Figure 1.
MIYAIRI 588 prevents progression of CDAA diet-induced liver steatosis to tumorigenesis.
Each group was investigated at 8, 16, and 50 weeks after completion of the diet regimen. MIYAIRI 588 was administered after CDAA diet feeding for 2 weeks. To confirm that the feeding of the CDAA diet caused the liver steatosis, the groups administered CSAA and CDAA were examined at 2 weeks after the commencement of this experiment. (A) Macroscopic histomorphology and (B) hematoxylin and eosin staining for microscopic histopathology were performed at the indicated time periods. Data are representative of 6 individual liver sections. Original magnification, ×25.
Figure 2.
MIYAIRI 588 reduced hepatic lipid deposition and insulin resistance.
Male Fischer 344 rats (n = 6 per group) were fed a control (choline-sufficient/L-amino acid-defined CSAA) diet, choline-deficient/L-amino acid-defined (CDAA) diet, or CDAA diet plus MIYAIRI 588 for 8 weeks. MIYAIRI 588 was administered after CDAA diet feeding for 2 weeks, as described in the Materials and Methods. Cont, control. (A) Lipid accumulation was evaluated by oil red O staining of the liver sections. Data are representative of 6 individual liver sections. Original magnification, ×40. (B) Total triacylglycerol (TG) content in the liver was measured and normalized to protein concentration. Results represent mean ± SD values. *p<0.05 versus the CDAA-diet-fed group. (C) AMPK activation and lipogenesis- or lipolysis-related protein expression were detected by western blotting. β-actin expression was used as a loading control. (D) Fasting blood glucose levels, (E) Fasting plasma insulin levels, and (F) HOMA-IR were assessed in the rats. The data are shown as mean ± SD values. **p<0.01 vs. the CDAA-diet-fed group. (G) Total and phosphorylated AKT (Ser473 and Thr308) were represented under regular feed conditions.
Figure 3.
MIYAIRI 588 improves endotoxin levels in the portal vein and restores tight-junction protein expression.
Rats were fed a control (CSAA) diet, CDAA diet, or CDAA diet plus MIYAIRI 588 for 8 or 16 weeks. MIYAIRI 588 was administered after 2 weeks of CDAA diet feeding. Cont, control. (A) Serum levels of endotoxin are shown. Mean ± SD values from 6 rats per group are indicated. *p<0.05, **p<0.01 compared to the CDAA-diet-fed group for the CDAA diet plus MIYAIRI 588 group. (B) The organization and distribution of tight junction proteins on intestinal tissues were examined by immunohistochemistry. Arrows indicate a disrupted intestinal barrier. Data are representative of 6 individual intestinal sections. Scale bars = 500 µm (upper panels) or 250 µm (lower panels). (C) Expression of ZO-1 and occludin were subjected to western blot analysis in the intestinal tissues. ZO-1 and occludin expression levels were measured by densitometric analysis. β-actin expression was used as a loading control. Data are expressed as mean ± SD values. *p<0.05 compared to the CDAA-diet-fed group for the CDAA diet plus MIYAIRI 588 group. (D) The nuclear levels of the p65 subunit of NF-κB were detected by western blotting analysis of the liver samples. Expression of p65 NF-κB was normalized as a ratio to β-actin expression as a loading control. Data are expressed as mean ± SD values. **p<0.01 vs. the CDAA-diet-fed group. (E) Serum ALT levels were determined from 6 individual samples from each group. (F) Hepatic TNF-α protein level was analyzed by an enzyme-linked immunosorbent assay. Mean ± SD values for 6 rats per group are indicated. *p<0.05 compared to the CDAA-diet-fed group for the CDAA diet plus MIYAIRI 588 group.
Figure 4.
MIYAIRI 588 suppressed hepatic oxidative stresses and induced nuclear factor erythoid 2-related factor 2 expression.
