Effects Of Leptin On The Expression Of HepG2BSEP Protein In Cell Signal Transduction Pathway

Primary intrahepatic stones (PIS) refers to the left andright hepatic duct stones confluence above each branch bile duct. Thepossible mechanism refers nutritional status, biliary infection,biliarystricture,bile metabolism, composition imbalance and other factors, butthe exact pathogenesis is not clear. Studies shown that bile acidconcentration in hepatolithiasis group was lower than the control group,so the bile acid may play an important role in the formation of PIS.Leptin as one of the upstream activating factors of Adenosinemonophosphate-activated protein kinase(AMPK), plays an important rolein energy metabolism and regulation etc. Recent studies have shown thatAMPK regulates the expression of bile salt export pump (BSEP) protein,and BSEP is the important channel that pumps intrahepatic bile acid intothe bile canaliculi, which can control the concentration of biliary bileacid; therefore, The study in effects of leptin on the expression of BSEPprotein in HepG2cells and the mechanism of action, further from themetabolic pathway of the pathogenesis of PIS and may provide newtreatment for PIS. Methods: Leptin as a stimulating factor, the culturedHepG2cells were divided into4groups: The normal control group(group NC: the culture medium not containing leptin),10-8mol/L leptingroup(group CT1, the culture medium containing10-8mol/L leptin), 10-7mol/L leptin group(group CT2, the culture medium containing10-7mol/L leptin),10-6mol/L leptin group(group CT3, the culture mediumcontaining10-6mol/L leptin); The CT1, CT2, CT3groups were culturedfor24H(CT1.1, CT2.1, CT3.1),48H (CT1.2, CT2.2, CT3.3),72H (CT1.3,CT2.3, CT3.3) and detect long leptin receptor (leptin recptor B, LepRb),AMPKa, Phosphorylated adenosine monophosphate activated proteinkinase the a subunit (p-AMPKa), BSEP protein levels with Western-blot,and the content of Alanine aminotransferase(ALT), aspartateaminotransferase(AST), total bile acid,(TBA) and prealbumin(PA) weremeasured in the cell culture supernatant. Comparison of different timeand leptin stimulation, The strongest BSEP expression point as group RB(medium containing10-6mol/L+10umol/L AMPK Compund C), BSEPprotein expression was detected by Western-blot. Results:1. Comparedwith the normal group, the leptin stimulated HepG2cells24H,48H, ALT,AST in the supernatant were increased, and leptin concentrations(10-6mol/L,10-7mol/L) were positively correlated (P<0.05), but nostatistical significance in72H and leptin concentrations of10-8mol/Lcompared with normal group (P>0.05), leptin increased content ofsupernatant TBA and PA(P<0.05);2. Compared with the normal group,different concentrations of leptin intervention HepG2cells24H,10-8mol/L leptin concentration group increased expression of LepRb butthe difference was not statistically significant (P>0.05), with the concentration increased the expression of LepRb protein decreasedgradually (P>0.05);48H,72H measured LepRb protein expression wastime and dose dependent increase gradually; in the72H when theconcentration of leptin was the strongest, compared with the normalgroup and other intervention groups, The expression of LepRb proteinwas statistically significant difference in the strongest (P<0.05).3.24Hand48H of different concentrations of leptin intervention in HepG2cells,the expression of AMPKa had no significant change(P>0.05), But in72H,AMPKa protein expression gradually increased with theconcentration of leptin increased, in leptin concentration(10-6mol/L) wasthe strongest AMPKa protein expression, with statistically significantdifference compared with the NC group (P<0.05);4. The expression ofp-AMPKa is measured in each group was lower than in NC group at24H,decreased with the leptin concentration increased, but without statisticalsignificance compared with the normal group and the difference betweentwo groups (P>0.05); the level of AMPKa phosphorylation was graduallyincreased in dose and time dependent at48H,72H compared with NCgroup, the difference was statistically significant (P<0.05);24H,48H,72H at the same concentration of leptin group of p-AMPKa proteinexpression increased gradually, CT3.3comparison with CT3.1and CT3.2group had significant difference (P<0.05);5. The BSEP proteinexpression was compared with the NC group were increased at24H, and it decreased with the increase of leptin concentration, but withoutstatistical significance with the normal group and the difference betweentwo groups (P>0.05); the expression of BSEP protein increased graduallyafter48H, the difference compared with the NC group CT3.2group and72H groups were statistically significant (P<0.05);24h,48h,72H at thesame concentration of leptin group of BSEP protein expression increasedgradually, CT3.3comparison of CT3.1and CT3.2group had significantdifference (P<0.05).6. CT3group, RB group compared with NC groupat72H, BSEP protein expression were increased (P<0.05), the expressionof BSEP protein in RB group compared with CT3group decreased(P<0.05). Conclusion:1. Leptin can promote the expression of BSEPprotein in HepG2cells by "leptin-AMPK-SRC2-FXR-BSEP" approach；2. Leptin intervention HepG2cell24h can reduce the expression ofLepRb protein and phosphorylation of AMPK, but24h leptin was doseand time dependent and promote the expression and phosphorylation ofAMPKa LepRb protein;3. Leptin intervention HepG2cell72H canincrease AMPKa protein expression.4. Leptin can promote prealbuminand bile acid secretion in HepG2cells, provide a theoretical basis forleptin in pathogenesis and treatment of PIS.