الفهرس 
NON-ALCOHOLIC STEATOHEPATITISOnly 14 pages are availabe for public view
 |  | from | 215 |  |  |
| | | from | 215 | | |
Abstract
on-alcoholic steatohepatitis (NASH) is the clinically aggressive variant of non-alcoholic fatty liver disease (NAFLD) that is the main cause of chronic liver disease in the Western world and a major health problem, NAFLD/NASH now considered the top reason for liver transplantation.
Contributing to the increasing global epidemic of obesity and type 2 diabetes (T2DM), a recent model has estimated a 178% increase in liver deaths related to NASH by 2030.NASH diagnosis depends on blood tests, imaging techniques and histopathological confirmation. Imaging techniques include US, CT, MRI and Elastography, however, the histopathological confirmation is still considered the gold standard for diagnosing patients with NASH.
Currently, there are no FDA-approved medical therapies for NASH or liver fibrosis. There is an urgent need for new therapeutic approaches that are not only effective in ameliorating fat accumulation, cell death, and inflammation, but also is effective at reducing or reversing fibrosis. Kefir is an acidic-alcoholic fermented milk, produced by kefir grains. Because of its microflora, and the existence of some metabolites as organic acids, it has several biological effects e.g., antibacterial, antioxidant, immunomodulatory, antidiabetic, and cholesterol lowering actions. Therefore, Kefir milk could improve liver injury of NASH.
The hippo pathway is an evolutionary conserved cascade that plays an essential role in restraining cell proliferation and promoting apoptosis. Hippo signaling pathway was reported to have a critical role in the regulation of hepatic size, proliferation, apoptosis, and stress response. Accordingly, its dysregulation could have an effect on NASH pathogenesis via liver dedifferentiation and tumorigenesis.
Due to previous stated information, we used an integrative approach based on bioinformatics analysis together with clinical validation to construct a genetic-epigenetic network linked to Hippo signaling as a modulator of NAFLD/NASH pathogenesis, then assess the effects of kefir Milk on this network and NASH progression.
First, in silico analysis was used to construct a genetic-epigenetic network linked to Hippo signaling. Second, an investigation using rats, including HSHFD induced NASH and Kefir treated animals, was designed. Experimental procedures included biochemical and histopathologic analysis of rat blood and liver samples. At the molecular level, the expression of genetic (SOX11, AMOTL2 and SMAD4 mRNAs) and epigenetic (miR6807-5p) network was measured by real-time PCR. Network expression was validated with immunohistochemistry detection of (HepPar1) and target effector proteins IL-6 and TGF-β estimation by ELISA.
The study was carried out at Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University (2020-2022) and was approved by Research Ethics Committee, Faculty of Medicine, Ain Shams University. (FMASU MD 85/2020), Assurance No. 000017585.
The study was performed on 40 adult male Wistar rats that were equally divided into three groups; where treatment was given orally by gavage:
(I) Normal control group (NC) (n= 8) fed a normal pellet diet.
(II) NASH model group (n= 16) this group was further subdivided into:
o group IIa (n= 8): (9-weeks NASH model).
o group IIb (n= 8): (12-weeks NASH model).
(III) Kefir treatment group (n=16) this group was further subdivided into:
o group IIIa (n= 8): (NASH/early Kefir-treated), rats were fed HSFD for 12 weeks and received probiotic treatment daily from day one for 12 weeks.
o group IIIb (n= 8): (NASH/late Kefir treated), rats were fed HSHF for 12 weeks and received probiotic treatment daily in the last 3 weeks.
At the end of the study, rats were fasted for 12 hrs. and anesthetized. Retro orbital blood samples were taken, then rats were sacrificed, and livers were collected. For each animal, blood samples were aliquoted and stored at −20 °C for subsequent biochemical analysis. Part of the hepatic tissues was immediately stored in −80 °C for RNAs and protein assessment whereas the remaining parts were rapidly fixed in 10% neutral buffered formalin for histopathological and immune-histochemical analyses.
Evaluation of SMAD4, SOX11 and AMOTL2 mRNAs in the liver tissues was performed by real time PCR in relation to GAPDH as the housekeeping gene in all samples. And miR-6807-5p was performed also by real time PCR in relation to Hs_SNORD72_11 as an internal control in all samples. Then the results were statistically analyzed by using GraphPad Prism.
The results of the current study revealed the following:
NASH group rats’ whole-body weights were significantly higher than normal controls. As well as Early Kefir group showed a significant decrease in body weight and the ratio of liver weight to body weight than NASH (12 weeks) group.
NASH groups had significantly higher serum levels of AST, ALT, GGT, ALP, total bilirubin, and direct bilirubin compared to the controls. On contrary, oral administration of either early or late Kefir treatment significantly reduced the elevated levels of these variables. Noticeably, this ameliorative effect was more remarkable when Kefir was administered prophylactically (Early Kefir).
SOX11, SMAD4 and AMOTL2 expression levels were assessed in the liver tissue of the experimental animals. NASH group animals showed a sharp significant decrease in SOX11 expression level; however, Kefir administration has normalized this significant decrease. Additionally, no significant change in hepatic SOX11 expression level was observed between early and late treated groups.
Meanwhile, NASH group recorded a remarkable significant increase in level of SMAD4 compared to normal controls which was significantly corrected with Kefir treatment, compared to NASH group animals.
Moreover, data manifested that hepatic AMOTL2 expression level in NASH group animals was significantly decreased in comparison to controls and was significantly corrected by Kefir administration.
MiR-6807-5p in the liver tissue of NASH group animals was significantly increased compared to controls. Furthermore, both early and late Kefir treatment normalized the expression of hepatic miR-6807-5p, compared with that of NASH groups.
In conclusion, by comparing the Kefir supplemented groups with the controls there was no significant difference detected regarding the expression of SMAD4, AMOTL2 and hsa-miR 6807-5p indicating the effectiveness of Kefir supplementation. Moreover, the hepatoprotective role of Kefir is mediated mostly by its anti-inflammatory and anti-fibrotic effect through a significant decrease of both IL-6 and TGF-β1 proteins respectively compared with NASH groups.
Kefir administration minimized biochemical and histopathologic alterations caused by NAFLD/NASH. HSC activation and expression of profibrogenic IL-6 and TGF-β effector proteins were reduced via inhibition of hippo pathway. This inhibition could be proved by decreased hepatocyte persistent proliferation and up-regulation of HepPar1-positive cells in the liver tissues.