| Micro ribonucleic acid （miRNA） was as a small molecule with regulative function,which specifically expressed in vascular endothelial cell （VEC）. miRNA couldprevent the translation or degrade the messager ribonucleic acid （mRNA）, throughspecific binding to the3’untranslated region （3’UTR） of target mRNA, whichparticipated in the regulation of the VEC morphology and function to play animportant role in the vascular pathphysiology process. The studies showed thatmicroRNA-126（miR-126） had specific expression which involved in the injury andrepair process of VEC in the cardiovascular system. The downregulation of miR-126level could lead to the upregulation of vascular cell adhesion molecule-1（VCAM-1）expression which induced by tumor necrosis factor-alpha （TNF-α）, resulted inmonocyte （MC） adhesion to VEC. However, the upregulation of miR-126expressionby its precursor overexpression, could decrease VCAM-1expression. Therefore,miR-126played a regulative role in gene expression through regulation of VCAM-1expression which intervened MC adhesion to VEC, thus prevented the occurrence anddevelopment of atherosclerosis （AS）.Paeonol （Pae） was one of the active ingredients which isolated from dried rootbark of Paeonia Suffruticosa Andr. and dried root and rhizome of CynanchumPaniculatum （Bge.） Kitag., which had a wide range of pharmacological effects suchas anti-arrhythmic, anti-hypertensive, anti-thrombotic, et al. In our studied team, thestudies found that Pae had significantly anti-AS in the experimental quail, protectionof the hyperlipidemic rat VEC, and suppression of lipid peroxidation in serum and inaortic of hyperlipidemic rat. Meanwhile, Pae also inhibited oxidative modification ofoxidized low density lipoprotein （ox-LDL） of healthy human serum in vitro. Inox-LDL-induced VEC inflammatory injury process, Pae could decrease thephosphorylation level of phosphatidylinositol3-kinase （PI3K）,protein-serine-threonine kinase （Akt） and nuclear factor-kabba B （NF-κB） signalingprotein and blocked cell inflammatory signal transduction, then blocked VCAM-1of cell adhesion factors expression and release, which suppressed MC adhesion to VEC,the VEC proliferation and migration to curb the AS inflammation formation anddevelopment.OBJECTIVETo explore miR-126specific expression in ox-LDL-induced VEC inflammation,and verification of its regulation of target gene VCAM-1expression, as well as theeffects on MC adhesion to VEC. Studied the Pae regulation of miR-126expressionwhich affected signaling protein phosphorylation and transduction ofPI3K/Akt/NF-κB pathway，and studied molecular mechanisms of Pae target onanti-AS.METHODSThe primary VEC was isolated and cultured from rat thoracic aorta withpre-digestion adherence technology, and also identified the obtained cell byimmunocytochemistry method. MTT assay was used to detect effect of ox-LDL onVEC activity and trypan staining method was used to detect cell membranepermeability. The expression of miR-126and VCAM-1mRNA were detected byreal-time quantitative SYBR Green PCR. The VCAM-1protein expression wasdetected by western blotting. miR-126mimic or inhibitor was transfected into theVEC by HiPerFect reagent respectively. miR-126targeted gene to regulate expressionof VCAM-1which was validated with Dual-luciferase reporter system. The RoseBengal reagent was used to detect MC adhesion to VEC.The protection of Pae on ox-LDL-damaged VEC was detected by MTT method,and also detected the lactic acid dehydrogenase （LDH） release. miR-126mimic orinhibitor was transfected into VEC by HiPerFect reagent. miR-126, VCAM-1mRNAexpression which inducd by ox-LDL were detected with real-time quantitative SYBRGreen PCR. The signaling protein expression of PI3K, Akt, p65, IκB pathway wasdetected by western blotting. The Rose Bengal reagent