Bilirubin, once a toxin but now an antioxidant alleviating non-alcoholic fatty liver disease in an autophagy-dependent manner in high-fat diet-induced rats: a molecular and histopathological analysis

Abstract

Background and purpose: As an endogenous antioxidant, bilirubin has surprisingly been inversely correlated with the risk of non-alcoholic fatty liver disease (NAFLD). Thereupon, the current evaluation was designed to assess the positive effects of bilirubin on the autophagy flux, as well as the other pathogenic processes and parameters involved in the expansion of NAFLD.

Experimental approach: Thirty adult male rats weighing 150-200 g with free access to sucrose solution (18%) were randomly subdivided into 5 groups (n = 6). Subsequently, the animals were euthanized, and their blood specimens and liver tissue samples were collected to measure serum biochemical indices, liver histopathological changes, intrahepatic triglycerides content, and tissue stereological alterations. Furthermore, the expression levels of autophagy-related genes (Atgs) were measured to assess the state of the autophagy flux.

Findings/results: Fasting blood glucose, body weight, as well as liver weight, liver-specific enzyme activity, and serum lipid profile indices markedly decreased in rats that underwent a six-week bilirubin treatment compared to the control group. In addition, histopathological studies showed that hepatic steatosis, fibrosis, inflammation, and necrosis significantly decreased in the groups that received bilirubin compared to the control animals. Bilirubin also caused significant alterations in the expression levels of the Atgs, as well as the Beclin- 1 protein.

Conclusion and implication: Bilirubin may have potential ameliorative effects on NAFLD-associated liver damage. Moreover, the beneficial effects of bilirubin on intrahepatic lipid accumulation and steatosis were comparable with the group that did not ever receive bilirubin.

Keywords: Autophagy; Bilirubin; Inflammation; Non-alcoholic fatty liver disease.

Fig. 1

Mean body weight of rats after 14 weeks of treatment. Normal rats were considered the control group. Values are expressed as mean ± SD. ^**P < 0.01 and ^***P < 0.001 indicate significant differences compared to the control group; ###P < 0.001 versus the HFD group. HFD, High-fat diet; BR, bilirubin.

Fig. 2

Comparison of the relative mRNA expression levels of Atgs. Hepatic mRNA expression levels of (A) Atg3; (B) Atg5; (C) Atg6; and (D) Atg7 were determined by quantitative real-time polymerase chain reaction and normalized to β-actin expression levels. Values are expressed as mean ± SD, n = 6. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 indicate significant differences compared to the control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus the HFD group. HFD, High-fat diet; BR, bilirubin.

Fig. 3

The effects of bilirubin on Atg6 (Beclin-1) protein expression levels in different rat groups were detennined by western blotting and normalized to GAPDH expression levels. (A) Western blot bands; (B) densitometric findings of western blotting analysis. HFD, High-fat diet; BR, bilirubin.

Fig. 4

Volumetric parameters of the liver’s structures. (A) Hepatocyte and (B) Sinusoids. Values are expressed as mean ± SD, n = 6. ^*P < 0.05 and ^**P < 0.01 indicate significant differences compared to the control group; ^##P < 0.01 versus the HFD group. HFD, High-fat diet; BR, bilirubin.

Fig. 5

Evaluation of the quantitative parameters that are responsible for the number of cells and nuclei inside the liver. (A) Hepatocytes’ nuclei and (B) Kupffer cells. Values are expressed as mean ± SD, n = 6. ^*P < 0.05 and ^***P < 0.001 indicate significant differences compared to the control group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus the HFD group. HFD, High-fat diet; BR, bilirubin.

Fig. 6

Histopathological effects of HFD and bilirubin therapy on the liver tissue. Microscopic images (magnification: 40 ×) of H&E-stained liver tissue sections from HFD, HFD-BR6, and HFD-BR14 groups demonstrate congestion, hepatocellular steatosis, inflammation, and stage 3 fibrosis in comparison with the liver sections of the rats of the control group. BR14 rats only demonstrated low degrees of inflammation and congestion compared to the control group. The steatosis, as well as the lobular inflammation, hepatocellular ballooning, and fibrotic lesions, were found to be improved in rats who received bilirubin (HFD-BR6 and HFD-BR14). Scale bar = 50 μm. HFD, High-fat diet; BR, bilirubin.