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العنوان
The potential protective effect of niclosamide in a model of experimentally-induced liver fibrosis in rats /
المؤلف
Esmail, Manar Mohamed.
هيئة الاعداد
باحث / منار محمد إسماعيل
مشرف / ريم نبيل أبو النجا
مشرف / نهي محمد سعيد عبدالعظيم
مشرف / هايدي عفت ميشيل
تاريخ النشر
2021.
عدد الصفحات
253 p. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
علم السموم
تاريخ الإجازة
1/1/2021
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - الأدوية والسموم
الفهرس
Only 14 pages are availabe for public view

from 253

from 253

Abstract

Cholestasis is considered as one of the most significant stimulus for fibrotic liver. It is characterized by severe increase in liver and serum bile acid levels that leads to liver toxicity followed by fibrosis and cirrhosis. Activation of cholangiocytes proliferation is thought to help initiate and progress cholestatic liver fibrosis. HPCs are the source of proliferating cholangiocytes and Notch signaling promotes the differentiation of HPCs to cholangiocytes. The proliferating cholangiocytes then secrete profibrotic factors which induces activation and proliferation of HSCs to produce collagen and α-SMA that induces ECM deposition and fibrotic progression. Additionally, there are other signaling pathways involved in cholangiocytes proliferation including non-canonical Wnt signaling pathway that functions independently from β-catenin. Till now, no therapy could effectively inhibit abnormal activation of cholangiocytes.
Niclosamide is an oral anthelmintic drug which is FDA approved for the treatment of tapeworm infection. It was shown that niclosamide has potential anti-cancer effect by inhibiting several intracellular signaling pathways including the NOTCH pathway.
Accordingly, this study was conducted to to investigate the role of NOTCH pathway in CLF pathogenesis and the potential hepatoprotective effect of niclosamide as a NOTCH inhibitor against CLF in rats. The effect of niclosamide on Wnt pathway was also investigated.
Cholestatic injury and fibrosis in rodents was induced using BDL model. This model causes modifications that strongly resemble to those observed in cholestasis and cirrhosis in humans from liver cell injury and elevated serum enzyme levels, to a severe inflammatory response in the liver to an advanced hepatic fibrosis three to four weeks after surgery.
Rats were randomly divided into five main groups (6 per group): sham, BDL, BDL/niclosamide 5, BDL/niclosamide 10 and niclosamide 10 only group. Niclosamide was administered i.p. for 4 weeks starting at the same day of surgery at doses 5 and 10 mg/kg. At the end of the fourth week, the blood was collected then all animals were euthanized and liver tissues were dissected out for further analysis.
The main findings of the present study are summarized as follows:
1- Histopathological examination of liver tissue stained with H&E was conducted to illustrate BDL-induced CLF. Liver sections obtained from the sham and niclosamide-only treated groups showed no histopathological alteration and normal histological structure of the central vein and the surrounding hepatocytes in the parenchyma. On the contrary, BDL group showed massive inflammatory and fibrotic cells infiltration and massive number of newly formed bile ductules in the portal area. Moreover, there was a congestion in the portal vein. The BDL/niclosamide 5 group showed mild inflammatory and fibrotic cells infiltration and a few numbers of newly formed bile ductules in the portal area. Notably, the higher dose of niclosamide 10mg/kg show no signs of fibrosis and no newly formed bile ductules.
2- Niclosamide (5 and 10 mg/kg) significantly reduced liver enzymes levels. It was found that after 4 weeks, AST, ALT, ALP and GGT were significantly increased in BDL group, as compared to the sham group. The co-treatment with niclosamide (5 and 10 mg/kg) in BDL group significantly decreased AST, ALT, ALP and GGT, as compared to the BDL group
