| Atherosclerosis （AS） is considered as a long-term comprehensive pathologicalprocesses which is characterized by multi-gene regulating and a number of factorsinvolvement. Damage and dysfunction of vascular endothelial cells （VEC） are keyfactors to initiate atherosclerosis. It is currently recognized that oxidized low densitylipoprotein （ox-LDL） is one of the risk factors induced VEC injury. Inflammatoryresponse causes endothelial cell dysfunction is considered to be an important part ofox-LDL-induced atherosclerosis development. p38MAPK is a member of themitogen-activated protein kinase （MAPK） family and play an important role in thestress and inflammtion response. p38MAPK converted from non-phosphorylated stateto phosphorylated one thus contributing to the phosphorylation of downstreamsubstrates to acheieve signal transduction. Thereby promoting the secretion ofinflammatory cytokines which mediate inflammation response and involved theformation of AS. However, the activated and upstream regulatory mechanism ofp38MAPK signal pathway has not yet been clarified.MicroRNA-21（miR-21）is a special miRNA which attracts much concern in the fieldof oncology with significant effect against cancer cell apoptosis. The further researchfound that miR-21also play important role in cardiovascular disease. It related tovascular inflammation, neointimal formation, vascular smooth muscle cells migrationand apoptosis. Besides, miR-21is high expression in the endothelial cells ofatherosclerosis in animal models of intimal injury and high glucose state. It has beenconfirmed that the tensin homolog deleted from chromosome-10（PTEN） is target geneof miR-21. And PTEN could inhibit the activation of p38MAPK signaling pathway.Therefore, the occurrence of AS may be related to miR-21expression in endothelialcells and regulating of downstream PTEN related signaling pathway.Paeonol is an active compound isolated from cortex Moutan. Our previous studieshave shown that Pae has many pharmacological activities. Pae inhibit the release of TNF-α and IL-1β in plaque of experimental AS in rabbit and reduce the immflamationreaction of plaque local. Further study also confirmed that Pae contained serum in vitroreduce the phosphorylation level of TNF-α and ox-LDL induced MAPK signalingpathway and LPS induced NF-κB signaling pathway. Simultaneity, Pae reduce adhesionmolecule expression in VECs and inhibit monocytes adhesion to rate aortic endothelialcell. These studies suggest that The effect of Pae against AS may via regulating miR-21expression to inhibit the inflammation reaction in VECs.In this study, ox-LDL was used as stimulator to induce VEC injuried cell model ofAS. It was planned to observe the expression of miR-21in injuried endothelial cells andexplore the way of miR-21regulating the function of endothelial cells. Simultaneity, itwas planned to investigate the correlation of miR-21with p38MAPK signalingpathway. It made further studies to investigate the effect of Pae on miR-21. This studyelucidated the pathogenesis of AS from the genetic level and provided theoretical basisfor new drug’s targets of Pae in clinical treatment on AS.OBJECTIVETo investigate the expression of miR-21in ox-LDL injuried VECs. To explorewhether Pae affect the miR-21expression to regulate PTEN/p38MAPK signalingpathway and thus participate in against inflammatory injury of endothelial cells.METHODSMiR-21expression level was detected after VEC stimulated by ox-LDL. Endothelialcells were transfected by miR-21mimic or inhibitor with Hiperfect transfection reagent.And then VEC were cocultured with ox-LDL for24h or pretreated with Pae for24h.RT-qPCR assay was used to determine miR-21and PTEN mRNA expression level inVEC. Western blotting assay was used to determine PTEN, Ras, MKK3/6, P-MKK3/6,p38and P-p38protein level in each group. ELISA assay was used to determine therelease of inflammatory cytokines TNF-α in VEC. RESULTS1Injury effect of ox-LDL on VECVEC were cultured with different concentration of ox-LDL(5,10,20,40,80mg·L-1)for different time（0,12,24,48h）.VEC growth were inhibited in dose-andtime-dependent manner. When ox-LDL concentration up to20mg·L-1and cultured for24h, VEC growth was inhibited significantly（P<0.01）.2Effect of ox-LDL on miR-21expression in VECmiR-21expression level was detected by RT-qPCR assay. The results showed thatmiR-21expression level was increased significantly in ox-LDL