Effect Of Sulforaphane On Lipid Droplets Formation And Regulatory Metabolism Of Mitochondria-Associated Membrane In Liver

Non-alcoholic fatty liver disease(NAFLD)is a disorder of lipid metabolism characterized by excessive deposition of lipid droplets(LDs)in liver,which could directly lead to nonalcoholic steatohepatitis,cirrhosis and finally develop into liver cancer.Sulforaphane(SFN),a phytochemical of isothiocyanates(ITCs),is rich in cruciferous vegetables and has a variety of biological functions.Previous studies found that SFN can improve metabolic diseases caused by disorders of glucose and lipid metabolism such as diabetes,obesity and alcoholic liver injury.However,whether SFN could improve NAFLD caused by high fat intake and the possible mechanism have not been reported.In this paper,a high-fat diet(HFD)was used to establish animal model of NAFLD.Fatty acid(FA)-induced HHL-5 cell was used as an in vitro model and SFN was used as an intervention factor.Mitochondria-associated membrane(MAM)was the main research core to investigate the effects of SFN on the formation of LDs and the molecular mechanism of improving NAFLD.HFD was fed to rats for 10 weeks and HHL-5 hepatocytes were treated with 250?mol/L FA for 5 d to successfully established hepatocyte steatosis model both in vivo and in vitro.Rats were oral gavage of SFN(5,10,20 mg/kg)for 10 weeks and HHL-5 cells were treated SFN(1,5,10,20 ?mol/L).SFN reduced the content of triglyceride(TG)and total cholesterol(TC)in serum and liver of rats and HHL-5 cells.Pathological observation by HE and oil red O staining found that SFN significantly improve liver pathological damage and reduce the number and average area of LDs.SFN could inhibit the expression of diacylgycerol acyltransferase 2(DGAT2)and acyl coenzyme A:cholesterol acyltransferasel(ACAT1),key enzymes to synthesize TG and TC,by western blot method.Real-time PCR,immunohistochemistry and western blot were used to detect the structure proteins of LDs surface.SFN inhibited the expression of PLIN2 and PLIN5 both on mRNA and protein levels in liver.After 10 d of FA induction,the expression of PLIN2 and PLIN5 increased gradually,and SFN down-regulated PLIN2/PLIN5 from d 3 to d 10.SFN inhibited PPAR?,transcriptor of PLIN2/5.TG content increased and PLIN2/PLIN5 up-regulated after treatment by PPAR? agonist Tro.SFN no longer inhibited PLIN2/PLIN5 after PPAR? stimulation and overexpression.These results suggest that SFN not only reduces the content of LDs lipid core,but also inhibits the expression of LDs peripheral proteins PLIN2 and PLIN5 by suppressing PPARy.FA is the substrate for the synthesis of LDs lipid core.Real-time PCR and Western blot analysis of FA synthesis related enzymes ACC1,SCD1 and FAS and its transcription factor SREBPlc expression.SFN down-regulated their mRNA and protein expression.Dynamic observation showed that SFN decreased SREBPlc after FA treatment for 24 h.After 3 d of FA treatment,TG content was significantly decreased.Endoplasmic reticulum(ER)is the key organelle for the synthesis of FA.Transmission electron microscopy(TEM)of hepatocytes showed that SFN could significantly repair the physiological morphology of ER and mitochondria(MT)and reduce ER swelling as well as short the circumference of ER.Endoplasmic reticulum stress(ERS)is an important pathway of regulating FA synthesis related enzymes.Western blot analysis found that SFN down-regulated the expression of ERS marker protein-GRP78,XBP1 and PERK both mRNA and protein expression.ERS inhibitor 4-PBA was treated to HHL-5 cells and XBP1 and SREBPlc mRNA expression was inhibited;TG content and formation of LDs was also significantly reduced.It can be seen that SFN was similar to the ERS inhibitor 4-PBA.In conclusion,SFN reduces lipid accumulation by two ERS pathways,XBP1-ACC1/SCD1 and PERK-SREBPlc-FAS.Morphology and function changes of MT and ER would form the structural coupling zone of them after ERS.TEM observation of liver and crude MT revealed that SFN attenuated the expansion of MT and increased the distance between MT and ER.SFN significantly down-regulated PACS2,the linker of MAM,which point outed that SFN had a tendency to promote the disintegration of MAM.SFN could significantly reduce the concentration of Ca2+in cytoplasm and MT,thereby reducing the signaling molecule activity in MAM.Western blot analysis revealed that SFN down-regulated the expression of IP3R,the Ca2+ release channel protein on ER membrane and mitochondrial calcium uniporter(MCU)responsible for Ca2+absorption in MT inner membrane.SFN inhibited DGAT2 and AC AT1,the key enzymes responsible for LDs lipid core synthesis in MAM.High fat intake could seriously impair mitochondrial DNA(mtDNA)synthesis,while SFN increased mtDNA expression,restored mitochondrial membrane potential and reduced ROS production,thereby reducing mitochondrial peroxidative damage.SFN could increase the activity of complex IV(COXIV)in mitochondrial oxidized respiratory chain,promote ATP synthesis,and improve energy metabolism.In conclusion,SFN can inhibit the formation of LDs in hepatocytes induced by high fat.The mechanism is related to inhibiting ERS,down-regulating Ca2+-related proteins in MAM and promoting oxidative phosphorylation of MT.