The Mechanism Of Zedoarondiol Regulating Platelet Exosome-Neovascularization In Stabilizing Atherosclerotic Plaque

BackgroudAtherosclerosis(AS)plaque rupture secondary to thrombosis is the main pathological basis for acute cardiovascular events,which directly impact the outcome and prognosis of the disease.Based on the academic thought of "activating blood circulation and removing blood stasis" by academician Chen Keji,and combining with modern research results,our group proposes that "blood and qi are blocked due to blood stasis" and "qi becomes poisonous due to stasis" in the process of AS lesion.Therefore,breaking blood and removing stasis is the key to solving the problem of mutual stasis and toxicity.Zedoarondiol is a monomer component extracted from the blood-breaking and stasis-removing Chinese medicine Curcuma.The results of previous studies have confirmed that Zedoarondiol has definite effects in protecting endothelial cell(EC)function,inhibiting abnormal proliferation of smooth muscle cells,and reducing inflammatory load within plaques.However,it has not been reported whether Zedoarondiol can regulate EC angiogenesis and stabilize AS plaques.In existing studies,it has been found that exosomes play an important role in the intercellular signaling pathway of AS and can regulate intraplaque vascularization through delivery of microRNAs.The aim of this study was to further validate the crosstalk mechanism between Zedoarondiol intervention exosomes and EC based on the previous research results,and to clarify the inhibition of AS plaque revascularization effect by Zedoarondiol.Therefore,we propose that Zedoarondiol regulates exosomal microRNA as a key mechanism.In this paper,we started our study from three aspects:clinical,animal and cellular.Firstly,we explored the correlation between circulating exosomal miR-let-7a and acute myocardial infarction through a small sample clinical control study.Secondly,we used high fat diet feeding ApoE-/mice to establish an AS model to observe the inhibitory effect of Zedoarondiol on intraplaque revascularization and the effect on circulating exosome miR-let-7a and its downstream proteins.Finally,the molecular mechanism of inhibition of platelet exosome(Pexo)-mediated miR-let-7a by Zedoarondiol was verified by platelet activation assay in vitro,and lumen generation assay.Study Ⅰ:Clinical study on the correlation between exosomal microRNA and acute myocardial infarctionObjective:To explore the correlation between exosomal miR-let-7a and acute myocardial infarction based on clinical samples.Methods:15 patients with acute myocardial infarction(MI group)and 15 healthy volunteers(healthy group)were recruited from Xiyuan Hospital,China Academy of Chinese Medical Sciences and Wangjing Hospital,China Academy of Chinese Medical Sciences,and their demographic data,medical history information,blood routine,cardiac enzyme test,blood biochemistry,and coagulation test were collected.The plasma exosomes were extracted by ultracentrifugation in both groups and the exosomes were assayed for miR-let-7a,miR-210 and miR-126,which are the key exosomal microRNAs affecting the stability of AS spots.Results:Compared with the healthy group,plasma levels of exosome miR-let-7a were increased in the MI group(P<0.05),miR-210 and miR-126 were not significantly changed(P>0.05),plasma expression levels of pro-angiogenic factors VEGF and MMP-9 were upregulated(P<0.01),APTT was shortened,and platelet activationrelated factors CD62P and 5-TH levels were increased(P<0.01);there was no significant difference in cGMP between the two groups(P>0.05).Conclusion:Patients in the MI group had increased plasma levels of exosomal miR-let-7a,upregulated levels of pro-angiogenic proteins,and increased levels of platelet activation.Exosome-mediated miR-let-7a may be a key gene in regulating AS plaque stability.Study Ⅱ:Effect of Zedoarondiol on intraplaque angiogenesis in ApoE-/-miceObjective:To investigate the effect of Zedoarondiol on intraplaque angiogenesis in AS plaques of mice.To explore the mechanisms involved through the exosomal miRlet-7a/THBS-1 pathway.Methods:ApoE-/-mice were randomly divided into 4 groups for continuous highfat diet feeding to establish AS model,model group:equal volume saline,ZEL group:Zedoarondiol 5mg/kg/d,ZEL group:Zedoarondiol lOmg/kg/d,atorvastatin group:atorvastatin calcium 10mg/kg/d,while C57BL/6J mice were used as the control group:equal volume of saline.Mice were fed with drugs and high-fat feeding with concurrently for 14 weeks.Sections of full-length aorta as well as aortic roots were stained with oil red O to observe plaque generation.Immunohistochemical staining was performed to detect the density of neovascularization within the plaques.Western blot(WB)was performed to detect the expression of THBS-1,CD36,VEGF and CD34,which are proteins related to aortic neovascularization.Biochemical assay for lipid levels.Elisa for plasma TNF-α,MMP-9,LDL,and HDL.qPCR for exosome miR-let7a levels.Platelet activation rate was measured by flow cytometry.Results:After 14 weeks,compared