Effects Of Pterostilbene On Lipid Accumulation And Oxidative Stress Of NAFLD

NAFLD(non-alcoholic fatty liver disease),is a kind of liver disease caused by non-alcohol and other liver damage or extreme lipid deposition,can lead to hepatic steatosis.It is now believed that oxidative stress and autophagy are involved in the development of various metabolic diseases,including NAFLD.Nuclear transcription factor-2p45(NF-E2)-related factor-2(Nrf2),plays an important role in the anti-oxidative stress system,and it transfers into nucleus which could resist oxidative stress injury.It is often used as a potential therapeutic target for liver diseases.Autophagy is the body's protective response,can phagocytize the body's metabolic waste and the accumulated toxic endogenous substances.Many studies have shown that natural small molecule compounds could activate Nrf2,which relieves pathological changes induced by disease.Pterostilbene(PTE)is a kind of active compounds extracted from blueberry,grape and palmwood.It has many biological effects,such as anti-inflammation,anti-tumor and anti-cognitive disorder.Therefore,it is important to explore whether PTE could protect NAFLD through Nrf2 and the molecular mechanism involved.In this study,free fatty acids,oleic acid(OA)and palmitic acid(PA),were used to induce HepG2 cells to establish lipid accumulation model in vitro.Molecular biological technologies,including CCK-8,oil red O staining,Western blot,immunofluorescence,mRFP-GFP-LC3 adenovirus transfection,were used to investigate the role of PTE in oxidative stress and autophagy in cells.The results showed that PTE(12.5 and 25 ?M)had no cytotoxicity on HepG2 cells.There was plenty of lipid accumulation in HepG2 cells induced by OA and PA,and PTE pretreatment effectively reduced the lipid content in HepG2 cells.PTE significantly enhanced the expression of Nrf2,PPAR? and HO-1,and promoted Nrf2 translation from cytoplasm to nucleus.PTE enhanced AMPK activity,inhibited mTORC1,S6,S6K and SREBP-1C,which plays a key role in regulating lipid synthesis.Furthermore,PTE activated PI3K,proteins in ATG family and promoted the transformation of LC3I to LC3II which suggested the activation of autophagy.However,PTE activation of AMPK was significantly weakened in Nrf2-/-cells.And the inhibition of mTORC,SREBP-1C and the promotion of PI3K were also eliminated after Nrf2 knockout.p62 was activated,and the regulation of lipid accumulation and autophagy was lost in Nrf2-/-cells.After the application of 3-MA,PTE had no regulatory effect on PI3K and SREBP-1C.Application of BAF A1,an autophagy inhibitor,made PTE fail to regulate ATG7,ATG16L1 and the transformation between LC31 and II,but promoted the expression of p62,which could accelerate the degradation of autophagy lysosomes.In vivo study,PTE can effectively reduce the levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein(LDL)and very low-density lipoprotein(VLDL)and increase the content of high-density lipoprotein in WT mice induced by tyloxapol.PTE decreased the content of reactive oxygen species(ROS),myeloperoxidase(MPO),and increase the content of superoxide dismutase(SOD)to protect the mice from oxidative stress damage.Through reducing the levels of matrix metalloproteinase(MMP-9)and vascular cell adhesion molecule-1(VCAM-1),PTE reduced the adhesion of blood to vascular wall.PTE reduced the accumulation of lipid and inflammation in mice hepatocytes though promoting the expression of Nrf2 in nucleus.It also can promote the phosphorylation of AMPK,ACC and AKT to facilitate the oxidation of fatty acids.PTE inhibited the activity of P-mTORC1,P-S6K,P-S6,SREBP-lc and enhanced the activation of ATG to promote autophagy.In Nrf2-/-mice,the regulatory effect of PTE on HDL and VLDL,which is the carrier of triglyceride transportation,was blocked.And the levels of lipid in blood of each group were higher than these in WT mice.The inhibitory effect on MPO and ROS and the evaluating effect on SOD of PTE disappeared.Nrf2 knockout also prevented the activation of P-AMPK and P-AKT,and then affected the militating on PI3K and mTORC,but induced the expression of P62 to reduce autophagy,which is consistent with the results in vitro.In conclusion,this study firstly elucidates the role and molecular mechanism of pterostilbene in steatosis through vivo and vitro experiments.In FFA induced HepG2 cells and tyloxapol stimulated C57BL/6 mice,PTE affected AMPK by targeting Nrf2,inhibited mTORC pathway,enhanced ATG protein activity to activate autophagy.It effectively alleviated oxidative stress damage induced by excessive lipid accumulation in hepatocyte,thus promoted the metabolism and decomposition of fatty acids to improve NAFLD.