| Objective: Non-alcoholic fatty liver disease(NAFLD)is a common chronic liver disease and rapidly becomes a major public health problem worldwide,accompanied by the high prevalence of obesity and metabolic syndrome.Currently,lifestyle intervention and adjuvant drug therapy is the main treatment measuires of NAFLD,but there is still no specific treatment in clinic.Thus,the development of potentially effective treatment drugs has important clinical and social value.Endoplasmic reticulum stress(ERS)plays a vital role in the pathological mechanisms of NAFLD.This study intended to firstly analyze the enrichment signaling pathways of NAFLD by bioinformatics,then observe the potential therapeutic effect of hesperidin(HSP)on NAFLD through experiments in animal model and cells,and explore its mechanism related to ERS induced inflammatory.Methods:1.The differentially expressed genes(DEGs)between NAFLD patients and healthy donors in NAFLD related gene expression dataset GSE48452 and GSE89632 were analyzed by bioinformatics.The biological functions and pathways of all DEGs were evaluated using GO and KEGG enrichment analysis.2.SD rats were randomly divided into 4 groups,namely normal diet(ND)group,ND+HSP group,high fat diet(HFD)group and HFD+HSP group.After one week of adaptive feeding,SD rats were fed with HFD for 12 weeks to establish NAFLD,withthose fed with ND used as control.During this period,rats were treated with HSP or normal saline(NS)every day according to body weight,respectively.Blood and liver tissues were collected after the experiment.Liver steatosis formation in rat liver tissue was observed by Oil red O staining.While triglyceride(TG),total cholesterol(TC)and low density lipoprotein(LDL)in serum were measured by biochemical analysis.Western blotting was applied to detect the expressions of GRP94,transcription activator 6(ATF6),transcription activator 4(ATF4),and the transcription activators of ATF6,ATF4,phosphorylated pancreatic el F-2 kinase elf-2kinase(PERK),and phosphorylatedi nositol requiring enzyme1α(IRE1α)in liver tissue.Furthermore,the levels of serum free fatty acid(FFA),TNF-α,IL-6 and IL-1β were measured using ELISA kit.3.Human THP-1 cells were stimulated to differentiate into macrophages by phorbo ester(PMA),and then treated by FFA to induce ERS.The cells were interfered with serum-free medium containing solvent + FFA or HSP+FFA or thapsigargin(a Ca2+-ATPase inhibitor widely used intracellular ERS inducer)+ FFA or HSP+ thapsigargin + FFA for 24 h,respectively.The expression of GRP94 was observed by immunofluorescence staining.The expressions of GRP94,ATF6,ATF4,p-PERK,p-IRE1 α,IL-1β,IL-6 and TNF-α were detected by Western blotting.To further observe the effect of HSP on liver lipid synthesis,co-cultured Hep G2 cells and THP-1 cells were treated with solvent + FFA or HSP+ FFA or thapsigargin + FFA or HSP+ thapsigargin+ FFA,respectively.Then the expressions of sterol-regulatory element binding proteins 1c(SREBP1c),SREBP 2 and CCAAT/enhancer binding protein(C/EBP)were detected by Western blotting.Results:1.In GSE48452 data set,there were 247 up-regulated DEGs and 165down-regulated DEGs in NAFLD;while in GSE89632 data set,there were298 up-regulated DEGs and 298 down-regulated DEGs in NAFLD.The results of GO biological function analysis showed that DEGs in GSE48452 data set were mainly related to oxidation-reduction process,IREL mediated folding protein reaction,ATF1-ATF4 transcription factor complex,inflammatory response,apoptosis and immune response signaling pathways.DEGs in THE GSE89632 data set were mainly related to oxidation-reduction process,lipid metabolism,exosomes,inflammatory responses,apoptosis and endoplasmic reticulum signaling pathways.KEGG pathway analysis showed that most DEGs in GSE48452 and GSE89632 data sets were