Peroxisome Proliferator-Activated Receptor-Gamma Reduces ER Stress and Inflammation via Targeting NGBR Expression

Increased Nogo-B receptor (NGBR) expression in the liver improves insulin sensitivity by reducing endoplasmic reticulum stress (ER stress) and activating the AMPK pathway, although it remains elusive the mechanisms by which NGBR is induced. In this study, we found that PPARγ ligands (rosiglitazone or pioglitazone) increased NGBR expression in hepatic cells and HUVECs. Furthermore, promoter analysis defined two PPREs (PPARγ-responsive elements) in the promoter region of NGBR, which was further confirmed by the ChIP assay. In vivo, using liver-specific PPARγ deficient (PPARγLKO) mice, we identified the key role of PPARγ expression in pioglitazone-induced NGBR expression. Meanwhile, the basal level of ER stress and inflammation was slightly increased by NGBR knockdown. However, the inhibitory effect of rosiglitazone on inflammation was abolished while rosiglitazone-inhibited ER stress was weakened by NGBR knockdown. Taken together, these findings show that NGBR is a previously unrecognized target of PPARγ activation and plays an essential role in PPARγ-reduced ER stress and inflammation.

Heme Oxygenase-1 Inhibits the Proliferation of Hepatic Stellate Cells by Activating PPARγ and Suppressing NF-κB

Background. Hepatic stellate cells (HSCs) are reported to play significant roles in the development of liver fibrosis. Heme oxygenase-1 (HO-1) is a key rate-limiting enzyme, which could decrease collagen synthesis and liver damage. Nevertheless, it was yet elusive towards the function and mechanism of HO-1. Methods. An HO-1 inducer Hemin or an HO-1 inhibitor ZnPP-IX was used to treat the activated HSC-T6, respectively. MTT assay was adopted to detect cell proliferation. Immunocytochemical staining was employed to test the levels of alpha-smooth muscle actin (α-SMA), peroxisome proliferator-activated receptor-γ (PPARγ), and nuclear factor-kappa B (NF-kappa B) levels in HSC-T6. HO-1, PPARγ, and NF-κB expression levels were measured by qRT-PCR and Western blotting. ELISA was then used to detect the levels of transforming growth factor- (TGF-) beta 1 (TGF-β1), interleukin-6 (IL-6), serum hyaluronic acid (HA), and serum type III procollagen aminopeptide (PIIIP). Results. HSC-T6 proliferation was inhibited in Hemin-treated HSCs. The levels of α-SMA, HA, and PIIIP and the production of ECM were lower in Hemin-treated HSCs, whereas those could be rescued by ZnPP-IX. NF-κB activation was decreased, but PPARγ expression was increased after HO-1 upregulation. Furthermore, the levels of TGF-β1 and IL-6, which were downstream of activated NF-κB in HSC-T6, were reduced. The PPAR-specific inhibitor GW9662 could block those mentioned effects. Conclusions. Our data demonstrated that HO-1 induction could inhibit HSC proliferation and activation by regulating PPARγ expression and NF-κB activation directly or indirectly, which makes it a promising therapeutic target for liver fibrosis.

Tanyu Tongzhi Formula Delays Atherosclerotic Plaque Progression by Promoting Alternative Macrophage Activation via PPARγ and AKT/ERK Signal Pathway in ApoE Knock-Out Mice

We previously demonstrated that the Tanyu Tongzhi Formula (TTF) significantly alleviated the clinical symptoms of patients with coronary heart disease and lowered serum lipid and inflammatory factor levels in patients with coronary heart disease and atherosclerosis model rats. However, the mechanism underlying TTF remains unknown. In this study, we examined the effect of TTF on atherosclerotic plaques in ApoE-/- mice and underlying mechanisms involved in macrophage polarization. Sixty male ApoE-/- mice were randomly divided into four groups. Mice in the control group were fed a regular diet, whereas experimental mice were fed a high-fat diet and received either saline (HFD group) or TTF at concentrations of 0.60 (TTF-L group) or 2.25 g/ml (TTF-H group) by daily oral gavage for 16 weeks. In the TTF-L and TTF-H groups, the levels of serum cholesterol, triglyceride, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were decreased, lipid content was significantly decreased, and percentage area of collagen/lipid increased in atherosclerotic plaque compared to in the HFD group. Moreover, we found TTF promoted the expression of alternative macrophage markers (Fizz1, Arg1, and Mrc) and suppressed the expression of M1 macrophage markers (TNF-α, IL-1β, and IL-6) by regulating peroxisome proliferator-activated receptor γ (PPARγ) expression and AKT/extracellular signal-regulated kinase (ERK) activation. We further investigated whether alternative macrophage was reduced when PPARγ was inhibited or the AKT/ERK signaling pathway was activated. TTF delayed atherosclerotic plaque progression by promoting alternative macrophage activation through increasing PPARγ expression and inhibiting AKT/ERK phosphorylation, providing a theoretical basis for its clinical application.

Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Background: Foam cells are markers of atherosclerosis and characterise advanced atherosclerotic plaque, stimulated by inflammation caused by high lipid levels in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition. Objective: we investigated the inhibiting role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor- (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages. Methods: Coffee and green tea were extracted by filtration and infusion respectively, and underwent decaffeination using active carbon and blanching methods, respectively. Cells were administered 160/160 and 320/320μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and the accumulation of lipids in macrophages with ELISA. Observations of lipid influx and efflux were determined through semiquantitative cluster differentiation 36 (CD36) and ATP binding cassette transporter A1 (ABCA1) expression through immunofluorescence. The inflammation process was quantified using inflammatory/anti-inflammatory markers, e.g., tumor necrosis factor α (TNFα) and interleukin 10 (IL10) with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with PCR and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR. Results: DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50ng/ml and oxLDL 50μg/ml. PPARγ expression (p=0.00) and activity (p=0.001), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased. Conclusion: DCGTE lowered foam cell numbers, possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms in M-CSF and oxLDL-stimulated Raw264.7 cells, via upregulation of PPARγ and miR-155. Our results suggest DCGTE may help prevent atherosclerosis-based diseases.

Hepatocarcinogenesis Prevention by Pirfenidone Is PPARγ Mediated and Involves Modification of Nuclear NF-kB p65/p50 Ratio

Targeted therapies for regulating processes such as inflammation, apoptosis, and fibrogenesis might modulate human HCC development. Pirfenidone (PFD) has shown anti-fibrotic and anti-inflammatory functions in both clinical and experimental studies. The aim of this study was to evaluate PPARγ expression and localization in samples of primary human tumors and assess PFD-effect in early phases of hepatocarcinogenic process. Human HCC tissue samples were obtained by surgical resection. Experimental hepatocarcinogenesis was induced in male Fischer-344 rats. TGF-β1 and α-SMA expression was evaluated as fibrosis markers. NF-kB cascade, TNFα, IL-6, and COX-2 expression and localization were evaluated as inflammation indicators. Caspase-3, p53, and PARP-1 were used as apoptosis markers, PCNA for proliferation. Finally, PPARα and PPARγ expression were evaluated to understand the effect of PFD on the activation of such pathways. PPARγ expression was predominantly localized in cytoplasm in human HCC tissue. PFD was effective to prevent histopathological damage and TGF-β1 and α-SMA overexpression in the experimental model. Anti-inflammatory effects of PFD correlate with diminished IKK and decrease in both IkB-phosphorylation/NF-kB p65 expression and p65-translocation into the nucleus. Pro-apoptotic PFD-induced effects are related with p53 expression, Caspase-3 p17 activation, and PARP-1-cleavage. In conclusion, PFD acts as a tumor suppressor by preventing fibrosis, reducing inflammation, and promoting apoptosis in MRHM.

Ginsenoside Re Improves Inflammation and Fibrosis in Hepatic Tissue by Upregulating PPARγ Expression and Inhibiting Oxidative Stress in db/db Mice

Ginsenoside Re (Re) is the main component of “Zhenyuan Capsule” (ZYC), which was wildly used in clinic in China for adjunctive treatment of coronary heart disease (CHD) and type II diabetes (T2DM). Nonalcoholic fatty liver disease (NAFLD) is one of the most important complications of T2DM, as well as an important risk factor of CHD. The aim of the present study was to investigate the effects of Re on NAFLD in db/db mice, one of the most recognized gene deficient animal models on T2DM. Sixteen db/db mice and sixteen wild-type mice were divided into four groups and orally administered Re or placebo in equal volume. According to the results, Re showed no obvious effect on blood glucose, lipids, or body weight of db/db mice. Histology pictures of hepatic tissue showed that Re did not improve steatosis, too. However, some evidence suggested that hepatic injury in db/db mice was attenuated by Re administering. Collagen deposition and aminotransferase elevation were significantly downregulated in the DB + Re group compared to those in the DB Group. The mechanisms of the protect effects of Re represented in db/db mice with NAFLD might be inhibiting oxidative stress and the reupregulation of peroxisome proliferator-activated receptor γ (pparγ) expression. The results of this study indicated that ZYC might be able to help T2DM patients with NAFLD to control the progress of NAFLD as an alternation of thiazolidinediones, synthetic agonists of PPARγ, whose side effects and adverse events should not be ignored.