3- Niclosamide (5 and 10 mg/kg) significantly reduced the BDL induced oxidative stress. After 4 weeks, there was a significant increase in the MDA level in the BDL group, as compared to the sham group. The co-treatment with niclosamide (5 and10 mg/kg) significantly ameliorated this elevation, as compared to the BDL group. Furthermore, tissue levels of GSH and SOD were significantly reduced in the BDL group, as compared to the sham group. However, the co-treatment with niclosamide at doses 5 and 10 mg/kg significantly increased GSH and SOD, as compared to the BDL group.
4- Niclosamide (5 and 10 mg/kg) significantly reduced the BDL-induced inflammation. It was found that TNF-α, IL-6 and NF-κB levels were significantly increased, as compared to the sham group. On the contrary, niclosamide (5 and 10 mg/kg) significantly reduced TNF-α, IL-6 and NF-κB levels compared to the BDL group.
5- Niclosamide (5 and 10 mg/kg) significantly suppressed BDL induced NOTCH activation. Jagged1 protein expression was significantly increased in the BDL group, as compared to the sham group. Interestingly, this elevation was decreased in the niclosamide-treated groups (5 and 10 mg/kg) by, as compared to the BDL group. In addition, HES1 expression was significantly increased in the BDL group, as compared to the sham group. In contrast, niclosamide co-treated groups (5 and 10 mg/kg) showed significant decrease in HES1 expression, as compared to the BDL group. Furthermore, the expression of NOTCH2 and NOTCH3 was significantly increased in the BDL group, as compared to the sham group. However, NOTCH2 and NOTCH3 expression was significantly decreased in the niclosamide co-treated groups (5 and 10 mg/kg), as compared to the BDL group. In addition, the BDL group exhibited a significant elevation in SOX9 ratio, as compared to the sham group. However, niclosamide co-treatment (5 and 10 mg/kg) significantly reduced SOX9, as compared to the BDL group.
6- Niclosamide (5 and 10 mg/kg) significantly reduced Wnt5B and Wnt10A expression. The BDL group exhibited a significant elevation in Wnt5B and Wnt10A protein expression, as compared to the sham group. On the other hand, niclosamide (5 and 10mg/kg) caused a significant lowering in Wnt5B and Wnt10A expression, as compared to the BDL group.
7- Niclosamide (5 and 10 mg/kg) significantly reduced the BDL-induced fibrosis. Immunohistochemical detection of TGF-β1 revealed a significant increase in TGF-β1 expression in the BDL group, as compared to the sham group. Co-treatment with niclosamide (5 and 10 mg/kg) significantly decreased TGF-β1 tissue level, as compared to the BDL group.
8- Besides TGF-β1, the immunohistochemical detection of α-SMA, as a marker of HSC activation, revealed a significant increase in its expression, in comparison with the sham group. In contrast, niclosamide (5 and10 mg/kg) significantly decreased the α-SMA expression, as compared to the BDL group.
9- The collagen production in liver tissue was estimated biochemically and by Masson’s trichrome stain where, biochemical assessment showed that a marked elevation in hydroxyproline level in the BDL group, as compared to the sham group. Nevertheless, the niclosamide-administered rats (5 and 10 mg/kg) showed a marked reduction in hydroxyproline content, as compared to the BDL group.
10- Masson’s trichrome stain of liver sections taken from the sham group and niclosamide 10 mg/kg only treated group without BDL showed no collagen fibres around the classical hepatic lobules. On the contrary, the BDL group showed severe blue colour in between the newly formed bile ductules, indicating a strong positive histochemical reaction for collagen fibers around the portal tract and central veins. On the other hand, the sections taken from niclosamide (5 mg/kg) co-treated group showed moderate blue staining, indicating a reduction of collagen deposition. Furthermore, the rats co-treated with 10 mg/kg niclosamide showed limited staining, indicating more reduction of collagen deposition.
Taken together, this study presents niclsoamide as a promising hepatoprotective agent against CLF. Additionally, the underlying protective mechanisms of niclosamide were highlighted focusing on its modulatory effect on NOTCH signaling pathway and Wnt signaling pathway. Indeed, more studies are needed to clarify the interaction between niclosamide and the Wnt pathway for inhibition of CLF as well as to clarify the effect of niclosamide on CLF at varying time points following BDL.