The Effects Of Armadillidium Vulgare On OPN-induced Vascular Smooth Muscle Cell Proliferation And Migration And Its Molecular Mechanism

Purpose: The proliferation of vascular smooth muscle cells （VSMCs） tosubendocardial migration is high blood pressure, atherosclerosis andangioplasty restenosis after vascular remodeling disease cytopathologyfoundation.Phenotypic changes of vascular smooth muscle cells, adhesion, thesubintimal migration, proliferation and secretion of extracellular matrix （ECM）is high blood pressure, atherosclerosis and vascular remodeling Restenosisafter percutaneous transluminal angioplasty in cellular pathologic basis ofdisease. Among them, VSMC adhesion, migration and proliferation in film,involved in vascular inflammatory hyperplasia, causes vascular remodeling,this process relies on micro-interactions between cells and ECM, dynamicbalance. Studies have shown that several growth factors and ECM componentssuch as the OPN, fibronectin （FN） are involved in the regulation of VSMCproliferation and adhesion, migration process. ECM-Integrin （Integrin）interaction effects are mainly generated by intracellular area raised through itsbeta-subunit protein kinase regulation of myosin phosphorylation State,cytoskeletal reorganization and cell migration.Integrin （Integrin） is made up of α and β subunits of the heterologousdimerization of transmembrane receptors, VSMC major expression of Integrinα v β3, its main ligand include OPN, fibronectin （FN） and vitronectin （VN）.Due to integration pigment itself does not has kinase activity, it needs throughbeta Asia units of cell within area and Talin, and paxillin, and mount spotkinase （FAK） and integration pigment connection kinase （ILK）, mutual role,makes its raised to cell film inside, common formed mount spot （focaladhesion） composite real, and started more article signal go guide way, to led to cell skeleton restructuring, caused VSMC of stuck echoed migration,participation vascular of inflammatory sexual hyperplasia and vascularremodeling process. We are at the early stage of experimental discovery ofOPN in adhesion and migration of vascular smooth muscle cells induced by,and FAK and ILK plays an important role in this process.Pillbugs （Armadillidium Vulgare）, also known as mouse negative,negative Pan mouse Regardless, mouse stick, and lice, is a the CrustaceaIsopoda woodlice Division pillbugs genus commonly known." Compendiumof Materia Medica " records pillbugs expelling blood, and asthma, passthrough, diuretic, detoxification, pain and other effects. Modern researchhas found that the role of anti-mouse arrhythmias, analgesic,anti-inflammatory pillbugs, pillbugs clinical application of the treatment ofhemorrhoids, common warts, moderate to severe cancer pain, stomatitis,tonsillitis, chronic bronchitis, post-operative pain, liver cancer, and so on.In a variety of vascular remodeling diseases, vascular inflammatoryhyperplasia clear led lumen stenosis, is a important cause of ischemic heartdisease, rat woman has the important function of the anti-inflammatory.Research has shown that women through inhibition of epoxide Hydrolase isthe rat （COX） play an anti-inflammatory effect and thus involved in theoccurrence and development of vascular remodeling diseases. But whether itaffects vascular inflammatory hyperplasia on proliferation and migration ofvascular smooth muscle cells have not been reported. This researchobservation rat woman for OPN induced of VSMC of migration, andproliferation, and apoptosis dead of effect, to and related of signal molecularprotein （FAK and ILK） expression activity of change, to discussion rat womanin effect VSMC proliferation and migration area of role, and research itsrelated of molecular mechanism, for prevention and treatment to VSMCproliferation and migration for cell pathology learn based of vascularinflammatory sexual hyperplasia sexual disease （as atherosclerosis, and PTCAHou again narrow,） provides reasonable of experimental pursuant to, laytheory based, And provide new therapeutic targets. Method:1Cell cultureSD rat aortic smooth muscle cells cultured in DMEM medium containing10%fetal bovine serum, penicillin100U/ml, streptomycin100ug/ml setsaturated humidity,37℃,5%CO₂incubator culture. SD rats were culturedthe thoracoabdominal aortic vascular smooth muscle cells, by conventionalmethods and to3-5cells selected for the experiment.2Flow cytometry to detect apoptosisPrimary cell cultures3-5generations later, the cells were divided intoseven groups, each group of3-5bottles, each bottle is added OPN20ug/ml;the pillbugs water extract （20mg/ml）25ul/ml of50ul/ml,100ul/ml; pillbugsethyl acetate extract （20mg/ml）12.5ul/ml25ul/ml,50ul/ml; continue37℃incubation for24hours, collecting the cells in each group, was washed twotimes with PBS, at room temperature centrifuged at2000rpm,8minutes, andthe supernatant was