Direct Inhibitory Effects Of Pioglitazone On Fetuin-a Expression Associated With NAFLD

Objectives To explore the effects and possible mechanisms of pioglitazone(PGZ)on m RNA and protein expression of fetuin-A(Fet A)in nonalcoholic fatty liver disease(NAFLD).Methods1.Hep G2 cells were incubated for different doses of oleic acid(OA).Lipid contents in cells were observed by the use of oil red O.Then we tested the Fet A protein secretion by enzyme linked immunosorbent assay(ELISA)after OA incubated.Besides,we analyzed c DNA sample of different doses of OA-incubated cells for Fet A m RNA expression by real-time polymerase chain reaction(real-time PCR)after OA incubated.2.Hep G2 cells were treated in the absence or presence of 25?M PGZ with or without preincubation with 0.25 m M OA.We analyzed c DNA sample of Hep G2 cells for Fet A m RNA expression by real-time PCR.Besides,Fet A protein secretion were tested by Western Blot after treated with PGZ.The groups are as follows.1 blank control group(Control): Hep G2 cells were incubated without OA for 24 hours,then treated without PGZ.2 negative control group(Control+PGZ): Hep G2 cells were incubated without OA for 24 hours,then treated with 25?M PGZ.3 positive control group(OA): Hep G2 cells were incubated with 0.25 m M OA for 24 hours,then treated without PGZ.4 experimental group(OA + PGZ): Hep G2 cells were incubated with 0.25 m M OA for 24 hours,then treated with 25?M PGZ.3.Hep G2 cells were transfected adenovirus vector for PPAR-? 1 si RNA to suppress the activity of PPAR-?.After transfection,Hep G2 cells were treated in the absence or presence of 25?M PGZ for 48 hours.we tested the PPAR-? and Fet A protein secretion by Western Blot.The experiments were divided into four groups.1 blank control group(Control): normal cells,and treated without PGZ.2 positive control group(Control+PGZ): normal cells,treated with 25?M PGZ.3 negative control group(si RNA): PPAR-?1 suppressed cells,and treated without PGZ.4 experimental group(si RNA+PGZ): PPAR-? 1 suppressed cells,treated with25?M PGZ.Results1.Lipid contents in cells were increased along with the increasement dose of OA.Besides,OA treatment significantly Fet A m RNA expression and protein secretion since dose 0.15 m M compared to control group(0.00 m M OA)(P<0.05).2.Fet A m RNA expression and protein secretion were decreased by PGZ treatment both in Hep G2 cells with or without OA treatment(P<0.05).The inhibition rate of Fet A m RNA expression in Hep G2 cells without or with OA treatment are 89.47% and87.38%,the difference is 2.09%.While the inhibition rate of Fet A protein secretion in Hep G2 cells without or with OA treatment are 51.97% and 67.82%,the difference is15.85%.3.The PPAR-? protein secretion of positive control group was more than blank Control group,while Fet A protein secretion was less.The PPAR-? protein secretion of negative Control group was less than blank Control group,while Fet A protein secretion was more.On the contrary,the PPAR-? protein secretion of experimental group was more than negative Control group,while Fet A protein secretion was less.Conclusions1.The level of lipid contents in cells is positively correlated with the dose of OA.2.The level of Fet A is positively correlated with the liver lipid contents.3.PGZ can directly inhibit Fet A expression in OA-incubated Hep G2 cells.4.The enhancement of Fet A expression induced by PPAR-?1 suppression can be reversed by PGZ.5.PGZ may suppress hepatocellular Fet A expression in NAFLD through the PPAR-? pathway.