was used to analysis theregulation of Pae on miR-126expression to impact on MC adhesion to VEC. RESULTS1miR-126had lower of the expression in ox-LDL-induced rat VECmiR-126had lower of the expression obviously in rat VEC inflammatory injurywhich stimulated by ox-LDL (20mg·L-1)（P <0.01）.2miR-126regulated the direct target of VCAM-1in rat VECmiR-126could regulate3’UTR of VCAM-1mRNA: miR-126mimic promotedmiR-126higher expression which decreased firefly luciferase activity by acting on the3’UTR of VCAM-1mRNA; when miR-126higher expression bound with the mutant3’UTR plasmid of VCAM-1mRNA, the activity of the firefly luciferase was notdifference.3Effects of miR-126on the MC adhesion to VECThe absorbance （A） value was higher significantly which compared with controlgroup （P<0.01）, the function of MC adhesion to VEC was significantly increased inox-LDL (20mg·L-1) group. Comparing with the ox-LDL group, the A value was lowersignificantly in miR-126mimic transfection group （P<0.01）.4Effects of Pae on miR-126expression in ox-LDL-injured VECmiR-126had a significant downexpression in the ox-LDL (20mg·L-1) group（P<0.01）. miR-126had a specific higher expression in Pae （60μM） group （P<0.05）,and miR-126also had higher expression in Pae （120,240μM） group （P<0.01）.Pae could promote miR-126higher expression which induced by miR-126mimic（P<0.05）; miR-126expression level was lower significantly in miR-126inhibitortransfection group.5Effects of Pae upregulation of miR-126on VCAM-1expression inox-LDL-induced VECVCAM-1expression had a hidher trend in ox-LDL (20mg·L-1) group. Pae （120μM） significantly reduced VCAM-1expression （P<0.05）. VCAM-1expression wasdecreased significantly in miR-126mimic transfection group （P<0.01）. However,VCAM-1expression had obviously higher trend in miR-126inhibitor transfectiongroup. 6Effects of Pae upregulation of miR-126expression on PI3K/Aktpathway in ox-LDL-induced VECPI3K and Akt phosphorylation levels were improved highly in ox-LDL (20mg·L-1)group. Pae was able to significantly reduce the level of PI3K and Akt phosphorylationwhich induced by ox-LDL. PI3K phosphorylation level was downregulated （P<0.05）,and inhibition of Akt phosphorylation （P<0.01） in miR-126mimic transfection group.7Effects of Pae upregulation of miR-126expression on NF-κBpathway in ox-LDL-induced VECThe inhibitory kappa B （IκB） phosphorylation level had a significant upward trendin ox-LDL (20mg·L-1) group. Pae significantly reduced IκB phosphorylation leveland downexpression of p65protein. The IκB phosphorylation level had downwardtrend in miR-126mimic group （P<0.05）, and had upward trend in miR-126inhibitorgroup （P<0.01）. LY294002was as a PI3K protein inhibitor, which could suppressphosphorylated IκB protein expression, and reduced the p65protein expression, sothat indicated NF-κB was the downstream signaling protein of the PI3K/Akt pathway.8Effects of Pae upregulation of miR-126expression on MC adhesionto ox-LDL-injured VECThe function of MC adhesion to VEC was increased obviously in ox-LDL (20mg·L-1) group （P<0.05）. Pae could obviously reduce the MC number of adhesion toVEC （P<0.05）, and the function of MC adhesion to VEC was significantly lower inmiR-126mimic transfection group （P<0.01）.CONCLUSIONS1. miR-126had a lower specific expression in ox-LDL-induced rat VEC inflammation.The VCAM-1was proved as negative regulation of target gene of miR-126expression,and downregulaion of miR-126could upregulate the VCAM-1expression and release,thus enhanced the MC adhesion to VEC.2. Pae could promote the miR-126expression to inhibit VCAM-1expression andrelease in ox-LDL-induced VEC. Meanwhile, Pae upregulation of miR-126expression blocked PI3K/Akt/NF-κB pathway transduction, and decreased MCadhesion to VEC. |
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