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Figure 4 | Molecular Medicine

Figure 4

From: p38 Mitogen-Activated Protein Kinase and Liver X Receptor-α Mediate the Leptin Effect on Sterol Regulatory Element Binding Protein-1c Expression in Hepatic Stellate Cells

Figure 4

p38 MAPK mediates leptin reduction of LXRα protein level and activity and LXRα binding to SREBP-1c promoter and the effect of leptin-induced inhibition of SREBP-1c expression on alpha1(I) collagen transcription in cultured HSCs. (A) Western blot analysis of LXRα protein level. HSCs were pretreated with SB203580 (SB) at different doses for 30 min before incubation with or without leptin (100 ng/mL) for an additional 24 h (n = 3). (B) Transfection assay for evaluating LXR activity. HSCs were cotransfected with pLXRE-Luc, pwtP38 or pdnP38 and the empty vector and treated with or without 100 ng/mL of leptin for 24 h (n = 6). The empty vector was used to ensure the equal amount of total DNA in transfection assay. *P < 0.05 versus the control (the first column on the left). **P > 0.05 versus cells without pwtP38, pdnP38 and leptin (the first column on the right). (C) ChIP assay for examining the binding of LXRα to SREBP-1c promoter. After HSCs were maintained in DMEM containing the adipogenic differentiation cocktail (MDI: 0.5 mmol/L isobutylmethylxanthine, 1 µmol/L dexamethasone and 1 µmol/L insulin) with 0.4% FBS for 24 h, cells were switched to the medium with or without SB203580 (10 µmol/L) and pretreated for 30 min before incubation with or without 100 ng/mL leptin for an additional 24 h. Chromatin fragments were immunoprecipitated by anti-LXRα antibody. Real-time PCR analysis was used to quantified a 260-bp segment of rat SREBP-1c promoter (n = 3). *P < 0.05, #P > 0.05 versus cells without treatment. **P < 0.05 versus cells with leptin alone (the second column on the left). (D) Real-time PCR analysis of alpha1(I) collagen mRNA level. HSCs in a 25-cm2 flask were transfected with 8 µg plasmid ptwist2 or empty vector. The empty vector was used to ensure the equal amount of total DNA. After incubation with MDI for 24 h, cells were switched to the medium with or without SB203580 (10 µmol/L) and pre-treated for 30 min before incubation with or without leptin (100 ng/mL) for an additional 24 h (n = 3). *P < 0.05, #P > 0.05 versus cells with no treatment (the first column on the left). **P < 0.05, #P > 0.05 versus cells with leptin alone (the second column on the left). ***P < 0.05 versus cells with SB plus leptin (the third column on the left).

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