Glucocorticoids Prevent The Occurrence And Progression Of Liver Failure By Upregulating P-gp Expression

ALF(acute liver failure), SALF(subacute liver failure) or ACLF(acute on chronic liver failure) is serious clinical syndrome, with rapid disease progression and high mortality rate. Unfortunately, except liver transplantation, there are no effective medical therapies that can improve the prognosis of ALF, SALF and ACLF. Glucocorticoids can rapidly inhibit excessive immune response and inflammatory reaction. Furthermore, glucocorticoids also have hepatoprotective effect. Our previous work and Japanese reports have shown that glucocorticoids are effective in improving liver function and prognosis of acute-on-chronic pre-liver failure(pre-ACLF) associated with HBV. Therefore, treating ALF, SALF and ACLF with glucocorticoids is a reasonable treatment decision. However, the effects of glucocorticoids in treating ALF or SALF remained controversial because the mechanism of glucocorticoids treatment in preventing the occurrence and progression of liver failure is still not clear, so it is hard to select the best responders and timing of glucocorticoids. Besides the inhibitory effect on immune response and inflammatory reaction, the improvement of hepatoprotection may be more important for glucocorticoids to prevent the occurrence and progression of liver failure. P-glycoprotein(P-gp) played an important role in cytoprotection. Glucocorticoids can increase the expression of P-gp in placenta, blood brain barrier and other tissues. We therefore hypothesize that glucocorticoids can increase the expression of P-gp in human hepatocytes and improve hepatoprotection through P-gp pathway, thereby prevent the occurrence and progression of liver failure.Methods:1. A retrospective analysis was performed based on 73 patients with ALF and 165 patients with SALF hospitalized in our department from 2000-2012. We evaluated the efficacy of glucocorticoids in improving spontaneous survival(SS) of ALF and SALF. The impact of serum ALT levels, MELD score, coma grade and the time interval from symptom appearance to initiation of glucocorticoids use on the therapeutic effect of glucocorticoids were all investigated. Then we tried to determine the best responders and timing of glucocorticoids.2. The levels of P-gp were observed in liver biopsies from As Cs(chronic asymptomatic carriers), patients with mild chronic hepatitis B, patients with pre-ALF-HBV(Acute pre-liver failure associated HBV) and patients with HBV-associated liver failure using immunohistochemistry. We analyzed the relationship between P-gp levels and clinical types of chronic HBV infection and try to thereby clarify if there is hepatoprotection in P-gp and the role of P-gp in the occurrence and progression of liver failure.3. We compared the P-gp levels in hepatic tissues of pre-ALF-HBV patients before and after the treatment of glucocorticoids. The relationship between P-gp levels and ALT, AST, TBil, Alb, PTA levels were also analyzed.4. The L-02 normal human liver cell was cultured in vitro. Efficacies of dexamethasone on P-glycoprotein expression and TRAIL-induced apoptosis were both determined. Then we analyze the impact of Tariquidar, a third-generation P-gp inhibitor, on the anti-apoptotic action of dexamethasone, to validate that dexamethasone can protect L-02 cell from apoptosis by upregulating P-gp expression.The major results as follows:1. Glucocorticoids improved SS in patients with liver failure(38.2% versus 20.2%, P=0.011).2. A significantly improved SS associated with glucocorticoids use was observed among patients who had ALT ≥303ULN(upper limit of normal) and coma grade <4 and MELD <35(65.0% versus 17.4%, P=0.002).3. SS associated with glucocorticoids use was significantly higher for patients with length of illness ≤2 weeks compared to patients with illness lasting>2 weeks(51.4% versus 15.0%, P=0.010).4. The P-gp expression level of patients with pre-ALF-HBV was significantly higher than that of patients with liver failure(P<0.01), As Cs and patients with mild chronic hepatitis B(P<0.01).5. As for 14 patients with pre-ALF-HBV, their P-gp expression level was higher after the treatment of glucocorticoids(P<0.01).6. The serum ALT leverl of pre-ALF-HBV patients during glucocorticoids treatment was significantly negatively correlated with the P-gp expression level(rs=-0.569, P<0.05), the serum AST leverl of pre-ALF-HBV patients during glucocorticoids treatment was significantly negatively correlated with the P-gp expression level(rs=-0.557, P<0.05), the serum TBil leverl of pre-ALF-HBV patients during glucocorticoids treatment was significantly negatively correlated with the P-gp expression level(rs=-0.565, P<0.05), the serum PTA leverl of pre-ALF-HBV patients during glucocorticoids treatment was significantly positively correlated with the P-gp expression level(rs=775, P<0.05).7. The results of immunocytochemistry, western blot analysis, flow cytometry, and q RT-PCR all verified that P-gp is existed in L-02 cell line.8. Dexamethasone upregulated the m RNA and protein levels of P-gp in a dose- and time-dependent manner.9. Dexamethasone protected human L-02 liver cell line from TRAIL-induced apoptosis. TUNEL assay showed that treatment with 10 μM dexamethasone for 48 h resulted in the lowest observed levels of apoptosis in L-02 cells(p<0.05).10. Tariquidar, a third-generation P-gp inhibitor, reduced the anti-apoptotic effects of dexamethasone in L-02 cell. When cultured with tariquidar for 50 n M, 24 h, the apoptosis level in L-02 cell increased dramatically compared to the group treated with only dexamethasone and TRAIL(p<0.05).Conclusions:1. Glucocorticoids improve the prognosis of patients with ALF and SALF. The best responders to glucocorticoids are patients with a relatively less severe liver failure and a potentially risk of rapid disease progression. The best timing of intervention with glucocorticoids is within 2 weeks of symptom appearance.2. Compensatory increase of P-gp can be observed in hepatic tissue of patients with more severe hepatic injury, indicating that P-gp has hepatoprotective action and P-gp play an important role in preventing cell death of hepatocyte and in preventing the occurrence and progression of liver failure.3. Glucocorticoids treatment can prevent the disease progression of patients with pre-ALF-HBV and this effect may associate with the increase of P-gp levels in these patients.4. Dexamethasone protects L-02 cell from TRAIL-induced apoptosis by upregulating P-gp expression.