The Protective Role Of PPARα In D-GalN/LPS-induced Acute Liver Failure And Its Mechanism In The Pathogenesis

Objective: Acute liver failure(ALF), an inflammation-mediatedhepatocellular injury process, the mechanisms by which the organ damageoccurs are not completely understood, the prognosis is extremely ominous.Peroxisome proliferator-activated receptor α(PPAR α) belongs to the nuclearhormone receptor superfamily of ligand-activated transcription factors. It hasbeen reported to be involved in a number of cellular processes including lipidmetabolism, apoptosis, and the inflammatory response. Although studies havedemonstrated that PPAR α exhibit potent anti-inflammatory activity. However,the functional role of PPAR α in the mechanism of ALF is still relativelylacking and need to be further explored. So, in the present study, Our purpose isto explore the protective role of PPAR α in acute liver failure and its mechanismin the pathogenesis.Methods: This experiment included the following two parts.1Animal ExperimentsTo induce ALF, the mice(except for the control) were injectedintraperitoneally with D-GalN(700mg/kg, Sigma, St Luis, MO) andLPS(10μg/kg, Invivogen, San Diego, CA). The PPAR α activatorWy-14,643(6mg/kg, Sigma, St Luis, MO) was administered via the tail veininjection at2hours prior to D-GalN/LPS exposure. Suppression of autophagywas achieved by tail vein injection of3-methyladenine(3-MA,10mg/kg, Sigma,St Luis, MO) or ATG7-specific siRNA(siAtg7,50μM/kg). At selected timepoints after D-GalN/LPS treatment, mice were anesthetized and blood wascollected. Serum samples were taken from the mice at6h after D-GalN/LPSinjection. Serum levels of alanine aminotransferase(ALT), aspartateaminotransferase(AST) as markers of hepatic damage were measured by using amultiparameteric analyzer(AU5400, Olympus, Japan), according to an automated procedure. Liver tissues were fixed in formalin and embedded inparaffin wax, and sections in5um were stained with hematoxylin andeosin(H&E) using a standard protocol, and then analyzed by light microscopy.Differential expressions of inflammatory cytokines(TNF-α, IL-1β, IL-6),chemokines(CXCL-1, CXCL-10) and autophagy related genes(ATG-5, ATG-7,LAMP-1) were detected by real-time quantitative PCR. Differential proteinexpressions of p-NF-κBp65, p-JNK, p-ERK, p-P38and LC3, ATG-5, ATG-7,LAMP-1in inflammatory and autophagy pathways were detected by Westernblotting.2Cell ExperimentsMurine bone marrow-derived macrophages(BMM) were differentiatedfrom bone marrow from6-to8-week-old C57BL/6mice by culturing in DMEMcontaining10%fetal bovine serum,1%penicillin/streptomycin and20%L929conditioned medium for6days. LPS(20ng/ml) was used to activate cells,Wy-14,643(50μM/L) was used to activate PPAR α, and3-MA(10mM/L) orsiAtg7(3mg/ml) was used to inhibit autophagy in the macrophages. Transienttransfection was performed with the GFP-LC3plasmid or siAtg7using FugeneHD(Roche) according to the manufacturer’s instructions. Differentialexpressions of inflammatory cytokines(TNF-α, IL-1β, IL-6) andchemokines(CXCL-1, CXCL-10) were detected by real-time quantitative PCR.Differential protein expressions of LC3, ATG-5, ATG-7, LAMP-1in autophagypathways were detected by Western blotting.Results:1The gene and protein levels of PPAR α were gradually reducedthroughout the procession of ALF.2Peroxisome Proliferator-Activated Receptor-α-mediated autophagyattenuates inflammatory response to protect against acute liver failure in mice.For gross morphology of the liver, compared with the model group,Wy-14,643-treated mice almost appears substantially the normal livermorphology, and the liver architecture was better preserved. Serumaminotransferase levels of Wy-14,643-treated group were significantly lower. We also found that PPAR α activation suppressed the expression ofproinflammatory cytokines, chemokines and the protein levels ininflammatory pathways. However, the expression of autophagy related geneand protein levels were significantly higher in the Wy-14,643-treated group,and the differences were statistically significant.33-MA or siAtg7reversed the liver protection by Wy-14,643in ALF mice,respectively. As compared with Wy-14,643treated ALF, the mice receiving3-MA or siAtg7suffered severe ALF again, evidenced by worse grossmorphology, worse preserved liver architecture by histology and significantlyhigher sALT, sAST levels. Thus, the hepatoprotective mechanism of PPAR αactivation depends on autophagy pathway.4PPAR α activation indeed suppressed the expression ofpro-inflammatory cytokines and chemokines triggered by LPS in vitro. Bonemarrow-derived macrophages(BMM) were stimulated with LPS in the absenceor presence of Wy-14,643(10μM,25μM,50μM). Remarkably, the expression ofcytokines(TNF-α, IL-6, IL-1β) and chemokines(CXCL-1, CXCL-10) byLPS-stimulated macrophages were decreased by Wy-14,643treatment, whichwere in does-dependent manner.5PPAR α activation can induce autophagy in vitro. Western blot resultsalso showed that Wy-14,643promoted the expressions of LC3, ATG-5, ATG-7,LAMP-1proteins. Moreover, we transfected GFP-LC3plasmid into BMM toobserve the formation of autophagosome. As shown in Fig.19, the GFP-LC3signal was weak in the absence of Wy-14,643, but it was more and more brightand punctate in the presence of Wy-14,643with the increase of dose.6LPS-induced pro-inflammatory cytokine and chemokines levels wererestored after adjunctive3-MA or siAtg7and Wy-14,643. Real-time quantitativePCR revealed that compared with only Wy-14,643-treated macrophage, theexpression of cytokines(TNF-α, IL-6, IL-1β) and chemokines(CXCL-1,CXCL-10) by LPS-stimulated macrophages were increased after adjunctive3-MA or siAtg7and Wy-14,643.Conclusion: 1PPAR α is suppressed in liver during the procession of ALF.2Peroxisome Proliferator-Activated Receptor-α-mediated autophagyattenuates inflammatory response to protects against acute liver failure in mice.3PPAR α Activation Protect ALF Depending on Autophagy Mechanism.4PPAR α will be a potential therapeutic target for ALF.