Study On The Effect Of Ursolic Acid On The Expression Of MDR1 MRNA And P-gp And Its Mechanism Based On NF-κB Signaling Pathway

Background:Ursolic acid(UA) has a variety of pharmacological activities and vast application prospects, but its very low oral bioavailability greatly limited its usage in clinical practice. It has been confirmed that efflux of P-gp should be the key factor for its low oral bioavailability. However, we were highly puzzled by the result that with the increasing concentration, the effect of UA on P-gp can be changed from inhibition to inducing. Although it had been earlier reported that UA can play a role in anti-inflammatory and antiviral by inhibiting the activity of the Nf-kappa B(NFk B)signaling pathway; whether the effect of UA on efflux of P-gp is inhibition or inducing, whether UA regulates the expression of MDR1 m RNA and P-gp up and down, and finally affect the function of P-gp through the NF-k B signaling pathway?All these doubts have not been reported at home and abroad to date and were worthy us to pay close attention and do in-depth researches.Therefore, this project aims to clarify the regulating mechanism of UA on the expression of P-gp from molecular level based on the NF-k B signaling pathway, and provide experiment and theoretical basis for the transporting research of pentacyclic triterpene compounds.Objectives:This project aims to investigate the influence of UA on the expression of MDR1 m RNA and P-gp systematically by successfully constructing K562/ADR cell mode with stable and high expression of MDR1 m RNA and P-gp; and deeply discuss the molecular mechanism of the effect of UA on the expression of MDR1 m RNA and P-gp based on the site of NF-k B P65 of the NF-k B signaling pathways.Methods:1. With a gradual increasing concentration of adriamycin to induce the resistance of K562/ADR cells, and ultimately to maintain the drug resisitance with 1.0μg/ml adriamycin, cultivate K562/ADR cells according to conventional methods, cultivateK562/ADR cells withdraw adriamycin for 2 weeks before the expreiment, observe the growth status of cells and draw the cells growth curve.2. To test the toxicity of adriamycin, PDTC and UA on K562/ADR cells in vitro by MTS, and choose the condition under which the cell living rate was higher than80% for the subsequent experiments.3. K562/ADR cell mode was incubated with different concentrations of NF-k B inducer adriamycin or NF-k B specific inhibitor PDTC alone or together for 48 h, they were taken as the probe conducting parallel control experiments, the variation of the expression level of MDR1 m RNA and P-gp were detected by RT-PCR and Western blotting, the condition of the expression of MDR1 m RNA and P-gp were estimated in K562/ADR cell mode in order to provide a stable and reliable cell mode for subsequent study of UA.4. K562/ADR cell mode was incubated with different concentrations of UA along or together with 5μM adriamycin for 48 h, the variation of the expression level of MDR1 m RNA and P-gp were detected by RT-PCR and Western blotting, and to estimate the effect of UA on the expression of MDR1 m RNA and P-gp in K562/ADR cell mode.5. K562/ADR cell mode was incubated with different concentrations of UA﹑PDTC alone or together with 5μM adriamycin for 48 h based on the NF-k B signaling pathway, the nuclear protein was extracted from K562/ADR cells and the variation of the expression level of the nuclear protein P65 was detected by Western blotting, and to explore the effect of UA on the expression of NF-k B P65 which was the upstream regulating site of the expression of MDR1 m RNA and P-gp deeply.6. Data processing and statistical analysis: each group of data was indicated as mean±standard deviation(mean±SD), SPSS17.0 statistical software was applied for F test, single factor analysis of variance(One-way ANOVA) and related analysis,least significant difference(LSD) method was used to compare the results between groups; the statistical results is P<0.05 as significant difference, the statistical results is P>0.05 as no significant difference.Results:1. K562/ADR cells were incubated with 0.5~15μM NF-k B inducer adriamycinfor 48 h, the expression of MDR1 m RNA was up-regulated by 14.26%、31.58%、39.32%、46.9% and 62.37% respectively; the expression of P-gp was up-regulated by12.41% ﹑ 22.63% ﹑ 34.31% ﹑ 43.8% ﹑ 57.66% respectively. The expression of MDR1 m RNA and P-gp could be up-regulated by 0.5~15μM adriamycin, and there had statistically significant differences between them(P<0.05). With the increasing concentration, the inducing effect of adriamycin on the expression of MDR1 m RNA and P-gp were also increased within the scope of 0.5~15μM(R2=0.8110;R2=0.8901).2. K562/ADR cells were incubated with 