| Backgroud:Radix Ophiopogonis is one of the two main parts of Shenmai Injection, which has been used for cardio-cerebrovascular disease, and herb-drug interactions may occur when combined with those chemical drugs commonly used for cardio-cerebrovascular diseases. Our previous studies indicate that ophiopogonin D, one of the principal components in Radix Ophiopogonis, is the substrate of OATP1B1, and rosuvastatin and rifampicin, inhibitors against OATP1B1, are able to decrease the uptake of ophiopogonin D in a competitive way. However, in rat primary hepatocytes, ophiopogonin D increases the uptake of rosuvastatin when at low concentration, reduces that when at high concentration. Therefore, it is necessary to further investigate the effects of the major active components, including Methylophiopogonanone A, Methylophiopogonanone B, ophiopogonin D and ophiopogonin D′, on functions of OATP1B1, and another important hepatic transporter OATP1B3 to provide theoretical and experimental basis for their clinical usages of Radix Ophiopogonis and its active components. Objective:The purpose is to investigate influence of four of the main active components in Radix Ophiopogonis, including Methylophiopogonanone A, Methylophiopogonanone B, ophiopogonin D and ophiopogonin D′, on the activities of both OATP1B1 and OATP1B3 using HEK293 T cells models overexpressing OATP1B1 and OATP1B3. Furthermore, the correlation between these four active components and OATP1B1 and OATP1B3 was also assessed for the explaination about their effect mechanism on functions of OATP1B1 and OATP1B3. Methods:Firstly, we established LC-MS and LC-MS/MS methods for RSV, ATV, TMST and MA, MB, OPD, OPD′, respectively, to determine those in cell sapmles. Then, we explored the respective impacts of serial times and concentrations on the uptakes of RSV and ATV mediated by OATP1B1 and those of RSV and TMST mediated by OATP1B3 to obtain the the optimum conditions of the uptake of RSV and ATV in OATP1B1-HEK293 T cells and RSV and TMST in OATP1B3-HEK293 T cells, which would be employed in the following interaction parts of experiment. Furthermore, we examined the changes in uptakes of RSV or ATV in OATP1B1-HEK293 T cells when simultaneously adding serial concentrations of MA, MB, OPD or OPD′ into the cells, and calculated the respective parameters EC50 or IC50 comparing with the control groups. Likewise, we examined the changes in uptakes of RSV or TMST in OATP1B3-HEK293 T cells when simultaneously adding serial concentrations of MA, MB, OPD or OPD′ into the cells, and calculated the respective parameters EC50 or IC50 comparing with the control groups. Finally, to assess the correlations between MA, MB, OPD, OPD′ and OATP1B1 or OATP1B3, we investigated the time-dependent and concentration-dependent influences on the uptakes of those four in OATP1B1-HEK293 T and OATP1B3-HEK293 T cells. Results:1. The specificity, accuracy and precision of LC-MS analysis methods for RSV, ATV and TMST and LC-MS/MS methods for MA, MB, OPD and OPD′ satisfy the relevant requirements and can be well used to quantitatively analyze the biological samples.2. The uptake of RSV in OATP1B1- HEK293 T cells is saturated at 20 min, and calculated kinetic parameters, Km and Vmax, of RSV are 23.73μM and 14.28 pmol/mg protein·min-1, respectively; Similarly, the uptake of ATV in OATP1B1- HEK293 T cells is saturated at 10 min,and the parameters, Km and Vmax, of ATV are 15.78μM and 132.5 pmol/mg protein·min-1, respectively. Hence, in the following interaction parts, the concentrations of RSV and ATV, as substrates of OATP1B1, are set to 25μM and 10μM, respectively, and the times are set to 20 min and 10 min, respectively.3. The uptake of RSV in OATP1B3- HEK293 T cells is saturated at 20 min, and calculated kinetic parameters, Km and Vmax, of RSV are 28.10μM and 9.384 pmol/mg protein·min-1, respectively, Similarly, the uptake of TMST in OATP1B1- HEK293 T cells is saturated at 10 min, and the parameters, Km and Vmax, of TMST are 6.406μM and 38.80 pmol/mg protein·min-1, respectively. Hence, in the following interaction parts, the concentrations of RSV and TMST, as substrates of OATP1B3, are set to 25μM and 5μM, respectively, and the times are set to 20 min and 10 min, respectively.4. The uptakes of RSV in OATP1B1-HEK293 T cells are facilitated by MA and OPD′ by different degrees, and EC50 are calculated via the software Graphpad Prisms, they are 3.353 and 3.863μM, respectively. There are no significant differences in the uptake of RSV in OATP1B1-HEK293 T cells between the control and experiment groups when test drug is MB or OPD; However, the uptakes of ATV in OATP1B1-HEK293 T cells are increased by MA, MB, OPD and OPD′ by varying degrees, and their EC50 are 4.470, 15.52, 11.34 and 1.25μM, respectively.5. MA, MB and OPD have no sensible effects on the uptake of RSV and TMST in OATP1B3-HEK293 T cells, while OPD′ exhibits some certain promotion effects on the uptake of RSV, and its EC50 is 16.63μM. Surprisingly, OPD′ exerts substantial inhibition effects on the uptake of TMST in OATP1B3-HEK293 T cells, and its IC50 is 3.291μM.6. MA, OPD and OPD′ may be potential substrates of OATP1B1, and Km, calculated via software Graphpad Prism, are 26.09, 22.99 and 22.89μM, respectively, and calculated Vmax are 139.2, 12.28 and 187.8 pmol/mg protein·min-1, respectively; OPD and OPD′ may be potential substrates of OATP1B3, and similarily, the calculated Km are 17.37 and 11.27μM, respectively, and Vmax are 6.583 and 146.5 pmol/mg protein·min-1, respectively. Conclusions:1. Among these four components, MA and OPD′ increase the uptake of RSV in OATP1B1-HEK293 T cells to different extents. On the other hand, the uptake of ATV in OATP1B1-HEK293 T cells could be raised to varying extents by MA, MB, OPD and OPD′, respectively. Hence, the influences of MA, MB, OPD and OPD′ on transport activities of OATP1B1 are substrate-selective.2. MA, MB, and OPD show no considerable impacts on the uptakes of RSV and TMST in OATP1B3-HEK293 T cells, whereas the uptake of RSV mediated by OATP1B1 could be stimulated to some certain degrees by OPD′, by contrast, that of TMST can be obviously inhibited by OPD′, thus the effects on activities of OATP1B3 are also substrate-selective.3. MA may be a potential substrate of OATP1B1, and OPD probably is a overlapped substrate of OATP1B1 and OATP1B3 as well as OPD′, OATP1B1 or OATP1B3 does not participate in the uptake of MB. |
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