administration of EVCX,LIG,Butylphthalide（BP）,Senkyunolide A/I,Levistilide A and Ligustrazine（TMP）,ferulic acid,respectively,at different concentrations on HUVECs with OGD-R injury were estimated by MTT assay to examine its cellular activities.Consecutively,after OGD-R injury,the MDA content and SOD activity in HUEVCs and LDH,ACP leakage in cell-culture medium were measured after treatment with 20、40、60μM of LIG or BP which was considered to be the active components of EVCX on protecting damaged HUVECs.The following experimental procedures were underwent,the LIG taken for example..2.Investigation on the potential mechanism of Ligustilide protecting VECs2.1.The protective effect of LIG on OGD-R-induced injured HUVECs upon the activation of PI3K-Akt signaling pathway:Model and pre-treatment methods were as above.Cells were divided into 5groups containing solvent group,LIG high（60μM）,mediate（40μM）and low（20μM）dose groups,and model group.NO content and NOS activity were detected,by kits,PI3K、PDPK1、HIF-1α、VEGF content in cells and cell cycle were measured by the usage of immunofluorescence of High Content Screen（HCS）system,and the tubulin contents in cell was determined by western blot.2.2.The inhibitory effect of PI3K-Akt signaling pathway on the protection of LIG on OGD-R-induced injured HUVECs. Model and pre-treatment methods were as above.That the cells subjected to OGD-R stimulation were added 10μM of LY294002 and 60μM of LIG was setted as inhibitor group.The approach of the resting groups was similar with the above description.P53 contents in cells were measured by HCS system using immunofluorescence.Akt/p-Akt,GSK 3β/p-GSK 3β,Bcl-2,Bax and Caspase-3/Cleaved Caspase-3 contents in cells were detected by western blot.2.3.Investigation the angiogenesis effect of LIG:24 hpf zebrafish embryos belonging to Tg（kdrl:eGFP）were collected and raised into 24-well culture plate with varied concentrations of VRI（10,50,100,200,300,400,500 nM）for 3 h.Then the medicated culture was removed and renewed with fresh culture medium for another 21 h.The model for zebrafish angiogenetic disorder induced by adding appropriate concentration of VRI was copyed successfully by the assessment of the morphology with the employment of fluorescence microscope.After a exposion of various contration of LIG（10,20,30,40,50,60,80,100μM）to prepared zebrafish embryos for 21 h in 24-well culture plate,the morphology and hatchability of zebrafish were observed by fluorescence microscope and stereomicroscope,respectively.Then,the appropriate concentration of LIG（10-60μM）as treatmental dose was chosen.The method was mentioned as above,and the sprouting of ISVs was calculated by fluorescence microscope to estimate the pro-angiogenic effect of LIG.VEGF A,Kdr,Kdrl and Flt-1 m RNA expressions in zebrafish were detected by RT-PCR as well.Results:1.Screening the active components of EVCX in the aspect of protecting endothelium.Reading the results from pre-experiment,adding the appropriate concentration of Na2S2O4 for establishing OGD-R model in HUVEC was 8 mM.MTT assay revealed that EVCX protected HUVEC from OGD-R injury in a concentration of 0.031%and0.016%with a elevated cell viability,while EVCX at higher concentrations,.5%, 0.25%and 0.125%for example,presented a toxic reaction with a decrease of cell viability（P<0.05）;the study on the protective effect of TMP（30-60μM）,LIG（30-60μM）,BP（30-60μM）,Senkyunolide I（60μM）and ferulic acid（40-60μM）were conducted,respectively.the results showed that,TMP,LIG and BP could significantly increase cell viability,Levistilide A caused a reduction of cell viability,and Senkyunolide A had no obvious effect on cell viability.Taking a good concentration manner,both LIG and BP（20-60μM）were chosen to receive further investigation on protecting the cellular injury.The date indicated that,LIG treatment could concentration-dependently reduce MDA contents（P<0.05）and LDH leakage（P<0.05）and at the dse of 40μM as well as 60μM increase SOD activity（P<0.05）,and BP treatment could concentration-dependently reduce MDA contents（P<0.05）and at the dse of 40μM as well as 60μM decline LDH leakage（P<0.05）and heighten SOD activity（P<0.05）,when compared to that of vehicle control.Besides,LIG and BP didn’t affect the elevated trend of ACP in UVECs with the OGD-R stimulation（P>0.05）.2.The potential mechanism on LIG-produced protection in VECs2.1.The protective effect of LIG on HUVECs with OGD-R injury involved in PI3K-Akt signaling pathway:After the stimulation with OGD-R in HUVECs,it exhibited a diminishment in NO contents（P<0.05）and NOS activities（P<0.05）,a up-regulation in PI3K p85 P<0.05),HIF-1α（P<0.05）and VEGF（P<0.05）expressions,and a promotion in cell division（P<0.05）,and presented a increased trend in PDPK1（P>0.05）and Tubulin（P>0.05）expressions.while administration of LIG（20,40,60μM）could remarkably increase the NO release and NOS activity,and promote cell division and PDPK1, ubulin,PI3K p85,HIF-1αand VEGF expressions in a dose-dependent manner,as compared to the that of model group（P<0.05 in all）.2.2.The effect of PI3K-Akt signaling pathway inhibitor on the protection of in HUVECs with OGD-R injuryOGD-R challenge markedly down-regulated Bcl-2 content,Bax,Cleaved aspase-3 and p53 contents in HUVECs（P<0.05 in all）,but didn’t modify Akt/p-Akt and GSK3β/p-GSK3βcontents（P>0.05 in both）,when compared to vehicle control,respectively.LIG could concentration-dependently increment p-Akt,p-GSK3β,Bcl-2expressions（P<0.05 in all）and down-regulated Bax,Cleaved Caspase-3 and p53 xpressions（P<0.05 in all）,but didn’t influence the Akt and GSK3βexpressions（P>0.05 in both）.Additionally,the combination of LIG and PI3K-Akt signaling pathway inhibitor LY294002 down-regulated p-Akt,p-GSK3β,Bcl-2 contents P<0.05 in all),up-regulated Bax,Cleaved Caspase-3 and p53 contents（P<0.05 in all）,and had indifferent impact on Akt and GSK3βcontents（P>0.05 in both）when ompared that in the group treated with LIG alone.2.3.The effect of LIG on zebrafish with angiogenetic disorder.The model of zebrafish with angiogenetic disorder was prepared using 300 nM of VRI.In vitro toxic test,100μM of LIG markedly inhibited the hatching rate of zebrafish embryo（P<0.05）and 80μM of LIG displayed a trend to inhibit the hatching rate（P>0.05）.Accordingly,the concentrations of LIG testified were 10,20,40,50,60μM in current study on pro-angiogenetic abilities.Adding LIG could dose-dependently reverse the declined sprouting rate of ISVs and VEGF A,Kdr,Kdrl and Flt-1 m RNA transcription in zebrafish with insufficient angiogenesis（P<0.05 in all）.Conclusions:1.The active compounds,Ligustilide,Butylphthalide and Senkyunolide I,from EVCX had a strongly protective effect of HUVEC from OGD-R-elicited reduction of cell viability,associated with the attribution of anti-oxidative damage and stabilization of the membrane’s structure,and the first two presented a good dose-dependent manner.2.LIG protecting HUVEC from OGD-R injury seems to be related to the activation of PI3K-Akt signaling pathway,and thus exerts anti-hypoxic activity.3.the pro-angiogenetic effect of LIG may be involved in the corresponding genic expressions of VEGF/VEGFR pathway. |
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