removed, fixed with70%ethanol,4℃refrigeratoroscillator mix,1week to send the province four hospital experimental Centerthe flow cytometry late apoptosis.3MTT assay value-added3.1Collected on the number of cells, adjusting the concentration of the cellsuspension to5X104/ml, coated96-well plates,5%CO₂,37℃for24hours.3.224hours abandoned to training liquid, joined no serum DMEM trainingliquid, each hole200ul, set3-5a complex hole, the group plus drug, setadjusting zero hole （training liquid, and MTT, and II methyl Asia sulfone）,against hole （cell, and OPN, and training liquid, and MTT, and II methyl Asiasulfone）, experimental hole （cell, and OPN, and different concentration ofdrug that mouse negative water extraction real0.5mg/ml, and1mg/ml, and2mg/ml; mouse negative acetic acid b ester extraction real0.25mg/ml, and0.5mg/ml, and1mg/ml; culture, MTT, dimethyl sulfoxide）5%CO₂,37℃incubation for24hours.3.3Each hole20ulMTT solutions （5mg/ml,0.5%MTT）, to continue to build4hours. 3.4Discard supernatant to put an end to training.3.5Each hole150ul dimethyl sulfoxide, reset the tilt bed speed oscillation10min, crystallization of fully dissolved. OD490nm enzyme-linkedimmunoassay instrument measuring the absorbency of the hole.3.6Analysis of experimental results, determine the armadillidium vulgareextract and ethyl acetate extract effective concentration.4Cells scratches experimental determination of cell migrationCell culture to3-5substituting coated6-well plates, and divided intothree groups, each hole2, each of the groups VSMC inoculated in sterileglass slides, the cells were grown to100%confluency, remove slides withsterile Blister head on the slide scratches after washing in PBS the cells werescraped, the slides placed in a culture solution containing OPN （20ug/ml）,and the blank control group without OPN, the experimental groups wereadded to the effective concentration mouse negative water extract and ethylacetate extract, and after24hours incubation was continued at lowmagnification to observe cell migration, counting the number of migratedcells as an expression of the cell migration activity.5Western bolt detection PCNA、FAK、ILK protein levelsCollection the Group smooth muscle cell�'PBS washing�'extractioncell cracking liquid�'protein quantitative�'SDS-PAGE electrophoresis�'nitrocellulose film transfer�'5%skim milk closed,37℃1-2H�'combined,the first antibody4℃overnight�'TTBS wash three times,10minutes/times�'combined rabbit anti-Rat IgG,37℃1-2H�'TTBS washthree times,10minutes/times, TBS wash once,5minutes/times�'developer,photography and coagulation rubber image analysis system determination theprotein article with of integral light density, Ratio calculation of proteins andbeta--actin in the expression.6Excel2010spreadsheet software records and preliminary processing of thedata, mapping, SPSS18.0statistical package for the statistical analysis ofdata. Measurement data±s. OUTCOME MEASURES between groups werecompared using one-way ANOVA test. Each test are P <0.05was considered statistically significant.Results:1the detection of apoptosisA poptosis using flow cytometry, and the results showed that littledifference in apoptosis between the control group and the concentration of themouse negative W Group, Group E mouse negative not statisticallysignificant, indicating that the experimental concentration range mousenegative water extract and ethyl acetate extract does not affect VSMCapoptosis.2MTT assay cell value2.1The aqueous extract mouse negative VSMC proliferation on the basis ofcultureThe MTT assay experimental results show that the various concentrationof the mouse negative W group does not affect the VSMC proliferationactivity under the conditions of the basic culture, no significant differences inabsorbance values between the experimental group and the control group, nostatistical significance, but also that this concentration range the mousenegative water extracts on VSMC cytotoxicity.2.2The mouse negative ethyl acetate extract VSMC proliferation on the basisof cultureResults similar to the2.1, each the concentration mouse negative Egroup no significant role in the foundation VSMC proliferation under theculture conditions, no significant difference also shows no toxic effect oncells.2.3The impact of the mouse negative water extract of OPN induced VSMCproliferationThe experimental results show that, mouse negative water extract of theOPN-induced VSMC proliferation inhibitory effect, but with the increase ofthe concentration of the water extract of the mouse negative, inhibitionreduced. Statistical analysis showed that, the mouse negative W Group（0.5mg/ml and1mg/ml） absorbance values were the OPN group85.5%and 90.9%, both with OPN group were statistically significant, P <0.05, lowDescription the concentration mouse negative water extract inhibit OPN-induced VSMC proliferation.2.4. Mouse negative ethyl