| Objective:Atherosclerosis(atherosclerosis,AS)is a common cardiovascular disease.With the improvement of people’s living standard,the incidence of AS is increasing year by year,which seriously endangers human health.However,The etiology of AS is complex and the mechanism is still not completely clear.AS the modern medicine describes,the lesion foundation of AS consists of lipid metabolic disorders,vascular intima damage,lipid and compound carbohydrate accumulation,hemorrhage and thrombosis,and fibrous tissue hyperplasia and calcinosis,atherosclerotic plaque formation,and a middle artery gradually disintegration and calcification,resulting in arterial wall thickening harden,vascular stenosis,finally in various cardiovascular events.There are various drugs for AS in clinic,but the effect is not satisfactory.Semen plantaginis,belonging to the diuretics for eliminating dampness,which has the efficacy of clearing heat and diuresis,Percolating,clearing liver and clear eyes,relieving cough and flooding phlegm,etc.Modern medical research has found that the Semen plantaginis has the effect of anti-atherosclerosis,anti-inflammatory,Hypolipidemic,etc,but the effective components and mechanism of which are not clear.In this paper,we have screened the effective compounds regulating the macrophage cholesterol metabolism in semen plantaginis by computer molecular docking technology and decided to select Geniposidic acid(GPA),an compound extracted from plantain seed,as the research object.We have totally explored the effect of GPA on the regulation of cholesterol in human mononuclear macrophage THP-1,and further discussed the possible mechanism of the antiatherosclerosis of GPA,which provide basic theoretical data forfurther research.Method:1.The docking of macrophage cholesterol regulation related proteins with Semen plantaginisComputer virtual screening was used for modeling.Fifty-five chemical compositions of the Semen Plantaginis are chosen as the ligand,and three current target proteins(ABCA1,CD36,LOX-1)which associated with the regulation of cholesterol metabolism in macrophages are selected as the receptors.Using SYBYL surflex-dock approach to ligand to the receptor,and the ligand which get the highest score in each docking group was selected to observe its hydrophobicity and the hydrogen bond donor and acceptor by MOLCAD.Lastly,the interactions between the ligands and receptors were analyzed by Discovery Studio2016,to predict their binding mode and affinity.2.The effect of GPA on the regulation of cholesterol in human monocyte macrophage THP-1 derived foam cells induced by Oxidized low-density lipoprotein(oxLDL).Human mononuclear cells(THP-1)were cultured in RPMI-1640 medium containing 10% fetal bovine serum and PMA(160nM)to induce human monocyte THP-1 to differentiate into macrophages.A component(GPA)with higher score by Total Score was selected from the compound of Semen Plantaginis as the research object.The first batch is divided into four groups:oxLDL(50mg/L)group,oxLDL+GPA(200mg/L)group,oxLDL+GPA(100mg/L),oxLDL+GPA(50mg/L)group.The second batch is divided into groups: Control group,oxLDL(50mg/L)group,GPA(200mg/L)+oxLDL group,GPA(200mg/L)+oxLDL+Compund C(10 u M)group.The changes of lipid droplets in foam cells were observed after cultured for 24 h by oil red O staining.RT-PCR was used to detect the transcriptional level of ABCA1,ABCG1,CD36,LOX-1,SR-A1,SR-B1 mRNA.The expression levels of ABCG1,ABCA1 and CD36 protein were detected by Western Blot.3.Effects of GPA on the secretion of inflammatory factorson induced by LPS on human monocyte macrophage cell line THP-1,were differentiated into macrophages cultured with RPMI-1640 culture medium containing 10% fetal bovine serum.Firstly,There were Control group,LPS(500ng/mL)group,GPA(200mg/L)+LPS group,GPA(200mg/L)+LPS+11-7082(10mM)group.The changes of lipid droplets in foam cells were observed after cultured for 24 h by oil red O staining.RT-PCR was used to detect the transcriptional level of ABCA1、ABCG1、CD36、LOX-1、SR-A1、SR-B1、TLR4、NF-κB、IL-6、TNF-α、CD206、IL-10 mRNA.The expression levels of ABCG1,ABCA1,CD36 protein were detected by Western Blot.The concentration of TNF-α and IL-6 in each group was detected by ELISA.Secondly,Control group,LPS(500ng/mL)group,GPA(200mg/L)+LPS