The Mechanism Of Hepatic Lipid Droplets Degradation By Sulforaphane Via Mitochondria And Autophagy

With the the westernization of diet and aggravation of sedentary life style in China,Nonalcoholic fatty liver disease(NAFLD)has become one of the important pulic health problems,which endanger people.There is curren tly no specific drug for treating NAFLD.Improving diet and changing lifestyle are still considered as the prior treatment for NAFLD.Sulforaphane(SFN)is a kind of isothiocyanate containg –N=C=S group,which results in having multiple physical function.Previous studies have confirmed that SFN effectively degraded lipid droplets(LDs)in liver and improved NAFLD.In this paper,the molecular mechanism of lipid droplets(LD)degradation by SFN was explored via targeting fatty acid profiles,mitochondria,macroautophagy and chaperone mediated autophagy.Rats(4 weeks,male)were fed with high fat diet(HFD)for 10 weeks and HHL-5 hepatocytes were treated with free fatty acid(FFA)250 ?mol/L for 4 days to induce hepatocyte steatosis in vitro and in vivo.SFN(20 mg/kg)reduced the content of various lipids in serum and liver of NAFLD rats,improved the tissue morphology of liver,and reduced the LDs content of liver.After treated with SFN(10 ?mol/L)for 48 h,triglyceride(TG)content and PLIN2 protein expression significantly decreased,and m RNA levels of adipose triglyceride lipase(ATGL)and hormone-sensitive triglyceride lipase(HSL)decreased.The content of fatty acids in liver and serum was determined by GC-MS.The results showed that SFN significantly reduced the content of n6 polyunsaturated free fatty acids,reduced ratio of n3/n6.Fatty acid undergoes mainly metabolism in mitochondira.Transmission electron microscopy,flow cytometry,q RT-PCR and Western blot were used to determine mitochondrial function,antioxidant capacity and mitochondrial biogenesis.Results showed that SFN improved hepatocyte antioxidant capacity,represented by a decrease of MDA level and elevated level of GSH,SOD,?detoxification enzymes(HO-1,GST,NQO1 and Trx R)protein,to alleviate the mitochondrial swelling and protect mitochondrial function.SFN increased levels of m RNA and protein of mitochondrial production-related genes(PGC-1?,NRF1 and TFAM)to promote mitochondrial biogenesis,including mitochondrial content,mt DNA copy number and respiratory chain genes(NDUF1,SDHA,COX41 and UCP2)m RNA levels increased.Western blot showed that SFN reduced the protein content of PLIN2 and PLIN3 in liver tissue and HHL-5 cells.Western blot and q RT-PCR presented that SFN increased the m RNA level and protein expression of LAMP-2A to improve the activity of chaperone mediated autophagy.The LDs were purified by gradient density centrifugation.Western blot of purified lipid droplets showed SFN promoted the chaperone mediated autophagy of LDs,which was reflected by reduced protein content of PLIN2 and PLIN3,and increased protein content of HSC70 and ATGL.The confocal microscope experiment showed SFN increased the co-localization of between PLIN2 and LAMP-2A.Lysosomes was purified by gradient density centrifugation and underwent Western blot.The results showed that the protein contents of PLIN2,PLIN3,LC3,HSC70 and LAMP-2A increased in SFN group.confirming that PLIN2 and PLIN3 were degraded in lysosomes.After si RNA interfered Nrf2 protein expression,LAMP-2A protein was reduced and SFN failed to improve the protein content of LAMP-2A.Transmission electron microscopy showed SFN increased autophagosomes.Western blot and q RT-PCR confirmed SFN increased m RNA levels of LC3,ATG4,ULK1,ATG7 and ATG5,and upregulated protein expression of LC3,ATG7 and ATG5 to improve macroautophagy activity.the confocal microscopy exper iment showed that more colocalizations of LC3-LDs,LAMP-1-LDs,ATG7-LDs and ATG5-LDs were found in SFN group.The Western blot for purified LDs by gradient density centrifugation showed more LC3,ATG7 and ATG5 in SFN group.After Nrf2 was silenced,the protein of ATG7 and ATG5 decreased and SFN failed to upregulate ATG7 and ATG5 expression.For HHL-5 hepatocytes preteated with FFA,Nrf2 silence increased TG content,while SFN failed to reduce TG content.In conclusion,SFN induced LDs degradation via impoving mitochondrial function,promoting mitochondrial biosynthesis,and upregulating the lipophapy and chaperone mediated autophagy of LDs.