Plant Sterol Eater Of α-linolenic Acid Prevents Non-alcoholic Fatty Liver Disease Through Improving Mitochondrial Dysfunction And Oxidative Stress Via AMPK Signaling

Objective:The present study was designed to explore the beneficial mitochondrial effects and anti-oxidative activities of plant sterol ester of α-linolenic acid(PS-ALA)through AMP-activated protein kinase(AMPK)signaling in the treatment of non-alcoholic fatty liver disease(NAFLD).Methods:Part Ⅰ: 50 C57BL/6J mice were randomly divided into five groups of ten animals each,including the control group(Control group,normal diet),high fat and high cholesterol-diet group(HFD group,high fat and high cholesterol-diet),plant sterol group(PS group,HFD+2% PS),α-linolenic acid group(ALA group,HFD+1.3% ALA),and plant sterol ester of α-linolenic acid group(PS-ALA group,HFD+3.3% PS-ALA).After16 weeks,the levels of TG,TC,MDA,GSH in serum and liver of mice were detected by GPO-PAP,COD-PAP,TBA and DTNB,respectively;the deposition of lipid droplets in liver was assessed by oil red O staining;the ultrastructure of hepatocyte mitochondria was observed by transmission electron microscopy;the levels of ROS in the liver were measured by flow cytometry and fluorescence staining;Immunohistochemical staining and Western blot were used to detect the expression levels of AMPK,p-AMPK and key signaling molecules of mitochondrial dynamics(Mfn2,Opa1,Drp1),mitochondrial biogenesis(PGC-1α,Nrf1,Tfam),mitochondrial fatty acid β-oxidation(PPARα,CPT1A),mitochondrial respiratory chain(COXⅠ,COXⅢ)and oxidative stress(Nrf2,HO-1).Part Ⅱ: HepG2 cells were cultured and divided into 5 groups: the control group(normal medium),the model group(OA),PS group(OA+PS),ALA group(OA+ALA)and PS-ALA group(OA+PS-ALA).The deposition of lipid droplets in hepatocytes was observed by oil red O staining;ROS level was detected by DCFH-DA fluorescent probe.HepG2 cells were then cultured and divided into 7 groups: the control group(normal medium),the model group(OA),PS-ALA group(OA+PS-ALA),AMPK inhibitor group(OA+CC),AMPK inhibitor+PS-ALA group(OA+CC+PS-ALA),AMPK activator group(OA+AICAR),AMPK activator + PS-ALA group(OA+AICAR+PS-ALA).Nile red staining was used to observe lipid droplet deposition;ROS level was detected by DCFH-DA probe;the number of mitochondria was assessed by Mito-Tracker Green fluorescent probe;mitochondrial membrane potential was measured with JC-1 probe;the expression levels of AMPK,p-AMPK,and the key signaling molecules of mitochondrial biogenesis(PGC-1α/Nrf1/Tfam),fatty acid β-oxidation(PPARα/CPT1A)and antioxidant(Nrf2/HO-1)were analyzed by immunofluorescence and Western blot analysis.Results:Part Ⅰ: Compared with the Control group,the mice in the HFD group showed a large amount of lipid deposition in the liver tissue of mice;significantly increased levels of TC and TG in liver and serum;obviously abnormal ultrastructure of mitochondria in hepatocytes;increased production of ROS;high levels of MDA and low levels of GSH in serum and liver;decreased protein expression of p-AMPK,PGC-1α,Nrf1,Tfam,PPARα,CPT1 A,Mfn2,Opa1,Drp1,COXⅠ,COXⅢ,Nrf2,HO-1 and enhanced protein expression of CYP2E1 in the liver(P < 0.05).Compared with HFD group,the mice in PS-ALA group exhibited reduced degree of liver steatosis;low level of TC and TG in serum and liver;obviously recovered ultrastructure of mitochondria;reduced ROS production in the liver;high levels of MDA and low levels of GSH in serum and liver;increased protein expression of p-AMPK,PGC1α,Nrf1,Tfam and PPARα,CPT1 A,Mfn2,Opa1,Drp1,COXⅠ,COXⅢ,Nrf2,HO-1 and decreased protein expression of CYP2E1 in the liver(P < 0.05).Part Ⅱ: The results of oil red O staining and ROS detection showed that,compared with the Control group,incubation with OA induced lipid droplet accumulation and excessive ROS production in HepG2 cells.PS-ALA intervention significantly decreased lipid droplets and reduced ROS levels in HepG2 cells,and its effect was superior to ALA and PS intervention alone.The data of AMPK expression level detection showed that the expression of p-AMPK protein was markedly reduced in OA-treated HepG2 cells,meanwhile,the intracellular lipid deposition was significantly increased.In addition,PS-ALA enhanced the expression of p-AMPK in the AIACR+PS-ALA intervention group,but a decrease was observed in the CC group.Concomitantly,the PS-ALA and PS-ALA+AICAR groups manifested less lipid droplet accumulation in HepG2 cells than that of the CC and PS-ALA+CC groups.Mito-Tracker Green and JC-1 results showed that the number of mitochondria and mitochondrial membrane potential in the OA group were significantly less than that in the Control group.PS-ALA intervention not only remarkably increased the mitochondria number,but also significantly increased the mitochondrial membrane potential of HepG2 cells.However,those above beneficial effects of PS-ALA were weakened by using AMPK inhibitor CC.Immunofluorescence and Western blot results showed that the key genes taking charge in mitochondrial biogenesis(PGC-1α/Nrf1/Tfam),mitochondrial fatty acid β-oxidation(PPARα/CPT1A),and oxidant stress(Nrf2/HO-1)were significantly down-regulated in OA group compared with the Control group.PS-ALA intervention remarkably increased the protein expression of the genes mentioned above,whereas this effect was disrupted by AMPK inhibition.Conclusion:The protective effects of PS-ALA on NAFLD appear to be associated with improving mitochondrial function and oxidative stress via activating AMPK signaling and up-regulating the PGC-1α/Nrf1/Tfam,PPARα/CPT1 A and Nrf2/HO-1 signaling pathways.