| (Nrf2) is an essential transcription factor that regulates an array of detoxifying and antioxidant defense genes expression in the liver. It is activated in response to electrophiles and induces its target genes by binding to the antioxidant response element (ARE).Therefore, the roles of the Nrf2-ARE pathway in liver diseases have been extensively investigated. Therefor we established two models to observe the role of Nrf2:(1) Non-alcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice by high fat diet feeding and liver fat-lowering effect of exogenous estrogen was subsequently assessed. (2) Wild-type and Nrf2-null mice were subjected to bile duct ligation (BDL) or sham operation. Various assessments were performed at different time point following surgery.Part I Nrf2 deficiency results in amplification of liver fat-lowering effect of estrogenBackground and AimsTranscription factor nuclear factor-E2-related factor 2 (Nrf2) regulates multiple biological processes including hepatic lipid metabolism Increasing evidence indicates the crosstalk between these two molecules. The aim of the study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism.Methods1. Animals model of NAFLD induced by HFD and estrogen treatment Twenty-five Nrf2+/+ and Twenty-five Nrf2-/- male mice were of a C57BL6/129SV mixed background were used for the study. To induce NAFLD, mice were given a high fat diet (HFD) (TD.88137, Harlan Laboratories, Madison, WI) for 16 weeks. Following that, mice received vehicle (sesame oil), estradiol-17? (E2,25 ?g/mouse, Sigma-Aldrich, St. Louis, MO) (Bourassa, Milos et al.199 progesterone (1 mg/mouse, Sigma-Aldrich, St. Louis, MO) (Walter, Rogers et al.2005), or a combination of these two steroid hormones (25 ?g of E2 and 1 mg of progesterone/mouse) subcutaneously once daily for three weeks, while they 6), were continuously fed the HFD. Animals were sacrificed four hours after the last dosing. Liver tissues were stained with hematoxylin-eosin (HE) and Oil O Red stain for histopathologic examination.2. The protein expression of FABP5 and TFF3 were measured by Immunohistochemical analysesParaffin sections were deparaffinized in xylene and rehydrated. Nonenzymatic antigen retrieval was done by heating the sections to 121 ? in sodium citrate buffer for 10min. Primary Abs used in this study included Fatty acid binding protein 5 (FABP5, Abcam,USA) and Trefoil factor family (TFF3,Sigma, USA). Finally, the sections were developed with 3,3'-diaminobenzidine counterstained with hematoxylin, and mounted under cover slips.3. Measurement of liver lipid contentHepatic triglyceride, total cholesterol, and free fatty acid were quantified using commercially available kits (ab65336, ab65359, ab65341, Abeam, Cambridge, MA) according to the manufacturer's instruction.4. Real-time quantitative RT-PCR was used to detect the mRNA level in hepatocytesTaqMan Universal PCR Master Mix and the primers and TaqMan MGB probes of mouse Nrf2 (Mm00477786_ml), NQO1 (MmO1253561_ml), ERa (Mm00433149_ml), ER? (Mm00599821_ml), TFF3 (Mm00495590_ml), FABP5 (Mm00783731_s1), and albumin (Mm00802090_ml) were purchased from Applied Biosystems (Foster City, CA). The amplification reactions were carried out with the ABI Prism 7900 sequence detection system (Applied Biosystems) with initial hold steps (50? for 2 min followed by 95? for 10 min) and 40 cycles of a 2-step PCR (92? for 15 seconds and 60? for 1 min).5. The protein expression of NQO1,-ER?,EABP5 were measured by Western Blot AnalysisLiver homogenates (10?g) were separated by polyacrylamide gel electrophoresis under reducing conditions. Proteins from the gels were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Antibodies against NQO1 (ab80588, Abcam), ERa (SC-514910, Santa CruzBiotechnology), FABP5 (GTX12109, GeneTex), and GAPDH (SC-130656, Santa Cruz Biotechnology) were used as probes. Immune complexes were detected using the enhanced chemiluminescence system (Pierce, Rockford, IL).Results1. Nrf2 absence results in strikingly enhanced liver fat-lowering effect of estrogen in mice with NAFLDThe animals were sacrificed at the end of 19 week. Livers were immediately excised and weighed. We found that, HFD induced liver enlargement and massive hepatic fat accumulation in both wild-type and Nrf2-null mice in vehicle control group. In comparison to vehicle control, exogenous estrogen resulted in decreases in liver size and hepatic fat deposition in both genotype groups of mice, exerting a liver fat-lowering effect as demonstrated by others. Notably, histological assessment revealed that this effect was evidently enhanced when Nrf2 was lacking, which was manifested by further reduction in liver fat accumulation in Nrf2-null mice. To further demonstrate the above findings, we performed Oil Red O staining on liver sections to visualize fat deposition. As a result, compared to vehicle control, estrogen administration reduced liver fat staining in wild-type mice and, strikingly, almost diminished that in Nrf2-null mice.2. Nrf2 absence causes TFF3 and FABP5 a marked increase in exogenous estrogen-induced hepatic estrogen signaling in mice with NAFLD Immunohistochemical analysis showed compared with normal chew feeding, HFD feeding increased hepatic FABP5 and TFF3 protein expression in both two genotypes. Remarkably, Nrf2 absence causes TFF3 and FABP5 a marked increase in exogenous estrogen-induced hepatic estrogen signaling in mice with NAFLD.3. Nrf2 absence results in hepatic triglyceride, free fatty acids and total cholesterol decrease effect of estrogen in mice with NAFLDRelative to normal chew, HFD induced excessive accumulation of triglyceride, total cholesterol, and free fatty acids in the liver, which were equivalent quantitatively between wild-type and Nrf2-null mice. In wild-type mice, relative to