group were setting.The effect of GPA on the activity of THP-1 in human mononuclear cells was detected by flow cytometry using DCFH-DA probe at 37°C for 30 minutes.The level of reactive oxygen species in the cells was observed by inverted fluorescence microscope.4.Study of GPA on cholester in regulation mechanism of PPAR γ signaling pathwayHuman monocyte THP-1 was cultured in RPMI-1640 medium containing 10%fetal bovine serum,160 nM PMA induces human mononuclear cell THP-1 to differentiate into macrophages.There were Control group,oxLDL(50mg/L)group,oxLDL+GPA(200mg/L)group,oxLDL+GPA(200mg/L)+GW9662(PPAR γinhibitor),oxLDL+GPA(200mg/L)+BRL(PPARγ activator).RT-PCR was used to detect the transcriptional level of PPARγ、LXR-α、ABCA1、ABCG1、CD36、LOX-1mRNA after 24-hour treatment.Result:1.By using the technology of computer virtual molecular docking,the GPA,which has a higher score,is selected as the research object.It is observed that there are three compounds derived from semen plantaginis,which produce various molecular inter-atomic forces binding small molecules firmly at the active site by SYBYL molecular docking method.Geniposidic acid(GPA),a compound extracted from semen plantaginis with a higher score though Total Score,was selected as the research object.According to data provided by the supplier,the purity of GPA used in this experiment was 99.3221%.The inhibition rate of cell proliferation showed that GPA with different doses(200mg/L,100mg/L,50mg/L)had no obvious effect on the proliferation inhibition rate of human monocyte macrophage derived foam cells.2.OxLDL induces a human mononuclear macrophage THP-1 to establish a foam cell model.GPA can reduce cholesterol content in foam cells by increasingABCA1,ABCG1 gene or protein expression levels and decreasing LOX-1,CD36 gene or protein expression levels.The above effect can be inhibited by AMPK inhibitor(Compund C).After exposed to oxLDL(50mg/L)for 24 h and stained with oil red O,it is shown that lots of intracellular red lipid droplets filled in human THP-1 becoming frothy and significantly different from the control group after extracted cholesterol by the isopropyl alcohol,which successfully established foam cell model;the results of RT-PCR revealed that expression of ABCA1,ABCG1 cells mRNA decreased while expression of LOX-1,CD36 increased in human THP-1 derived foam cells,and the difference was statistically significant(P<0.05 or P<0.01).Results of oil red O staining showed that GPA(200mg/L,100mg/L)could significantly reduce the content of lipid droplet in THP-1;the results of RT-PCR displayed that GPA(200mg/L,100mg/L)could improve the expression level of ABCA1,ABCG1 mRNA and reduce the expression level of CD36,LOX-1 of which was statistically significant(P< 0.05 or P<0.01).However,expression level of SR-A1,SR-B1 mRNA showed no obvious effect(P> 0.05).The AMPK inhibitor(Compund C)can inhibit the increase of ABCA1 and ABCG1 and decrease CD36 and LOX-1 exposed to GPA(200mg/L),and the difference is statistically significant(P<0.05).Compared with the oxLDL group,GPA(200 100mg/L)can improve the expression level of ABCA1,ABCG1 mRNA(P< 0.05),to reduce the expression level of CD36,LOX-1(P< 0.05),the expression level was no significant changes of SR-B1,SR-A1(P>0.05).Compared with oxLDL group,GPA(200 100mg/L)can improve the expression level of protein on ABCA1,ABCG1,decreasing the expression level of protein on CD36.3.LPS stimulates the inflammatory response of human mononuclear macrophage THP-1,GPA can reduce the expression level of NF-κB、IL-6、TNF-α,improve intracellular oxidative stress,reduce the concentration of IL-6、TNF-α in the cell supernatant.The RT-PCR results of TLR4、NF-κB、IL-6、TNF-α、CD206、IL-10:RT-PCR was conducted to detect the expression level of mRNA in LPS induced THP-1 after treated with GPA、GPA+BAY11-7082 for 24 hours.Compared with the control group,the expression level of TLR4,IL-6,TNF-α,NF-κB mRNA of LPS group increased significantly(P< 0.05),while the expression level of CD206 andIL-10 decreased obviously(P<0.01).Compared with LPS group,the expression of IL-6,TNF-α,NF-κB mRNA decreased obviously and the expression levels of CD206 and IL-10 increased in GPA(200mg/L)group,which has statistically difference(P<0.05 or P<0.01),conversely,made no effect