The Studies Of The Molecular Mechanism Of P-glycoprotein Expression Regulated By Dexamethasone In L02 Cells

Background & ObjectiveGlucocorticoid(GC) is a kind of steroid hormone which is synthesized and secreted by adrenal cortex, widely existing in human body. Synthetic glucocorticoids are widely used in the therapy of diseases at present. In our preliminary study and the research of Japanese scholar, we observed that glucocorticoid can prevent the occurrence of hepatitis B virus(HBV)-related acute liver failure effectively, however, the exact mechanism has not been completely clarified. P-glycoprotein(P-gp), which is coded by multi-drug resistance gene 1(MDR1), widely exists in human blood brain barrier, blood placental barrier, liver cells, intestinal epithelial tissue and so on. P-gp acts as multi-resistant transporter on the cell membrane and it can enhance the protective function of liver cells. As we all know, the damage and the protective function of liver cells are the two main keys to determin the survival of liver cells. As the past studies show, glucocorticoids can increase the expression of P-gp among various cells, therefore, we speculate that glucocorticoids may enhance the protective function of liver cells to prevent the occurrence of HBV-ralated acute liver failure by increasing the expression of P-gp. In addition, glucocorticoids can activate phosphatidylinositol 3 kinase(PI3K) /protein kinase B(Akt), nuclear fac tor-κ-gene binding(NF-κB) and pregnane X receptor(PXR) signal pathways, which participate in the regulation of P-gp in plenty of cells. As a consequence, we speculate that glucocorticoids may increase the expression of P-gp via activation of PI3K/Akt, NF-κB and PXR pathways.In this study,we cultured human normal liver cell line L02 cells in vitro to investigate the effects of dexamethasone on P-gp expression in L02 cells, and the roles of PI3K/Akt, NF-κB and PXR pathways in the regulation of P-gp. We analyzed the results by Real time q PCR, Western-blot and RNA interference to address the effects of dexamethasone on P-gp expression in L02 cells and the primary molecular mechanism.Methods1. Human normal liver cell line L02 cells were cultured in vitro. We set up normal controls and treated L02 cells with dexamethasone in different concentrations for 24 h. Then we collected cells of the normal controls and experimental groups to extract total RNA and protein.2. The protein levels of P-gp, Akt and NF-κB of L02 cells in normal controls and experimental groups treated with dexamethasone after 24 h were detected by Western-blot analysis.3. The m RNA levels of MDR1 of L02 cells in normal controls and experimental groups treated with dexamethasone after 24 h were measured by Real time q PCR(SYBR).4. L02 cells were cultured in vitro. We set up normal controls and treated L02 cells with LY294002, PDTC and dexamethasone alone or conbined for 24 h. Then we collected cells of the normal controls and experimental groups to extract total RNA and protein.5. The protein expressions of P-gp, Akt and NF-κB in normal controls and experimental groups treated with LY294002, PDTC and dexamethasone alone or conbined after 24 h were analyzed by Western blotting.6. We detected the m RNA expressions of MDR1 in normal controls and experimental groups treated with LY294002, PDTC and dexamethasone alone or conbined after 24 h by using Real time q PCR(SYBR).7. We cultured L02 cells in vitro and transfected L02 cells with PXR si RNA and control si RNA, then treated cells with dexamethasone for 24 h. We collected cells of the normal controls and experimental groups to extract total RNA and protein.8. The protein levels of P-gp in normal controls, PXR si RNA transfected groups and control si RNA transfected groups after treated with dexamethasone for 24 h were analyzed by Western blotting.9. The m RNA levels of MDR1 of L02 cells in normal controls, PXR si RNA transfected groups and control si RNA transfected groups after treated with dexamethasone for 24 h were measured by Real time q PCR(SYBR).Results1. The levels of P-gp, Akt and NF-κB expressions were significantcly increased for 60.8%(P=0.0267), 25.0%(P=0.0358) and 24.1%(P=0.0026) in L02 cells treated with 10μM dexamethasone for 24 h, compared to control L02 cells.2. The levels of MDR1 m RNA expression were significantly increased for 36.3%(P=0.0004) in L02 cells treated with 10μM dexamethasone for 24 h, compared to normal control.3. The levels of P-gp expression were positive correlated with the levels of Akt(pearson correlation coefficient=0.87, P<0.01) and NF-κB(pearson correlation coefficient=0.72, P<0.01) expressions in L02 cells.4. Both the levels of P-gp expression and MDR1 m RNA in LY294002(20μM) treated group were significantly decreased for 33.8%(P=0.0184) and 81.0%(P<0.0001) compared with those in untreated cells; And Both the levels of P-gp expression and MDR1 m RNA in PDTC(30μM) treated group were significantly decreased for 36.3%(P=0.0018) and 69.0%(P<0.0001) compared to normal control.5. The levels of P-gp expression in dexamethasone alone treated group were 163.5±18.4% of untreated cells, and the levels of P-gp expression in group treated with combination of dexamethasone and LY294002 were 130.1±22.0% of untreated cells, the difference between them were significant(P=0.0313). Moreover, The levels of P-gp expression in dexamethasone alone treated group were 150.7±9.8% of untreated cells, and the levels of P-gp expression in group treated with combination of dexamethasone and PDTC were 123.7±3.9% of untreated cells, the difference between them were significant(P=0.0423).6. The levels of MDR1 m RNA in group treated with combination of dexamethasone and LY294002 were significantly decreased for 92.7%(P < 0.0001) compared with dexamethasone alone treated group; While the levels of MDR1 m RNA in group treated with combination of dexamethasone and PDTC were significantly decreased for 59.0%(P<0.0001) compared with dexamethasone alone treated group.7. The levels of P-gp expression in PXR si RNA transfected group were significantly decreased for 58.4%(P=0.0039) conpared with control si RNA transfected group.8. The expressions of MDR1 at m RNA levels were significantly decreased for 86.0%(P<0.0001) compared with control si RNA transfected group.ConclusionIn these studies, we observed that glucocorticoids can increase the levels of MDR1 m RNA and P-gp in human normal liver cell line L02 cells, which indicated that glucocorticoids can enhance the protective function of liver cells by increasing the expression of P-gp.In addition, we observed that glucocorticoids can increase the ex pression of P-gp via activation of PXR, PI3K/Akt and NF-κB signaling.