Comparative Effects of Luteolin and Quercetin on Adipogenesis in 3T3-L1 Cells

Purpose: Quercetin has been reported as a more potent inhibitor of fat accumulation than other flavonoids. However, little information is available regarding the strength and mechanism of the repressive action of luteolin on fat accumulation. Therefore, the aim of the present study was to evaluate the comparative effects of luteolin and quercetin on the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Methods: 3T3-L1 preadipocytes were differentiated by treatment with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the presence of luteolin or quercetin. Alterations in triacylglycerol (TG) levels, lipid-filled adipocyte quantity, and the mRNA and protein expression levels of CCAAT-enhancer-binding protein α (C/EBPα) and peroxisome proliferator–activated receptor γ (PPARγ) were measured. Results: Both luteolin and quercetin reduced TG levels, the number of lipid-filled adipocytes, and the mRNA expression levels of C/EBPα and PPARγ; however, these effects occurred with lower concentrations of luteolin than quercetin. Conclusions: These results suggest that luteolin may be more potent than quercetin in inhibiting adipocyte differentiation. These effects may be explained by differences in the inhibitory effects of the two compounds on C/EBPα and PPARγ expression. This study suggests that luteolin might be a beneficial dietary supplement for obesity and lifestyle-related diseases.

PAK1 Silencing Attenuated Proinflammatory Macrophage Activation and Foam Cell Formation by Increasing PPARγ Expression

Macrophage polarization in response to environmental cues has emerged as an important event in the development of atherosclerosis. Compelling evidences suggest that P21-activated kinases 1 (PAK1) is involved in a wide variety of diseases. However, the potential role and mechanism of PAK1 in regulation of macrophage polarization remains to be elucidated. Here, we observed that PAK1 showed a dramatically increased expression in M1 macrophages but decreased expression in M2 macrophages by using a well-established in vitro model to study heterogeneity of macrophage polarization. Adenovirus-mediated loss-of-function approach demonstrated that PAK1 silencing induced an M2 macrophage phenotype-associated gene profiles but repressed the phenotypic markers related to M1 macrophage polarization. Additionally, dramatically decreased foam cell formation was found in PAK1 silencing-induced M2 macrophage activation which was accompanied with alternation of marker account for cholesterol efflux or influx from macrophage foam cells. Moderate results in lipid metabolism and foam cell formation were found in M1 macrophage activation mediated by AdshPAK1. Importantly, we presented mechanistic evidence that PAK1 knockdown promoted the expression of PPARγ, and the effect of macrophage activation regulated by PAK1 silencing was largely reversed when a PPARγ antagonist was utilized. Collectively, these findings reveal that PAK1 is an independent effector of macrophage polarization at least partially attributed to regulation of PPARγ expression, which suggested PAK1-PPARγ axis as a novel therapeutic strategy in atherosclerosis management.

Rosiglitazone requires hepatocyte PPARγ expression to promote steatosis in male mice with diet-induced obesity

Thiazolidinediones (TZD) are peroxisome proliferator-activated receptor γ (PPARγ agonists that could reduce hepatic steatosis through their effects in adipose tissue, and therefore have been assessed as potential therapies to treat non-alcoholic fatty liver disease (NAFLD) in humans. However, some studies suggest that expression and activation of hepatocyte PPARγ promotes steatosis and that would limit the benefits of TZD as a NAFLD therapy. To further explore this possibility, we examined the impact of short-term rosiglitazone maleate treatment after the development of moderate or severe diet-induced obesity, in both control and adult-onset hepatocyte-specific PPARγ knockout (Pparg △Hep) mice. Independent of the level of obesity and hepatic PPARγ expression, the TZD treatment enhanced insulin sensitivity, associated with an increase in white adipose tissue (WAT) fat accumulation, consistent with clinical observations. However, TZD treatment increased hepatic TG content, only in control mice with severe obesity. Under these conditions, Pparg △Hep reduced diet-induced steatosis and prevented the steatogenic effects of short-term TZD treatment. In these mice, subcutaneous WAT was enlarged and associated with increased levels of adiponectin, while hepatic levels of phosphorylated AMP-activated protein kinase (AMPK) were also increased. In addition, in mice with severe obesity, the expression of hepatic Cd36, Cidea, Cidec, Fabp4, Fasn, and Scd-1 was increased by TZD in a PPARγ-dependent manner. Taken together, these results demonstrate that hepatocyte PPARγ expression offsets the anti-steatogenic actions of TZD in mice with severe obesity. Therefore, in obese and insulin resistant humans, TZD-mediated activation of hepatocyte PPARγ may limit the therapeutic potential of TZD to treat NAFLD.