on TLR4(P>0.05).Compared with GPA(200mg/L)group,the NF-κB inhibitor(BAY11-7082)group can further reduce the expression level of IL-6,TNF-α,NF-κB mRNA,and further improve the expression level of CD206,of which the difference was statistically significant(P<0.05).What’s more,it is tend to improved the expression level of IL-10 but make no effect on TLR4(P>0.05).with treatment of LPS,the level of ROS in human mononuclear macrophage THP-1 was significantly enhanced compared with the control group.After adding GPA(200mg/L)for 24 hours,the intracellular ROS level was decreased.Oil red O staining After intracellular cholesterol extraction by isopropanol,cholesterol of LPS group tend to increase compared with the control group.Compared with LPS group,GPA group(200mg/L)showed a tendence to decrease the intracellular cholesterol level.The RT-PCR results of ABCA1,ABCG1,CD36,LOX-1,SR-A1 and SR-B1:Compared with the control group,the expression level of ABCA1,ABCG1 mRNA decreased while the expression level of CD36 and LOX-1 increased,of which the difference was not statistically significant(P>0.05).Moreover,there was no significance in the expression of SR-B1 and SR-A1(P>0.05).Ccompared with the oxLDL group,the GPA(200mg/L)group tend to improve the expression level of ABCA1,ABCG1 mRNA and reduce expression level of CD36 and LOX-1 mRNA,of which the difference was not statistically significant(P>0.05)and there was no significance in the expression of SR-B1 and SR-A1(P>0.05).Compared with the GPA(200mg/L)group,the NF-κB inhibitor(BAY11-7082)group had no effect on expression of ABCA1,ABCG1,CD36,SR-B1,and SR-A1(P>0.05).4.oxLDL induced human monocyte macrophage THP-1 to establish foam cell model.GPA can enhance the level of PPARγ and LXR-α gene expression,thereby improving the expression level of ABCA1 and ABCG1,and decreasing the expression level of CD36 and LOX-1.RT-PCR was used to detect mRNA expression level of OxLDL induced human monocyte macrophage THP-1 derived foam cell with treatment of GPA 、GPA+GW9662 and GPA+BRL for 24 hours.The RT-PCR results of LXR-α,PPARγ: Compared with the control group,the expression level of PPARγ and LXR-α mRNA in the oxLDL group decreased significantly,of which the difference was statistically significant(P<0.01);compared with the oxLDL group,the expression level of PPARγ,LXR-α mRNA significantly increase in the GPA(200mg/L)group and the differences were statistically significant(P<0.01);Compared with the GPA(200mg/L)group,the GW9662(PPARγ inhibitor)group can inhibit the improvement of the expression level of PPARγ and LXR-αmRNA resulting from GPA and the difference was statistically significant(P<0.01);while BRL(PPARγ activator)made no effect on expression level of PPARγ,LXR-αmRNA and there was no significant difference(P>0.05).The RT-PCR results of ABCA1,ABCG1,CD36,LOX-1: Compared with the oxLDL group,it is shown that the expression of ABCA1 ABCG1 mRNA obviously increased while expression of CD36 and LOX-1 mRNA were significantly decreased in the GPA(200mg/L)group and the difference was statistically significant(P<0.05 or P<0.01).Compared with GPA(200mg/L)group,it is shown that the expression level of ABCA1 ABCG1 mRNA decreased significantly while expression level of CD36 and LOX-1 mRNA was significantly increased in the GPA+GW9662 group and the difference was statistically significant(P<0.05 or P<0.01).Besides,compared with the GPA(200mg/L)group,there was improvement of expression level of ABCA1,ABCG1 mRNA in GPA(200mg/L)+oxLDL+BRL group(P<0.05),however,the expression of CD36 and LOX-1 mRNA made no significant changes(P>0.05).Conclusion:In this study,we have screened the components of the Semen plantaginis by the computer virtual molecular docking technology,and mainly explored GPA,the compound with higher scores combined with the receptor ABCA1.In vitro assay,it is discovered that GPA can influence cholesterol metabolism in human monocyte /macrophage THP-1 derived foam cells,which probably associated with APMK,PPARγ mediated cholesterol controlling signal path.Meanwhile,The GPA can inhibit the secretion of inflammatory factors in human mononuclear macrophage THP-1,playing a role in the NF-κB-mediated inflammatory signaling pathways. |
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