Based On CircRNAs, We Explored The Molecular Mechanism Of Ligusticum Chuanxiong-Red Paeoniae Rubra's Active Ingredients In Intervening AS Plaque Angiogenesis

Atherosclerosis（AS）plaque rupture is the main cause of death in cardio-cerebrovascular diseases,while angiogenesis in plaques would accelerate the progress of plaques and induce plaques rupture and hemorrhage.Therefore,the inhibition of plaque angiogenesis has been considered as a potential therapy to inhibit progress of plaques and to increase the stability of plaques.Activating blood circulation and removing blood stasis（ABCRS）prescriptions and drugs have the clinical efficacy in the treatment of AS.Ligusticum chuanxiong Hort.and Radix Paeoniae Rubra herb pair in Chinese herbal medicine have anti-atherosclerosis effect.While the effect of their major active ingredients,tetramethylpyrazine（TMP）and paeoniflorin（PF）,on angiogenesis in AS plaque has not been studied.Circular RNAs（circRNAs）regulate the transcription and post-transcriptional levels of genes.CircRNAs can act as microRNAs（miRNAs）sponges by competitive binding to miRNAs,and then regulate target genes of miRNAs.While whether combination of TMP and PF（TMP-PF）could improve angiogenesis in AS plaque by regulating circRNAs and the relevant target genes is not clear.1.PurposeThis study aims to identify specific molecular markers associated with AS and angiogenesis,and to evaluate the effect of TMP and PF on angiogenesis in AS plaque.On that basis,the molecular mechanism and target for TMP and PF intervention in AS plaque angiogenesis were further explored by targeting at circRNAs.2.Method（1）AS related differentially expression circRNAs screening and angiogenesis related circRNA-miRNA-mRNA networks constructionBased on the transcriptomics sequencing results of clinical AS blood stasis syndrome patients and healthy people from Xiyuan Hospital of China Academy of Chinese Medical Sciences and the results of literature review analysis,the related circRNAs in association with AS were screened in present study,and the miRNAs and mRNAs corresponding to the target circRNAs were predicted.Then circRNA-miRNA-mRNA networks in association with AS and angiogenesis were established,and circRNAs,miRNAs,mRNAs and target proteins associated with AS and angiogenesis were preliminary obtained.（2）The effect and mechanism of TMP and PF on ox-LDL-induced angiogenesisHuman umbilical vein endothelial cells（HUVECs）were induced by different concentrations of oxidized low-density lipoprotein（ox-LDL）,and AS angiogenesis model was established.Then HUVECs were divided into normal group,model group,TMP group,PF group and TMP+PF group.Different drug groups administrated with different concentrations of TMP and PF（10,1,0.1,0.01μmol/L）,either alone or in combination.After drug intervention for 24h,cell proliferation was measured by MTT assay.The synergic effect of TMP and PF was analyzed by Chou-Talalay combination index method,and the optimal compatibility ratio of TMP and PF was preliminary obtained.Then,ox-LDL-induced HUVECs were treated with TMP,PF,or an optimal combination of TMP and PF.HUVECs were divided into normal group,model group,TMP group,PF group,TMP+PF group,and simvastatin group.After ox-LDL induced,HUVECs were intervened with 1 μmol/L TMP,10μmol/L PF,1 μmol/L TMP+10μmol/L PF and 1 μmol/L simvastatin for 24h.Cell proliferation was detected by MTT assay.Cell migration was measured by wound healing assay.Cell tube formation was measured by cell tube formation assay.The expression of vascular endothelial markers von willebrand factor（vWF）and CD31 were examined by immune fluorescence assay.The expression of vascular endothelial growth factor（VEGF）was detected by ELISA.The expression of VEGF receptor2（VEGFR2）and the related proteins of Notch pathway（Jagged1,Notch1 and Hes1）are detected by Western Blot.（3）Exploring the molecular mechanism and targets of TMP-PF intervention in AS angiogenesis based on circRNAsOx-LDL induced HUVECs to construct the in vitro angiogenesis model.Then ox-LDL-induced HUVECs were treated with TMP-PF（1 μmol/L+10 μmol/L）.The expression of screened circRNAs was detected by real-time quantitative PCR（qPCR）,from which a target circRNA was selected and applied with further RNA interference study.HUVECs were divided into normal group,model group,TMP+PF group,TMP+PF+circRNA intervention（siRNA）group,TMP+PF+circRNA intervention（siRNA）+miRNA intervention（inhibitor）group.After cell transfection with siRNA（100nmol/L）or inhibitor（100 nmol/L）for 24h,ox-LDL induction and TMP-PF intervention for 24h,cell proliferation was detected by MTT assay.Cell migration was measured by wound healing assay.Cell tube formation was measured by cell tube formation assay.The expression of circRNAs,miRNAs and mRNAs were measured by qPCR.The expression of target proteins was detected by Western Blot.