| Objective:To investigate whether SIP specific inhibitor or S2P specific inhibitor or inhibition of S1P and S2P gene expression regulates endogenous SREBP-2 processing to affect the expression of autophagy-associated genes and autophagic flux.Methods:1.Firstly,HL-7702 and HepG2 cells were treated with SIP specific inhibitor PF-429242(1,10,and 30 ?M)or S2P specific inhibitor NFV(5,10,and 20 ?M)for 0,4,8,and 24 h,respectively.Relative mRNA and protein levels of autophagy-related genes,SREBP-2,SIP,and S2P were determined by qRT-PCR and Western blot.Then,HL-7702 and HepG2 cells were transfected with the GFP-mRFP-LC3 double-tagging plasmid for 24 h,followed by PF-429242(30 ?M)or NFV(20 ?M)or/and CQ(50 ?M)for 24 h.Total protein levels of LC3B and GAPDH were analyzed by Western blot,and cell autophagic flux was evaluated by laser confocal scanning microscopy.2.AML-12 cells were transfected with S1P and S2P shRNAs,respectively.Relative mRNA and protein levels of autophagy-related genes,SREBP-2,S1P,and S2P were determined by qRT-PCR and Western blot.Results:1.We found the mRNA levels of autophagy-associated genes were increased in a time-and dose-dependent manner;however,SREBP-2,S1P,and S2P mRNA levels were remained unchanged by PF-429242 or NFV treatment in HL-7702 and HepG2 cells.Meanwhile,with the extension of treatment time,a gradual increase of P-SREBP-2 protein and decrease of N-SREBP-2 protein was observed in both cell lines.Western blot analysis showed that LC3-II/LC3-I ratio was additionally enhanced at 24 h after the co-treatment with CQ and PF-429242 in HL-7702 and HepG2 cells.In addition,PF-429242 or NFV mono-treatment significantly increased amount of red dots compared with the control group.However,CQ and PF-429242 or NFV treatment exhibited significantly increased amount of yellow dots compared with PF-429242 or NFV mono-treatment group.2.Inhibition of S1P and S2P gene expression increased the mRNA levels of the autophagy related genes,but had no impact on SREBP-2 mRNA expression level.However,compared with the NC-shRNA group,Western blot data showed increased P-SREBP-2 accumulation,reduced N-SREBP-2 amounts,and increased LC3-?/LC3-? ratio in SIP and/or S2P in shRNA-transfected AML-12 cells.Conclusions:These results suggested that the S1P or S2P specific inhibitor by altering S1P or S2P protein bioactivity regulated SREBP-2 protein expression to induce expression levels of autophagy related genes and autophagic flux.Furthermore,the inhibition of SIP and S2P gene expression could regulate SREBP-2 protein expression so as leading to increased autophagy.Objective:To investigate the changes of SREBP-2 expression levels and autophagy activity in HL-7702 and HepG2 cells with steatosis induced by palmitic acid and the liver tissue of HFD-induced mice and NAFLD patients,and whether macrolipophagy is regulated via inhibition of nuclear translocation of SREBP-2.Methods:1.Liver biopsy samples of nineteen NAFLD patients and sixteen normal people were analyzed by Western blot for P-SREBP-2,N-SREBP-2,SCAP,LC3B and LAMP1.Paraffin-embedded sections were subjected to double immunofluorescent stainings for LC3B and LAMP 1.2.Firstly,HL-7702 and HepG2 cells were treated with PA to built up cell model of fatty liver,then treated with NFV or/and PF-429242.Total protein levels of P-SREBP-2,N-SREBP-2,SCAP,LC3B,ATG5,Beclinl,LAMP1 and SQSTM1 were analyzed by Western blot.Then,HL-7702 and HepG2 cells were transfected with the GFP-mRFP-LC3 double-tagging plasmid for 24 h,followed by PA in combination with NFV or/and PF-429242 or CQ.Cell autophagic flux was evaluated by laser confocal scanning microscopy.Furthermore,Lipid droplet,autophagosome and autolysosome were detected by electron microscopy in HL-7702 cells exposed to PA in combination with NFV or/and PF-429242.In addition,Intracellular lipid accumulation was visualized by microscopy after Oil red O staining and quantitated on a spectrophotometer at 510 nm.Enzymatic methods were used to detect TG and TC content in total cell lysates.3.Having been fed with HFD for 19 weeks,male C57BL/6 mice were injected with lentivirus vector-based shRNA to silence hepatic S1P and S2P expression,respectively.After two weeks of continuous feeding,all mice were sacrificed.Relative mRNA levels of SIP and S2P were determined by qRT-PCR from all samples;total protein levels of P-SREBP-2,N-SREBP-2,SCAP,LAMP1,SQSTM1,ATG5,Beclin1 and LC3B were analyzed by Western blot;double immunostaining was used to detect co-localization and expression of SREBP-2 and SCAP or LC3 and LAMP1;lipid droplet,autophagosome and autolysosome were detected by electron microscopy;intracellular lipid accumulation was visualized by microscopy after Oil red O staining;enzymatic methods were used to detect the average serum levels of TG,TC,GOT and GPT.Results:1.We found that significantly increased N-SREBP-2 and SCAP protein levels were detected in NAFLD patients compared with the control group.Meanwhile,a noticeable increase of LC3-II/LC3-1 ratio,and remarkable reduction of LAMP1 protein levels were found in NAFLD patients compared with normal individuals.LC3 puncta numbers were significantly increased,and LAMP1 puncta numbers were slightly decreased in NAFLD patients.2.Western blot analysis showed that N-SREBP-2 and SCAP protein expression levels were significantly increased,while no statistically significant difference of P-SREBP-2 protein level was detected between the PA-treated and control cells.Furthermore,LC3-II/LC3-I ratio was increased and the protein levels of other two autophagy related genes,ATG5 and Beclin 1,were also slightly increased in PA-treated cells.However,protein levels of SQSTM1 were also increased.In addition,protein levels of LAMP1 were decreased.Interestingly,NFV or/and PF-429242 could lead to reduced protein expression levels of N-SREBP-2 and SCAP,and increased protein expression levels of P-SREBP-2 compared with the PA-treatment group.Importantly,PA-treated cells exposed to NFV or/and PF-429242 showed significantly increased LC3-?