Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antiox-idant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four ex-perimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfu-sion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the op-eration. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a signif icant increase in serum AST and ALT levels (AST: 260.13 ± 40.17 U/L vs 186.00 ± 24.21 U/L, P < 0.01; ALT: 139.63 ± 11.35 U/L vs 48.38 ± 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 ± 40.17 U/L vs 216.63 ± 22.65 U/L, P < 0.05; ALT: 139.63 ± 11.35 U/L vs 97.63 ± 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In ad-dition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevat-ed liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Pachymic Acid Ameliorates Pulmonary Hypertension by Regulating Nrf2-Keap1-ARE Pathway

Objective:Pulmonary hypertension(PH)is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death.The purpose of this study was to investigate the mechanism by which pachymic acid(PA)pretreatment affects PH and pulmonary vascular remodeling in rats.Methods:PH was induced via hypoxia exposure and administration of PA(5 mg/kg per day)in male Sprague-Dawley rats.Hemodynamic parameters were measured using a right ventricular floating catheter and pulmonary vascular morphometry was measured by hematoxylin-eosin(HE),a-SMA and Masson staining.MTT assays and EdU staining were used to detect cell proliferation,and apoptosis was analyzed by TUNEL staining.Western blotting and immunohistochemistry were used to detect the expression of proteins related to the Nrf2-Keapl-ARE pathway.

Low- and high-dose hydrogen peroxide regulation of transcription factor NF-E2-related factor 2

Background Reactive oxygen species （ROS） may play both physiological and pathophysiological roles. Transcription factor NF-E2-related factor 2 （Nrf2） regulates antioxidant response element （ARE）-mediated genes expression and coordinates induction of chemoprotective proteins in response to physical and chemical stresses. The exact role of Nrf2 in cellular responses to different levels of oxidative stresses remains unknown. Methods Rat pulmonary microvascular endothelial cells were cultured and treated with 0 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L and 2.0 mmol/L hydrogen peroxide solution for 2 hours. Nrf2 gene expression was assayed by reverse transcription-PCR, Nrf2-ARE binding activity was assayed with electrophoretic mobility shift assay （EMSA）, and localization of Nrf2 was detected with immunohistochemistry. Results Low and moderate （0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L） doses hydrogen peroxide exposure of rat pulmonary microvascular endothelial cells led to the nuclear accumulation of Nrf2, increased activity of transcription regulation and up-regulation of ARE-medicated gene expression. In contrast, high doses of hydrogen peroxide （1 mmol/L 2 mmol/L） exposure of the cells led to the nuclear exclusion of Nrf2, decreased activity transcription regulation and down-regulation of ARE-mediated gene expression. Conclusion Low and moderate doses of hydrogen peroxide play protective roles by increasing transcription activity of Nrf2, whereas high- dose hydrogen peroxide plays a deleterious role by decreasing transcription activity of Nrf2.

Effects of extracts from soothing-liver and invigorating-spleen formulas on the injury induced by oxidative stress in the hepatocytes of rats with non-alcoholic fatty liver disease induced by high-fat diet

OBJECTIVE:To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress,mediated by the Nuclear factor-like 2(Nrf2)-Antioxidant response element(ARE) pathway,in the hepatocytes of rats with non-alcoholic fatty liver disease(NAFLD) induced by high-fat diet.METHODS:Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups:normal,model,soothing-liver formula group(SLG),invigorating-spleen formula group(ISG),integrated formula group(IG).The rat model of NAFLD was induced by feeding rats a high-fat diet(HFD).After 16 weeks,the hepatic tissue was examined following Haematoxylin-Eosin(H&E) staining and with Transmission electron microscopy(TEM).Levels of hepatic lipids,serum lipids and hepatic functions were measured using a biochemical analyser.Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry,cellular immunofluorescence and flow cytometry.The expression levels of Nrf2,Kelch-like epichlorohydrin-associated protein 1(Keap-1),haeme oxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase 1(NQO1) m RNAs were assessed by real-time fluorescence quantitative PCR.Nrf2,Keap-1,HO-1 and NQO1 proteins were measured by Western blotting.Finally,the levels of oxidative stress factors Superoxide Dismutase(SOD),malonaldehyde(MDA) and Glutathione peroxidase(GSH-Px) in hepatocytes were measured by WST-1,TBA and colorimetry.RESULTS:The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features.The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased.Compared with the model group,the levels of hepatic lipids,serum lipids and hepatic function in the treatment groups were ameliorated to different degrees.The yields of purified hepatocytes in each rat were 4-5 × 108.The viability of the isolated hepatocytes was higher than 95%,with a purity over 93.2%.Cellular immunohistochemistry analysis showed that the hepatocytes were brown,while in the cellular immunofluorescence analysis,the hepatocytes showed green fluorescence.The expression levels of Nrf2,Keap-1,HO-1 and NQO1 m RNA and protein in the hepatocytes were significantly higher in the model group than in the normal group(P < 0.05,P < 0.01).Compared with the model group,the expression of Nrf2,Keap-1,HO-1 and NQO-1 m RNAs and proteins in all treatment groups increased,especially in the IG(P < 0.01).CONCLUSION:The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway,which may be the mechanism having the potential for prevention and treatment of NAFLD.