（4）The effect and mechanism of TMP and PF on angiogenesis in AS plaque of ApoE-/-miceC57BL/6J mice were used as the controls,ApoE-/-mice were divided into model group,low dosage group（1 mg/kg/d+10mg/kg/d）,medium dosage group（2mg/kg/d+20mg/kg/d）,high dosage group（5 mg/kg/d+50 mg/kg/d）,very high dosage group（10 mg/kg/d+100 mg/kg/d）,simvastatin（2.5mg/kg/d）group,TMP group and PF group.The respective dosages of TMP group and PF group were derived from optimal compatibility dosages of TMP+PF group.The controls were given ordinary diet,while ApoE-/-mice were intraperitoneally inj ected with TMP and PF or intragastrically administrated with simvastatin after fed with high-fat diet for 2 months and were continually given high-fat diet till the end of 3rd month.The serum triglyceride（TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）,very low-density lipoprotein（VLDL-C）and high-density lipoprotein cholesterol（HDL-C）were detected by automatic biochemical analysis.The areas of plaques were observed by HE staining.The expression of vWF and CD31 in aorta was detected by immune fluorescence assay to observe vessel density.The expression of VEGF in serum was determined by ELISA.The expression of VEGFR2,Notch1 and target proteins in aorta was detected by Western Blot.3.Results（1）Five circRNA-miRNA-mRNA networks associated with AS and angiogenesis were obtainedThree major AS-related differentially expressed circRNAs were obtained from transcriptome sequencing results,namely circRNA₀6206（circRNA of stimulator of chondrogenesis 1,circSCRG1）,hsa_circ₀004417 and hsa_circ₀041555.Two circRNAs were obtained from literature review analysis,namely has_circ₀000284 and hsa_circ₀003575.The circRNA-miRNA-mRNA networks in association with AS and angiogenesis were established,and the relevant target genes and target proteins were selected for the further study.（2）TMP-PF inhibited ox-LDL-induced in vitro angiogenesisOx-LDL induced HUVECs proliferation,and different concentrations of TMP and PF（10,1,0.1 μmol/L）inhibited ox-LDL-induced HUVECs proliferation（P<0.05）.The results of synergistic analysis indicated that TMP and PF（1:10）displayed more effective synergistic activity at different dose and better dose response relationship than did TMP and PF administered in the other ratios,and this combination had a CI value of less than 1 at most Fa levels.In addition,1 μmol/L TMP and 10 μmol/L PF exert the best effects.Therefore,compatibility ratio 1:10 for TMP and PF was selected for the further study.The present study suggests that TMP-PF（1 μmol/L TMP+10 μmol/L PF）as well as simvastatin（1 μmol/L）inhibited ox-LDL-induced HUVECs proliferation,migration and tube formation,and decreased the expression of CD31,VEGF,VEGFR2 and Notchl（P<0.05）,while had no effect on the expression of vWF（P>0.05）.In addition,TMP-PF could also decrease the expression of Jagged1 and Hes1 in HUVECs（P<0.05）,while TMP alone or PF alone could not decrease the expression of Jagged1 and Hes1（P>0.05）.It is preliminary indicated that TMP-PF could inhibit ox-LDL-induced in vitro angiogenesis,and the mechanism might be related to inhibiting the VEGF pathway and the Notch pathway.（3）TMP-PF inhibited ox-LDL-induced angiogenesis by regulating circSCRG1After HUVECs were induced by ox-LDL,the expression of circSCRGl decreased and the expression of hsa_circ₀003575 increased,while TMP-PF could increase the expression of circSCRG1（P<0.05）.Therefore,circSCRGl was selected for the further interference study.This study found that TMP-PF inhibited ox-LDL-induced HUVECs proliferation,migration and tube formation,and decreased the protein expression of nucleus receptor 4A1（NR4A1）（P<0.05）.Simultaneous administration of siRNA to inhibit the expression of circSCRGl,TMP-PF had no anti-angiogenesis effects in HUVECs,and the protein expression of NR4A1 was not decreased（P>0.05）.When continually administrated with hsa-miR-1268b inhibitor,TMP-PF exerted anti-angiogenesis effect again.Therefore,TMP-PF inhibited ox-LDL-induced angiogenesis,and the molecule mechanism of anti-angiogenesis effect might be related to TMP-PF’s regulation on circSCRGl,which accordingly regulates the expression of target protein NR4A1 of hsa-miR-1268b.（4）TMP-PF inhibited plaques angiogenesis with anti-atherosclerosis effectThe weight,and the levels of serum TG,TC,LDL-C and VLDL-C were increased while the level of serum HDL-C was decreased in AS model mice（P<0.05）.In addition,the expression of CD31,vWF,VEGFR2 and NR4A1 in aorta was increased while hypoxia-inducible factor prolyl hydroxylase 2（PHD2/EGLN1）,SAM and SH3 domain containing 1（SASH1）were decreased in AS model mice（P<0.05）.Also,the AS model mice have the increased density of the new vessels in plaques and increased plaque areas（P<0.05）.High dosage of TMP-PF and simvastatin reduced the weight and the level of serum TG,reduced the ratio of plaques areas and lumen areas,and decreased the expression of CD31 in aorta（P<0.05）,which indicated the decreased density of the new vessels in plaques.In addition,high dosage of TMP-PF also reduced the level of serum LDL-C and the expression of VEGFR2 and the target protein NR4A1 in aorta（P<0.05）.These results preliminary suggest that TMP-PF might has anti-atherosclerosis effect by inhibiting plaques angiogenesis,and the main targets are VEGFR2 and NR4A1.4.ConclusionTMP-PF could inhibit ox-LDL-induced angiogenesis in HUVECs and angiogenesis in aortic plaques in ApoE-/-mice,so as to exert the effect of anti-atherosclerosis.The candidate molecule mechanism of anti-angiogenesis effect might be related to TMP-PF’s regulation on circSCRG1,which accordingly regulates the expression of target protein NR4A1 of hsa-miR-1268b,and then inhibit the VEGF/VEGFR2 pathway.