Korean J Hepatobiliary Pancreat Surg.  2013 Aug;17(3):89-108. 10.14701/kjhbps.2013.17.3.89.

Fenofibrate, a peroxisome proliferator-activated receptor alpha-agonist, blocks lipopolysaccharide-induced inflammatory pathways in mouse liver

• Won TW

• KMID: 1472524
• DOI: http://doi.org/10.14701/kjhbps.2013.17.3.89

Abstract

BACKGROUNDS/AIMS
During the acute phase response, cytokines induce marked alterations in lipid metabolism including an increase in serum triglyceride levels and a decrease in hepatic fatty acid oxidation, in bile acid synthesis, and in high-density lipoprotein levels.
METHODS
Peroxisome proliferator-activated receptors (PPARs: PPARalpha, beta/delta, and gamma) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Proinflammatory profiles including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) are the important pathological factors in inflammatory responses during the pathological progression of the acute phase response. Lipopolysaccarides (LPS) induced the expression of TNF-alpha, IL-1beta, and IL-6. LPS-induced inflammation decrease the expression of peroxisome proliferator-activated receptor alpha (PPARalpha), PPARbeta/delta, PPARgamma, and coactivators PPARgamma co-activator 1 alpha (PGC-1alpha), PGC-1beta messenger RNA (mRNA) in the liver of Balb/c mouse. In addition, LPS-induced inflammation diminishes the protein level of PPARalpha, PPARbeta/delta, and PPARgamma. Proinflammatory cytokines including TNFalpha, IL-1beta, and IL-6 are the principal reducer of PPARs. However, the knockout mouse model against TNFalpha and IL-6 does not block decrease of PPARs in serum and liver. The mice were pretreated with fenofibrate at 100 mg/kg for 2 days.
RESULTS
These treatment protocols increased the amount of PPARs mRNA in the liver. Fenofibrate inhibited LPS-induced TNF-alpha, IL-1beta, and IL-6 production in the serum and liver. Similar results were obtained when human hepatoma HepG2 cells exposed to LPS were co-incubated with fenofibrate. LPS-treated HepG2 cells decreased expression of IkappaB. Moreover, activation of PPARs abrogated LPS-induced degradation of IkappaB, thus suppressing LPS-induced NF-kappaB activities.
CONCLUSIONS
Therefore, fenofibrate decreases the expression and secretion of TNF-alpha, IL-1beta, and IL-6 via the NF-kappaB signaling pathway, thus serving as therapeutic targets to attenuate inflammation that is involved in hepatic pathological progression.

Keyword

Peroxisome proliferator activated receptors alpha agonist

MeSH Terms

Animals
Bile
Carcinoma, Hepatocellular
Cell Proliferation
Clinical Protocols
Cytokines
Fenofibrate
Glucose
Hep G2 Cells
Homeostasis
Humans
Inflammation
Interleukin-1beta
Interleukin-6
Lipid Metabolism
Lipoproteins
Liver
Mice
Mice, Knockout
NF-kappa B
Peroxisome Proliferator-Activated Receptors
Peroxisomes
PPAR alpha
PPAR-beta
PPAR delta
PPAR gamma
RNA, Messenger
Tumor Necrosis Factor-alpha
Cytokines
Fenofibrate
Glucose
Interleukin-1beta
Interleukin-6
Lipoproteins
NF-kappa B
PPAR alpha
PPAR-beta
PPAR delta
PPAR gamma
Peroxisome Proliferator-Activated Receptors
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Schematic representation of in vivo experiment.

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  Fig. 2 Body weight change in LPS-injected mice. Balb/c mice were injected intraperitoneally with lipopolysaccharide 5 mg/kg once. Mice were weighed after 20 and 40 h.

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  Fig. 3 Histopathological changes of liver sections of mice under LPS treatment. Hematoxylin and eosin stained liver sections from mice liver (original magnification, ×100). Arrowhead indicates leukocytes. Arrow indicates an inflammatory focus.

