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Abstract The current work was designed to investigate the potential antifibrotic effect of honokiol in Con A immunological model of liver fibrosis as well as its underlying molecular mechanisms by studying its effects on some key events such as oxidative stress, inflammation and fibrogenesis signaling pathway. To fulfill these goals, this study was divided into two parts: I. Screening the hepatoprotective dose of honokiol against Con A induced acute hepatotoxicity: animals were divided randomly into five groups and treated for 5 days as follow: honokiol once daily at doses of (5, 10 & 20 mg/kg) orally and/or Con A (20 mg/kg; IV) on day 5. The following parameters were investigated: 1-Assessment of Liver enzymes: ALT&AST. 2-Histopathological examination of liver tissue using H&E stain. The results of the present study can be summarized as follows: 1- A single Con A injection induced acute hepatotoxicity represented by significant ALT and AST increase associated with histopathological alterations such as hepatocytes degeneration and inflammatory cells infiltration. 2- Screening different doses of honokiol (5, 10 & 20 mg/kg) revealed that, all honokiol doses significanlty reduced serum ALT and AST as compared to Con A group. Summary and Conclusions - 151 - 3- Histopathological examination using H&E stain revealed that 10 mg/kg dose almost preserved the normal architecture of the liver. Collectively, the results of the acute study proved that, honokiol at dose of 10 mg/kg is more protective than 5 & 20 mg/kg. Therefore the chronic study was carried out using the 10 mg/kg dose. II. Studying the potential antifibrotic mechanisms of honokiol against chronic Con A model of liver fibrosis: animals were divided randomly into four groups and treated for 4 weeks as follow: honokiol five times weekly orally at dose (10 mg/kg) and/or Con A (20 mg/kg; IV) once weekly. The following parameters were investigated: 1-Hepatotoxicity indices: Liver index (liver weight / body weight) x100, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and albumin. 2- Oxidative stress markers: Liver content of reduced glutathione (GSH), Superoxide dismutase activity (SOD) and lipid peroxides (MDA). 3- Inflammatory markers: Liver contents of Tumor necrosis factor alpha (TNF-α), Interferon gamma (INF-γ), nuclear factor kappa b NF- κB (p65) subunit expression and inducible nitric oxide synthase enzyme expression (iNOS). 4-Liver fibrosis markers: Liver content of hydroxyproline and alpha smooth muscle actin expression (α-SMA). Summary and Conclusions - 152 - 5- TGF-β/SMAD pathway markers: Liver content of TGF-β and p.samd2/3 expression. 6- Histopathological examination: Hematoxylin and Eosin for routine histological examination, Masson’s trichrome for detection of collagen fibers. The results of the present study can be summarized as follows: 1- In Con A group, relative liver weight, serum ALT and AST were significantly elevated while serum albumin was significantly reduced when compared to control group. However, co-treatment with honokiol restored all the levels of these parameters. 2- chronic Con A injection induced oxidative stress as it significantly reduced GSH level by 34 % and SOD activity by 36 % while significantly increased lipid peroxides level by 44% as compared to control values. Treatment of animals with honokiol concomitantly with Con A afforded significant protection against Con A intoxication as it normalized the values of GSH, SOD and lipid peroxides levels as compared to control group. 3- Con A also showed pro-inflammatory response evidenced by marked elevation in TNF-α and INF-γ levels by 159% and 89 % respectively as compared to control group while concomitant Summary and Conclusions - 153 - treatment with honokiol prevented this elevation. The inflammatory effect of Con A was further confirmed by the immunohistochemical detection of NF-κB and iNOS as shown by intense brown staining. 4- Liver fibrosis was evaluated histologically by visualizing the blue color of collagen fibers using Masson’s trichrome stain followed by morphometric analysis. In Con A group, collagen fibers were heavily deposited around portal tract and central vein forming pseudolobules. Morphometric analysis revealed an area percent of 14.6%. Interestingly, honokiol co-treatment prevented the abnormal collagen fibers deposition and induced a significant decrease in the area percent as compared to Con A group. 5- Biochemical measurement of hydroxyproline supported these results in which Con A induced a significant increase in the liver hydroxyproline content reaching 1.5 fold as compared to control group. On the other hand, co-treatment with honokiol induced a significant decrease the hydroxyproline content as compared to Con A group. Additionally, immunohistochemical detection of α-SMA revealed minimal staining in the blood vessels of the control group while marked expression was observed periportally and perisinusoidally in the Con A group as shown by the intense brown staining. Honokiol co-treatment markedly attenuated this elevated expression. Summary and Conclusions - 154 - 6- Additionally, Con A activated TGF-β/SMAD signalling as evidenced by high TGF-β levels as well as increased p.samd2/3 expression. In Con A group, TGF-β was significantly increased by 40.5% as compared to control group. Moreover, p.samd2/3 was highly expressed as shown by intense nuclear brown staining that quantitated by 55.5% increase in optical density as compared to control group. On the other hand, honokiol cotreatment attenuated the activation of the pathway. 7- Histopathological examination using H & E stain showed heavy cells infiltration around most portal areas, sever congestion and fibroblastic cells proliferation in the periductal tissue surrounding the bile ducts in the portal area in the Con A group. Interestingly, honokiol preserved the normal architecture of the hepatic parenchyma when given concurrently with Con A. In conclusion, the present study clearly demonstrates that honokiol could be promising antifibrotic agent against Con A induced liver fibrosis. Furthermore, this study sheds light on the mechanisms involved in its antifibrotic effect. It guards against oxidative stress induced by Con A by restoring cellular defense mechanism, through restoring the liver GSH content, SOD activity and normalizing lipid peroxidation. In addition, the antifibrotic effect was mediated through attenuating the TGF-β/SMAD signalling as well as decreasing the expression of NF-κB p65 and subsequent production of proSummary and Conclusions - 155 - inflammatory enzyme iNOS and pro-inflammatory cytokines (TNF-α and INF-γ) (Fig.30). Further investigations are warranted to establish the clinical applicability of honokiol in patients with chronic liver diseases, especially those associated with active fibrogenesis |