Anti-atherosclerosis Effect Of Ginsenoside Rg3 Combined With Rosuvastatin Based On Different Endothelial Protective Mechanisms

Atherosclerosis（AS）,is one of the most vital causes,which leads to unstable coronary syndrome and sudden cardiac death.Epidemiological statistics indicate that the incidence of cardiovascular diseases flow from abnormal lipid metabolism induced AS has risen sharply in a number of Asian countries since 2015,and prove to be an unless infectious disease with higher mortality outside the disease.Endothelial cells are the main component cells of vascular intima which are the first important barrier to protect blood vessels.Vascular intimal abnormality is a typical pathological change of AS,which is manifested by the decrease of endothelial cell healing ability and anti-inflammatory cell infiltration ability,as well as cell apoptosis,then lead to further subintimal damage.Ginsenoside Rg3 is the fundamental active component of ginseng,which possess pharmacological effects such as anti-tumor and immune regulation as well.With the advances of studies,what has been demonstrated is that ginsenoside Rg3 play preventive as well as therapeutic effects on cardiovascular diseases.However,the anti-AS effect of Rg3 and related mechanisms remains to be further thrashed out;Rosuvastatin,which is much popular used because of its advantages,such as stronger regulation of blood lipid,shorter half-life and higher bioavailability than other statins,however,the mechanism of their anti AS and whether they are related to endothelial protection need further study.In accordance with project of Natural Science Foundation of Jilin Provincial Science and Technology Department:"The intervention effect and molecular mechanism of rosuvastatin on endoplasmic reticulum stress induced by ox-LDL"（No.20150101200JC）,this study be conducted to grope for the role of rosuvastatin in abating AS induced by abnormal lipid metabolism.On the one hand,not only the endothelial protective effect and mechanism of rosuvastatin were explored,but the endothelial protective and anti-AS effects and mechanisms of Rg3were compared,and results suggested that the rosuvastatin and Rg3 may play the role of endothelial protection through different mechanisms.Furthermore,some patients could not tolerate the full dose of statins or accompany with the increase of liver enzyme activity,leading to additional treatment might be needed to provide more effective coronary care.Therefore,in vivo experiments were conducted to compare the effects of rosuvastatin,Rg3 or their combination on AS of ApoE^-/-mice,in order to supply theoretical basis for clinical application of Rg3 in the treatment of AS especially when the effect of statins was not acceptable.The cardinal research procedures are as below:1.Effect and mechanism of ginsenoside Rg3 on ox-LDL-induced endothelial dysfunctionFor the sake of determining whether ginsenoside Rg3 is able to inhibit ox-LDL-induced endothelial cell dysfunction,endothelial cell injury model induced by ox-LDL（200μg/mL）was applied.HUVECs were pretreated with ginsenoside Rg3（15,30μmol/L）.Scratch test,monocyte adhesion test as well as Western blotting were utilized.In order to investigate the effect of Rg3 on ox-LDL-induced HUVECs dysfunction.The outcome revealed that Rg3 is capable of enhancing significantly the healing ability of HUVECs induced by ox-LDL,decreasing the adhesion of monocytes,reducing the phosphorylation of FAK,inhibiting the expression of adhesion molecules ICAM-1 and VCAM-1,and restraining the expression of MMP-2and MMP-9.The results presented that Rg3 are in a position to inhibit ox-LDL-induced HUVECs dysfunction.It is related to the inhibition of FAK-mediated cytokine expression.For elucidating the mechanism of Rg3 on ox-LDL-induced HUVECs dysfunction,the methods and techniques of migration as well as monocyte adhesion probes,Western blotting,immunofluorescence and qPCR were wielded to detect the related indicators.The conclusion displayed that Rg3 could significantly improve the inhibition of PPAR gamma expression by ox-LDL in HUVECs,inhibit the activation and nuclear translocation of NF-kappa B in damaged endothelial cells,and repress the mRNA levels of inflammatory factors（MCP-1 and IL-6）.GW9662（PPAR gamma specific inhibitor）pretreatment of HUVECs restrict the enhancement of HUVECs migration by Rg3 dramatically.Meanwhile,Rg3’s down-regulation of FAK activation,adhesion factor and matrix metalloproteinase expression,NF-kappa B activation and inflammatory factors were astricted as well,suggesting that Rg3 probably play a protective role in HUVECs dysfunction induced by ox-LDL through up-regulation of PPAR gamma expression in endothelial cells.2.Protective effect as well as mechanism of rosuvastatin on ox-LDL-induced apoptosis of endothelial cellsFor the reason that resolving whether the anti-AS effect of rosuvastatin is related to the inhibition of endothelial cell apoptosis,endothelial cell injury model induced by ox-LDL were put to use.HUVECs were pretreated with rosuvastatin（0.01,0.1,1μmol/L）.MTT,flow cytometry,DAPI staining and Western blotting were made use of exploring the effects of