5~100μM NF-k B specific inhibitor PDTC for 48 h, the expression of MDR1 m RNA was down-regulated by 16.81%、32.74% 、 43.36% ﹑ 54.87% and 66.37% respectively; the expression of P-gp was down-regulated by 29.02%﹑45.85%﹑64.51%﹑74.61%﹑82.12% respectively. The expression of MDR1 m RNA and P-gp could be down-regulated by 5~100μM PDTC,and there had statistically significant differences between them(P<0.05). With the increasing concentration, the inhibiting effect of PDTC on the expression of MDR1 m RNA and P-gp were also increased within the scope of 5~100μM(R2=0.7708;R2=0.6583).3. K562/ADR cells were incubated with 5~50μM NF-k B specific inhibitor PDTC together with 5μM adriamycin for 48 h, the expression of MDR1 m RNA could be down-regulated by 25μM PDTC and 50μM PDTC by 26.60% 、 48.94%respectively and the expression of P-gp by 35.73% ﹑ 35.73% respectively. The expression of MDR1 m RNA and P-gp which were induced by adriamycin could obviously be down-regulated by 25μM PDTC and 50μM PDTC and there had statistically significant differences between them(P<0.05).4. Compared with the blank group, K562/ADR cells were incubated with5~50μM UA for 48 h, the expression of MDR1 m RNA was down-regulated by 9.04%﹑ 15.58% 、 28.75% and 44.21% respectively; the expression of P-gp was down-regulated by 23.36% 、 37.23% 、 43.07% and 64.23% respectively. The expression of MDR1 m RNA could be down-regulated by 10~50μM UA, the expression of P-gp could be down-regulated by 5~50μM UA and there had statistically significant differences between them(P < 0.05). With the increasing concentration, the inhibiting effect of UA on the expression of MDR1 m RNA andP-gp were also increased within the scope of 5~50μM UA(R2=0.9636;R2=0.8310).5. K562/ADR cells were incubated with 5~50μM UA together with 5μM adriamycin for 48 h, the expression of MDR1 m RNA could be down-regulated by10μM UA﹑25μM UA and 50μM UA by 24.06%﹑28.72% and 34.53% respectively;the expression of P-gp by 20.96%﹑24.36%﹑31.2% respectively. The expression of MDR1 m RNA and P-gp which were induced by adriamycin could obviously be down-regulated by 5~50μM UA and there had statistically significant differences between them(P<0.05).6. Studies which aim at the regulation of MDR1 m RNA and P-gp had shown that K562/ADR cells were incubated with 5~50μM UA for 48 h, the expression of their upstream regulatory nuclear protein P65 was down-regulated by 13.73%﹑28.92%﹑44.07%﹑56.77% respectively; the expression of P65 was down-regulated by 3.66%﹑39.8%﹑53.76%﹑65.59%﹑ 80.65% respectively within the scope of specific inhibitor PDTC 5~100μM. With the increasing concentration, the inhibiting effect of UA and PDTC on the expression of P65 were also increased(R2=0.8924,R2=0.7548).7. K562/ADR cells were incubated with 5~50μM UA or PDTC together with5μM adriamycin for 48 h, the expression of nuclear protein could be down-regulated by 10μM UA﹑25μM UA and 50μM UA by 32.62%﹑36.9%﹑45.45% respectively;the expression of nuclear protein could be down-regulated by 25μM PDTC and 50μM PDTC by 42.86%﹑56.98% respectively. The expression of P65 which were induced by adriamycin could obviously be down-regulated by UA and PDTC within the same scope of 5~50μM.Conclusions:1. The expression of MDR1 m RNA and P-gp were stable﹑specific and high in K562/ADR cell mode constructed by this research, could provide a suitable and reliable cell mode for studying the effect of UA on the expression of MDR1 m RNA and P-gp based on the site of NF-k B P65.2. The expression of MDR1 m RNA and P-gp in K562/ADR cell mode could be obviously inhibited by UA from the level of gene and protein, the effect of UA on the expression of MDR1 m RNA and P-gp was similar to PDTC which was the specific inhibitor of the activation of the NF-k B pathway, and both of them could competewith the role of induction of inducer adriamycin, these studies had suggested that the mechanism of its inhibition effect was possible that UA could inhibit the activity of the NF-k B signaling pathway.3. The expression of nuclear protein P65 which was the upstream regulatory site of MDR1 m RNA and P-gp in K562/ADR cell mode could be significantly inhibited by UA, the inhibition effect of UA on the expression of P65 was similar to PDTC which was the specific inhibitor of the site of NF-k B nuclear protein P65 although together with the inducer adriamycin, these studies had indicated that the inhibition effect of UA on the expression of MDR1 m RNA and P-gp possibly by inhibiting the experssion of NF-k B signaling pathway P65, reducing the nuclear transfer of NF-k B,thus inhibiting the activity of NF-k B and interfering the transcription of MDR1 gene,eventually leading to the down-regulate of the amount of the expression of MDR1 m RNA and P-gp.