| Objective:For the menthol as a small intestine absorption enhancer used in clinical lay the foundation,this article is intended to explore the mechanism by which menthol as a small intestine absorption enhancer,and investigate the relationship between menthol and P-glycoprotein,and whether the expression of P-gp was regulated by NF-?B signaling pathway.Methods: 1.Study on the Toxicity of drugs on Caco – 2 cellsDifferent concentration gradient of menthol,P-gp inhibitor,verapamil,NF-?B signaling pathway inhibitor,PDTC(Ammonium pyrrolidinedithiocarbamate),were preparated.And administering Caco-2 cells for toxicity experiments.The OD value was determined by a microplate reader,and The concentration of each drug administered to Caco-2 cells can make Caco-2 cell's survival rate not less than 90%.At the same time,the three groups of high,medium and low concentrations of menthol were set.2.Study on the Efficacy of Drugs on Caco-2 Cells at different points in timeMethol,verapamil,and PDTC,as defined in the previous chapter,were used to study the time-sensitive toxicity of the sensitive drug dose for Caco-2 cells.Each group of drug effects on Caco-2 cells and CCK-8 was added at 5 time points.The OD values of each group were determined.Finally,the survival rate of Caco-2 cells at different time points after administration was observed.3.Effects of menthol on expression of MDR1 and P-gp protein in Caco-2 cellsCaco-2 cells were treated with low concentration menthol group,medium concentration menthol group,high concentration menthol group,verapamil group and PDTC group.At 5 time points(1h,2h,12 h,24h,48h)The m RNA and total protein of each group were extracted,and the expression of MDR1 and P-gp protein was observed by fluorescence quantitative PCR and western blot.4.Effects of menthol on NF-?B signaling pathway in Caco-2 cellsCaco-2 cells were injected into the three concentrations of menthol,verapamil and PDTC respectively.RNA and total protein were extracted at 5 time points(1h,2h,12 h,24h,48h),and quantified by fluorescence,Western blot were used to determine the expression of Ik B? gene and protein.In addition,the nucleus and cytoplasmic protein of Caco-2 cells were detected at 5 time points after drug administration,and the migration of p65 protein in the cytoplasm was observed by western blot.The effects of different concentrations of menthol on NF-?B Signal path.Results: 1.Determination of the concentration of Caco-2 cells in each drugThe CCK-8 drug toxicity test showed that the survival rate of Caco-2 cells was> 90% when the concentration of menthol was not higher than 16 ?M.Therefore,the low,medium and high concentrations of menthol were 2 ?M,8 ?M and 16 ?M,respectively group.The survival rate of Caco-2 cells was> 90% when verapamil and PDTC concentrations were not higher than 25 ?M and 100 ?M,respectively.2.Effects of each group of drugs on aging toxicity of Caco-2 cells.Caco-2 cells were administered with low,medium and high menthol groups,verapamil 25?M and PDTC 100?M respectively.After 5 time points(1h,2h,12 h,24h,48h),the corresponding Caco-2 cell survival rate of not less than 90%.3.Effects of menthol on expression of MDR1 gene and P-gp protein in Caco-2 cellsThe results of fluorescence quantitative PCR showed that the expression of MDR1 was significantly inhibited in the verapamil group and PDTC group at 5 time points(1h,2h,12 h,24h,48h)compared with the control group(P <0.01(P <0.01).The inhibitory effect of the low concentration of menthol group on the first three time points(1h,2h,12h)was obviously,but The inhibitory effect was decreased at the last two time points(24h,48h)(P <0.05).The concentration of menthol in the first three time points on the expression of MDR1 have a certain inhibitory effect(P <0.05),however,the inhibitory effect on MDR1 expression was not obvious at 24 h and 48 h.The effect of high concentration of menthol on MDR1 was not statistically significant compared with the control group.Western blot results showed that the effect of different concentrations of menthol on P-gp expression at different time points was consistent with that of MDR1.4.Effects of menthol on NF-?B signaling pathway in Caco-2 cellsThe results of fluorescence quantitative PCR showed that the expression of Ik B? gene was significantly induced in the low concentration of menthol,verapamil and PDTC groups at the first 3 points(1h,2h,12h)compared with the control group,but the induction effect of Ik B? gene was decreased(P <0.05)in the low concentration of menthol group at the last two time points(24h,48h),while the induction effect of verapamil group and PDTC group was still significant(P <0.01).The induction of Ik B? gene expression in the medium concentration of menthol and high concentration of menthol was not statistically significant at 5 time points.Western blot analysis showed that the expression of Ik B? protein was significantly induced by low concentration of menthol in the first 4 time points(1h,2h,12 h,24h)compared with the control group(P <0.01).When the time was prolonged to 48 h,The induction effect has been weakened(P<0.05).The expression of Ik B? protein was significantly induced in the verapamil group and PDTC group at 5 time points(1h,2h,12 h,24h,48h)(P <0.01).The expression of Ik B? protein was induced by the medium concentration of menthol and the high concentration of menthol in the first three time points(1h,2h,12 h,).The induction of Ik B? protein was not significant at 24 h and 48 h.In addition,compared with the control group,western blot results also proved that low concentration of menthol,verapamil group,PDTC group at 5 time points(1h,2h,12 h,24h,48h)can effectively inhibit p65 The protein migrated from the cytoplasm to the nucleus(P <0.01).While the medium concentration of menthol and high concentration of menthol group which made p65 protein from the cytoplasm to the nucleus of the migration process is not obvious.Conclusions: 1.In the high,medium and low menthol groups,the verapamil group and the PDTC group did not exert toxic effects on Caco-2 cells at 5 time points(1h,2h,12 h,24h,48h).Its survival rate of not less than 90%2.Low concentration of menthol can significantly inhibit the expression of MDR1 and P-gp protein in Caco-2 cells,while the medium concentration of menthol and high concentration of menthol have a certain effect on the expression of MDR1 and P-gp protein.3.Low concentration of menthol can significantly induce the expression of Ik B? gene and protein.The medium concentration of menthol and high concentration of menthol can inhibit the expression of Ik B? gene and protein in different degree.4.Low concentration of menthol can inhibit the migration of p65 protein from cytoplasm to cytoplasm,while medium concentration of menthol and high concentration of menthol can induce p65 protein migration from cytoplasm to nucleus.5.Low concentration of menthol may be through the NF-?B signaling pathway to inhibit the expression of P-gp.6.Low concentration of menthol is expected to be an effective absorption enhancer for low bioavailability drugs in the small intestine. |
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