with the control group,the model group showed significant plaque generation in the full-length aorta and aortic root,a significant increase in the density of neovascularization within the aortic root plaque in mice(P<0.01),a decrease in the levels of anti-neovascular protein THBS-1 and its receptor CD36 in aortic tissue(P<0.01),an increase in the levels of pro-neovascular proteins VEGF and CD34(P<0.01),increased plasma concentrations of TG,TC,LDL,TNF-α,and MMP-9(P<0.01).downregulated HDL levels(P<0.01),and increased platelet activation rate and exosome-derived miR-let-7a levels(P<0.01).Compared with the mod group,after Zedoarondiol intervention,the aortic root plaque area ratio was reduced in the ZEH group of mice(P<0.05),the intraplaque neovascularization density was decreased(P<0.01),the expression of THBS-1 and CD36 in the aorta was increased(P<0.01,P<0.05),VEGF and CD34 were decreased(P<0.01,P<0.05),plasma concentrations of TC,LDL,TNF-α,and MMP-9 decreased(P<0.05),while TG and HDL levels did not change significantly(P>0.05),and platelet activation rate and plasma exosome miR-let-7a levels decreased in mice(P<0.01).Conclusions:Zedoarondiol inhibited aortic plaque formation and reduced intraplaque angiogenesis in mice with AS,upregulated the angiogenesis inhibitory protein THBS-1 and downregulated the angiogenesis promoting protein VEGF in aortic tissues,reducing plasma TC,LDL,MMP-9 and TNF-α concentrations.It also inhibited platelet activation and decreased plasma exosome miR-let-7a levels in mice.It was shown that the anti-angiogenic effect of Zedoarondiol was associated with the regulation of exosomal miR-let-7a and platelet activation.Study Ⅲ:In vitro study of Zedoarondiol modulation on Pexo-derived miR-let-7a affects endothelial cell luminal formationObjective:To observe the effects of Zedoarondiol in inhibiting platelet activation in vitro and down-regulating Pexo-derived miR-let-7a levels.And to explore the mechanism of Zedoarondiol inhibition of human umbilical vein endothelial cell(HUVEC)angiogenesis through Pexo/miR-let-7a/THBS-1 signaling pathway.Methods:(1)Platelet activation experiment in vitro:platelets were obtained from healthy volunteers and divided into rest group,model group and Zedoarondiol group.rest group:incubated with equal volume of PBS without activator;Zedoarondiol group:incubated with Zedoarondiol 15μg/mL for 10min and then activated by adding ADP;model group:incubated with equal volume of PBS for 10min and then activated by adding ADP.After the intervention,platelets were fixed with paraformaldehyde and labeled with FITC-CD61 and PE-CD62P,and the platelet activation rate was detected by flow cytometry.qPCR was performed to detect the miR-let-7a level in each group of Pexo.(2)Cell transfection:HUVEC was transfected with miR-let-7a minics to establish miR-let-7a overexpression cell model,and ineffective NC transfection was used as negative control.qPCR and WB verified the transfection model.(3)HUVEC lume formation assay:Intervention with miR-let-7a enriched Pexo after successful transfection.Cells were grouped:HU group:normal HUVEC,HU+Pexo group:normal HUVEC+Pexo 100μg/mL,HU+Pexo+minics group:HUVEC transfected with miRlet-7a minics+Pexo 100μg/mL,HU+Pexo+NC group:HUVEC transfected with NC+Pexo 100μg/mL.24h later,cells from each group were collected for lumen generation assay to observe their tube-forming ability.After the experiment,qPCR was performed to detect miR-let-7a as well as THBS-1 mRNA levels in the cells,and WB was performed to detect THBS-1 and CD36 levels.Results:(1)In the platelet activation assay,the platelet activation rate and the level of Pexo-derived miR-let-7a were significantly higher in the model group compared with the rest group(P<0.01).Compared with the model group,the platelet activation rate decreased(P<0.01)and Pexo-derived miR-let-7a decreased(P<0.01)after Zedoarondiol intervention.(2)In the cell transfection experiment,miR-let-7a expression was increased and THBS-1 protein content was decreased in miR-let-7a minics transfected cells compared with normal HUVEC(P<0.01,P<0.05),while there was no significant difference in NC transfected cells compared with rest group(P>0.05),indicating the successful establishment of miR-let-7a over expression cell model.(3)In the lumen generation assay,compared with the HU group,lumen length increased(P<0.01),miR-let-7a levels increased(P<0.01),THBS-1 mRNA decreased(P<0.01),THBS-1 and CD36 protein expression decreased(P<0.01,P<0.05),with no significant difference between the HU+Pexo and HU+Pexo+NC groups(P>0.05),and the indices were further altered in the HU+Pexo+minics group compared with the HU+Pexo group(P<0.05).Conclusion:Zedoarondiol inhibited platelet activation in vitro and downregulated Pexo miR-let-7a levels.Pexo miR-let-7a was able to inhibit THBS-1 mRNA expression and promote HUVEC tubulogenesis.It is shown that Zedoarondiol inhibits platelet activation,downregulates Pexo miR-let-7a,attenuates its interference with THBS-1 mRNA,promotes THBS-1 protein expression,and thus inhibits angiogenesis.