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  Fig. 4 Lipopolyssacharide-induced inflammation profiles in Balb/c. Mice were injected intraperitoneally with LPS. After 20 h, the liver was collected and analyzed for Western blot, reverse transcriptase PCR, real time PCR, and immunohistochemistry. (A) Induction of pro-inflammatory cytokines, TNF-α, and stress inducible protein, heme oxygenase-1 (HO-1) in LPS-treated mouse. (B) Expression levels of TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR in LPS injected mouse liver and observed by agarose-gel electrophoresis under UV illumination staining with ethidium bromide. (C) Liver lysates were prepared. Whole lysates (40 µg of protein) were separated by electrophoresis on SDS 10% polyacrylamide gels and then the protein was transferred onto nitrocellulose membrane. Each membrane was immunoblotted with antibody specific for phosphorylated or tatal JAK and STAT. (D) Levels of VCAM and ICAM mRNA were determined by semi-quantitative real time PCR in LPS injected mouse liver (Cont: *p<0.05, LPS: †p<0.01). (E) Liver from Balb/c mice injected with PBS or LPS were removed and embedded in paraffin, and then 4 µm sections were prepared. For immunohistochemistry studies, an immunohistochemistry kit was used, and all the procedures were performed according to the instructions of the manufacturer. The anti-TNF-α and anti-Tall like receptor (TLR) 4 were used. All results were representative of three separate experiments.

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  Fig. 5 Secretion of TNF-α, IL-1β, and IL-6 in serum from LPS-injected Balb/c. Secretory protein levels of TNF-α, IL-1β, and IL-6 from the serum were measured by the ELISA method for the indicated times. All results were representative of three separate experiments. (A) injection of LPS resulted in a marked increase of TNF-α which was observed as early as at 2 h and reached a primary peak level with 1,591.15±5.798 pg/ml at 2 h, and thereafter gradually decreased in a time-dependent manner. (B) LPS also increased in secretion of IL-1β, which was already reached a peak level with 183.2604±6.542 pg/ml at 4 h, thereafter sustained its level up to 12 h, and gradually decreased in a time-dependent manner. (C) IL-6 secretion was already reached a peak level with 766.3889±8.214 pg/ml at 2 h, and thereafter gradually decreased in a time-dependent manner.

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  Fig. 6 Expression level of PPARs, PGC1β, ACOX1, SREBP1α, SREBP1c, and SREBP2 mRNA was determined by qPCR (normalized to GAPDH). Balb/c mice were injected with LPS. Total RNA from the liver was isolated. Two micrograms of total RNA was reverse transcribed to cDNA. PPARs (A), PGC1β and ACOX1 (B), SREBP1α, SREBP1c, and SREBP2 (C) mRNA expression was measured by real-time QPCR as described under "Methods". QPCR data were normalized using GAPDH mRNA as the invariant control for all experiments. Data (mean±SD) are expressed as a relative unit.

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  Fig. 7 Expression level of PPARs protein was determined by Western blot and IHC. Mice were injected intraperitoneally with LPS. After 20 h, the liver was collected and analyzed for Western blot (A) and immunohistochemistry (B).

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  Fig. 8 Body weight change in LPS-injected mice. TNF-α (A) and IL-6 (B) knockout mice were injected intraperitoneally with lipopolysaccharide 5 mg/kg once. Mice were weighed after 20 and 40 h.

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  Fig. 9 Secretion of TNF-α, IL-1β, and IL-6 in serum from LPS-injected TNF-α and IL-6 knockout mice. Secretory protein levels of TNF-α, IL-1β, and IL-6 from the serum were measured by the ELISA method for the indicated times. All results were representative of three separate experiments. (A-C) TNF-α knockout mice; (D-F) IL-6 knockout mice.

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  Fig. 10 LPS induced inflammation profiles in TNFa, IL-6 knockout mice. Level of VCAM and ICAM mRNA were determined by semi-quantitative real time PCR in LPS or PBS injected knockout mouse liver (A: TNF-α KO, C: IL-6 KO). Expression levels of TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR in LPS injected mouse liver and observed by agarose-gel electrophoresis under UV illumination staining with ethidium bromide (B: TNF-α KO, D: IL-6 KO).

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  Fig. 11 In vivo effect of LPS on TNF-α and TLR4 expression in the liver of TNF-α (A), IL-6 (B) knockout mice. Liver from knockout mice injected with PBS or LPS were removed and embedded in paraffin, and then 4 µm sections were prepared. For immunohistochemistry studies, an immunohistochemistry kit was used, and all the procedures were performed according to the instructions of the manufacturer. The anti-TNF-α and anti-Tall like receptor (TLR) 4 were used. All results were representative of three separate experiments.

