The Study Of Chuanxiong-Chishao Herb Pair And Its Active Components On Inhibiting Angiogenesis In Plaque By Regulating Mir-126

Atherosclerosis(AS)plaque angiogenesis could result in intraplaque bleeding,plaque rupture,and acute cardio-cerebrovascular events.The inhibition of plaque angiogenesis may offer avenues for stabilizing AS plaques.Modern studies have found that the syndrome of blood stasis in traditional Chinese medicine is strongly correlated with angiogenesis in atherosclerotic plaques.Therefore,promoting blood circulation and removing blood stasis may be a new approach to treat angiogenesis in atherosclerotic plaque.Chuanxiong-Chishao herb-pair(CX-CS)is a classical herb pair for promoting blood circulation and removing blood stasis,which has been demonstrated to have beneficial effects on AS by both clinical and basic studies,but the related mechanism is still unclear.MicroRNA-126(miR-126)is a kind of miRNAs primarily expressed in endothelial cells.Whether CX-CS plays a role in inhibiting angiogenesis and anti-AS by targeting miR-126 remains unclear.1.PurposeTo investigate the effect of CX-CS and its active components tetramethylpyrazine and paeoniflorin(TMP-PF)on plaque angiogenesis in AS and explore the molecular mechanism of CX-CS and its active components TMP-PF intervention in plaque angiogenesis by targeting miR-126.2.Methods(1)The effect and mechanism of CX-CS on plaque angiogenesis in ApoE-/-miceC57BL/6J mice were used as control group(control),ApoE-/-mice were randomly divided into model group(model),CX-CS group,CX-CS+miR-126 antagonist group(CX-CS+miR-126 antagonist)and simvastatin group.ApoE-/-mice were fed with high-fat diet for 3 months and administered with CX-CS extractum or simvastatin for 1 month.CX-CS+miR-126 antagonist group was injected with an adeno-associated virus(AAV)-mediated miR126a-5p antagonist before drug administration.Biochemical detection of serum lipids levels:triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C).The expression of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)in serum was detected by ELISA.Plaque area and lipid in plaque were observed by HE staining and oil red O staining.The expression of CD31 was examined by immunofluorescence.The expression of miR-126 in aortic tissue was detected by PCR.The expression of VEGF and VEGF receptor 2(VEGFR2),bFGF and FGF receptorl(FGFR1)in aortic tissue was detected by western blot.(2)To explore the effect and mechanism of CX-CS active components(TMP-PF)on oxidized-low density lipoprotein(ox-LDL)-induced angiogenesis in human umbilical vein endothelial cells(HUVECs)by targeting miR-126Angiogenesis model was established with ox-LDL-induced HUVECs in vitro.The active components of CX-CS(TMP-PF),tetramethylpyrazine(TMP)and paeoniflorin(PF)combination(1μmol/L TMP+10μmol/L PF)was administrated to ox-LDL-induced HUVECs.HUVECs were divided into control group,model group,CX-CS active components group(TMP-PF),CX-CS active components+miR-126 inhibitor group(TMP-PF+miR-126 inhibitor),and miR-126 inhibitor group(miR126 inhibitor).HUVECs was transfected with miR-126 inhibitor for 24 hours,induced with ox-LDL for 12 hours,and treated with 1 μmol/L TMP+10 μmol/L PF for 24 hours.Endothelial cell proliferation was detected by MTT assay.Cell migration was measured by wound-healing assay.Cell tube formation was detected by cell tube formation test.The expression of VEGF,VEGFR2,bFGF and FGFR1 in endothelial cells was detected by western blot.The expression of VEGF in supernatant was detected by ELISA,and the expression of miR-126 in endothelial cells was detected by PCR.3.Results(1)CX-CS inhibits plaque angiogenesis by up-regulating miR-126Compared with those of control group,the levels of TC,TG and LDL-C in model group were significantly increased;HDL-C levels were significantly decreased;plaque area and lipid infiltration in plaque was increased;neovascularization density(CD31 expression)in plaque was increased;the expression of VEGF,bFGF,VEGFR2 and FGFR1 in aorta was significantly upregulated;and the expression of miR-126 was significantly down-regulated in model group(P<0.05).Compared with those of model group,the levels of TG and LDL-C in CX-CS group were significantly decreased;plaque area and lipid infiltration were decreased;the density of new vessels(CD31 expression)in plaque was decreased;the expression of VEGF,bFGF,VEGFR2 and FGFR1 in aortic tissue was decreased;and the expression of miR-126 was up-regulated in CX-CS group(P<0.05).After combined with miR-126 antagonist,the effects of CX-CS on improving blood lipid,plaque area and plaque angiogenesis were not significant;miR-126,VEGF and VEGFR2 expression was not significantly changed compared with those of model group(P>0.05).In simvastatin group,TG levels,the aortic plaque area and plaque lipid infiltration were decreased,and the expression of CD31,bFGF and FGFR1 in aortic tissue was significantly decreased(P<0.05).In short,CX-CS up-regulated the expression of miR-126 and inhibited the expression of VEGF and VEGFR2 in aorta,reduced serum LDL-C levels and the lipid infiltration in plaque,inhibited plaque angiogenesis and reduced the plaque area,thereby delaying the pathological process of AS.(2)Active components of CX-CS(TMP-PF)inhibits ox-LDL-induced angiogenesis in vitroCompared with control group,ox-LDL significantly induced proliferation,migration and tube formation of HUVECs in model group.And the expression of VEGF in cell culture supernatant was significantly increased;the expression of VEGF,VEGFR2,bFGF and FGFR1 was significantly increased;and the expression of miR-126 was significantly down-regulated in model group compared with those of control group(P<0.05).Compared with model group,the active components of CX-CS(TMP-PF)significantly inhibited the proliferation,migration and tube formation of HUVECs,decreased the expression of VEGF in cell culture supernatant,down-regulated the expression of VEGF,VEGFR2,bFGF and FGFR1 while upregulated the expression of miR-126(P<0.05).After combining with miR-126 inhibitor,cell proliferation,migration,tube formation,and VEGF expression in cell culture supernatant,and both VEGF and VEGFR2 expression were not significantly affected by TMP-PF(P>0.05).Compared with model group,simvastatin significantly inhibited the proliferation,migration and tube formation of HUVECs,decreased the expression of VEGFR2 and bFGF while increased the expression of miR-126(P<0.05).Therefore,TMP-PF might inhibit angiogenesis in vitro by upregulating miR-126 expression and inhibiting VEGF/VEGFR2 signaling pathway.4.Conclusions:CX-CS and its active components TMP-PF could inhibit plaque angiogenesis by up-regulating miR-126 expression and inhibiting the VEGF/VEGFR2 signaling pathway,thereby further reducing the AS plaque area,stabilizing plaque and delaying the pathological process of AS.