Myeloid-derived growth factor alleviates non-alcoholic fatty liver disease alleviates in a manner involving IKKβ/NF-κB signaling

Figures

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  Fig. 1. Myeloid cell-specific MYDGF deficiency exacerbated inflammation, lipogenesis, and hepatic steatosis in NAFLD mice. KO and WT mice aged 4–6 weeks were divided into two groups (WT and KO) and fed a HFD for 12 weeks (n = 6 mice per group). The control group indicates WT mice fed a NCD. A Representative images showing HE staining and oil red O staining of liver sections (n = 6). B Liver/body weight (n = 6). C Hepatic TG content (n = 6). D mRNA levels of genes related to fat metabolism in isolated hepatocytes (n = 6). E Serum ALT and AST levels (n = 6). F Representative images showing F4/80 staining of liver sections (n = 6). G Levels of inflammatory factors in isolated hepatocytes (n = 6). H Levels of inflammatory factors in KCs (n = 6). I Levels of inflammatory factors in serum (n = 6). Data are presented as the mean ± SEM. Black scale bar: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 2. BMT alleviated inflammation, lipogenesis, and hepatic steatosis in NAFLD mice. BMT was performed in KO and WT mice aged 4–6 weeks, which were fed a HFD for 12 weeks as indicated in Fig. S4C. A Representative images showing HE staining and oil red O staining of liver sections (n = 6). B Liver/body weight (n = 6). C Hepatic TG content (n = 6). D mRNA levels of genes related to fat metabolism in isolated hepatocytes (n = 6). E Serum ALT and AST levels (n = 6). F Representative images showing F4/80 staining of liver sections (n = 6). G Levels of inflammatory factors in isolated hepatocytes (n = 6). H Levels of inflammatory factors in KCs (n = 6). I Levels of inflammatory factors in serum (n = 6). Data are presented as the mean ± SEM. Black scale bar: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 3. Bone marrow-specific overexpression of MYDGF alleviated inflammation, lipogenesis, and hepatic steatosis in NAFLD mice. Intramarrow AAV-MYDGF injection was performed in KO and WT mice aged 4–6 weeks, which were fed a HFD for 12 weeks as indicated in Fig. S6E. A Representative images showing HE and oil red O staining of liver sections (n = 6). B Liver/body weight (n = 6). C Hepatic TG content (n = 6). D mRNA levels of genes related to fat metabolism in isolated hepatocytes (n = 6). E Serum ALT and AST levels (n = 6). F Representative images showing F4/80 staining of liver sections (n = 6). G Levels of inflammatory factors in isolated hepatocytes (n = 6). H Levels of inflammatory factors in KCs (n = 6). I Levels of inflammatory factors in serum (n = 6). Data are presented as the mean ± SEM. Black scale bar: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 4. MYDGF improved hepatocyte inflammation, lipogenesis, and fat deposition in co-culture experiments. The experiments were performed as indicated in Fig. S9C, D. A Fat content (determined by oil red O staining) and p65 nuclear translocation in rMYDGF-treated PMHs. B Quantification of TG levels in rMYDGF-treated PMHs. C mRNA levels of genes related to fat metabolism in rMYDGF-treated PMHs. D Levels of inflammatory factors in hepatocytes and supernatants after rMYDGF treatment. E Fat content (determined by oil red O staining) and p65 nuclear translocation in PMHs co-cultured with BMCs. F Quantification of TG levels in PMHs co-cultured with BMCs. G mRNA levels of genes related to fat metabolism in PMHs co-cultured with BMCs. H mRNA levels of TNFα, IL-1β, and IL-6 in PMHs co-cultured with BMCs. I Levels of P-p65, p65, P-IκBα, IκBα, pSREBP1c, nSREBP1c, FAS, ACC1, and SCD1 proteins in PMHs co-cultured with BMCs. Black scale bar: 20 μm, white scale bar: 10 μm. Each experiment was repeated five times. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s…