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  Fig. 12 Expression level of PPARs, PGC1β, ACOX1, SREBP1α, SREBP1c, and SREBP2 mRNA was determined by QPCR (normalized to GAPDH) in the liver of KO mice. TNF-α (A, B) and IL-6 (C, D) KO mice were injected with LPS. Total RNA from liver was isolated. Two micrograms of total RNA was reverse transcribed to cDNA. PPARs (1), PGC1β and ACOX1 (2), SREBP1α, SREBP1c, and SREBP2 (3). mRNA expression was measured by real-time QPCR as described under "Methods." QPCR data were normalized using GAPDH mRNA as the invariant control for all experiments. Data (mean±SD) are expressed as a control level.

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  Fig. 13 In vivo effect of LPS on PPARs expression in the liver of TNF-α and IL-6 KO mice. Expression level of PPARs protein was determined by immunohistochemistry. TNF-α (A) and IL-6 KO (B) mice were injected intraperitoneally with LPS. After 20 h, the liver was collected and analyzed for immunohistochemistry.

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  Fig. 14 Effects of fenofibrate on LPS-induced inflammation in Balb/c mice. Hematoxylin and eosin stain (×100) shows intracellular lipid accumulation and inflammation.

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  Fig. 15 Effect of fenofibrate on proinflammatory cytokine mRNA and protein level in Balb/c mice. LPS-induced expression levels of mRNA (A) and protein (B) were determined by RT-PCR and Western blot in the presence and absence of fenofibrate as described under "Methods." The amplified gene products were observed by agarose-gel electrophoresis under UV illumination staining with ethidium bromide. TNF-α protein level was detected by Western blot. (C) Western blot analyses of IκBα and IκBβ in liver treated with PPAR-α agonist fenofibrate (100 mg/kg) and followed by LPS treatment (5 mg/kg).

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  Fig. 16 Effect of fenofibrate on proinflammatory cytokine mRNA levels in serum of Balb/c, TNF-α KO, and IL6 KO mice. All mice were fed fenofibrate (100 mg/kg) or methylcellulose. After LPS injection, the pro-inflammatory cytokines were measured by the ELISA method for the indicated times. All results were representative of three separate experiments. (A-C) Balb/c mouse, (D-F) TNF-α KO mouse; (G-I) IL-6 KO mouse.

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  Fig. 17 Effect of fenofibrate on PPARs (A), catalase, PMP70, and PGC1b (B) expression in treated liver tissue of Balb/c. After pretreatment with fenofibrate, LPS was injected intraperitoneally. Whole liver lysates (40 µg of protein) were separated by electrophoresis on SDS 10% polyacrylamide gels and then the protein were transferred onto a nitrocellulose membrane. Each membrane was immunoblotted with antibody specific for PPARs, catalase, PGC1, PMP70, and actin. The results were representative of three separate experiments.

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  Fig. 18 Effect of fenofibrate on proinflammatory cytokine mRNA level in HepG2. Expression levels of TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR. PCR products observed by agarose-gel electrophoresis under UV illumination staining with ethidium bromide (TNF-α KO; IL-6 KO).

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  Fig. 19 Effect of fenofibrate on PPARs mRNA level in the liver of Balb/c. Fenofibrate (100 mg/kg) or its vehicle (water containing 0.5% methylcellulose) were administered. After LPS injection, total RNA from the liver was isolated. Two micrograms of total RNA was reverse transcribed to cDNA. PPARs mRNA expression was measured by real-time QPCR as described under "Methods." QPCR data were normalized using GAPDH mRNA as the invariant control for all experiments. Data (mean±SD) are expressed as a relative unit.

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  Fig. 20 PPARα is a modulator of the inflammatory response in the liver.

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  Fig. 21 Cellular mechanism of the LPS in the liver and interaction of PPARα at several levels of the inflammatory signaling pathway. Interaction with the NFκB pathway. LPS increases cytokines including TNF-α, IL-1β, and IL-6 through theNFκB pathway. Fenofibrate, a PPARα agonist, inhibits the NFκB pathway through blocking translocation of NFκB into nucleus. (1) Inhibition of phosphorylation of IκB. (2) Inhibition of nuclear translocation of inflammatory transcription factors. (3) Interference with activation of the transcription initiation complex via cofactor interaction.

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