vehicle control, exogenous estrogen decreased hepatic triglyceride and free fatty acids, but did not significantly affect total cholesterol. When Nrf2 was absent, exogenous estrogen reduced hepatic triglyceride, free fatty acids and total cholesterol.4. Nrf2 absence causes the level of mRNA changed in exogenous estrogen-induced hepatic estrogen signaling in mice with NAFLDIn wild-type mice, relative to normal chew feeding, HFD feeding highly activated Nrf2, which was manifested upregulation in Nrf2 and NQO1 gene expression respectively. In wild-type mice with NAFLD, compared to vehicle control, estrogen administration did not significantly alter Nrf2, while marginally increased NQO1, transcript level.In normal chew-fed mice, Nrf2 deficiency caused ER? and TFF3 mRNA expression increase. Compared with normal chew feeding, HFD feeding increased and reduced hepatic ERa mRNA expression in wild-type and Nrf2-null mice respectively, resulting in equivalent ERa mRNA levels between the two genotypes. Remarkably, relative to vehicle control in wild-type mice, E2 administration elevated hepatic TFF3 mRNA expression by 122 fold in the presence of Nrf2 and by 480-fold in the absence of Nrf2.Conclusions1. Nrf2 absence results in strikingly enhanced liver fat-lowering effect of estrogen in mice with NAFLD.This finding indicates that Nrf2 counteracts with estrogen signaling in hepatic lipid metabolism.2. Nrf2 absence causes TFF3 mRNA and protein a marked increase in exogenous estrogen-induced hepatic estrogen signaling in mice with NAFLD.3. Nrf2 absence results in hepatic triglyceride, free fatty acids and total cholesterol decrease effect of estrogen in mice with NAFLD.4. Nrf2 absence causes the level of FABP5 mRNA and protein remarkable increased in exogenous estrogen-induced hepatic estrogen signaling in mice with NAFLD.Part? Nrf2 Deficiency Results in Expansion of CD133+Hepatocytic-appearing Liver Parenchymal Cells during CholestasisBackground and AimsTranscription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a central regulator of cellular defense against oxidative stress and inflammation and is also involved in regulating liver regeneration. CD133 is widely as markers for hepatic progenitor cells, however whether Nrf2 signal can repair cholestasis liver injury and regulat the expression of CD 133 is unclear. The aim of the study is to evaluate whether Nrf2 mediates hepatic repair response during cholestasis.Metheds5. Animals model of BDLFifty Nrf2+/+ and Fifty Nrf2-/- male mice were randomly divided into the BDL group and the control group. Standard rodent chow and water were provided ad libitum throughout the entire feeding period. The surgical procedures of BDL and sham operation were performed under aseptic conditions. Mice were anesthetized with inhaled anesthetic agent isoflurane. The common bile duct was ligated with two ligatures separated by 2 mm. A cut was made between the two ligatures. Control mice underwent a sham operatiotion that consisted of exposure but no ligation of the common bile duct. Mice were sacrificed at various time points after the surgeries for collecting livers. Livers were immediately excised and weighed.6. The protein expression of Ki67, Laminin, CK19 and CD133 were measured by Immunohistochemical analysesParaffin sections were deparaffinized in xylene and rehydrated. Nonenzymatic antigen retrieval was done by heating the sections to 121 ? in sodium citrate buffer for 10min. Primary Abs used in this study included Ki67 (BS1454, Bioworld), laminin (L-9393, Sigma-Aldrich), CD133 (14-1331, eBioscience), and CK19 (SC-33111, Santa Cruz Biotechnology). Finally, the sections were developed with 3,3'-diaminobenzidine counterstained with hematoxylin, and mounted under cover slips.7. The protein expression of NQO1, CD133, CK19 were measured by Western Blot AnalysisLiver homogenates (10 ?g) were separated by polyacrylamide gel electrophoresis under reducing conditions. Proteins from the gels were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Antibodies against NQO1 (2618-1, Epitomics), CK19 (SC-33111, Santa Cruz), CD133 (PAB12663, Abnova), and glyceraldehyde 3-phosphate dehydrogenase (GADPH) (SC-25778, Santa Cruz Biotechnology) were used as probes. Immune complexes were detected using the enhanced chemiluminescence system.Results1. Nrf2 deficiency does not cause increased liver injury after BDLThe lack of Nrf2 does not significantly affect BDL-induced liver size adjustment, hepatocyte proliferation, and hepatic fibrotic response.2. BDL indices persistent activation of hepatic Nrf2NQO1 is a typical Nrf2 target gene and has been shown to be solely regulated by Nrf2 in BDL-induced cholestasis. Our study found that hepatic NQO1 protein expression was increasingly elevated as the cholestasis progressed in wild-type mice, which was fully prevented due to Nrf2 absence. The finding indicates that hepatic Nrf2 exhibits persistent and strong activation in response to cholestasis.3. The lack of Nrf2 results in massive expansion of CD133+ hepatocyte-like cells after BDLImmunohistochemical and western blot analysis both showed that genetic deletion of Nrf2 causes marked expansion of hepatocyte-like CD133+ cells during cholestasis, revealing Nrf2 as a critical regulator of liver progenitors.Conclusions1. Nrf2 deficiency does not cause increased liver injury after BDL.2. We found that hepatic NQO1 protein expression was increasingly elevated as the cholestasis progressed in wild-type mice, which was fully prevented due to Nrf2 absence. The finding indicates that BDL indices persistent activation of hepatic Nrf2.3. The lack of Nrf2 results in massive expansion of CD133+hepatocyte-like cells after BDL revealing Nrf2 as a critical regulator of liver progenitors. |
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