The M1/M2 Macrophage Polarization and Hepatoprotective Activity of Quercetin in Cyclophosphamide-Induced Experimental Liver Toxicity

Abstract

Background: Chemotherapy drugs may lead to hepatic injury, which is considered one of the limitations of these drugs.

Objectives: The aim of this study was to evaluate the effect of quercetin (QUE) on M1/M2 macrophage polarization and hepatoprotective effect in cyclophosphamide (CTX)-induced liver toxicity.

Methods: Twenty-four mice were divided into four groups (Control, QUE, CTX, CTX + QUE). The CTX and CTX + QUE groups received 200 mg/kg CTX. The animals in the QUE and CTX + QUE groups received 50 mg/kg QUE. All animals were sacrificed, and serum and liver samples were used for laboratory analyses.

Results: Examinations indicated that CTX exposure led to disruption of liver functions and morphological degenerations. Tissue pro-apoptotic Bax and caspase 3, pro-inflammatory TNF-α and IL-1β, transcription factor NF-κB, and M1 macrophage polarization marker CD86 were upregulated significant (p < 0.05) in this group. In addition, CTX exposure led to significantly (p < 0.05) upregulation of the Bax/Bcl-2 mRNA ratio and DNA fragmentations. The PCNA-positive hepatic cell ratio and anti-apoptotic Bcl-2 expression are remarkably suppressed (p < 0.05). Immunohistochemical analyses are also indicated that M2 macrophage polarization marker CD163 is slightly but remarkably (p < 0.05) downregulated in the CTX group compared to the Control and QUE groups. The morphological and biochemical disruptions were alleviated in QUE-treated animals in the CTX + QUE group. Liver function test results, apoptosis, inflammatory, transcription factor NF-κB, regeneration/proliferation, and apoptotic index results in this group were similar (p > 0.05) to the control and QUE groups. The M1 cell surface marker expression of CD86 is significantly (p < 0.05) downregulated, and M2 macrophage polarization marker expression of CD163 is upregulated significantly (p < 0.05) compared to the CTX group.

Conclusions: This study indicates that QUE has the potential to downregulate CTX-induced hepatic injury and regulate M1/M2 macrophage polarization to the M2 side, which indirectly demonstrates activation of anti-inflammatory signalling and tissue repair.

Keywords: apoptosis; cyclophosphamide; inflammation; liver toxicity; quercetin.

FIGURE 1

Representative micrographs of the groups. Irregular and oedematous parenchyma (*), increased pyknotic cell nuclei (arrow), and increased inflammatory cell accumulation in the CTX group. Pathological changes significantly improved in QUE‐treated animals. A: Control, B: QUE, C: CTX, D: CTX + QUE. Staining: Haematoxylin and eosin. Magnification: 40×.

FIGURE 2

Representative immunohistochemistry micrographs of Bax, Bcl‐2, caspase 3, TNF‐α, PCNA, NF‐κB, CD86 and CD163. Brown staining indicates the immunopositivity and density of the protein of interest. The sections were counterstained to determine the immunodensity of the interest protein. Counterstaining: Haematoxylin. Bar: 50 µm.

FIGURE 3

Representative micrographs of TUNEL Assay of the groups. Green fluorescence nuclei indicate double‐strand DNA break. All nuclei were counterstained with DAPI, and the micrographs were merged to obtain distribution of DNA fragmented hepatocyte ratio over total nuclei in hepatic lobules. Staining: TUNEL Assay (fluorescein), Counterstain: DAPI, Magnification: 40×.

FIGURE 4

Graphical demonstration of the statistical analysis of tissue TNF‐α, IL‐1β levels of ELISA Assay, tissue immunodensity of Bax, Bcl‐2, caspase 3, TNF‐α, positivity of PCNA and apoptotic index among the groups. In addition to, graphs indicate statistically significance of Bax/Bcl‐2 mRNA ratio among the groups. Different superscripts on each or between the columns indicate differences between the groups. *p < 0.05, **p < 0.01.