Rats were fed a control (CSAA) diet, CDAA diet, or CDAA diet plus MIYAIRI 588 for 8 weeks. MIYAIRI 588 was administered after CDAA diet feeding for 2 weeks. Cont, control. (A) 4-HNE-stained sections of liver specimens have been shown. The data are representative of 6 individual liver sections. Original magnification, ×40. Quantitative analysis of 4-HNE protein adducts was performed by counting the 4-HNE-positive cells for every 5 centrilobular areas of the liver tissue sections obtained for each group (lower panel). (B) Hepatic MDA levels were measured in 3 groups. The data show mean ± SD values. **p<0.01 vs. the CDAA-diet-fed group. (C) Hepatic superoxide generation was detected with dihydroethidium staining in 3 groups. (D) Immunostaining of Nrf2 was shown in the liver of the rats of each group. Data are representative of 6 individual liver sections. Original magnification, ×40 (upper panels) or ×100 (lower panels). (E) Expression of Nrf2 and its targeted genes encoding enzymes, such as HO-1, NQO1, and BSEP, was examined by western blot analysis in the liver samples of each group. β-actin expression was analyzed as a loading control.
Figure 5.
Sodium butyrate (NaB) induced Nrf2 expression and nuclear accumulation.
(A) Dose-dependent effects of NaB on the expression of Nrf2 protein in HepG2 cells maintained in serum-free medium for 24 h and then treated with NaB at described concentrations for 6 h. Lamin B expression was used as a loading control. (B) Expression of Keap1, HO-1, NQO1, and TRX was examined by western blot analysis at 6 h. HepG2 cells were treated with 1.5 mM NaB at the indicated time points. (C) Nuclear accumulation of Nrf2 was stimulated by 1.5-mM NaB treatment of cells for the indicated time. Cytoplasmic and nucleic extracts were prepared and subjected to western blot analysis. Anti-α-tubulin and anti-histone H3 antibodies were used as markers for the cytoplasmic and nuclear extracts, respectively. (D) Ubiquitination of endogenous Nrf2 was assessed in HepG2 cells treated with DMSO, 1.5 mM NaB or 100 µM tBHQ for 9 h, along with 10 µM MG132. Nrf2 was immunoprecipitated with an anti-Nrf2 antibody and ubiquitinated Nrf2 was detected with an anti-ubiquitin antibody. (E) Post-transcriptional regulation of both the steady-state level and half-life of Nrf2 protein was evaluated. CHX (100 µM) was added to block protein synthesis. Cells were lysed at the indicated time points, and cell lysates were subjected to western blot analysis with anti-Nrf2 and anti-β-actin antibodies (upper panels). Lower panels depict the natural logarithm of the relative levels of the Nrf2 protein as a function of CHX chase time in the absence or presence of 1.5 mM NaB. The protein half-life has been determined in the linear range of the degradation curve.
Figure 6.
Nrf2 expression is regulated by AMPK and AKT activation and mTORC2 modification underlying NaB treatment.
(A) Serum-starved HepG2 cells were treated with 1.5 mM NaB for the indicated time periods. Western blot analysis was performed with the indicated antibodies. β-actin expression was used as the loading control. (B, C) Serum-starved cells were pretreated for 1 h with AMPK agonist compound C (CC; 20 µM) or the PI3K-specific inhibitor LY294002 (LY; 25 µM) and then incubated with 1.5 mM NaB or 1 mM AMPK activator AICAR for 6 h. Western blot analysis was performed with the indicated antibodies. (D) Cells were transfected with indicated siRNAs for 48 h and then incubated with 1.5 mM NaB for 6 h under serum-starved conditions. Western blot analysis was performed with the indicated antibodies. α-tubulin expression was used as the loading control. (E) Serum-starved cells were pretreated for 1 h with 20 µM CC or 25 µM LY and then incubated with 1.5 mM NaB for 6 h. Cell lysates and mTOR immunoprecipitates (IPs) prepared from the total cell lysates were analyzed by western blotting for the levels of mTOR and rictor. (F) Cells were transfected with the indicated siRNAs for 48 h and then incubated with 1.5 mM NaB or 1 mM AICAR for 6 h under serum-starved conditions. Cell lysates and mTOR immunoprecipitates (IPs) prepared from the total cell lysates were analyzed by western blotting for the levels of mTOR and rictor. (G) Nuclear accumulation of SIRT1 or rictor was examined by western blot analysis. Cells were treated with NaB for 6 h at the indicated concentration. Anti-α-tubulin and anti-lamin B antibodies were used as markers for the cytoplasmic and nuclear extracts, respectively. (H) Serum-starved cells were pretreated for 1 h with 20 µM CC or 25 µM LY and then incubated with 1.5 mM NaB for 6 h. Nuclear accumulation of SIRT1 or rictor was examined by western blot analysis. α-tubulin and lamin B were evaluated for expression levels as markers for the cytoplasmic and nuclear extracts, respectively. (I) Cells were transfected with indicated siRNAs for 48 h and then incubated with 1.5 mM NaB for 6 h under serum-starved conditions. Western blot analysis was performed with the indicated antibodies. α-tubulin and lamin B expression were used as the loading control of cytoplasmic and nuclear extract proteins, respectively.
