The Protective Effect Of L-carnitine On Hepatocytes Damaged By Hydrogen Peroxide In Human HL7702

Objective:L-carnitine?L-carnitine,LC?is a vitamin-like essential nutrient.It is abundant in animal foods.Clinical studys found that LC was insufficient in liver disease patients and exogenous LC complement has adjuvant therapy for variety of liver disease.The experimental study also demonstrated that LC had protective effects against liver injury induced by various factors.Currently,the molecular mechanism in the hepatoprotective effect of LC has not been elucidated.In this study,hydrogen peroxide?H₂O₂?-induced cell damage model of human HL7702 hepatocytes will be established to investigate the molecular mechanism of hepatoprotective effect of L-carnitine?LC?through antioxidation and cell signal transduction molecules?Nrf₂,MAPK,PI3K/Akt,PPAR-?,AMPK,and GSK-3??.Methods:1.Effect of LC on cell viability and antioxidant capacity in H₂O₂-treated HL7702 cells: The experimental design is as follows: control group,H₂O₂ model group and LC group?0.1,0.5,1.0mmol/L?.The cell viability were determined by MTT and lactate dehydrogenase?LDH?methods;Superoxide dismutase?SOD?activity,catalase?CAT?activity and malondialdehyde?MDA?content were assayed by enzyme chemistry methods.Intracellular reactive oxygen species?ROS?levels were detected by flow cytometry using 2,7-Dichlorofluorescin diacetate?DCFH-DA?.2.Modulation of LC on the nuclear factor erythroid 2-related factor 2?Nrf₂?/heme oxygenase 1?HO-1?signaling pathway: The change of Nrf₂ and HO-1 protein expressions upon H₂O₂ exposure in HL7702 cells were detected by Western blot;The translocation of Nrf₂ was observed by immunofluorescence staining;The Nrf₂-DNA binding activity was evaluated by Electrophoretic Mobility Shift Assay?EMSA?;The relationship between Nrf₂ and HO-1 and the roles of Nrf₂ in LC's protection were analyzed by Nrf₂ si RNA.3.Modulation of LC on mitogen-activated protein kinase?MAPK?and phosphatidylinositol3-kinase/protein kinase B?PI3K/Akt?signaling pathway: The change of MAPK and PI3K/Akt pathway in H₂O₂-treated HL7702 cells and the effects of LC on them were determined by Western blot.The roles of JNK,ERK and p38 MAPK in H₂O₂-induced damage of HL7702 cells were analyzed by their inhibitors respectively;Using Akt inhibitor IV to evaluate whether Akt is involved in the protective effect of LC and the activation of Nrf₂/HO-1 pathway by LC.4.Effect LC on the expressions of fatty acid metabolism-related and energy metabolism-related genes in H₂O₂-treated HL7702 cells: The change of peroxisome proliferator-activated receptors-??PPAR-??protein expression over time in H₂O₂-treated HL7702 cells were detected by Western blot;The effects of LC on PPAR-?,palmitic acid carnitine transferase 1?CPT1?and fatty acyl coenzyme Aoxidase?ACOX?were investigated in m RNA and protein levels;PPAR-? agonist or inhibitor was used to analyze the roles of PPAR-? in the protective effect of LC and the correlation between PPAR-? and SOD or CAT.The change of AMP-activated protein kinase?AMPK?and glycogen synthase kinase-3??GSK-3??in H₂O₂-treated HL7702 cells and the effects of LC on them were determined by Western blot;Roles of AMPK in the protective effect of LC and the correlation between PI3K/Akt,AMPK and GSK-3? were analyzed by inhibitors.Results1.Treatment of HL7702 cells with 0.3 mmol/L H₂O₂ for 12 h successfully eatablished cell damage model.0.1¹.0 mmol/L LC inhibited H₂O₂-induced cell death and LDH release dose-dependently?P<0.05,P<0.01?.Compared with control group,SOD and CAT protein levels decreased significantly in H₂O₂ group?P<0.01?,SOD and CAT activities decreased by 30.07% and 38.26% respectively,ROS and MDA levels increased to 3.8 times and 1.7 times;LC effectively suppressed H₂O₂-induced decrease of SOD,CAT protein expressions and activities,and suppressed H₂O₂-induced increase of ROS and MDA levels?P<0.05,P<0.01?.2.In the Nrf₂ expression analysis,nuclear Nrf₂ expression increased at 1 h after H₂O₂ exposure and gradually reduced,HO-1 expression gradually increased;LC pre-incubation significantly enhanced H₂O₂-induced Nrf₂ nuclear translocation and the increase of Nrf₂-DNA binding activity and HO-1protein expressions?P<0.05?;After the silence of Nrf₂ expression by Nrf₂ si RNA,the increase of cell viabilities and HO-1 expression induced by LC were inhibited.3.p-JNK,p-ERK and p-p38 MAPK expressions increased rapidly after H₂O₂ exposure,p-Akt expression increased firstly and then decreased;LC effectively inhibited H₂O₂-induced increase of p-JNK and p-p38MAPK?P<0.05,P<0.01?,but had no effect on p-ERK expression;LC not only enhance the increase of p-Akt at 1 h?P<0.01?,but also suppressed its decrease at 12 h?P<0.01?.Inhibitors for JNK,p38 MAPK or Akt can effectively inhibit H₂O₂-induced cell death except ERK inhibitor?P<0.05?;Akt inhibitor also suppressed the activation effect of LC on Nrf₂/HO-1 pathway.4.The study firstly found that PPAR-? protein expression decreased gradually in hepatocytes after H₂O₂ treatment.LC pre-incubation enhanceed the expressions of PPAR-? and its downstream genes CPT1 and ACOX?P<0.05,P<0.01?;PPAR-? agonists effectively inhibited H₂O₂-induced decrease of SOD and CAT activity and cell viabilities;PPAR-? inhibitor blocked the increase of SOD and CAT expression and cell viabilities induced by LC.Upon H₂O₂ exposure,p-AMPK and p-GSK-3?expressions gradually reduced,and LC had inhibitory effect on it?P<0.01?.LC also suppressed the decrease of activities of liver kinase B1?LKB1?and acetyl-Co A carboxylase?ACC?in H₂O₂-treated cells?P<0.05,P<0.01?,which are upstream kinase and downstream substrates of AMPK respectively.AMPK inhibitor blocked the enhancement of cell viabilities and p-GSK-3? expression by LC,but Aktinhibitor had no effects on the increase of p-GSK-3? expression induced by LC.Conclusions1.The protective effects of LC against H₂O₂-induced damage in HL7702 cells attributed to increased SOD,CAT activities and decreased ROS,MDA levels.2.LC protected HL7702 cells from H₂O₂-induced injury through activation of Akt/Nrf₂/HO-1pathway.3.The activation of JNK and p38 MAPK were involved in H₂O₂-induced HL7702 cell damage.The protective effects of LC were associated with the inhibition of JNK and p38MAPK;ERK were not involved in H₂O₂-induced cell damage and the protective effects of LC.4.PPAR-? was involved in H₂O₂-induced HL7702 cell damage.LC increased expressions of PPAR-? and its downstream genes?CPT1 and ACOX?,activated LKB1/AMPK/ACC pathway,and inhibited GSK-3? activity,thereby inhibited HL7702 cell damage induced by H₂O₂.The elevated PPAR-? expression contributed to the increase of SOD and CAT expressions.