Study On Mechanism Of Transportation Of Danshensu In Primary Rat Hepatocytes

Background:Danshensu (Salvianic acid A) is a phenolic aromatic acid, isolated from Sal via, one of the most important components in aqueous extract from Salvia, because it is a free acid and not stable, it will be made into sodium salt to enhance its stability and purity. Danshensu has obvious pharmacological activities, mainly for cardiovascular diseases, it can be quickly absorbed in the body, but the elimination rate is slower. Transportation of danshensu is mediated by P-gp with Caco-2 model, danshensu is a substrate of P-gp, at the same time, it also maintains a high concentration in the liver tissue, it is necessary to probe the mechanism of transportation of danshensu in hepatocyte cells.Objective:Rat primary hepatocytes cell model which is simulated the uptake of the liver can be used to study drug transporting. Rifampicin is selected as a probe to study and research drug transportation of danshensu, further to clarify the relationship between Organic anion transporting polypeptide Ib2 and danshensu, in order to clarify the molecular mechanism of danshensu transporting.Methods:1 Establish steady, feasible and reliable separation approach of rat primary hepatocytes and hepatocytes incubation conditions. Using this model to observe the absorptive and transporting process, properties of rat primary hepatocytes cell is built up and made a full evaluation.2 To explore and establish a LC-MS method of detecting biologic samples about hepatocytes cells.3 To inspect the influences of temperature, time and concentration on the uptake transport of Danshensu in hepatocytes incubation conditions.4 The inhibitor Rifampicin is used for considering the uptake of Danshensu transporting in hepatocytes cell. The twelve groups were separated in this experiment part. Between danshensu and Rifampicin were set up three different concentrations. Otherwise, the control group is necessary. Among the different hepatocyte cells groups, to authenticate the uptake of danshensu.5 All data are demonstrated by mean±standard deviation (Mean±SD) using SPSS 12.0 software for data processing, the results is P<0.05 as a significant difference.Results:1 The established LC-MS method of the hepatocytes cells samples has higher sensitivity and specificity, and methods of detection meet the satisfaction of biological samples.2 The study constructed model separation technique of rat hepatocytes, and made sure incubate system in the best condition system, which meets the requirement of the drug transport experiment and settles a solid foundation for the study.3 Inspect the effect of temperature, time and concentration on the uptake transport of Danshensu. The amount of Danshensu uptake into the liver cells is increased almost linearly in 0-20min, but after 20th minute, then growed gradually. Among the concentration of 62.5-1000μM, it is raised within range, the intake of transport increased with the increase of drug concentration, when the concentration is up to 1000μM, it is slowly increasing; Then system temperature is 4℃,25℃and 37℃, which the intake of Danshensu were 26.88±0.53,26.44±1.98,28.97±1.55 nmol·mg-1 protein, the result of statistical analysis is P>0.05, showing that there is no significant difference, therefore, the uptake of Danshensu in the primary hepatocytes is not affected by temperature.4 Inhibitor effect of rifampicin on Danshensu:when the concentration of rifampicin is 0.5μM, the inhibition ratio on three different concentrations of Danshensu (0.1 mM,0.5mM, 1.0mM) is respectively 23.64%,16.61% and 6.69%; then when the concentration of rifampicin is 1.0μM, the inhibition ratio on three different concentrations of Danshensu (0.1 mM,0.5mM, 1.0mM) is respectively 31.62%,35.21% and 7.12%; eventually when the concentration of rifampicin is 2.0μM, the inhibition ratio on three different concentrations of Danshensu (0.1 mM, 0.5mM,1.0mM) is respectively 52.09%,46.45% and 14.48%; With the concentration of rifampicin adding, the inhibition on Danshensu is more and more obvious. Compared with the control group, t test results that the difference was statistically significant.Conclusions:In this subject, studying Danshensu transportation in liver cells systematically internationally can be the innovation of this article. The current study results suggest that Danshensu is transported to the liver with organic anion transporter polypeptide by the way of active transport; Rifampicin may competitively inhibit the transporting of danshensu, in the same time, danshensu has a protective effect on liver cells; Rifampicin, as a probe, the site of organic anion transporting polypeptide is Oatplb2. The results show that rifampin may inhibit the effect of danshensu. And it may be mediated by Oatp1b2, transport mechanism in rat primary hepatocytes model about danshensu is preliminary obtained, Oatplb2 path may be one of the ways for danshensu in liver, danshensu may be actually the substrate of Oatp1b2.