Evaluation of the role of kefir in management of non-alcoholic steatohepatitis rat model via modulation of NASH linked mRNA-miRNA panel

Figures

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  Figure 1. Flow chart showing the experimental design of the animal studies. NASH nonalcoholic steatohepatitis, HSHF high sucrose and high fat.

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  Figure 2. Liver/body ratio in different study groups. Values are mean ± SEM; number of animals = 8 rats/each group. One-Way ANOVA test followed by Tukey’s multiple comparison test. *P < 0.001 compared to control group, δP < 0.001 compared to NASH (12 weeks) group.

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  Figure 3. The effect of early and late Kefir treatment on hepatic steatosis, inflammation and fibrosis using HE and Masson’s trichrome staining (Magnifications: × 100). (A) Control group showed no pathological changes, (B) NASH (9 weeks) group developed mainly micro vesicular steatosis, mild hepatic lobular inflammation and mild fibrous septa(star) (C) NASH (12 weeks) group showed diffuse and extensive micro and macro-vesicular steatosis, severe lobular inflammation, focal necrosis, hepatocellular ballooning, marked expansion of facultative hepatic progenitor cells (arrows), dense fibrous septa and collagen fibers (star) (D) Early Kefir and (E) Late Kefir groups showed a lower degree of steatosis, inflammation and fibrosis, where Early Kefir showed more remarkable histological improvements.

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  Figure 4. Effect of Kefir on the expression level of hepatic m-RNAs SOX11, SMAD4 and AMOTL2 (A − C), hepatic miR 6807-5p (D). Values are mean ± SEM; number of animals = 8 rats/each group. *P < 0.001 compared to control group, #P < 0.001 compared to NASH (9 weeks) group, δP < 0.001 compared to NASH (12 weeks) group. One-way ANOVA followed by Tukey’s multiple comparison test. RQ relative quantification, SOX11 SRY-Related HMG-box gene 11, SMAD4 SMAD family member 4, AMOTL2 Angiomotin-like 2, hsa-miR 6807-5p homo sapiens miR 6807-5p.

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  Figure 5. Immunohistochemical staining for hepatocyte specific antigen (HepPar1). (A) Controls showed a strong signal with Hep Par 1 (hepatocyte marker) producing distinct granular, cytoplasmic staining of hepatocytes, whereas in both (B) NASH (9 weeks) and (C) NASH (12 weeks) groups were mostly negative with HepPar-1. By comparison, (96%) hepatocytes in (D) Early Kefir group were immunoreactive for HepPar-1 and (E) Late Kefir group tended to show weaker, patchy positivity (HepPar1 × 100) indicated to hepatocyte differentiation.

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  Figure 6. The effect of Kefir treatment on (A) Level of IL-6 (B) Level of TGF-β1 in liver tissues from various groups. Values are mean ± SEM, number of animals = 8 rats/each group. *P < 0.001 compared to control group, #P < 0.001 compared to NASH (9 weeks) group, δP < 0.001 compared to NASH (12 weeks) group, €P < 0.05 compared to control group. One-way ANOVA followed by Tukey’s multiple comparison test. IL6 Interleukin 6, TGFB1 transforming growth factor beta 1.

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  Figure 7. Proof of concept map of the study hypothesis.