The role of Sirtuin 1 in regulation of fibrotic genes expression in pre-adipocytes

Abstract

Purpose: Considering inhibition of pre-adipocyte cells differentiation in adipose tissue fibrosis, we aimed to explore whether Sirt1 and Hif-1α in pre-adipocytes have a significant effect on fibrotic gene expression.

Methods: 3T3-L1 pre-adipocytes were transfected with SIRT1-specific siRNA, confirmed by real-time polymerase chain reaction (RT-PCR) and western blotting. Additionally, cells were treated with varying concentrations of resveratrol and sirtinol as the activator and inhibitor of Sirt1, respectively. Involvement of Hif-1α was evaluated by treatment with echinomycin. Subsequently, we assessed the gene and protein expressions related to fibrosis in the extracellular matrix of adipose tissue, including collagen VI (Col VI), lysyl oxidase (Lox), matrix metalloproteinase-2 (Mmp-2), Mmp-9, and osteopontin (Opn) in pre-adipocytes through RT-PCR and western blot.

Results: The current study demonstrated that Sirt1 knockdown and reduced enzyme activity significantly increased the expression of Col VI, Lox, Mmp-2, Mmp-9, and Opn genes in the treated 3T3-L1 cells compared to the control group. Interestingly, resveratrol significantly decreased the gene expression related to the fibrosis pathway. Inhibition of Hif-1α by echinomycin led to a significant reduction in Col VI, Mmp-2, and Mmp-9 gene expression in the treated group compared to the control.

Conclusion: This study highlights that down-regulation of Sirt1 might be a predisposing factor in the emergence of adipose tissue fibrosis by enhancing the expression of extracellular matrix (ECM) components. Activation of Sirt1, similar to suppressing of Hif-1α in pre-adipocytes may be a beneficial approach for attenuating fibrotic gene expression.

Supplementary information: The online version contains supplementary material available at 10.1007/s40200-024-01389-4.

Keywords: Echinomycin; Fibrosis; Knockdown; Pre-adipocytes; Resveratrol; Sirtinol; Sirtuin 1; siRNA.

Fig. 1

Transfection efficiency of PEI- FAM-labeled siRNA complexes. Percentage of 3T3L-1 cells transfected with PEI-siRNA complexes (A) compared to control group (B) PEI: polyethyleneimine, siRNA, small interfering RNA, cy3: Cyanine 3

Fig. 2

The Sirt1 levels in transfected 3T3L1 cell line with siRNA-Sirt1. The Sirt1 mRNA 3 replicates (A) and protein 2 replicates (B). Results are presented as mean ± SD of at least two replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

The genes and protein expression related to fibrosis in transfected 3T3-L1 cells with siRNA-SIRT1 compared to the control group. The results of ColVI (A), Lox (B), Mmp-2(C), Mmp-9(D), and Opn (E) mRNA levels obtained by real-time PCR and ColVI (F) and Opn (G) protein levels obtained by western blot in cells transfected with 40 nm of siRNA for 72 h after 2 h of starvation. The results are presented as mean ± SD of at least three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 4

The effect of inhibition of Sirt1 activity by Sirtinol on gene and protein levels of adipose tissue ECM in treated pre-adipocyte cells compared to untreated control. The results of ColVI (A), Lox (B), Mmp-2(C), Mmp-9 (D), and Opn (E) mRNA levels and ColVI (F) and Opn (G) protein levels in 3T3-L1 cells treated with 25 μm of Sirtinol for 36 h after 2 h of starvation. The results are presented as mean ± SD of at least three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 5

Effects of enhanced Sirt1 activity on gene and protein levels in 3T3-L1 cells stimulated or not with resveratrol. Quantification of ColVI (A), Lox (B), Mmp-2 (C), Mmp-9 (D), and Opn (E) mRNA levels and ColVI (F) and Opn (G) protein levels in 3T3-L1 cells treated with 40 μm of Resveratrol for 24 h after 2 h of starvation. The results are presented as mean ± SD of at least three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 6

The effect of inhibition of Hif-1α activity by Echinomycin on protein and mRNA levels of adipose tissue ECM in treated 3T3-L1 cells compared to the control group. The results of ColVI (A), Lox (B), Mmp-2 (C), Mmp-9 (D), and Opn (E) mRNA and ColVI (F) and Opn (G) protein levels in 3T3-L1 cells treated with 10 nm of Echinomycin for 12 h after 2 h of starvation. The results are presented as mean ± SD of at least three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001