Non-alcoholic fatty liver disease (NAFLD) has become a very common disease because of the prevailing increase in obesity worldwide. Currently, several NAFLD therapies are being targeted to improve insulin resistance (IR), but there is no effective treatment . Probiotic treatment was shown to improve NASH through modulating insulin resistance, the key factor which plays a major role in the development of a serious liver condition . Due to correlation between small intestinal bacterial overgrowth (SIBO) and NAFLD observed in experimental and clinical studies , , probiotics could also delay disease progression and prevent complications by modulating intestinal flora, intestinal permeability, and inflammatory response  .
In this context, a high fat/high sucrose diet (HFD) induced NASH model experiment was conducted to assess the beneficial effects of the Kefir formula on the degree of hepatic fibrosis and steatosis, inflammation, and body composition via modulation at both epigenetic and genetic level. Previous studies have showed that kefir improved NAFLD regarding to BW, energy expenditure and basal metabolic rate through inhibition of the lipogenesis pathway . This may explain our findings where the supplementation with Kefir prophylactically or therapeutically along with HFD, decreased the percentage and stage of steatosis, inflammation and fibrosis seen in NASH rats. Moreover, the serum levels of ALT and AST, and the hepatic content of fat, TGFB 1 and IL 6 proteins were significantly reduced with the treatment of the Kefir formula compared to NASH groups. In agreement with the previous finding that Kefir can diminish the inflammatory response in association with a decrease in tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and transforming growth factor beta (TGF-β) cytokines .
Hepatic stem/progenitor cells (HPCs or HpSCs, in humans) or oval cells (in rodents) are bipotential stem cells that can differentiate into mature hepatocytes and cholangiocytes . They represent a reserve compartment that can be activated to reactive ductulus (Or ductular reaction: DR) only when the mature epithelial cells of the liver are continuously damaged or blocked in their replication or in cases of serious cell loss .
Hippo pathway could promote cell death and differentiation and inhibit cell proliferation, however, the regulatory mechanisms for this signaling pathway are not clearly understood . The downstream effector YAP1 is shown to be involved in hepatic cell proliferation, survival, development and differentiation . Activated YAP1 leads to activation of hepatic stellate cells (HSC), prolonged activation of these cells causes liver fibrogenesis . Therefore, a key objective is to understand the mechanisms that stimulate the switch of quiescent HSCs in a healthy liver to activated, myofibroblastic HSCs in NASH.
In this sense, variety of public microarray databases and computation algorithms have been investigated in the current work, for the selection of hepatic mRNA-miRNA panel linked to NAFLD/NASH Hippo signaling pathway and gut microbita regulated genes. We identified 3 mRNAs (SOX11, SMAD4 and AMOTL2), their epigenetic regulator (miR-6807) and their target effector proteins (TGFB, IL6 and HepPar1). Protein -protein interaction between the selected mRNAs protein products and HIPPO target effectors was obvious by STRING database as shown in (supplementary figure 4A).
Cytosolic AMOTL2 proteins can attach YAP1 and WWTR1 (TAZ) in their unphosphorylated states, providing a Hippo-independent mechanism to down-regulate the activities of these proteins . AMOTL2 mRNA is downregulated in the current study in the rat liver tissue of NASH compared to NC or treated groups (P < 0.001), which indicated the freeing of YAP1 with subsequent activation of hepatic stellate cells and fibrogenesis participating in the pathogenesis of NASH. On the other hand, AMOTL2 is up regulated by the intake of kefir in rats which possibly ameliorates the features of NASH in the treated rat group by bounding YAP1.
SMAD4 interacts with SMAD2/3 and participates in the intracellular TGF-β signaling pathway. Knockout of SMAD4 from mesangial cells resulted in inhibition of TGF-β1-produced ECM synthesis .These previous published results confirmed that SMAD4 has a significant role in the pathogenesis of fibrosis by controlling the ability of SMAD3 to activate transcription of a number of fibrogenic genes (collagens), markers (α-SMA and E-cadherin)  and explain the significant overexpression of SMAD4 in NASH group than NC and treated groups observed in the current study (P < 0.001).
SOX-11(SRY-related HMG-box) is a member of the group C SOX transcription factor family involved in the regulation of embryonic development and in the determination of the cell fate . Its mRNA shows decreased expression in NASH versus other study groups conducted in the current study (P < 0.001). This agrees with a previous study that validated hepatic expression of a subset of mRNAs and reported downregulation of SOX11 in Obesity-Related Nonalcoholic Steatohepatitis relative to normal tissue .
MiRNAs are reported to play important role in mediating the development of diseases, including NASH . MiR 6807-5p is shown to be a novel microRNA biomarker for detecting gastric cancer (GC) but no previous reports have correlated its expression with the incidence or progression of NASH or with other liver diseases . Our bioinformatics data analysis revealed its expression in liver (supplementary figure 6A) and its correlation to Hippo signaling pathway (supplementary.figure 6B). Moreover, SOX11, SMAD4 and AMOTL2 are shown to be direct mRNA targets of miRNA-6807-5p as was retrieved from miRwalk database.(supplementary figure 5A-C). Our results revealed that there was a significant increase in miR 6807-5p concomitant with marked increase in SMAD4 and significant down regulation of SOX11, and AMOTL2 mRNA levels in the liver tissue of NASH group compared to NC or treated groups. This deregulated molecular profile reverts to normal after treatment with kefir milk.
TGF-β was significantly increased in NASH as compared with other groups (P < 0.001) which is explained by previous work reporting that activation of TGF‐β signaling pathway was involved in the occurrence and development of NASH through motivating HSCs and the formation of extracellular matrix (ECM) ,.
Continuous increased triacylglycerols buildup produces reactive oxygen species (ROS) and proinflammatory cytokines e.g IL6, which induces NASH. IL-6 expression was significantly increased in the hepatic biopsy of patients with nonalcoholic steatohepatitis (NASH) than patients having simple steatosis or normal biopsies. Morever, there was a positive correlation between fibrosis staging and inflamation degree confirming the presence of hepatic IL-6 expression in human NASH . Our results revealed a marked increase in IL-6 level in rat NASH liver when compared to NC (P < 0.001) but it was returned back to normal levels after kefir treatment.
In the present study, proliferation of oval cells was enhanced in NASH groups and decreased in Kefir groups. Previous studies found that HPC activation and the expansion of DR have been correlated with progressive fibrosis in adult and pediatric NASH and in HCV associated cirrhosis .
In addition, upon differentiation towards hepatocytes, HPCs were previously shown to gradually lose their biliary features, including markers such as keratin 19 (K19) and keratin 7 (K7)  as well as Hepatocyte Paraffin 1(Hep Par1). For the best of our knowledge, Hep Par1 was used for the first time as a marker of hepatocellular differentiation in NASH where it is shown to be mostly negative in the liver of NASH group while 96% of hepatocytes were immunoreactive for HepPar-1. with kefir treatment (figure 6).
Taken all together, our experimental model hypothesized that treatment with probiotic Kefir down-regulated miR 6807-5p with subsequent upregulation of of SOX 11, and AMOTL2 and downregulation of SMAD4. Accordingly, YAP1 is negatively regulated with its subsequent sequestration in the cytosol. This cytoplasmic translocation of YAP1 inhibited cell proliferation and suppressed the expression of inflammatory IL6 and fibrotic TGF-β1 (figure 8).