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  Fig. 5. MYDGF inhibited NF-κB signaling and lipogenesis in vivo and in vitro. A Levels of NF-κB signaling and lipogenesis proteins in the livers of KO mice (n = 3). B Levels of NF-κB signaling and lipogenesis proteins in AAV-MYDGF-treated livers of KO mice (n = 3). C Levels of NF-κB signaling and lipogenesis proteins in rMYDGF-treated PMHs. D Levels of P-IKKβ, P-JNK, P-p38MAPK, and P-ERK proteins in the livers of KO mice (n = 3). E Levels of P-IKKβ, P-JNK, P-p38MAPK, and P-ERK proteins in AAV-MYDGF-treated livers of KO mice (n = 3). F Levels of P-IKKβ, P-JNK, P-p38MAPK, and P-ERK proteins in rMYDGF-treated PMHs. Each in vitro experiment was repeated five times. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 6. IKKβ silencing inhibited NF-κB signaling and lipogenesis and alleviated inflammation and fat deposition in vivo. A–G WT and KO mice aged 4–6 weeks were treated with siIKKβ and siCon and fed a HFD for 12 weeks. A Levels of NF-κB signaling and lipogenesis proteins in the livers of siIKKβ-transfected WT and KO mice (n = 3). B Representative images showing HE staining, oil red O staining, and F4/80 staining of liver sections (n = 6). Black scale bar: 50 μm. C Hepatic TG content (n = 6). D mRNA levels of genes related to fat metabolism in isolated hepatocytes (n = 6). E Levels of inflammatory factors in isolated hepatocyte (n = 6). F Levels of inflammatory factors in KCs (n = 6). G Levels of inflammatory factors in serum (n = 6). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 7. Luciferase and ChIP under IKKβ silencing conditions in PMHs. PMHs were transfected with siIKKβ or siCon for 12 h, before using for luciferase and ChIP experiments. A PMHs were transfected with NF-κB-luciferase or SV40–Renilla after treatment with siCon or siIKKβ. Cells were left untreated or treated with rMYDGF for 24 h, and/or PA for 24 h before luciferase and Renilla assessment. B PMHs were transfected with siCon or siIKKβ, and then treated with or without rMYDGF for 24 h, and/or PA for 24 h. De novo fatty acid biosynthesis was measured before or after PA stimulation. C Nuclear protein was analyzed for SREBP1c by immunoblotting, and GAPDH was used as the normalization control; nSREBP1c denotes nuclear SREBP1c. D, E PMHs were transfected with siCon and siIKKβ, and then treated with or without rMYDGF for 24 h, and/or PA for 24 h. IgG, p65, and histone antibodies were used as a reference substrate. ChIP and RT-PCR was performed to determine SREBP1c (D) and IkBα (E) promoters. Data are presented as the mean ± SEM. Each experiment was repeated five times. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA…

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  Fig. 8. p65 silencing inhibited the expression of SREBP1c and alleviated inflammation and fat deposition in vivo. A–G WT and KO mice aged 4–6 weeks were treated with sip65 or siCon and fed a HFD for 12 weeks. A Expression of SREBP1c proteins in the livers of sip65-transfected WT and KO mice (n = 3). B Representative images showing HE staining, oil red O staining, and F4/80 staining of liver sections (n = 6). Black scale bar: 50 μm. C Hepatic TG content (n = 6). D mRNA levels of genes related to fat metabolism in isolated hepatocytes (n = 6). E Levels of inflammatory factors in isolated hepatocytes (n = 6). F Levels of inflammatory factors in KCs (n = 6). G Levels of inflammatory factors in serum (n = 6). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 9. PKCθ is involved in the MYDGF-induced regulation of IKKβ phosphorylation in hepatocytes. A Levels of PKC signaling proteins in the livers of KO mice (n = 3). B Levels of PKC signaling proteins in the livers of AAV-MYDGF-treated KO mice (n = 3). C Levels of PKC signaling proteins in rMYDGF-treated or PKCθ inhibitor-treated PMHs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significant differences were determined by Student’s t-test or one-way ANOVA followed by Tukey’s post-test.

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  Fig. 10. Schematic showing that MYDGF plays a protective role in NAFLD via the IKKβ/NF-κB signaling pathway.