enriched in inflammatory response and IRE1α activation signaling pathways.2.After 3 months of feeding,compared with the ND(ND + NS)group,the weight of rats in HFD(HFD + NS)group was significantly increased,and the levels of serum FFA,TC,TG and LDL were significantly elevated.Meanwhile,oil red O staining results showed that obvious hepatic steatosis occurred in rats of HFD group,with the liver tissues obviously red stained.After 4 weeks of HSP treatment,the body weight of rats in HFD group(523.5± 7.2g)was significantly lower than that of control group(563.3 ± 9.2g,P <0.05).Meanwhile,serum FFA level and positive staining area of liver tissue were significantly decreased(P < 0.05).But HSP had no significant effect on serum TC,TG and LDL levels of rats(P > 0.05).Also,HSP alone had no significant effect on body weight,serum FFA,TC,TG and LDL levels or hepatic steatosis in rats of ND group.The red fluorescence of GRP94 in rat liver tissues of HFD group was more bright than ND group(P < 0.05),while HSP significantly darken the fluorescence of GRP94(P < 0.05).Meanwhile,the expression of GRP94,ATF6,ATF4,p-PERK and p-IRE1α in rat liver tissues of HFD group were significantly higher than those of ND group(P < 0.05),and HSP treatment significantly reduced the expression levels of these proteins(P < 0.05).And the expression of IL-6,TNF-α and IL-1β in rat liver tissue of HFD group were significantly higher than ND group,while HSP significantly decreased their levels(P < 0.05).3.FFA could induce the expressions of GRP94,p-PERK,p-IRE1α,ATF6,IL-6,TNF-α and IL-1β,with low levels of GRP94(red fluorescence)and p-PERK(green fluorescence)observed,to activate ERS and induce inflammatory response in THP-1 cells.Its effect can be further enhanced by thapsigargin.HSP could significantly inhibit the expressions of GRP94,p-PERK,p-IRE1α,ATF6,IL-6,TNF-α,IL-1β(P < 0.05),and decrease the immunofluorescence intensity of GRP94 and P-PERK in cells(P < 0.05).However,these effects of HSP on ERS related proteins and inflammatory factors could be eliminated by the addition of thapsigargin.4.FFA could induce the expression of SREBP1 c,SREBP 2,C/EBP in Hep G2 cells co-cultured with THP-1 cells,and activate the lipid synthesis process.And the expressions of SREBP1 c,SREBP 2 and C/EBP could be significantly enhanced by thapsigargin.HSP could inhibit the expression of SREBP1 c,SREBP 2 and C/EBP in Hep G2 cells(P < 0.05),but its inhibition effect could be eliminated by thapsigargin(P > 0.05).Conclusions:1.Bioinformatics analysis showed that ERS and inflammatory response were the enriched signaling pathways of DEGs in NAFLD.2.HFD could activate ERS in rat liver tissue and induce inflammatory response,leading to weight gain and liver steatosis in vivo.HSP could significantly supress the expression of ERS-related protein and secretion of inflammatory cytokines in liver tissue of NAFLD rats,thereby inhibiting weight gain and liver steatosis.In addition,HSP could inhibit the expression of lipid synthetic proteins induced by FFA and inflammatory cytokines in liver cells.3.FFA could induce ERS-related protein expression and secretion of inflammatory cytokines in THP-1 cells,and enhance the expression of lipid synthetic proteins in Hep G2 cells co-cultured with THP-1 cells.HSP could suppress FFA induced expression of ERS-related protein and secretion of inflammatory cytokines in THP-1 cells,and inhibit the expression of lipid synthetic proteins in FFA stimulated Hep G2 cells co-cultured with THP-1 cells,while these effects could be eliminated by thapsigargin.4.In summary,HSP could inhibit lipid synthesis and liver tissue steatosis via inhibiting ERS-mediated inflammatory response in vivo and in vitro,which has potential therapeutic value for NAFLD. |
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