Cyanate Induces Oxidative Stress Injury and Abnormal Lipid Metabolism in Liver through Nrf2/HO-1

Abstract

Chronic kidney disease (CKD) is problem that has become one of the major issues affecting public health. Extensive clinical data suggests that the prevalence of hyperlipidemia in CKD patients is significantly higher than in the general population. Lipid metabolism disorders can damage the renal parenchyma and promote the occurrence of cardiovascular disease (CVD). Cyanate is a uremic toxin that has attracted widespread attention in recent years. Usually, 0.8% of the molar concentration of urea is converted into cyanate, while myeloperoxidase (MPO) catalyzes the oxidation of thiocyanate to produce cyanate at the site of inflammation during smoking, inflammation, or exposure to environmental pollution. One of the important physiological functions of cyanate is protein carbonylation, a non-enzymatic post-translational protein modification. Carbamylation reactions on proteins are capable of irreversibly changing protein structure and function, resulting in pathologic molecular and cellular responses. In addition, recent studies have shown that cyanate can directly damage vascular tissue by producing large amounts of reactive oxygen species (ROS). Oxidative stress leads to the disorder of liver lipid metabolism, which is also an important mechanism leading to cirrhosis and liver fibrosis. However, the influence of cyanate on liver has remained unclear. In this research, we explored the effects of cyanate on the oxidative stress injury and abnormal lipid metabolism in mice and HL-7702 cells. In results, cyanate induced hyperlipidemia and oxidative stress by influencing the content of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), superoxide dismutase (SOD), catalase (CAT) in liver. Cyanate inhibited NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and the phosphorylation of adenosine 5'monophosphate-activated protein kinase (AMPK), activated the mTOR pathway. Oxidative stress on the cells reduced significantly by treating with TBHQ, an antioxidant, which is also an activator of Nrf2. The activity of Nrf2 was rehabilitated and phosphorylation of mTOR decreased. In conclusion, cyanate could induce oxidative stress damage and lipid deposition by inhibiting Nrf2/HO-1 pathway, which was rescued by inhibitor of Nrf2.

Keywords: HL-7702; Nrf2/ ho-1; ROS; cyanate; lipid metabolism.

Figure 1

Cyanate decreased body weight and caused dyslipidemia. (A) Mice body weights decreased by drinking 1% cyanate water. (B)The levels of ALT, AST, ALP increased in cyanate group (p < 0.05). (C) The levels of CHE and LDH decreased in cyanate group. The levels of total cholesterol, high-density lipoprotein increased in cyanate group (p < 0.05). (D) The changes of TBIL, DBIL, IBIL were not statistically significant. (E) The level of HDL had decreased, and the levels of TC, LDL were increased in cyanate group (p < 0.05).

Figure 2

Cyanate reduced the antioxidant capacity of the liver. (A) SOD activity decreased significantly after cyanate stimulation (p < 0.01). (B) The activity of CAT decreased after cyanate treatment (p < 0.01). (C) The activity of MDA increased significantly after cyanate stimulation (p < 0.01). (D) After cyanate treatment, the content of NO increased (p < 0.01). n ≥ 8 per group, and the experiment was repeated three times. Values are means and standard errors (* p < 0.05 and ** p < 0.01 versus control).

Figure 3

Cyanate caused liver injury and lipid accumulation. (A) Hematoxylin and eosin staining of liver tissues. (B) PAS staining of liver tissues. (C) MASSON staining of liver tissues. (D) Oil Red O staining of liver tissues. (E,F) Western blotting showed the levels of HMGCR and LDLR proteins in the liver of mice treated with or without cyanate (p < 0.01). n ≥ 8 per group, and the experiment was repeated three times. Values are means and standard errors (* p < 0.05 and ** p < 0.01 versus control).

Figure 4

Cyanate decreased the expression of Nrf2 in the Liver. (A,B) The levels of Nrf2 and HO-1 in the liver were significantly decreased in the cyanate group. (C,D) The levels of p-AMPK decreased and p-mTOR, p-S6K, and p-S6 increased in liver after mice treated with cyanate. n ≥ 8 per group, and the experiment was repeated three times. Values are means and standard errors (* p < 0.05 and ** p < 0.01 versus control).

