VSL#3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet-induced nonalcoholic steatohepatitis model in mice

Abstract

Nonalcoholic fatty liver disease (NAFLD) and its advanced stage, nonalcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease in the United States. NASH features the metabolic syndrome, inflammation, and fibrosis. Probiotics exhibit immunoregulatory and anti-inflammatory activity. We tested the hypothesis that probiotic VSL#3 may ameliorate the methionine-choline-deficient (MCD) diet-induced mouse model of NASH. MCD diet resulted in NASH in C57BL/6 mice compared to methionine-choline-supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased tumor necrosis factor alpha levels, and fibrosis. VSL#3 failed to prevent MCD-induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced up-regulation of serum endotoxin and expression of the Toll-like receptor 4 signaling components, including CD14 and MD2, MyD88 adaptor, and nuclear factor kappaB activation. In contrast, VSL#3 treatment ameliorated MCD diet-induced liver fibrosis resulting in diminished accumulation of collagen and alpha-smooth muscle actin. We identified increased expression of liver peroxisome proliferator-activated receptors and decreased expression of procollagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up-regulation of transforming growth factor beta (TGFbeta), a known profibrotic agent. In the presence of VSL#3, the MCD diet-induced expression of TGFbeta was maintained; however, the expression of Bambi, a TGFbeta pseudoreceptor with negative regulatory function, was increased. In summary, our data indicate that VSL#3 modulates liver fibrosis but does not protect from inflammation and steatosis in NASH. The mechanisms of VSL#3-mediated protection from MCD diet-induced liver fibrosis likely include modulation of collagen expression and impaired TGFbeta signaling.

Fig. 1

VSL#3 treatment failed to prevent MCD diet-induced liver injury. Mice were fed MCS or MCD diet for 10 weeks; VSL#3 was administered for the last 9 weeks of the MCD diet. Liver-to-body weight ratio was determined. Data are shown from 6 mice per experimental group. Liver histology was assessed after hematoxylin and eosin staining of liver tissue (A, representative picture, magnification 100X; the * indicates inflammatory foci); liver/body ratio (D), liver triglycerides (TG) (B), liver TNFα (C), and serum ALT (E) were determined from n=6/group. (F) Liver sections were analyzed for collagen expression with trichrome (top panel) and Sirius Red (medium panel) staining; liver collagen I protein content was quantified in Western blot using equal amounts of total liver proteins from each animal; one representative blot and densitometric analysis from n=6/group (bottom panel) are shown. (G) Liver sections were analyzed for α-SMA expression using immunohistochemistry (top panel) staining; liver α-SMA protein content was quantified in Western blot using equal amounts of total liver proteins from each animal; one representative blot and densitometric analysis from n=6/group (bottom panel) are shown.

Fig. 2

VSL#3 induces expression of peroxisome proliferator-activated proteins. The expression of liver peroxisome proliferator-activated receptor (PPAR)-α (A), PPAR-γ (B), and PPAR-γ coactivator 1 α (PGC-1α) (E) were assessed using quantitative PCR. Data are shown as fold increase of MCD or MCD+VSL#3 group over control MCS diet with 6 mice/group. (C) Equal amounts of nuclear proteins were analyzed in EMSA for binding to the PPAR response element (PPRE). One sample was pre-incubated with cold PPRE oligonucleotide prior to EMSA as specificity control (C); a representative EMSA gel (top) and the densitometric analysis from 6 mice/group (bottom panel) are shown. (D) Equal amounts of nuclear proteins were analyzed pre-incubated with anti-PPARα, -PPARγ or –RXR antibodies and subjected to EMSA for binding to the PPAR response element (PPRE); a cold competition control (Comp) was included as above. A representative EMSA gel (top) and the densitometric analysis from 6 mice/group (bottom panel) are shown.

Fig. 3

VSL#3 limits the expression of MCD diet-induced matrix metalloproteinase in the liver. The liver RNA levels of liver procollagen I-α1 (A), matrix metalloproteinase (MMP)-2 (B), and MMP-9 (C), and the 18S control were analyzed using qPCR. Data are shown as fold increase of MCD or MCD+VSL#3 group over control MCS diet, all adjusted to 18S internal controls, with 6 mice/group.

Fig. 4

VSL#3 failed to protect from MCD diet-induced endotoxemia. Mice were fed MCD or MCD diet for 10 weeks; VSL#3 was administered for the last 9 weeks of the MCD diet. Serum levels of endotoxin were analyzed at the end of the 10-week feeding period using a Limulus Amebocyte Lysates assay. Mean±SE data from 6 mice/group are shown.

Fig. 5

VSL#3 augments MCD diet-induced modulation of the LPS signaling complex. The liver mRNA levels of CD14 (A), MD-2 (C), MyD88 (C), toll-like receptor (TLR) 4 (D), and 18S were analyzed using qPCR. Data are shown as fold increase of MCD or MCD+VSL#3 group over control MCS diet, all adjusted to corresponding 18S housekeeping controls, with 6 mice/group. (E) Mice were fed MCS or MCD diet for 10 weeks; VSL#3 was administered for the last 9 weeks of the MCD diet. At the end of the 10 week-feeding period, the animals were challenged with LPS (0.5mg/kg body weight, i.p. for 1.5 hours). Liver nuclear extracts were analyzed for NF-κB binding activity in EMSA using specific radioisotope-labeled oligonucleotides; 20x excess of unlabelled oligonucleotide was used for cold competition (Comp). A representative gel is shown on the top and the densitometric analysis from 6 mice/group is shown on the bottom of each panel. * represents p<0.01 compared to the saline group with the same diet feeding.

Fig. 6

VSL#3 modulates TGFβ pathway The liver RNA levels of TGFβ (A), Bambi (B) and 18S were analyzed using qPCR. Data are shown as fold increase of MCD or MCD+VSL#3 group over control MCS diet, all adjusted to corresponding 18S housekeeping controls, with 6 mice/group.

Fig. 7

Hypothetical model of the effects of VSL#3 on MCD-induced NASH model.