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Comparative Study
. 2018 Jul 24;19(8):2165.
doi: 10.3390/ijms19082165.

Silibinin Ameliorates O-GlcNAcylation and Inflammation in a Mouse Model of Nonalcoholic Steatohepatitis

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Free PMC article
Comparative Study

Silibinin Ameliorates O-GlcNAcylation and Inflammation in a Mouse Model of Nonalcoholic Steatohepatitis

Su Jin Lee et al. Int J Mol Sci. .
Free PMC article

Abstract

The mechanisms underlying the progression to non-alcoholic steatohepatitis (NASH) remain to be elucidated. In the present study, we aimed to identify the proteins involved in the pathogenesis of liver tissue inflammation and to investigate the effects of silibinin, a natural polyphenolic flavonoid, on steatohepatitis. We performed comparative proteomic analysis using methionine and choline-deficient (MCD) diet-induced NASH model mice. Eighteen proteins were identified from the two-dimensional proteomic analysis, which are not only differentially expressed, but also significantly improved, by silibinin treatment. Interestingly, seven of these proteins, including keratin cytoskeletal 8 and 18, peroxiredoxin-4, and protein disulfide isomerase, are known to undergo GlcNAcylation modification, most of which are related to structural and stress-related proteins in NASH model animals. Thus, we primarily focused on how the GlcNAc modification of these proteins is involved in the progression to NASH. Remarkably, silibinin treatment alleviates the severity of hepatic inflammation along with O-GlcNAcylation in steatohepatitis. In particular, the reduction of inflammation by silibinin is due to the inhibition of the O-GlcNAcylation-dependent NF-κB-signaling pathway. Therefore, silibinin is a promising therapeutic agent for hyper-O-GlcNAcylation as well as NASH.

