Ginkgo biloba regulate Nrf2-Keap1-ARE signaling pathway to ameliorate liver injury in endemic arsenic poisoning rats induced by coal burning / 中华地方病学杂志

ABSTRACT

Objective:To explore the effects of Ginkgo biloba on regulating NF-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway in liver injury induced by coal-burning-borne endemic arsenic poisoning in rats.Methods:Group design method was adopted, according to body weight (80-100 g), a total of 30 Wistar rats were divided into 5 groups (6 rats in each group, half males and half females) by random number table method. The normal control group was fed with normal diet ad libitum for 4.5 months; the Ginkgo biloba control group was fed with Ginkgo biloba (25 mg/kg, 6 d/week) for 1.5 months after normal feeding for 3 months; the drinking water arsenic poisoning group and the arsenic contaminated grain group were fed respectively with 100 mg/L arsenic trioxide (As 2O 3) solution and 100 mg/kg arsenic-containing feed for 3 months, and then fed with normal diet for 1.5 months; the Ginkgo biloba treatment group was fed with 100 mg/kg arsenic-containing feed for 3 months, and then was given Ginkgo biloba (25 mg/kg, 6 d/week) for 1.5 months. After sacrificing the animals, the content of malondialdehyde (MDA), the activity of copper zinc superoxide dismutase (SOD1) and the activity of glutathione peroxidase (GPx) in serum were detected by thiobarbituric acid colorimetry, xanthine oxidase method and dimercaptodinitrobenzoic acid reduction method, respectively. The mRNA and protein expressions of indicator genes of Nrf2-Keap1-ARE signaling pathway in liver tissues were detected by quantitative real-time PCR, immunohistochemistry and Western blotting. Correlation between the indexes was analyzed by Pearson. Results:In drinking water arsenic poisoning group, arsenic contaminated grain group and Ginkgo biloba treatment group, the contents of MDA in serum were (3.54±0.51), (3.83±0.87) and (2.93±0.84) μmol/L, respectively, which were higher than that in normal control group [(1.85±0.36) μmol/L, P < 0.05]; and SOD1 activities [(68.21±4.37), (64.53±9.96), (73.09±5.43) U/ml] and GPx activities [(486.41±40.45), (458.24±42.25), (539.79±79.43) U/L] in serum were lower than those in normal control group [(81.47±5.73) U/ml, (747.86±80.33) U/L, P < 0.05]. Compared with the arsenic contaminated grain group, the content of MDA in serum in Ginkgo biloba treatment group was decreased, the activities of SOD1 and GPx in serum were increased ( P < 0.05). Compared with normal control group, the mRNA expressions of SOD1 and GPx1 in the liver tissues in drinking water arsenic poisoning group, arsenic contaminated grain group and Ginkgo biloba treatment group were significantly higher ( P < 0.05). Compared with arsenic contaminated grain group, the mRNA expressions of SOD1 and GPx1 in the liver tissue in Ginkgo biloba treatment group were increased ( P < 0.05). Compared with the normal control group, the protein expression of SOD1 in liver tissue in arsenic contaminated grain group was decreased ( P < 0.05), the protein expressions of GPx1 were decreased in the liver tissues in drinking water arsenic poisoning group, arsenic contaminated grain group and Ginkgo biloba treatment group ( P < 0.05). Compared with the arsenic contaminated grain group, the protein expressions of SOD1 and GPx1 were increased in the liver tissue in Ginkgo biloba treatment group ( P < 0.05). Compared with the normal control group and arsenic contaminated grain group, the protein expression of Keap1 was decreased in the liver tissue in Ginkgo biloba treatment group ( P < 0.05). Compared with the normal control group, the protein expressions of Nrf2 and phosphorylation of Nrf2 (pNrf2) were increased in the cytoplasm in drinking water arsenic poisoning group, arsenic contaminated grain group and Ginkgo biloba treatment group ( P < 0.05). Compared with the arsenic contaminated grain group, the protein expression of pNrf2 was decreased in the cytoplasm in Ginkgo biloba treatment group ( P < 0.05). The protein expressions of Nrf2 and pNrf2 in the nucleus in drinking water arsenic poisoning group, arsenic contaminated grain group and Ginkgo biloba treatment group were also higher than those in normal control group ( P < 0.05). Compared with the arsenic contaminated grain group, the protein expressions of Nrf2 and pNrf2 were increased in the nucleus in Ginkgo biloba treatment group ( P < 0.05). The results of correlation analysis revealed that the protein expressions of Nrf2 and pNrf2 in the nucleus were negatively correlated with Keap1 protein expression ( r=-0.523,-0.401, P < 0.05), and positively correlated with the mRNA expressions of SOD1 and GPx1 ( r=0.658, 0.530, 0.555, 0.603, P < 0.05). In addition, the protein expressions of SOD1 and GPx1 were positively correlated with their enzyme activities ( r=0.472, 0.629, P < 0.05). Conclusions:Arsenic could induce oxidative stress and liver injury. Ginkgo biloba could reduce the protein expression of Keap1, and promote nuclear translocation of Nrf2, which might induce the up-regulation of mRNA expressions of SOD1 and GPx1, and partially reverse the posttranscriptional regulation of arsenic on SOD1 and GPx1, and then increase their protein expressions and enzyme activities, thereby improve arsenic induced oxidative stress and liver injury.