SOCS2 Suppresses Inflammation and Apoptosis during NASH Progression through Limiting NF-κB Activation in Macrophages

Figures

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  Figure 1. SOCS2 in macrophages was decreased in macorphages during NASH progression and negative related to NASH level. A. The heatmap from RNA-Seq of human liver macrophages with NASH (n=5) or Normal (n=5). B. The related markers expression were detect by RT-qPCR in human liver macrophages with NASH (n=6) or Normal (n=6). C. The related mRNA level of SOCS2 was detected by RT-qPCR in liver tissue macrophage samples of human subjects without steatosis, with simple steatosis and with NASH. D. Immunoblotting of SOCS2 and β-actin (loading control throughout) in liver tissue macrophage samples of human subjects without steatosis (n=3), with simple steatosis (n=3) and with NASH (n=3). E. Pearson comparison analyses of the correlation between SOCS2 mRNA level and NASH (r = -0.9471), BMI (r = -0.8542), serum TG content (r = -0.8908), serum γ-GT concentrations (r = -0.8406), serum AST concentrations (r = -0.9207) and serum ALT concentrations (r = -0.9112) (n = 153). P < 0.0001 for all of these correlations by Spearman′s rank correlation coefficient analysis.

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  Figure 2. SOCS2 suppresses inflammation via limiting the activation of NF-κB signaling pathway. A. The related protein and mRNA level of SOCS2 was detected by Western blot and RT-PCR in RAW cell lines overexpreesing SOCS2 (RAW-Ctrl/ SOCS2+). B. The related protein and mRNA level of SOCS2 was detected by Western blot and RT-PCR in RAW cell lines knockdown SOCS2 (RAW-Ctrl/ shSOCS2). C. The related mRNA level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by RT-PCR in RAW-Ctrl or -SOCS2+ or -shSOCS2 treated with FFAs for 24 hr. D. The level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by ELISA in RAW-Ctrl or -SOCS2+ or -shSOCS2 treated with FFAs for 24 hr. E. Immunoblotting of phospho-IKKα, IKKα, phospho-IKKβ, IKKβ, phospho-P65, P65, SOCS2, and β-actin (loading control) in RAW-Ctrl or -SOCS2+ or -shSOCS2 treated with FFAs for 24 hr. F. The levels of IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18 in cell culture supernatants from RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or PDTC for 24 hr were detected by ELISA.

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  Figure 3. SOCS2 in macrophages suppresses inflammation via limiting the activation of inflammasomes signaling pathway. A. Immunoblotting of NLRP3, Pro-Caspase-1, Caspase-1 p20/p22 and β-actin (loading control) in RAW-Ctrl or -SOCS2+ or -shSOCS2 treated with FFAs for 24 hr. B. The levels of IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18 in cell culture supernatants from RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or MCC950 for 24 hr were detected by ELISA. C. The related mRNA level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by RT-PCR in RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or MCC950 for 24 hr were detected by ELISA.

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  Figure 4. SOCS2 in macrophages suppresses apoptosis via limiting the activation of NF-κB signaling pathway. A. Immunoblotting of BAX, BCL-2, SOCS2 and β-actin (loading control) in RAW-Ctrl or -SOCS2+ or -shSOCS2 treated with FFAs for 24 hr. B. The level of apoptosis of RAW-Ctrl / SOCS2+ treated with FFAs or PDTC for 24 hr, detected by Flow cytometry.

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  Figure 5. Overexpression of SOCS2 in macrophages restrains the development of NASH in vivo. A. The Western diet-induced NASH model in BMT mice (n = 6 per group). B. The liver from the mice described in Figure 5A. Liver sections were stained with Hematoxylin and Eosin (H&E), Oil-Red O, Masson and IHC (anti-F4/80), Original magnification, ×20. Bar = 200 μm. C. Quantitative analysis for Figure 5B of staining was shown. D. The level of serum TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 were determined in the mice described in Figure 5B by ELISA assay (n = 6 per group). E. Immunoblotting of phospho-IKKα, IKKα, phospho-IKKβ, IKKβ, phospho-P65, P65, SOCS2, and β-actin (loading control) in macrophage isolated from mice induced NASH (n = 6 per group).