Palmitic Acid Increases Hepatocyte Apoptosis By Mitochondrial Pathway With Growth Hormone Deficiency

Background and aims: Adult growth hormone deficiency is an endocrine disease caused by pathological damage to the anterior pituitary gland or genetic mutation.It is often accompanied with negative changes in body composition ratio,glucolipid metabolism disorders and significantly higher incidence of non-alcohol fatty liver disease than normal.Our previous studied have shown that palmitate(PA)could increase lipid deposition in hepatocyte under growth hormone deficiency situation.In this study,we investigated that the specific molecular mechanisms of PA-induced mitochondrial oxidative stress and apoptosis in cultured hepatocyte.Methods: the sequences of growth hormone receptor(GHR)gene was cloned to downregulated GHR expression.Hepatic L02 cells were treated with different concentrations of PA(75umol,150 umol,or 300umol)for 12 h,24h,and 48 h,and cell viability was measured.L02 cell were transinfected with GHR-lentiviral vector or control vector.Cells were treated with PA(150umol)for 24 hours.Oil red "O" and boron-dipyrromethene(BODIPY)fluorescence staining were used to observe lipid deposition;flow cytometry was used to detect apoptosis and ROS level;JC-1 was used to detect changes in membrane potential to assessed mitochondrial injury.DHE fluorescence to detect Adenosine Triphosphate(ROS)levels in cells and MitoSox fluorescence to detect mitochondrial ROS levels;Western blot was applied to detect mitochondrial-mediated caspase-activition apoptotic pathways in GHR-knockdown hepatocytes and normal hepatocytes.Immunofluorescence observation of co-localized expression of mitotracker and bax protein;Pretreating with targeted mitochondrial antioxidant,mitoTEMPO(100nmol)for 1h,hepatic L02 cells were observed whether it could change PA-induced changes in ROS levels and apoptosis.Results: PA inhibited the survival rate of hepatic L02 cells in a concentration-dependent manner.Treatment with 150 umol PA for 12 h,24h,and 48 h,it was found that the cell survival rate of the GHR-knockdown group was significantly lower than that of the Vector group.And the determination of cellular and mitochondrial ROS,MitoSox fluorescence intensity was significantly higher than the Vector group.The flow cytometry apoptosis results showed that proportion of apoptotic cells in GHR-knockdown group was significantly higher than that of Vector group.Oil red "O" staining and BODIPY fluorescence confirmed that GHR-knockdown group was more prone to lipid deposition.we investigated primary mitochondria-mediated apoptosis signaling pathway.Under the stimulation of PA,western blot showed an increasing expression of bax protein and a decreasing expression of B cell lymphoma-2(Bcl-2)in GHR-knockdown group than that of Vector group.The levels of cytochrome c protein,Cleaved Caspase 9,Cleaved Caspase 3,and polymerase(PARP)were significantly higher than those in the Vector group.Immunofluorescence demonstrated that more Bax protein was translocated from cytoplasm to mitochondria under the stimulation of PA by GHR-knockdown group.Pretreating cells with targeted mitochondrial antioxidant mitoTEMPO significantly ameliorated GHR-knockdown induced cells apoptosis.Conclusion: 1.A hepatic L02 cell line which stably low expressing growth hormone receptor is established.2.In the state of growth hormone deficiency,hepatocyte lipid deposition and hepatocyte apoptosis are aggravated and PA induced hepatic L02 apoptosis through mitochondrial caspase pathway and mitochondrial ROS played an important role in this process.