Figure 7.
NaB improves insulin signaling and insulin-resistance conditions.
(A) Serum-starved cells were incubated in a medium containing either normal or high glucose concentration for 24 h and then treated with or without 1.5 mM NaB for 6 h. Subsequently, the treated cells were stimulated with 100 nM insulin for 20 min. Western blot analysis was performed with the indicated antibodies. (B) Normal or insulin-resistance conditions were established, and cell lysates and mTOR immunoprecipitates (IP) prepared from the total cell lysates were analyzed by immunoblotting for the levels of mTOR, rictor, and raptor. (C) Lipid accumulation was examined by oil-red O staining in insulin-resistance conditions. Serum-starved cells were incubated in a medium containing high glucose concentration for 24 h and then treated with or without 1.5 mM NaB for 9 h in the absence and presence of insulin. Quantification of the extent of oil-red O staining was done by counting red-stained regions for every 3 centrilobular areas of the cells in each group (lower panel). The values represent the mean ± SD values. **p<0.01 compared with the group exposed to high glucose levels in the absence insulin and NaB.
Figure 8.
MIYAIRI 588 prevents the progression of CDAA-diet-induced liver fibrosis and cirrhosis.
Male Fischer 344 rats (n = 6 per group) were fed a CSAA diet (Cont), CDAA diet, or CDAA diet plus MIYAIRI 588 for 8 or 16 weeks. MIYAIRI 588 was administered after CDAA diet feeding for 2 weeks. (A) Hepatic fibrosis was assessed by Azan-Mallory staining. Data are representative of 6 individual liver sections. Original magnification, ×40. The fibrosis area was assessed using image analysis techniques to calculate the ratio of connective tissue to the whole area of liver sections stained with Azan-Mallory. The data are expressed as mean ± SD values. ** p<0.01 compared with the CDAA-diet-fed group. (B) Immunostaining of α-SMA expression is shown. Data are representative of 6 individual liver sections. Original magnification, ×40. (C) α-SMA and collagen I expression was examined by western blot analysis at the indicated time points. β-actin expression was used as the loading control.
Figure 9.
MIYAIRI 588 inhibits development of CDAA-diet-induced hepatocarcinogenesis.
Rats were fed a CSAA diet (Cont), CDAA diet, or CDAA diet plus MIYAIRI 588 for 8 or 16weeks. MIYAIRI 588 was administered after CDAA diet feeding for 2 weeks. (A) Representative GST-P-positive preneoplastic foci in the liver of rats were shown. The GST-P-positive area was assessed by calculating the ratio of GST-P foci to the whole area of liver sections (lower panels). Data are representative of 6 individual liver sections. The data are expressed as mean ± SD values. Scale bars were indicated in each photograph. **p<0.01 compared with the CDAA-diet-fed group. (B) GST-P expression was detected by western blot analysis at the indicated time points. β-actin expression was used as the loading control. (C, D) Tumor number (≥1.0 mm) and maximal tumor size (diameter in mm) in the livers of rats fed on a CDAA diet (n = 8) and CDAA diet plus MIYAIRI 588 (n = 9) rats. Cont, control. ND, not detected. The data are expressed as mean ± SD values. *p<0.05, **p<0.01 compared with the CDAA-diet-fed group.
Figure 10.
The hypothetical model of the effects of MIYAIRI 588 on the progression of NAFLD.
Schematic representation of the mechanisms showing that probiotic MIYAIRI 588 prevents the progression of NAFLD through the intestine/liver axis and systematic signaling activation. MIYAIRI 588 designates the AMPK activation as a starting point and regulates different pathophysiological events, such as lipid and energy metabolism, insulin sensitivity, and oxidative stress response, tight-junction modification through consistent and systematic signaling pathways, thereby causing pronounced suppression of NAFLD progression.