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Peroxisome proliferator-activated receptors gama ameliorates liver fibrosis in non-alcoholic fatty liver disease by inhibiting TGF-β/Smad signaling activation
doi: 10.2478/fzm-2024-0002
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Abstract:
Background Nonalcoholic fatty liver disease (NAFLD) is a chronic condition characterized by a progressive decline in liver function, leading to disruptions in liver integrity and metabolic function, resulting in lipid deposition and excessive accumulation of extracellular matrix (ECM). The pathogenesis of NAFLD is complex and not yet fully understood, contributing to the absence of specific therapeutic strategies. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor pivotal in regulating lipid and glucose metabolism. However, the impacts of PPARγ on NAFLD remains insufficiently explored. Thus, this study aimed to investigate the role of PPARγ in NAFLD and its underlying molecular mechanisms. Methods Chemical detection kits were utilized to quantify collagen content, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) level variations. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess alterations in extracellular matrix-related genes and inflammatory response genes in liver tissue and HepG2 cells, while western blotting was conducted to analyze the levels of both PPARγ and the TGF-β/Smad signaling pathway. Results Our findings unveiled significantly reduced PPARγ expression in a rat model of NAFLD, leading to subsequent activation of the TGF-β/Smad signaling pathway. Furthermore, PPARγ activation effectively mitigated NAFLD progression by inhibiting inflammation and fibrosis-related gene expression and collagen production. On a cellular level, PPARγ activation was found to inhibit the expression of extracellular matrix-related genes such as matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9), along with inflammatory response genes interleukin (IL)-1β and IL-6. Additionally, PPARγ activation led to a significant decrease in the levels of ALT and AST. At the molecular level, PPARγ notably down-regulated the TGF-β/Smad signaling pathway, which is known to promote liver fibrosis. Conclusion These groundbreaking findings underscore PPARγ activation as a promising therapeutic approach to delay NAFLD progression by targeting the TGF-β/Smad signaling pathway in hepatic cells. This highlights the potential of PPARγ as a promising therapeutic target for NAFLD management in clinical settings. -
Key words:
- NAFLD /
- PPARγ /
- TGF-β/Smad /
- liver fibrosis
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Figure 1. Fibrosis and collagen deposition in a rat model of nonalcoholic fatty liver disease (NAFLD). (A) Triglycerides (TG), total cholesterol (TCH), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) concentrations in the control and NAFLD groups. (N = 4-6), *P < 0.05, **P < 0.01, vs. Control. (B) The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the control and NAFLD groups. (N = 5-6), **P < 0.01, vs. Control. (C) Collagen contents in the liver homogenization samples, indicating collagen deposition in liver tissues in the control and NAFLD groups. (N = 3), **P < 0.01, vs. Control. (D) Oil red O staining of tissue sections showing the lipid deposition in the liver. Scale bar indicates 200 μm. (N = 3). (E) type Ⅰ collagens (Col-1), type Ⅲ collagens (Col-3), matrix metalloproteinase 2 (MMP2), and matrix metalloproteinase 9 (MMP9) mRNA expression in the control and NAFLD groups. (N = 3-11), **P < 0.01, vs. Control. Data are presented as means ± SEM.
Figure 2. The expression of peroxisome proliferator-activated receptor gamma (PPARγ) was decreased in nonalcoholic fatty liver disease (NAFLD). (A, B) Relative mRNA and protein levels of PPARγ in the control and NAFLD groups. (N = 9), **P < 0.01, vs. Control. (C) Immunofluorescence results indicating the expression of PPARγ in the control and NAFLD groups. Scale bars: 100 μm.
Figure 3. Effect of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on hepatic fibrosis in nonalcoholic fatty liver disease (NAFLD). (A) Relative protein levels of PPARγ in the control, free fatty acid (FFA), and FFA+Rosiglitazone groups. (N = 6), *P < 0.05, **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (B) Immunofluorescence results indicating the expression of PPARγ in the control, FFA, and FFA+Rosiglitazone groups. Scale bars: 50 μm. (C, D) ALT and AST concentrations in the control, FFA, and FFA+Rosiglitazone groups. (N = 5), *P < 0.05, **P < 0.01, vs. Control; #P < 0.05, vs. FFA. (E) Collagen contents in the control, FFA, and FFA+Rosiglitazone groups. (N = 5), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (F-I) Col-1, Col-3, matrix metalloproteinase 2 (MMP2) and MMP9 mRNA levels in the control, FFA, and FFA+Rosiglitazone groups. (N = 4-5), *P < 0.05, **P < 0.01, vs. Control; #P < 0.05, ##P < 0.01, vs. FFA. (J) The mRNA levels of connective tissue growth factor (CTGF) in the control, FFA, and FFA+Rosiglitazone groups. (N = 4), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (K) Tumor necrosis factor-α (TNF-α) mRNA levels in the control, FFA, and FFA + Rosiglitazone groups. (N = 4), **P < 0.01, vs. Control; #P < 0.05, vs. FFA. (L)Interleukin (IL)-1β mRNA level in the control, FFA, and FFA + Rosiglitazone groups. (N = 4), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (M) The mRNA levels of IL-6 in the control, FFA, and FFA + Rosiglitazone groups. (N = 4), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. Data are presented as means ± SEM.