induced VEC（P<0.05）.3miR-21target gene verifyDual-luciferase gene reports experiments used to verify if PTEN were target geneof miR-21in VEC. The results showed that the fluorescence intensity ofpGL3-PTEN-3’URT group was decreased significantly. And the fluorescence intensityof pGL3-PTEN-3’URT-mut group was increased significantly compared with controlgroup.4Effect of miR-21mimic or inhibitor on PTEN expression in VEC. PTEN mRNA of total RNA in each group were detected by RT-qPCR assay. Theresults showed PTEN mRNA was decreased significantly in single ox-LDL stimulatedgroup compared with control group. PTEN mRNA was decreased significantly in groupof transfecting miR-21mimic as well. And the level was lower than single ox-LDLstimulated group. The results was turned by miR-21inhibitor significantly.PTEN protein expression were detected by western blotting assay. The resultsshowed PTEN protein was decreased significantly in single ox-LDL stimulated groupcompared with control group. PTEN protein was decreased significantly in group oftransfecting miR-21mimic as well. And the level was lower than single ox-LDLstimulated group. The results was turned by miR-21inhibitor.5Effect of miR-21mimic or inhibitor on TNF-α expression in VECTNF-α in cell supernatant was detected by ELISA. The results showed that TNF-αwas overexpression in ox-LDL induced VEC. The level was also significantly increasedin miR-21mimic group, but decreased in miR-21inhibitor group. 6Pae increased ox-LDL induced VEC survival rateVEC were pretreated with different concentrations of Pae（15,30,60,120,240,480μM）for different time （0.5,6,12,24,36,48h）. Ox-LDL(20mg·L-1)were culturedwith VEC for another24h. Compared with ox-LDL group, Pae increased VEC survivalrate in dose-and time-dependent manner. Pae concentration of30μM and coculturedwith VEC for24h could significantly increased the survival rate（P<0.05）.When Paeconcentration up to120μM and cocultured with VEC for24h, the protective effect wasstrongest（P<0.01）.7Pae inhibited ox-LDL induced VEC expression of miR-21VEC were pretreated with Pae（60,120,240μM）for24h. Then cocultured withox-LDL for another24h. Total RNA in each group were extracted. RT-qPCR assaywas used to detect relative miR-21level. The results showed that Pae couldsignificantly decrease miR-21expression induced by ox-LDL in dose-andtime-dependent manner.8Effect of Pae on PTEN expression on VECAfter cocultured with Pae concentration of120μM, PTEN mRNA expression levelwas significantly increased compared with ox-LDL group. The level was also increasedin Pae combined with miR-21mimic group but not significantly. While the PTENmRNA level was significantly increased in Pae combined with miR-21inhibitor group.And the expression level was the highest.Pretreated VEC with Pae concentration of120μM, PTEN protein expression levelwas significantly increased compared with ox-LDL group. The level was also increasedin Pae combined with miR-21mimic group but not significantly. While the PTENprotein level was significantly increased in Pae combined with miR-21inhibitor group.9Effect of Paee on p38MAPK signaling pathway in VECWestern blotting assay was used to determine Ras, MKK3/6, P-MKK3/6, p38andP-p38expression in each group. Compared with ox-LDL group, Pae significantlyinhibited expression of Ras, P-MKK3/6and P-p38protein. When Pae combined with miR-21inhibitor, the effect was strongest. When Pae combined with miR-21mimic, theRas, P-MKK3/6and P-p38protein level was also decreased but not significantly.10Pae inhibited TNF-α expression in ox-LDL induced VECCompared with ox-LDL group, after pretreated with Pae （120μM）, the TNF-α levelwas significantly decreased. The effect was strongest when cells were treated with Paecombined with miR-21inhibitor.CONCLUSION1. Ox-LDL could induce VEC expression of miR-21.2. MiR-21regulated PTEN expression in VEC. PTEN was target gene of miR-21. Thedown-expression of PTEN lead to p38MAPK signaling pathway activation. MiR-21maybe mediate ox-LDL induced inflammation reaction of VEC which lead toatherosclerosis ultimately via regulating PTEN/p38MAPK signaling pathway.3. Pae could inhibit miR-21expression to improve the target gene PTEN expression.Overexpression of PTEN inhibited p38MAPK signaling pathway. The release ofinflammation cytokines was reduced. Thereby the development of AS was inhibited. |
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