Investigation of HO-1 Regulation of Liver Fibrosis Related to Nonalcoholic Fatty Liver Disease Through the SIRT1/TGF-ß/Smad3 Pathway

Abstract

Background and aims: Heme oxygenase 1 (HO-1) has an influential yet insufficiently investigated effect on Sirtuin 1 (SIRT1), a histone deacetylase activated by nicotinamide adenine dinucleotide, which may impact the transforming growth factor-β (TGF-ß)/Smad3 pathway in nonalcoholic fatty liver disease (NAFLD)-related liver fibrosis. This study aimed to elucidate the regulation of NAFLD-related liver fibrosis induced by HO-1 through the SIRT1/TGF-ß/Smad3 pathway.

Methods: HO-1 induction and inhibition were established in C57BL/6J mice fed a methionine- and choline-deficient (MCD) diet. Additionally, wild-type mice were fed either a normal diet or an MCD diet. Hematoxylin and eosin, Masson's trichrome, and Sirius Red staining were used to assess hepatic steatosis, inflammation, and fibrosis. In vitro, plasmid overexpression and small interfering RNA silencing of HO-1 were performed in LX-2 cells. Cell viability was assessed using the Cell Counting Kit-8, and apoptosis was evaluated via terminal deoxynucleotidyl transferase dUTP nick-end labeling and immunofluorescence. Flow cytometry was employed to assess apoptosis and reactive oxygen species production. Western blot and real-time quantitative reverse transcription polymerase chain reaction were used to analyze the mRNA and protein expression of genes related to HO-1, SIRT1, the TGF-ß signaling pathway, and fibrosis.

Results: MCD-fed mice developed significant liver damage, including steatosis, inflammatory infiltration, and pericellular fibrosis. Zinc protoporphyrin treatment exacerbated these conditions. Corroborating these findings, silencing HO-1 in LX-2 cells increased the expression of fibrosis-related genes. Furthermore, HO-1 overexpression not only increased SIRT1 expression but also reduced the activity of key regulatory factors in the TGF-ß signaling pathway, suggesting a potential interaction between HO-1 and the SIRT1/TGF-ß pathway.

Conclusions: HO-1 inhibits the activation of the TGF-ß/Smad3 pathway in NAFLD-related liver fibrosis through SIRT1. These findings provide insights into new therapeutic strategies for treating NAFLD-associated liver fibrosis.

Keywords: HO-1; Heme oxygenase-1; Non-alcoholic steatohepatitis-related liver fibrosis; SIRT1; Sirtuin 1; TGF-ß pathway.

Fig. 1. Mice fed with MCD diet developed steatohepatitis-related liver fibrosis.

(A) liver /body weight. (B) Body weight. Serum (C) ALT and (D) AST in each group. (E) Images of H&E (×200 magnification) staining of representative liver sections and (F) the NAFLD activity score. (G) Images of Masson (×200 magnification) staining of representative liver sections and (H) the Metavir score. (I) Images of Sirius (×200 magnification) red staining of representative liver sections and (J) Relative collagen content (%). Values are the mean ± SD (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MCD, methionine-and-choline-deficient diet; NAFLD, nonalcoholic fatty liver disease; H&E, hematoxylin-eosin. ALT, alanine aminotransferase; AST, aspartate transaminase; Znpp, zincprotoporphyria.

Fig. 2. HO-1 agonist ameliorates steatohepatitis-related liver fibrosis in MCD diet-fed mice.

(A–F) The expression levels of HO-1, SIRT1, TGF-β, Smad3, Collagen1 and α-SMA were measured by RT-qPCR. (G) Immunofluorescence (×200 magnification) double staining between of HO-1 and SIRT1 in liver tissue. (H–K) Immunofluorescence double staining semi-quantitative analysis. GAPDH served as the loading control. Data are presented as representative results of three independent experiments. Values are the mean ± SD (n = 6 per group). ***p < 0.001, ****p < 0.0001. MCD, methionine-and-choline-deficient diet; HO-1, heme oxygenase 1; SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Znpp, zincprotoporphyria.

Fig. 3. HO-1 overexpression upregulates SIRT1 expression and exacerbates liver fibrosis in MCD mice.

(A, C–G) Western blot analysis of HO-1, SIRT1, TGF-β, P-Smad2/3, and Smad2/3 in liver tissue. (B, H, I) Western blot analysis of α-SMA and Collagen1 in liver tissue. Values are the mean ± SD (n = 6 per group). **p < 0.01, ***p < 0.001, ****p < 0.0001. MCD, methionine-and-choline-deficient diet; HO-1, heme oxygenase 1; SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Znpp, zincprotoporphyria.

Fig. 4. HO-1 mediates NAFLD-related liver fibrosis in vitro by regulating the SIRT1/TGF-β/Smad3 signaling pathway.

(A, B) Western blot analysis of HO-1 overexpression and silencing in LX2 cells. (C, D) Western blot analysis of LX2 cells treated with SIRT1 activators and inhibitors. (E–I) Western blot analysis of SIRT1, TGF-β, P-Smad2/3, Smad2/3 in LX2 cells. (J–L) Western blot analysis of α-SMA and Collagen I in LX2 cells. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MCD, methionine-and-choline-deficient diet; HO-1, heme oxygenase 1; SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OE HO-1, overexpressing HO-1; SiHO-1, small interfering RNA silencing of HO-1.

Fig. 5. SIRT1 inhibits the activation of hepatic stellate cells.

(A–C) Immunofluorescence (×200 magnification) double staining of SIRT1 and α-SMA in LX2 cells. (D–E) Immunofluorescence (×200 magnification) showing α-SMA expression in LX2 cells. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; OE HO-1, overexpressing HO-1; SiHO-1, small interfering RNA silencing of HO-1; DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 6. The effects of HO-1 knockdown and overexpression, SIRT1 activation, and inhibition on LX2 cell proliferation, apoptosis, and ROS production.

(A, B) The proportion of apoptotic cells was evaluated by flow cytometry. (C) The CCK-8 assay was used to detect cell viability. (D,E) The ROS production was analyzed using flow cytometry. *p < 0.05, ****p < 0.0001. ROS, reactive oxygen species; SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; OE HO-1, overexpressing HO-1; SiHO-1, small interfering RNA silencing of HO-1.

Fig. 7. SIRT1 promotes apoptosis in LX2 cells.

(A, B) Detection of apoptotic cells by TUNEL assay (×200 magnification) following treatment with SIRT1 agonist and inhibitor. (C, D) Apoptotic cells were detected by TUNEL assay (×200 magnification). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SIRT1, Sirtuin 1; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; OE HO-1, overexpressing HO-1; SiHO-1, small interfering RNA silencing of HO-1; DAPI, 4′,6-diamidino-2-phenylindole.