Figure 4. Changes of the transforming growth factor (TGF)-β/Smad signaling pathway in nonalcoholic fatty liver disease (NAFLD). (A) Protein–protein interaction (PPI) network of peroxisome proliferator-activated receptor gamma (PPARγ), TGF-β, Smad2, Smad3, and Smad7 exported from STRING database (
https://string-db.org ) (version 12.0). (B, C) Relative mRNA and protein levels of TGF-β in the control and NAFLD groups. (N = 4-5), **P < 0.01, vs. Control. (D) Relative protein levels of smad7 in each group. (N = 5-8). **P < 0.01, vs. Control. (E) Total smad2/3 protein levels in each group. (N = 3). (F) Phosphorylated protein levels of smad2/3 in each group. (N = 10), **P < 0.01, vs. Control. Data are presented as means ± SEM.
Figure 5. Peroxisome proliferator-activated receptor gamma (PPARγ) inhibits the activation of the transforming growth factor (TGF)-β/Smad axis in nonalcoholic fatty liver disease (NAFLD). (A) PROMO database showing the sequences of the potential binding sites in the proximal region (2000 bp upstream) of TGF-β promoters. (B, C) Relative mRNA and protein levels of TGF-β in the control, free fatty acid (FFA), and FFA+Rosiglitazone groups. (N = 4-5), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (D) Relative protein levels of Smad7 in the control, FFA, and FFA+Rosiglitazone groups. (N = 4), **P < 0.01, vs. Control; ##P < 0.01, vs. FFA. (E, F) Relative protein levels Smad2/3 and p-Smad2/3 expression in the control, FFA, and FFA+Rosiglitazone groups. (N = 3-4), **P < 0.01, vs. Control; #P < 0.05, vs. FFA. Data are presented as means ± SEM.
Figure 6. Effect of peroxisome proliferator-activated receptor gamma (PPARγ) on the transforming growth factor (TGF)-β/Smad signaling pathway. (A) The mRNA expression levels of Col-1, Col-3, matrix metalloproteinase 2 (MMP2) and MMP9 in the control, rosiglitazone and GW9662 groups. (N = 3-5), *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, vs. Rosiglitazone. (B) The protein levels of PPARγ in the control, rosiglitazone, and GW9662 groups. (N = 3), *P < 0.05, vs. Control; #P < 0.05, vs. Rosiglitazone. (C, D) Relative TGF-β mRNA and protein levels measured by real-time PCR and western blot analysis, respectively, in the control, rosiglitazone, and GW9662 groups. (N = 4-6), *P < 0.05, vs. Control; #P < 0.05, vs. Rosiglitazone. (E) Relative protein levels of Smad7 in the control, rosiglitazone, and GW9662 groups. (N = 3), *P < 0.05, vs. Control; #P < 0.05, vs. Rosiglitazone. (F) Total smad2/3 protein in the control, rosiglitazone and GW9662 groups. (N = 3). (G) Phosphorylation of smad2/3 in the control, rosiglitazone, and GW9662 groups. (N = 4), *P < 0.05, vs. Control; #P < 0.05, vs. Rosiglitazone. Data are presented as means ± SEM.
Figure 7. Schematic diagram of the regulation of PPARγ on nonalcoholic fatty liver disease.
Table 1. The sequences of primers for used for qRT-PCR in this study.
Gene Forward primer Reverse primer PPARγ ACCACTCCCATTCCTTTG CACAGACTCGGCACTCG TGF-β GCGAGCGAAGCGACGAGGAG TGGGCGGGATGGCATCAAGGTA Col-1 CAATGGCACGGCTGTGTGCG CACTCGCCCTCCCGTCTTTGG Col-3 TGAATGGTGGTTTTCAGTTCAG GATCCCATCAGCTTCAGAGACT MMP2 AGCTTTGATGGCCCCTATCT GGAGTGACAGGTCCCAGTGT MMP9 CACTGTAACTGGGGGCAACT CACTTCTTGTCAGCGTCGAA IL-1β CCCTGCAGCTGGAGAGTGTGG TGTGCTCTGCTTGAGAGGTGCT IL-6 TTCCTACCCCAACTTCCAATG ATGAGTTGGATGGTCTTGGTC TNFα CCTCTCTCTAATCAGCCCTCTG GAGGACCTGGGAGTAGATGAG CTGF CAGCATGGACGTTCGTCTG AACCACGGTTTGGTCCTTGG Timp1 CTTCTGCAATTCCGACCTCGT ACGCTGGTATAAGGTGGTCTG GAPDH AAGAAGGTGGTGAAGCAGGC TCCACCACCCAGTTGCTGTA PPARγ, peroxisome proliferator-activated receptor gamma; TGF-β, transforming growth factor beta 1; Col-1, collagen, type Ⅰ, alpha 1; Col-3, collagen type Ⅲ alpha 1; MMP2, matrix metallopeptidase 2; MMP9, matrix metallopeptidase 9; IL-1β, interleukin 1 beta; IL-6, interleukin 6; TNFα, tumor necrosis factor-α; CTGF, connective tissue growth factor; Timp1, Tissue inhibitor of metalloproteinases-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 
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