ω-3 Pufas Alleviate Stress-induced Liver Injury Through GPR120 Mediated Pathway

Non-alcoholic fatty liver disease（NAFLD）is a widespread chronic metabolic disease characterized by ectopic fat storage and steatosis.A large number of studies have pointed out that chronic stress is an important inducer of NAFLD,which can promote peripheral lipolysis or insulin resistance as well as impair liver lipid metabolism,thus leading to oxidative stress and inflammation which would induce liver cell apoptosis.ω-3 polyunsaturated fatty acids（ω-3 PUFAs）are an important class of fatty acids that rich in fish oil,flaxseed oil et.al.A large number of studies have reported that supplementation ofω-3 PUFAs can effectively improve the symptoms of NAFLD through anti-inflammatory and regulating lipid metabolism.However,the anti-stress effect ofω-3PUFAs in liver is less studied,and its effect on stress-induced NAFLD remains to be further explored.In our study,we investigated the effects ofω-3 PUFAs on lipid deposition induced by stress in vivo and in vitro,and further revealed the protective effect ofω-3 PUFAs on lipid toxicity and the mechanism of alleviating oxidative stress in the liver.1 ALA Protects Glucocorticoid-induced Hepatic Steatosis by Rescuing GPR120We treated AML12 cells with corticosterone（CORT）for 2,12,or 24 h,and found that CORT-treatment inhibited the activating of GPR120 as well as induced its transposition from cell membrane to cytoplasm.The Western-Blotting results also showed that CORT-treatment significantly increased the expression ofβ-arrestin 2（which mediates desensitization of GPR120）and cyto-GPR120,decreased the expression of memb-GPR120,thereby inducing dysfunction of GPR120 and blocking the downstream ERK1/2-signaling pathway（P<0.05）.Whetherα-linolenic acid（ALA）exerts a certain protective effect on abnormal lipid deposition induced by stress is unknown,and the exact underlying mechanism of action remains unclear.Herein we employed a dexamethasone（DEX）-treated mouse model and administered ALA by gavage to investigate whether its addition could relieve the lipid disturbance caused by glucocorticoids,and to ascertain the potential pathway responsible.Furthermore,we used the ALM12 cell line in vitro to clarify the potentially direct action of glucocorticoids on GPR120 function.Animals were allocated to four groups:control,DEX-treated,ALA-treated,and DEX+ALA-treated.Our results demonstrated a significant lipid deposition in the DEX group,while the DEX+ALA group effectively relieved this deposition.The Fatty Acid Binding Protein 1（FABP1）and Hormone-Sensitive Lipase（HSL）in the DEX group were significantly downregulated in liver,while ALA addition increased their expression and significantly decreased the level of the lipid-synthesis-related protein FASN（P<0.05）.The GPR120/ERK-signaling pathway was also inhibited by DEX treatment,and its reactivation with ALA treatment improved the disordered hepatic lipid metabolism.Our findings showed that GCs impaired the function of GPR120 and ALA could ameliorate stress-induced liver steatosis via rescue the GPR120 dysfunction.They also suggest an important role of GPR120 played inω-3 PUFAs against stress-induced hepatic steatosis.2 Effect of DHA on cell damage induced by palmitic acidIn order to further explore the effect ofω-3 PUFAs on liver lipid deposition and liver injury induced by stress,we treated AML12 cells with 300μM PA to induced lipid deposition or 50μM DHA co-treatment for 24h in vitro to detect the level of cell injury.We found that DHA treatment significantly alleviated the decreased cell viability and apoptosis induced by PA treatment,as well as inhibited the activity of caspase-3 enzyme,and reduced excessive ROS production（P<0.05）.Therefore,we also detected the gene and protein expression of lipid peroxidation related enzymes.The results showed that the gene and protein expression of COX2 was significantly increased in the PA group,but relatively decreased in the PA+DHA group（P<0.05）.And the gene expression of ALOX12 and CYP2C70 in DHA group was significantly higher than that in PA group（P<0.05）.Furthermore,we examined the effect of PA and DHA treatment on intracellular oxylipin,and the results showed that PA treatment significantly decreased the expressions ofα-linolenic acid,γ-linolenic acid,EPA,FA22:4,FA20:3,PGD2,9-HODE and 13-HODE.The expression of 18-HETE,20-HETE,11-HDHA,13-HDHA,20-HDHA,19,20-EDP in DHA+PA group was significantly increased.In conclusion,lipotoxicity of PA activates the apoptotic pathway and oxidative stress,and DHA treatment effectively alleviates these damages caused by lipotoxicity of PA.In addition,PA treatment led to a decreasing of unsaturated fatty acids