acetate extract of OPN induced VSMC proliferationMTT assay results showed that the mouse negative ethyl acetate extract adisincentive to OPN-induced VSMC proliferation, and this inhibition isenhanced with the increase in the concentration of mouse negative adose-dependent manner. When the mouse negative Group E concentration of1mg/ml absorbance value of0.22667, which is81.6%of the OPN group,statistically significant difference between the two （P <0.05）. Compare themouse negative W group （0.5mg/ml） and Group E （1mg/ml） on cellproliferation, found little difference between the two, was not statisticallysignificant, and both can inhibit OPN-induced VSMC proliferation, no betteror worse.3Wound healing experimental determination of cell migrationThe experimental results show that, the scratch after24h control groupVSMC to the intermediate transfer to less, after OPN stimulation, a largenumber of VSMC migration from the wound two sides to the middle, thewound area reduced greatly; its water extract and give0.5mg/ml, migration ofcells was inhibited, the number decreased cell migration for the OPN group76.7%; similarly, cell migration mouse negative ethyl acetate extract to1mg/ml can also inhibit OPN induced cell migration,69.3%for OPN group,which is greater than the mouse negative W group. That cell migration mousenegative water extract and ethyl acetate extract can inhibit OPN induced by1mg/ml, and its ethyl acetate extract effect more obvious.4Western bolt detection PCNA、FAK、ILK protein levels4.1Western bolt detection PCNA protein levels: WB results show that, thecontrol group, PCNA protein expression level relatively low, giving OPNstimulation, its protein content was significantly increased, the control group1.59times, but by giving mouse negative PCNA expression decreased thatOPN+each concentration mouse negative W group and OPN+concentration mouse negative group E can reduce the level of expression of PCNA, both adose dependent manner with the MTT assay results, wherein a0.5mg/ml themouse negative W group and1mg/ml mouse negative group E most obviousinhibition, OPN group were77.8%and74.1%. This is further evidence thatthe mouse negative both extract can inhibit OPN-induced proliferation ofVSMC.4.2Western bolt detection ILK protein levels: To OPN stimulation is given,the ILK expression levels were significantly increased, which is consistentwith our previous experimental results. In OPN+mouse negative W groupexperiment, we observed that with the increase of the concentration of themouse negative ILK expression level is gradually increased, i.e., the lowconcentration （0.5mg/ml） the mouse negative W group inhibit OPN inducedILK expression more obvious, is the OPN group81.4%; However, in OPN+mouse negative experiments of group E, with the increase of theconcentration of mouse negative, ILK expression levels did not changesignificantly, indicating that the mouse negative water extract can inhibit ILKto influence the OPN-induced VSMC proliferation and migration.4.3Detected by Western bolt FAK protein expression levels: The experimentalresults show that, OPN can significantly induced FAK expression, which isalso consistent with our previous experimental results. In the OPN+mousenegative W set of experiments, the low concentration （0.5mg/ml） the mousenegative W Group inhibit OPN-induced FAK expression role, which is89.1%of the OPN group; while in the OPN+mouse negative E groupexperiments, high concentration （1mg/ml） group E is more evident for82.2%of the OPN group. This shows that the mouse negative of the water extractcan affect both the FAK way can also affect ILK pathway, while the mousenegative ethyl acetate extract by adjusting the FAK pathway to affect OPN-induced VSMC proliferation and migration process.Conclusion:1the experimental concentration range mouse negative water extract and ethylacetate extract does not affect the process of VSMC apoptosis, no cytotoxicity of VSMC.2The mouse negative water extract and ethyl acetate extract does not affectthe basic culture of VSMC proliferation.3The mouse negative water extract and ethyl acetate extract can inhibit OPN-induced VSMC proliferation.4The mouse negative water extract and ethyl acetate extract can inhibit OPN-induced VSMC migration.5The mouse negative two extracts can inhibit OPN-induced increase in PCNAexpression.6The mouse negative water extract can inhibit OPN induced ILK expressionincrease this effect, and the ethyl acetate extract.7The mouse negative water extract and ethyl acetate extract increase OPN-induced FAK expression was inhibited.8. The mouse negative ethyl acetate extract was purified by FAK pathwaythrough the FAK and ILK two-way, and the pillbugs water extract to inhibitOPN-induced VSMC proliferation and migration, thereby reducing vascularinflammatory, for the treatment of related heart vascular disease provideexperimental basis.