/LC3-? ratio and the protein expression levels of ATG5,Beclinl and LAMP1 compared with untreated controls.Increased SQSTM1 protein expression by PA was significantly reduced after NFV or/and PF-429242 treatment.In addition,PA mono-treatment only increased the amount of yellow dots;however,PA in combination with NFV or/and PF-429242 treatment for 24 h exhibited significantly increased amount of both yellow and red dots.Electron microscopy analysis showed that large amounts of lipid droplets and a few autophagy vesicles were found in PA-treated HL-7702 cells.Significantly,double membrane structures(autophagosome vesicles)and monolayer membrane structures(autolysosome vesicles)showed high accumulation in HL-7702 cells exposed to PA in combination with NFV or/and PF-429242.In addition,some autophagosome vesicles engulfing lipid droplets were observed contributing to lipid droplets breakdown.A significant reduction in lipid accumulation was found in both cells treated with PA in combination with NFV or/and PF-429242 compared with PA-treated cells as evaluated by Oil red O staining and quantitateing.Similarly,a significant reduction in TG and TC contents between cells treated with PA in combination with NFV or/and PF-429242 compared with PA-treated cells as evaluated by enzymatic methods.3.We found SIP and S2P mRNA levels were reduced by approximately 56%and 52%,respectively,in treated groups compared with CHD control group.Additionally,both Western blot and double immunostaining showed that SREBP-2 nuclear trafficking and cytoplasmic amount of SCAP were higher in the NC group than those in CHD control group.A clear upregulated P-SREBP-2 and downregulated N-SREBP-2 and SCAP expression levels were observed in knockdown mice.Notably,in the knockdown groups,LC3-II/LC3-I ratio and protein levels of ATG5,Beclinl and LAMP1 were increased compared with the CHD control group.Protein level of SQSTM1 was markedly decreased,and LAMP1 amounts significantly increased,in the knockdown groups compared with the NC group.Furthermore,double immunofluorescent staining showed increased green dots in the NC group,with increased red dots in the knockdown groups.In addition,compared with the CHD control group,double membrane structures were increased in the NC group,and both double and single membrane structures were increased in the knockdown groups,as shown by electron microscopy.Meanwhile,the number and dimension of intracellular lipid droplets were decreased in the knockdown groups compared with the NC group.Oil red O staining showed the amount of HFD-induced large lipid droplets significantly decreased in the knockdown groups.In the knockdown mice compared with the NC group,the average serum levels of TG,GOT and GPT enzymes were markedly reduced and the average serum levels of TC were slightly decreased.Conclusions:1.In HL-7702 and HepG2 cells with steatosis induced by palmitic acid and the liver tissue of HFD-induced mice and NAFLD patients,there were SREBP-2 over-activation and impaired autophagy.2.In PA-treated cells exposed to NFV or/and PF-429242,inhibition of SREBP-2 activity and accumulation of its precursor protein can promote macrolipophagy to increase lipid degradation and reduce lipid content.3.In HFD-fed mice,inhibition of SREBP-2 abnormal activation via SIP and S2P knockdown can also promote macrolipophagy to increase lipid degradation and reduce lipid content.Objective:To investigate the influence of regulating SREBP-2 intracellular trafficking to improve autophagic flux on endoplasmic reticulum stress(ERS)in both cell and animal models of NAFLD.Methods:1.The ER-associated stress sensor proteins PERK,P-PERX,EIF2?,P-EIF2?were assessed by Western blot in PA-treated cells after being treated with NFV or/and PF-429242 or CQ.Furthermore,we evaluated the role of autophagic flux in cell apoptosis by DAPI staining of nuclei and quantification of fragmented nuclei.2.Having been fed with HFD for 19 weeks,male C57BL/6 mice were injected with lentivirus vector-based shRNA to silence hepatic SIP and S2P expression,respectively.After two weeks of continuous feeding,all mice were sacrificed.Total protein levels of PERX,P-PERK,EIF2a,P-EIF2a were analyzed by Western blot.Results:1.Western blot analysis showed that NFV or/and PF-429242 blunted the PA-induced increases of P-PERK and P-EIF2a protein levels.Nevertheless,CQ significantly increased P-PERK and P-EIF2a protein expression levels.In addition,PA treatment increased the number of apoptotic nuclei;however,NFV or/and PF-429242 significantly decreased the number of apoptotic nuclei compared with PA alone treatment.2.Western blot analysis showed that protein levels of P-PERK and P-EIF2a were markedly decreased in the knockdown groups compared with the NC group.Conclusion:These results suggested that restoration of autophagic flux by regulating SREBP-2 intracellular trafficking via two specific inhibitors or shRNA targeting SIP or S2P could ameliorate ERS and reduce cell apoptosis in cell and mouse models of NAFLD. |
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