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. 2017 Jun 20;8(25):40982-40993.
doi: 10.18632/oncotarget.17334.

Dietary luteolin attenuates chronic liver injury induced by mercuric chloride via the Nrf2/NF-κB/P53 signaling pathway in rats

Haili Zhang  1 , Xiao Tan  1 , Daqian Yang  1 , Jingjing Lu  1 , Biying Liu  1 , Ruiqi Baiyun  1 , Zhigang Zhang  1
Affiliations

Dietary luteolin attenuates chronic liver injury induced by mercuric chloride via the Nrf2/NF-κB/P53 signaling pathway in rats

Haili Zhang et al. Oncotarget. .

Abstract

Mercury exposure is a common cause of metal poisoning which is biotransformed to highly toxic metabolites thus eliciting biochemical alterations and oxidative stress. Luteolin, a phenolic compound found in many natural products, has multiple biological functions. Our study was aimed to explore the biological effects of luteolin in a liver injury model induced in rats by mercuric chloride (HgCl2). Criteria for injury included liver enzyme, glutathione and malondialdehyde levels, histopathology, TUNEL assay, hepatocyte viability and reactive oxygen species levels. The results showed that luteolin protected against HgCl2-induced liver injury. Luteolin increased total nuclear factor-erythroid-2-related factor 2 (Nrf2) levels in the presence of HgCl2. Upregulation of its downstream factors, heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1, was also observed. This suggested that protection by luteolin against HgCl2-induced liver injury involved Nrf2 pathway activation. Luteolin also decreased expression of nuclear factor-κB (NF-κB) and P53. HgCl2 exposure led to increased Bcl-associated X protein (Bax), and decreased Bcl-2-related protein long form of Bcl-x (Bcl-xL) and B-cell leukemia/lymphoma-2 (Bcl-2) expression, leading to an increased Bax/Bcl-2 ratio. Taken together, our data suggested that decreasing oxidative stress is a protective mechanism of luteolin against development of HgCl2-induced liver injury, through the Nrf2/NF-κB/P53 signaling pathway in rats.

Keywords: HgCl2; NF-κB; Nrf2; hepatotoxicity; luteolin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
(A and B) illustrate AST and ALT activities respectively in normal and experimental groups of rats. HgCl2 administration increased ALP and ALT levels compared to the normal, while treatment with luteolin significantly restored this change. Data are expressed as means ± SEM (n = 8). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 2
Figure 2. Effects of luteolin given to rats prior to HgCl2 administration on concentration of MDA and GSH in liver tissue
Results of MDA activity (A) GSH activity (B) and GSH/GSSG ratio (C) in the liver are expressed as means ± SEM (n = 8). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 3
Figure 3. The histological of livers changed after different treatments
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Typical images were chosen from each experimental group (original magnification 200×): (A) control group. (B) Rats treated with luteolin. (C) Rats treated with HgCl2. (D) Rats treated with the luteolin and HgCl2.
Figure 4
Figure 4. Apoptosis index was determined using TUNEL assays
(A) Representative photographs of TUNEL staining in control, LUT, HgCl2 and HgCl2 + LUT groups. Scale bar = 20 μm. (B) Quantitative analysis of TUNEL-positive cells. Luteolin treatment significantly decreased the percentage of hepatocyte apoptosis after HgCl2 treatment. Data are expressed as means ± SEM (n = 8). * P < 0.05 compared to rhe control group; #P < 0.05 compared to HgCl2-treated group.
Figure 5
Figure 5
(A) The survival cells were determined by CCK-8 assay. (B) The levels of intracellular ROS in hepatocytes were determined by DCF-DA as described in Material and Methods. The results are expressed in percentage of control and presented as the means ± SEM (n = 6). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 6
Figure 6. RT-PCR analysis of FoxO3 gene expression levels after HgCl2 treatment and/or luteolin and β-actin was used as an internal control
Data are expressed as means ± SEM (n = 4). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 7
Figure 7. Treatment with luteolin activated the Nrf2 pathway and protected the liver against HgCl2-induced injury
(A) The protein levels of Nrf2, HO-1 and NQO1. (B, C and D) Quantifed protein levels were shown. GAPDH was used as an internal control. Data are expressed as means ± SEM (n = 4). *P < 0.05 compared to the control group; #P < 0.05 compared to the HgCl2-treated group.
Figure 8
Figure 8
(A) Western blot to evaluate the expression levels of Bcl-xl (B), Bcl-2 (C), Bax (D) and Bax/Bcl-2 ratio (E) in liver tissue and gray value analysis. GAPDH was used as an internal control and data are expressed as means ± SEM (n = 4). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 9
Figure 9
(A) Effects of luteolin on P53, NF-κB and TNF-α activaties induced by HgCl2 in the liver. These activities were detected by immunoblotting and GAPDH was used as loading control. (B, C and D) Quantifed protein levels were shown. Data are expressed as means ± SEM (n = 4). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 10
Figure 10. Mercury content analysis was done in the liver and mercury exposed rats as described in materials and methods
Tissues were digested in HNO3, followed by analysis by atomic fluorescence spectrometry. Data are expressed as means ± SEM (n = 8). *P < 0.05 compared to the control group; #P < 0.05 compared to HgCl2-treated group.
Figure 11
Figure 11. Summary indicating the mechanisms of luteolin attenuated liver injury induced by HgCl2
Hg(II) promoted ROS formation in cytoplasm inducing oxidative stress, finally led to apoptosis. However, luteolin triggered the activation of NF-κB/Nrf2/P53 signaling pathway and prevented liver injury induced by Hg(II). Green line denotes stimulatory or inhibitory effect of luteolin, and red line denotes inhibitory effect.
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