| Objective: The purpose of this study was to investigate themechanism of action atractylenolide I(AO-I) reduce paclitaxel-inducedIL-6,VEGF,Survivin expression of TLR4/MyD88+people ovarian cancerskov3cell.Methods:1、After paclitaxel (PTX), lipopolysaccharide (LPS),atractylenolide I (AO-I), AO-I plus paclitaxel, AO-I plus LPS-inducedculture SKOV3cell6hours,detect conformation of TLR4/MD2dimer byflow cytometry.2、After inducing culture human ovarian cancer MyD88+SKOV3cell24h with a fixed concentration atractylenolide I, differentconcentrations of paclitaxel (PTX), a fixed concentration ofatractylenolide I combined different concentrations of paclitaxel,respectively, detecte NF-κB P65and P65phosphorylation of proteinsamples in human ovarian cancer SKOV3expression by Western blot.3、After inducing culture MyD88+SKOV3human ovarian cancer cells andMyD88-A27806h,12h,24h with a fixed concentration atractylenolide I,a fixed concentration of PTX, a fixed concentration ofLPS,atractylenolide I plus paclitaxel, atractylenolide I plus LPSrespectively, detecte NF-κB P65and P65phosphorylation of proteinsamples in human ovarian cancer SKOV3and A2780expression byWestern blot.4、After PTX, LPS, atractylenolide I, AO-I plus paclitaxel,AO-I plus LPS-induced culture SKOV3/A2780cell12h and24h,detecte G2/M phase of the distribution of the cells by flow cytometry.5、Statistical software SPSS17.0was used for statistical analysis.Results:1、After paclitaxel (PTX)（2μmol/L,10μmol/L）, lipopolysaccharide(LPS)(1μg/ml), atractylenolide I (AO-I)(100μmol/L),AO-I pluspaclitaxel,AO-I plus LPS-induced culture SKOV3cell6h, detectconformation of TLR4/MD2dimer by flow cytometry:Compared with thecontrol group,LPS group, the amount of dimer formation increased,statistically significant (P<0.05);AO-I group,the amount of dimerformation reduced, statistically significant (P<0.05); Respectively, afterPTX2μmol/L,10μmol/L induced culture, conformation of dimerincrease, statistically significant (P<0.05). Compared with LPS,AO-Icombined with LPS group, the amount of dimer formation reduced,statistically significant (P <0.05); Compared with single-agent PTX,AO-I, respectively, combined with PTX2μmol/L,10μmol/L, theamount of dimer formation reduced, statistically significant (P <0.05).2、After Fixed concentration of AO-I100μmol/L induced MyD88+SKOV3human ovarian cancer cells24h, NF-κB P65phosphorylationlevels lower than the control group,statistically significant(P<0.05).Different concentrations of PTX0.01μmol/L,0.02μmol/L,0.05μmol/L, NF-κB P65phosphorylation higher level than the controlgroup;compared with the paclitaxel group, Atractylenolide I combineddifferent concentrations of paclitaxel,NF-κB P65phosphorylation level was significantly reduced.3、Fixed concentration of AO-I100μmol/L,PTX0.01μmol/L, LPS1μg/ml, AO-I combined PTX, AO-I combinedLPS, induce MyD88+SKOV3human ovarian cancer cells6h,12h,24h,results show: AO-I group, NF-κB P65phosphorylation levels6h,12h,24h lower than the control group in the corresponding timepoints,statistical significance(P<0.05);LPS group, NF-κB P65phosphorylation levels6h,12h,24h higher than the control group in thecorresponding time points, statistically significant(P<0.05). Comparedwith LPS,AO-I combined LPS group, NF-κB P65phosphorylation levels6h,12h,24h lower than LPS group in the corresponding time points,statistically significant (P<0.05). AO-I combined PTX group, NF-κB P65phosphorylation levels6h,12h,24lower than PTX group in thecorresponding time points,statistically significant (P<0.05). Drug-inducedMyD88-A2780human ovarian cancer cells6h,12h,24h, results show:compared with the control group, AO-I group, LPS group, NF-κB P65phosphorylation levels are no significant difference (P>0.05). PTX group,NF-κB P65phosphorylation levels lower than the control group,statistically significant (P<0.05). Compared with LPS group,AO-Icombined LPS group, NF-κB P65phosphorylation levels were notstatistically significant (P>0.05). Compared with LPS group, AO-Icombined PTX group,NF-κB P65phosphorylation levels were notstatistically significant (P>0.05).4、After PTX (0.01μmol/L), LPS (1 μg/ml), AO-I (100μmol/L), AO-I plus paclitaxel, AO-I plus LPS inducedSKOV3cells12h and24h, detecte G2/M phase of the distribution of thecells by flow cytometry: after drug-induced cuture cells12h and24h, theproportion of G2/M phase of single AO-I group were lower than thecontrol group, the proportion of G2/M phase of single PTX,LPS grouphigher than the control group; Respectively,compared with monotherapyPTX group, LPS group,AO-I plus PTX group, AO-I plus LPS group, theproportions of G2/M phase were lower; Compared with the effect of12hand24h, the proportions of G2/M phase of AO-I group in24h lowerthan12h. The proportions of G2/M phase of PTX group in24h increaserthan12h. The proportions of G2/M phase of LPS group in24h increaserthan12h. The proportions of G2/M phase of AO-I combined LPS groupin24h lower than12h.The proportions of G2/M phase of AO-I combinedPTX group in24h lower than12h. Suggest that AO-I play a role ofblocking PTX/LPS-induced cells into mitosis in SKOV3cells. Afterdrug-induced culture A2780cells12h and24h,the distribution of G2/Mphase display: no significant differences in the proportion of G2/M phasedistribution between different experimental group (P>0.05); Nosignificant difference in the same time between different experimentalgroups (P>0.05).Conclusion:1、After paclitaxel (PTX)2μmol/L,10μmol/L induced culture SKOV3cells6h, can detecte conformation ofTLR4/MD2dimers; AO-I100μmol/L combination with paclitaxel group,AO-I can reduced paclitaxel-induced conformation of TLR4/MD2dimers.2、 AO-I can reduce phosphorylation of NF-κB P65inTLR4/MyD88+SKOV3human ovarian cancer cells, AO-I plus paclitaxelgroup, AO-I can reduce paclitaxel-induced phosphorylation of NF-κBP65in TLR4/MyD88+SKOV3human ovarian cancer cells.However,AO-I can not lower phosphorylation of NF-κB P65in MyD88-A2780ovarian cancer cell.3、AO-I can block MyD88+SKOV3human ovariancancer cells into cell divion, strengthen the prevention mitosis of Taxolfor ovarian cancer. |
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