rosuvastatin on endothelial cell apoptosis.The results came to conclusion that rosuvastatin could significantly enhance NO secretion in HUVECs stimulated by ox-LDL,inhibit oxidative stress,strengthen PI3K/Akt/eNOS pathway activation and enhance Bcl-2/Bax ratio in HUVECs.MTT results manifested that rosuvastatin inhibited ox-LDL-induced HUVECs injury and significantly increased the survival rate of damaged endothelial cells;flow cytometry results made it clear that rosuvastatin significantly inhibited ox-LDL-induced HUVECs apoptosis rate;DAPI staining results stated that rosuvastatin significantly inhibited ox-LDL-induced HUVECs.What have been mentioned,suggest that rosuvastatin can inhibit ox-LDL-induced HUVECs damage and apoptosis.In addition,endothelial cells are rich in endoplasmic reticulum,suggesting that they may be more sensitive to endoplasmic reticulum stress related apoptosis,so as to clarify the mechanism of rosuvastatin inhibiting ox-LDL-induced apoptosis in HUVECs,the endoplasmic reticulum stress-related pathways were detectedand,rosuvastatin significantly inhibit the levels of CHOP,sXBP1,Caspase-12 and GRP78,inhibit PERK and IRE1 alpha activation,as well as activation of e IF2 alpha,suggesting that rosuvastatin may prevent the apoptosis of HUVECs from inducing by ox-LDL by inhibiting endoplasmic reticulum stress-related pathways.3.Study on the effect of ginsenoside Rg3 combined with rosuvastatin on AS induced by high-fat diet in ApoE^-/-miceTo determine whether Rg3 combined with rosuvastatin can more effectively inhibit the occurrence and development of AS,12 w of ApoE^-/-mice were fed with high-fat diet（40%fat,1.25%cholesterol）to replicate the atherosclerosis model.Model group（Mod）,Rg3（Rg3）,rosuvastatin group（RSV）and Rg3 combined with rosuvastatin group（Rg3+RSV）,C57BL/6J mice to keep feeding common diets as normal controls,4 w later,detected T-CHO,TG,LDL-C,HDL-C and CRP levels in mice serum,HE staining for observating aorta foam cells,MASSON staining to detecting the contents of aortic collagen,immunofluorescence staining for aortic endothelial cell apoptosis in mice;the contents of aortic macrophages and smooth muscle cells were detected by immunohistochemical staining,and then the nuclear cap ratio and vulnerability index of AS plaque in mice in each group were computed.The brief summary of this study is given,compared with the normal control group,T-CHO,TG,LDL-C,HDL-C and CRP levels in the Mod group rose apparently.Cells in the intima and subintima of aorta were arranged in disorder,and a large amount of cytoplasm was vacuolated,and the lumpy fibrous plaque layer in the intima of aorta was severely infiltrated by inflammatory cells.The apoptosis of aortic endothelial cells built up distinctly and the content of collagen fiber decreased significantly.The expression of macrophages in aorta and plaque rose palpably,on the other hand,the smooth muscle cells declined significantly,and the nuclear cap ratio and vulnerability index of AS plaque in mice multiplied evidently.In the first place,paralleled with Mod group,Rg3+RSV and RSV group were sharply lower than levels of LDL-C,aortic endothelial cell apoptosis,and do not exist overt difference between the two groups,furthermore,Rg3+RSV group significantly reduced in mice serum level of T-CHO,but still much higher than normal,TG level of RSV group dropped tangibly,prompt RSV to act a pivotal part in decreading serum lipoprotein and anti endothelial cell apoptosis in mice.In the next place,both Rg3+RSV group and Rg3 group reduced the CRP levels of AS mice significantly,and there was no remarkable discrepancy between the two groups,suggesting that Rg3 may take a prominent effect for reducing the inflammation.In addition,mouse aorta fiber content in Rg3+RSV group and RSV group augmented noteworthy,macrophage expression in Rg3+RSV group and Rg3 group abated greatly,compared with that in the model group,and alpha-SMA positive expression gain in Rg3+RSV group prominently,making the nuclear cap ratio and vulnerability index of AS plaque cut down significantly in Rg3+RSV group.There is no denying that Rg3 tend to strengthen blood vessel endothelial cell’s ability to resist inflammation,cell adhesion,reduce the inflammatory factors and promote the level of cytokines AS be associated perhaps with regulating PPAR gamma/FAK signal pathway,and Rg3 abate the AS level of inflammation in mice aortic intima and the AS plaque macrophages infiltration,signifies that Rg3 inhibit inflammatory infiltration induced endothelial injury may be the key mechanism of resistance to AS.In addition,rosuvastatin cut back the apoptosis of vascular endothelial cells in AS mice excepting improve the lipids,which stands a good chance of being related to enhancing the activation of PI3K/Akt/eNOS pathway of vascular endothelial cells and inhibiting the endoplasmic reticulum stress-related pathway.Rg3combined with rosuvastatin can more effectively inhibit the occurrence and development of AS.When the clinical application of statins for the treatment of AS is not passable or statin intolerance,the integrated application of Rg3 may be more efficacious for the treatment of AS.