Keywords: O-GlcNAcylation; inflammation; non-alcoholic steatohepatitis; proteomics; silibinin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of silibinin on hepatocellular injury. Mice were fed ND, MCD/LPS diet, or MCD/LPS diet + silibinin for three weeks. (A) The histology of the liver sections from these three groups was assessed by hematoxylin-eosin (H&E) staining (original magnification, 200×). (B) Serum AST, ALT values and the expression levels of the proteins involved in the development of steatohepatitis, such as TGFβ1, TNF-α, IL6, and iNOS, were measured by qRT-PCR. Genes were normalized to GAPDH RNA as an internal standard. Fold-increase data are expressed. Data are the mean ± SD (n = 7/group). * p < 0.05 vs. ND group, and # p < 0.05 vs. MCD/LPS group. (C) Liver sections of the mice fed ND, MCD/LPS, or MCD/LPS + silibinin were subjected to immunohistochemical analysis for α-SMA (original magnification, 100×).
Figure 1
Figure 1
Effect of silibinin on hepatocellular injury. Mice were fed ND, MCD/LPS diet, or MCD/LPS diet + silibinin for three weeks. (A) The histology of the liver sections from these three groups was assessed by hematoxylin-eosin (H&E) staining (original magnification, 200×). (B) Serum AST, ALT values and the expression levels of the proteins involved in the development of steatohepatitis, such as TGFβ1, TNF-α, IL6, and iNOS, were measured by qRT-PCR. Genes were normalized to GAPDH RNA as an internal standard. Fold-increase data are expressed. Data are the mean ± SD (n = 7/group). * p < 0.05 vs. ND group, and # p < 0.05 vs. MCD/LPS group. (C) Liver sections of the mice fed ND, MCD/LPS, or MCD/LPS + silibinin were subjected to immunohistochemical analysis for α-SMA (original magnification, 100×).
Figure 2
Figure 2
Validation of the differentially expressed protein. Liver tissue extracts were prepared from the mice fed ND, MCD/LPS, or MCD/LPS + silibinin. (A) The left panel shows the hepatic expression levels of annexin A5 and regucalcin, as determined by immunoblotting using equivalent amounts of total liver protein. The expression levels were normalized relative to GAPDH. The right panel shows the relative expression levels of CK8, CK18, and PRX4. Data represent three independent experiments. (B) Liver sections were subjected to immunohistochemical analysis using antibodies to CK8. ×100 magnification. (C) O-GlcNAc was immunoprecipitated with anti-O-GlcNAc antibody, and then immunoblotted against CK8, CK18, or peroxiredoxin-4, respectively.
Figure 3
Figure 3
O-GlcNAcylation and the expression levels of the proteins associated with the HBP pathway. (A) The upper panel shows the hepatic expression levels of O-GlcNAcylation in ND, MCD/LPS, or MCD/LPS + silibinin groups. The middle and lower panels represent the relative expression levels of OGT and GAPDH, respectively, as observed by immunoblotting. In the two graphs on the right, the intensity of O-GlcNAcylation and OGT was determined by densitometry. Data are representative of three independent experiments. (B) Liver sections of ND, MCD/LPS, or MCD/LPS + silibinin-fed mice were subjected to immunohistochemical analysis with antibodies against OGT. ×200 magnification. (C) Relative expression levels of the proteins associated with the HBP pathway were determined by qRT-PCR analysis. Differential levels of GFAT, GLUT4, and XBP1 mRNA are shown as fold-increase. Gene expression was normalized to 18 s RNA as an internal standard, and the data were presented as the mean value ± SE of seven samples. * p < 0.05 vs. ND group, and # p < 0.05 vs. MCD/LPS group.
Figure 4
Figure 4
Silibinin inhibits the NF-κB-signaling pathway of NASH. (A) Liver tissue extracts were prepared from mice fed the ND, MCD/LPS, or MCD/LPS + silibinin diets. Hepatic mRNA levels of TLR4, myd88, and the NF-κB-mediated inflammatory cytokines IL-6 and iNOS were measured by qRT-PCR. Genes were normalized to 18sRNA as an internal standard, and the data are expressed as fold-increase. * p < 0.05 vs. ND group, and # p < 0.05 vs. MCD/LPS group. All images shown are representative of three independent experiments. (B) Raw 264.7 cells were untreated or pretreated with OSMI-1 (50 µg/mL) or silibinin (100 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for 1 h. Immunofluorescent staining of NF-κB p65 or DAPI in raw 264.7 cells was performed with a kit, including ProLong® Gold and SlowFade® Gold Antifade. The right panel represents the relative nuclear expression levels of P65, respectively, as observed by immunoblotting. The secondary antibody was conjugated with a fluorescent green dye (Alexa Fluor 488). ×200 magnification. (C) Raw 264.7 cells were untreated or pretreated with OSMI-1 (50 µg/mL) or silibinin (100 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for 1 h. The upper left panel shows the extent of OGT expression by immunoblotting. Immunoprecipitation analysis using P65 antibody was performed, as shown in the upper right panel, followed by immunoblotting using GlcNAc antibody. Immunoprecipitation analysis was performed using whole cell lysate, and the GlcNAc levels were assessed using the same amount of precipitates. The lower two graphs show the relative amount of the above proteins as fold-increase. Data represent three independent experiments. * p < 0.05 vs. mock, and # p < 0.05 vs. LPS alone in each treatment.
Figure 4
Figure 4
Silibinin inhibits the NF-κB-signaling pathway of NASH. (A) Liver tissue extracts were prepared from mice fed the ND, MCD/LPS, or MCD/LPS + silibinin diets. Hepatic mRNA levels of TLR4, myd88, and the NF-κB-mediated inflammatory cytokines IL-6 and iNOS were measured by qRT-PCR. Genes were normalized to 18sRNA as an internal standard, and the data are expressed as fold-increase. * p < 0.05 vs. ND group, and # p < 0.05 vs. MCD/LPS group. All images shown are representative of three independent experiments. (B) Raw 264.7 cells were untreated or pretreated with OSMI-1 (50 µg/mL) or silibinin (100 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for 1 h. Immunofluorescent staining of NF-κB p65 or DAPI in raw 264.7 cells was performed with a kit, including ProLong® Gold and SlowFade® Gold Antifade. The right panel represents the relative nuclear expression levels of P65, respectively, as observed by immunoblotting. The secondary antibody was conjugated with a fluorescent green dye (Alexa Fluor 488). ×200 magnification. (C) Raw 264.7 cells were untreated or pretreated with OSMI-1 (50 µg/mL) or silibinin (100 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for 1 h. The upper left panel shows the extent of OGT expression by immunoblotting. Immunoprecipitation analysis using P65 antibody was performed, as shown in the upper right panel, followed by immunoblotting using GlcNAc antibody. Immunoprecipitation analysis was performed using whole cell lysate, and the GlcNAc levels were assessed using the same amount of precipitates. The lower two graphs show the relative amount of the above proteins as fold-increase. Data represent three independent experiments. * p < 0.05 vs. mock, and # p < 0.05 vs. LPS alone in each treatment.

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