and their metabolites,which may be related to the increasing of ROS and COX2.After DHA treatment,the level of COX2 and ROS was inhibited,and the total amount of unsaturated fatty acids also increased,especiallyω-3 PUFAs.These results suggested that the liver apoptosis induced by DHA on PA may be related to its antioxidant effect.3 DHA alleviates liver oxidative stress by activating the Nrf2 signaling pathwayOxidative stress plays an important role in the occurrence and development of NAFLD,which produce excessive ROS to damage organelles and induced apoptosis.Through the intervention of liver oxidative stress,reducing the production of ROS,could effectively alleviate liver injury.Although many studies have suggested that DHA supplementation can improve the symptoms of NAFLD,the effect of DHA on liver oxidative stress has not been determined,and its specific mechanism still unclear.The purpose of this study was to investigate the antioxidant effect of DHA in liver and elucidate the mechanism.In vitro,AML12 cells were pretreated with DHA or GPR120 agonist TUG891 for 12h,followed by H₂O₂ stimulation for 2h to induce oxidative stress.Then the ROS level and antioxidant related indexes were detected.Our results showed that DHA inhibited H₂O₂ induced apoptosis and ROS production,and activated the antioxidant response,the protein expression of SOD1 and HO-1 significantly increased（P<0.05）;The upstream transcription factor Nrf2was also activated by DHA treatment,and the total protein and nuclear expression of Nrf2was significantly increased（P<0.05）.In addition,the ERK1/2 signaling pathway was inhibited during oxidative stress,but reactivated after DHA treatment.After inhibiting ERK1/2 expression by U0126,the protective effect of DHA on oxidative damage of hepatocytes disappeared,and the activation of Nrf2 was also inhibited.In conclusion,DHA improved oxidative damage in hepatocytes induced by H₂O₂,and reduce ROS production and apoptosis.In this study,we found that DHA activated the ERK1/2 signaling pathway through GPR120,which mediated the nuclear transfer of Nrf2,as well as regulated the expression of its downstream antioxidant enzymes.4 Protective effect and mechanism of DHA on mitochondrial injury induced by oxidative stressAs the main organelle to produce ROS,mitochondrial were vulnerable to ROS attacking during oxidative stress,resulting in their dysfunction.In Chapter 7 of this paper,our dates confirmed that DHA alleviated liver oxidative stress by promoting cellular antioxidant response,but there are few studies on whether DHA can improve liver mitochondrial injury.In this study,the protective effect of DHA on mitochondrial damage induced by oxidative stress and its mechanism were investigated.In vitro,mouse AML12 cell lines,DHA and TUG8191 were pretreated for 12h,followed by H₂O₂ stimulation for 2h to induce oxidative stress in hepatocytes,then detected mitochondrial function and potential regulatory mechanisms.The result showed that DHA restored oxidative stress-impaired autophagic flux and promoted protective autophagy.In addition,PINK/Parkin-mediated mitophagy has been activated by DHA in AML12 cells and alleviated mitochondrial dysfunction.The ERK1/2 signaling pathway was inhibited during oxidative stress,but reactivated by DHA treatment.It was proved that the expression of ERK1/2 was involved in the regulation of mitophagy by using ERK1/2 inhibitor.Subsequently,the protective effect of DHA on liver was further verified in vivo.Eighteen 8-week-old C57BL/6J male mice were randomly divided into 3 groups（n=8）:CON group（CON）;CCl₄ injection group（CCl₄）,and DHA gavage+CCl₄ injection group（DHA+CCl₄）.DHA-treated group intragastric with 50mg/kg DHA which prepared as emulsion with 1%Arabic gum.Intragastric administration lasted for one week.2 hours after the last time of gavage,DHA and CCl₄ group were intraperitoneal injected with 10%CCl4（v/v）in olive oil at 2ml/kg body weight to construct acute liver injury model.Control group were intraperitoneal injected with equal olive oil.After 24h,we sacrificed the animals.The result showed that DHA effectively alleviated liver oxidative damage,intriguingly,autophagy also has been activated and DHA promoted the expression of autophagy-related proteins LC3II and Beclin1 in liver（P<0.05）.In vivo studies also showed that DHA reduced CCl₄-induced the level of AST（P=0.06）,ALT,and LDH(（P<0.05）in plasma and significantly increased the level of SOD and GSH in liver（P<0.05）.In conclusion,our data suggest that DHA activates the PINK1/Parkin-related mitochondrial autophagy pathway through the GPR120/Erk1/2 signaling pathway,thereby improving mitochondrial function and protecting hepatocytes from oxidative damage.