The PPARα Agonist Fenofibrate Prevents Formation of Protein Aggregates (Mallory-Denk bodies) in a Murine Model of Steatohepatitis-like Hepatotoxicity

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.

Formalin-fixed tissue sections from control mice, 10 weeks DDC treated mice and DDC+Fenofibrate co-treated mice (n=5) were stained with PicroSirius red and quantified using ImageJ. Data are represented as mean ± SD. Student's t-test was performed to evaluate significance, *: p < 0.05; **: p < 0.01

Preparation of mouse liver mitochondria
Animals (4-6 per group) were fasted overnight and sacrificed by cervical dislocation around 9.00 a.m.. Livers were excised, rinsed and minced in ice-cold mitochondrial isolation medium (10 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA) to remove blood and bile. All following steps were performed on ice or at 4°C. Livers were homogenized in a Potter-Elvehjem homogenizer with a loosely fitting pestle in mitochondrial isolation medium containing 1 mg/ml fatty acid-free bovine serum albumin and 1 mM dithiothreitol (freshly added, to prevent accidental oxidation of a sensitive cysteine residue on complex I). The homogenate was centrifuged at 800 x g for 10 min in a Sorvall RC-5B high-speed centrifuge using a Sorvall SS-34 rotor (DuPont Instruments, Inula, Vienna, Austria) to remove nuclei and cell debris. The supernatant was centrifuged for 15 min at 6000 x g to pellet mitochondria. The pellet was washed once by re-suspending in 20 ml isolation medium (without EDTA), re-pelleted and re-suspended in little isolation medium to give an approximate protein concentration of 70 mg/mL. Protein content was determined by the Lowry method using the Bio-Rad protein reagent kit (Bio-Rad, Vienna, Austria).

Determination of respiratory control coefficient
Mitochondrial respiration was measured using a CLARK electrode (Oxygraph, Anton Paar,

Determination of mouse liver tissue protoporphyrin IX (PPIX)
For determination of PPIX we adapted an albumin-enhanced fluorescence method 1 to allow determination of PPIX without interference by bilirubin, extracting tissue by a slightly modified FOLCH procedure 2 . Briefly, frozen tissue (20 mg) was homogenized in a reaction tube in a total volume of 200 µL potassium phosphate buffer (KP i ; 100 mM, pH = 7.4), followed by 500 µL of methanol and 100 µL 5% trichloroacetic acid in H 2 O. Tissue was homogenized with an immersion blender and then 800 µL CHCl 3 are added. All steps were performed on ice. The mixture was vigorously shaken for at least one minute, repeated two times with an interval of 5-10 minutes on ice. Subsequently, phase separation was accelerated by centrifugation (100 x g, 1 min, 4°C). The lower phase containing PPIX was collected and dried in vacuum. The dry residue was dissolved in Tris-HCl-albumin buffer (100 mM, pH = 9.3, 300 µM bovine serum albumin) and fluorescence measured at 410/630 nm (ex/em) in a black microplate. For quantification, a standard series of authentic PPIX was used (0 .. 10 µM PPIX). was then incubated for 60 min, followed by polyclonal rabbit-anti-rat Immunglobulins/HRP for 30 minutes and K5007 (DAKO) Envision+, also 30 minutes. As chromogen, AEC Substrate Chromogen ready to use (Dako) was incubated for 10 minutes. Counterstaining was Mayer`s haematoxylin for 30 seconds, followed by washing with tap water.

Staining with PicroSirius red (Collagen)
PicroSirius Red staining was performed to evaluate liver collagen. For quantification of the stained sections, the ImageJ software was used. The sections were scanned and a threshold defined by a certain intensity of staining was applied. The numerical value corresponding to the area of stained tissue above threshold represents the percentage of collagen deposition within the area.