Peroxisome Proliferator-activated Receptor-α Activation Protects Against Endoplasmic Reticulum Stress-induced HepG2Cell Apoptosis

Objective Endoplasmic reticulum stress （endoplasmic reticulum stress, ERS） isanother apoptotic pathway different from the death receptor pathway or mitochondrialdamage pathways. The growing numbers of studies have found that endoplasmicreticulum stress-induced apoptosis is closely related to viral hepatitis, alcoholic liverdamage, fatty liver, liver ischemia-reperfusion injury, liver damage and, thus inhibitingthe endoplasmic reticulum stress-induced apoptosis plays an important part in theprevention and treatment of liver injury. Peroxisome proliferator-activated receptor-α（PPARα） is one of the three subtypes of ligand-activated nuclear receptors PPARsfamily. WY14643is a selective agonist of PPARα, and MK886is a selective antagonistof PPARα. Studies have shown that WY14643can inhibit apoptosis vital organs such asthe heart, liver, brain and kidney ischemia/reperfusion injury and play a protective role,while MK886can induce apoptosis. Our previous studies have indicated that WY14643could protect liver against ischemia-reperfusion injury and rat hepatocytes cells againsthypoxia reoxygenation injury. However, whether PPARα affect to ER stress inhepatocytes remains unclear. The aime of this study is to investigate the effects ofPPARα on ER stress-induced apoptosis in HepG2cells.Methods To ensure the appropriate working concentration of H₂O₂to HepG2cells, theHepG2cells were exposed to0.1mM,1mM and3mM H₂O₂to induce ER stress andapoptosis. Meanwhile, control group, WY14643alone group, DMSO alone group,WY14643+various concentration of H₂O₂group, and DMSO+various concentration of H₂O₂group were set, and Methyltetrazolium （MTT） assay and combined PI-DAPIstaining were used to evaluate cell viability.According to the above experimental results, control group, DMSO alone group,WY14643group, H₂O₂alone group, WY14643+H₂O₂group and DMSO+H₂O₂groupwere set. ALT, AST and MDA elevation were tested to evaluate liver function andoxidative stress level. RT-PCR and western blot analysis was used to detect mRNA andprotein expression of PPARα, BiP and CHOP.Additionally, DMSO alone group, MK886alone group, H₂O₂alone group,WY14643+H₂O₂group and MK886+WY14643+H₂O₂group were set. The ALT, ASTand MDA elevation were tested to evaluate liver function and oxidative stress level;apoptotic cell death was labeled with Annexin V/PI and tested by flow cytometry;RT-PCR and western blot analysis was used to detect mRNA and protein expression ofPPARα, BiP and CHOP; fillaly, the immunofluorescence was adopted to observe theintracellular localization of CHOP.Statistical analysis: All data are expressed as the means±SD. To determine thesignificance of differences between the means of two groups, an unpaired two-tailedStudent’s t-test was applied to study the relationship between the different variables. Todetermine the significance of differences among the means of several groups, onewayanalysis of variance （ANOVA） followed by Scheffe’s post-hoc tests were applied.Statistical significance was determined via ANOVA followed by Scheffe’s post-hoc tests.A p-value of<0.05was considered to be significant.ResultsCompared with the control group, the cell viability of HepG2cells did not alter in DMSO alone group, WY14643alone group and0.1mM H₂O₂alone group, but the cellviability significantly reduced in1mM and3mM H₂O₂alone group （P <0.05）;compared1mM H₂O₂alone group, the cell viability increased from59.6%±9.6%and56.1%±5.6%to92.6%±14.2%in WY14643+1mM H₂O₂（P <0.05）, the cellviability did not alter in DMSO+1mM H₂O₂group; compared with3mM H₂O₂alonegroup, the cell viability increased from30.2%±3.5%and32.0%±4.7%to45.6%±4.0%and52.8%±6.0%in WY14643+3mM H₂O₂group （P <0.05）, while the viablilitydid not alter in DMSO+3mM H₂O₂group.Compared with the control group, ALT and AST activity of cell culture supernatantenhanced （P<0.05）, MDA content increased （P<0.05）, the espression of mRNA andprotein of BiP and CHOP increased significantly （P<0.05） and PPARα proteinexpression level raised （P<0.05） in H₂O₂alone group; there were no change inWY14643alone group except the up-regulation of PPARα （P<0.05）; all of indexes didnot alter in DMSO alone group. Compared with the H₂O₂alone group, ALT and ASTactivity of cell culture supernatant decreased （P<0.05）, MDA content decreased（P<0.05）, the espression of mRNA and protein of BiP and CHOP redused significantly（P<0.05） in WY14643+H₂O₂group; while, there was no alternation in DMSO+H₂O₂group.Compared with DMSO alone group, ALT and AST activity of cell culture supernatantenhanced （P<0.05）, MDA content increased （P<0.05） and the espression of mRNA andprotein of PPARα, BiP and CHOP increased significantly （P<0.05） in H₂O₂alone group;ALT and AST activity of cell culture supernatant enhanced （P<0.05）, MDA contentincreased （P<0.05）, the espression of mRNA and protein of BiP and CHOP increasedsignificantly （P<0.05） and down-regulation of PPARα in MK886alone group andMK886+WY14643+H₂O₂group. Compared with the H₂O₂alone group, ALT and AST activity of cell culture supernatant decreased （P<0.05）, MDA content decreased（P<0.05）, the espression of mRNA and protein of BiP and CHOP redused significantly（P<0.05） in the WY14643+H₂O₂group. Compared with the WY14643+H₂O₂group,ALT and AST activity of cell culture supernatant enhanced （P<0.05）, MDA contentincreased （P<0.05）, the espression of mRNA and protein of BiP and CHOP increasedsignificantly （P<0.05） and down-regulation of PPARα in MK886+WY14643+H₂O₂group.Compared with the control group, the cell apoptosis increased significantly in H₂O₂alone group and MK886alone group （P <0.05）; compared with the H₂O₂group, thecell apoptosis decreased significantly in WY14643+H₂O₂group （P <0.05）; comparedwith the WY14643+H₂O₂group, the cell apoptosis rose again inMK886+WY14643+H₂O₂group （P <0.05）. At last, we found that CHOP mainlylocated in the cytoplasm in control group and WY14643alone group, and translocatedfrom the cytoplasm to the nucleus in H₂O₂alone group and MK886group. CHOPprotein translocation was inhibited in WY14643+H₂O₂group. However, the most ofCHOP protein located in the nucleus again in MK886+WY14643+H₂O₂group.Conclusion The activation of PPARα could protect HepG2cells against H₂O₂-inducedapoptosis; its mechanism may be associated with suppression of excessive ER stress.PPARα antagonist-induced ER stress pathway activation may at least partly contributeto hepatocyte apoptosis.