Figure 5

Cyanate decreased ROS levels and lipid deposition in HL-7702 cell. (A) HL-7702 cells were incubated with increasing concentrations of cyanate (0, 0.5, 1, and 2 µM) for 24 h. Cell Counting Kit-8 (CCK-8) assay was performed to detect the cytotoxic effect of cyanate. (B) Morphological changes of cells under inverted microscope. (C) Oil Red O staining of HL-7702 cells after being treated with cyanate for 24 h. The results showed that the cell lipid droplets increased significantly after 24 h of cyanate treatment, and the lipid droplets of the cells treated with cyanate were reduced after pre-treatment with TBHQ for 2 h before exposure to cyanate. (D)The results of DCFH-DA assay showed that the intracellular ROS increased significantly after 24 h of cyanate treatment, while there were normal ROS level in the cyanate-treated cells by TBHQ pre-treatment for 2 h before exposure to cyanate. (E) The ultrastructure of the cells was observed under an electron microscope.

Figure 5

Cyanate decreased ROS levels and lipid deposition in HL-7702 cell. (A) HL-7702 cells were incubated with increasing concentrations of cyanate (0, 0.5, 1, and 2 µM) for 24 h. Cell Counting Kit-8 (CCK-8) assay was performed to detect the cytotoxic effect of cyanate. (B) Morphological changes of cells under inverted microscope. (C) Oil Red O staining of HL-7702 cells after being treated with cyanate for 24 h. The results showed that the cell lipid droplets increased significantly after 24 h of cyanate treatment, and the lipid droplets of the cells treated with cyanate were reduced after pre-treatment with TBHQ for 2 h before exposure to cyanate. (D)The results of DCFH-DA assay showed that the intracellular ROS increased significantly after 24 h of cyanate treatment, while there were normal ROS level in the cyanate-treated cells by TBHQ pre-treatment for 2 h before exposure to cyanate. (E) The ultrastructure of the cells was observed under an electron microscope.

Figure 6

TBHQ alleviates oxidative stress caused by cyanate in HL-7702 cells. (A,B) TBHQ can rescue decreased levels of CAT and SOD in HL-7702 cells caused by cyanate. (C,D) TBHQ can rescue increased MDA and NO content in HL-7702 cells caused by cyanate. Each group was a mixture of cells collected three times (n = 3) and the experiment was repeated three times. Values are means and standard errors (* p < 0.05 and ** p < 0.01 versus control).

Figure 7

TBHQ rescue the inhibition of the activity of the Nrf2 pathway and the activation of the mTOR pathway caused by cyanate. (A,B) The western blotting results showed that the expression levels of Nrf2, Keap1, and HO-1 in cytoplasmic protein. (C,D) The results of western blotting showed the relative expression of AMPK and mTOR pathway in cytoplasmic protein. (E,F) The results of immunofluorescence showed that TBHQ regulates the inhibition of the activity of the Nrf2 and HO-1 caused by cyanate in HL-7702 cell. (G) Mean fluorescence intensity of Nrf2 and HO-1 was quantified and is presented as means ± SD. Each group was a mixture of cells collected three times (n = 3) and the experiment was repeated three times. Values are means and standard errors (* p < 0.05 and ** p < 0.01 versus control).

Figure 8

Model of Nrf2 signaling cascade. Cyanate may reduce the release of Nrf2 into the nucleus by inhibiting the uncoupling of the Nrf2-Keap1 complex, thus the expression of antioxidant enzymes (including HO-1) and a series of detoxifying substances are reduced, resulting in excessive release of ROS. Further impairs mitochondrial damage and abnormal energy metabolism. This results in a decrease in the level of p-AMPK and activation of the mTOR pathway, causing lipid deposition to further damage the liver. More importantly, these signaling pathways play a key role in oxidative stress and inhibition of lipid accumulation.