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. 2024 Jan 27;16(1):75-90.
doi: 10.4254/wjh.v16.i1.75.

Rifaximin on epigenetics and autophagy in animal model of hepatocellular carcinoma secondary to metabolic-dysfunction associated steatotic liver disease

Matheus Truccolo Michalczuk  1   2 , Larisse Longo  2   3 , Melina Belén Keingeski  2   3 , Bruno de Souza Basso  3 , Gabriel Tayguara Silveira Guerreiro  2   3 , Jessica T Ferrari  2 , José Eduardo Vargas  4 , Cláudia P Oliveira  5 , Carolina Uribe-Cruz  2   3   6 , Carlos Thadeu Schmidt Cerski  2   7 , Eduardo Filippi-Chiela  2   8   9 , Mário Reis Álvares-da-Silva  1   2   3   10
Affiliations

Rifaximin on epigenetics and autophagy in animal model of hepatocellular carcinoma secondary to metabolic-dysfunction associated steatotic liver disease

Matheus Truccolo Michalczuk et al. World J Hepatol. .

Abstract

Background: Prevalence of hepatocellular carcinoma (HCC) is increasing, especially in patients with metabolic dysfunction-associated steatotic liver disease (MASLD).

Aim: To investigate rifaximin (RIF) effects on epigenetic/autophagy markers in animals.

Methods: Adult Sprague-Dawley rats were randomly assigned (n = 8, each) and treated from 5-16 wk: Control [standard diet, water plus gavage with vehicle (Veh)], HCC [high-fat choline deficient diet (HFCD), diethylnitrosamine (DEN) in drinking water and Veh gavage], and RIF [HFCD, DEN and RIF (50 mg/kg/d) gavage]. Gene expression of epigenetic/autophagy markers and circulating miRNAs were obtained.

Results: All HCC and RIF animals developed metabolic-dysfunction associated steatohepatitis fibrosis, and cirrhosis, but three RIF-group did not develop HCC. Comparing animals who developed HCC with those who did not, miR-122, miR-34a, tubulin alpha-1c (Tuba-1c), metalloproteinases-2 (Mmp2), and metalloproteinases-9 (Mmp9) were significantly higher in the HCC-group. The opposite occurred with Becn1, coactivator associated arginine methyltransferase-1 (Carm1), enhancer of zeste homolog-2 (Ezh2), autophagy-related factor LC3A/B (Map1 Lc3b), and p62/sequestosome-1 (p62/SQSTM1)-protein. Comparing with controls, Map1 Lc3b, Becn1 and Ezh2 were lower in HCC and RIF-groups (P < 0.05). Carm1 was lower in HCC compared to RIF (P < 0.05). Hepatic expression of Mmp9 was higher in HCC in relation to the control; the opposite was observed for p62/Sqstm1 (P < 0.05). Expression of p62/SQSTM1 protein was lower in the RIF-group compared to the control (P = 0.024). There was no difference among groups for Tuba-1c, Aldolase-B, alpha-fetoprotein, and Mmp2 (P > 0.05). miR-122 was higher in HCC, and miR-34a in RIF compared to controls (P < 0.05). miR-26b was lower in HCC compared to RIF, and the inverse was observed for miR-224 (P < 0.05). There was no difference among groups regarding miR-33a, miR-143, miR-155, miR-375 and miR-21 (P > 0.05).

Conclusion: RIF might have a possible beneficial effect on preventing/delaying liver carcinogenesis through epigenetic modulation in a rat model of MASLD-HCC.

Keywords: Animal model; Autophagy; Epigenetic; Hepatocellular carcinoma; Metabolic dysfunction-associated steatotic liver disease; Rifaximin.

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Conflict of interest statement

Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.

Figures

Figure 1
Figure 1
Experimental design. The control group (n = 8) received a standard diet, water without diethylnitrosamine (DEN) and gavage with vehicle (Veh) solution; hepatocellular carcinoma-group (n = 7) received a high-fat and choline-deficient (HFCD) diet, water with DEN and gavage with Veh solution; rifaximin (RIF)-group (n = 7) received a HFCD diet, water with DEN and gavage with RIF. After all the animals were euthanized. DEN: Diethylnitrosamine; HCC: Hepatocellular carcinoma; HFCD: High-fat and choline-deficient; RIF: Rifaximin; Veh: Vehicle.
Figure 2
Figure 2
Hepatic gene expression of autophagy markers. A: Autophagy-related factor LC3A/B; B: P62/sequestosome-1; C: Beclin-1; D: Enhancer of zeste homolog-2; E: Coactivator associated arginine methyltransferase-1. Data expressed as median (25th-75th percentile), Kruskal-Wallis test. Different letters indicate a significant difference between groups (P < 0.05). aP < 0.05, bP < 0.05. Different letters indicate a significant difference between groups. Becn1: Beclin-1; Carm1: Coactivator associated arginine methyltransferase-1; Ezh2: Enhancer of zeste homolog-2; HCC: Hepatocellular carcinoma; Map1 Lc3b: Autophagy-related factor LC3A/B; p62/Sqstm1: Sequestosome-1; RIF: Rifaximin.
Figure 3
Figure 3
Protein concentration of autophagy markers. A: Protein expression bands obtained through the Western blot technique; B: p62/SQSTM1 expression (densitometry analysis); C: MAP1LC3B expression (densitometry analysis). Data expressed as median (25th-75th percentile), Kruskal-Wallis test. Different letters indicate a significant difference between groups (P < 0.05). aP < 0.05, bP < 0.05. Different letters indicate a significant difference between groups. HCC: Hepatocellular carcinoma; MAP1LC3B: Autophagy-related factor LC3A/B, p62/SQSTM1: Sequestosome-1; RIF: Rifaximin.
Figure 4
Figure 4
Histological evaluation of hepatic tissue in different experimental groups and percentage description for the presence of microvesicular steatosis, macrovesicular steatosis, hypertrophy, inflammation, fibrosis and hepatocellular carcinoma. A and B: The images correspond to hematoxylin & eosin (H&E) and picrosirius red staining’s in the control group, showing a normal hepatic parenchyma with no significant changes; C and D: In the hepatocellular carcinoma (HCC)-group, animals exhibited macrovesicular steatosis and hypertrophy; E and F: Tumoral lesions with multiple fibrosis septa were observed in the HCC-group and rifaximin-group, as evidenced by H&E and picrosirius stained slides, respectively; G: Percentage description for the presence of microvesicular steatosis, macrovesicular steatosis, hypertrophy, inflammation, fibrosis, and HCC. Images A-D are magnified at 100 ×, and images E-F are magnified at 40 ×. HCC: Hepatocellular carcinoma; H&E: Hematoxylin & eosin; RIF: Rifaximin.
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