الفهرس 
Liver FailureOnly 14 pages are availabe for public view
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Abstract
Liver cell failure is a growing health problem and one of the main causes of death worldwide. Regenerative medicine as stem cells therapy is a newly rapidly developing field in which diseased tissues are regenerated. This study aim to evaluate the effectiveness of bone marrow derived mesenchymal stem cell on liver degeneration and fibrosis induced by Chlorpyrifos and comparing its efficacy with silymarin and colchicine as antifibrotic agents used in liver fibrosis on animal model.
The animals (mice) were randomly divided into four groups (A, B, C, D) of 15 animals each. group (A) was control, normal, non-diseased mice for sure of healthy environment and nutritional status to be compared with the diseased and treated groups. group (B) was diseased mice with liver cell failure due to Chlorpyrifos oral administration (LD50 / 80th of 60 mg/ Kg Body Weight/ 3 times per week) for 8 weeks. group (C) was treated with combination of oral silymarin and colchicine for four weeks (Silymarin dose was 150 mg/ Kilogram Body Weight/ 3 times per week, Colchicine dose was 200 ìg / Kilogram Body Weight/ 3 times per week) after induction of liver degeneration and fibrosis with chlorpyrifos. group (D) was treated with bone marrow derived mesenchymal stem cells (single intra-peritoneal injection of labeled BM-MSC 106 cells/ mouse intra-peritoneal) after induction of liver degeneration and fibrosis with chlorpyrifos. However, during the experiment 10 animals only were left in groups B, C, D due to the death induced from CPF.
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After 4 weeks of treatment, all mice were scarificed and, blood was collected for chemical analysis (liver functions tests: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Albumin, Bilirubin). Fibrosis index in liver tissue were assessed histopathologically by: H& E stain for evaluation and grading by Ishak scoring system, computed quantitative morphometric measurements were performed for Sirius red stained to determine the percentage of collagen (type I, III), and immunohistochemical staining [Matrix metalloproteinase-2 )MMP -2) and its tissue inhibitor (TIMP -2)] for confirmation of fibrosis.
Biochemical evaluation due to long term exposure to CPF (8 weeks) (group B) led to significant elevation of serum liver enzymes (ALT, AST) indicate degeneration of hepatocytes, with significant elevation of serum bilirubin level which indicate cholestatic liver disease due to fibrosis of portal tracts with obstructions of bile flow from chronic toxicity and fibrosis, and significant reduction of serum albumin level due to loss of synthetic function of liver tissue in comparison to control group (A).
Histological evaluation revealed, liver degeneration and fibrosis induced from eight weeks of oral chlorpyrifos (group B). Showed necrosis and vacuolar degeneration of zone 3 cells with pyknotic nuclei, fatty changes varies from droplets to diffuse involvement of liver cells with displacement of the nuclei, bile duct proliferation, marked inflammatory activity in portal zone, and appearance of congested blood vessels with moderate fibrosis around portal tracts and loss of hepatic architecture. Ishak scoring for this
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group in H& E Stain was (HAI 15, F3) indicated sever inflammatory reactions with moderate fibrosis. These confirm chronic toxicity of CPF effects on liver tissue.
This result was confirmed by Picosirius Red staining and immunohistochemical assays revealing MMP-2 and TIMP-2 expression. The percentage of the collagen proportionate area in Picosirius red stain was measured by Computed quantitative morphometry. There was elevation in collagen deposition in CPF group (group B) compared to control group. The content of hepatic fibrosis was significantly elevated.
Immunohistochemical assays revealing MMP-2 and TIMP-2 expression confirmed liver fibrosis. The expression of MMP-2 was moderate around portal tracts due to Activation of hepatic stellate cells and released of MMP-2 in compared to control group in which the expression of MMP-2 was negative. The expression of TIMP-2 in CPF group was strong all over the hepatic tissue due to collagen deposition and fibrosis in compared to control group in which the expression of TIMP-2 was moderate due to normal liver matrix.
CPF deteriorated liver architecture and initiated fibrous tissues deposition with significant elevation of serum liver enzymes (ALT, AST) and serum bilirubin level indicate degeneration of hepatocytes, loss of liver architecture and fibrosis, with significant reduction serum albumin level due to loss of synthetic function of liver tissue confirmed there was toxic effects of CPF on liver tissue.
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Treating liver degeneration and fibrosis with colchicine and silymarin (group C) was evaluated after four weeks of treatment. Biochemical evaluation of group C resulted in significant reduction of serum liver enzymes (ALT, AST) indicate regeneration of hepatocytes, and significant elevation of serum albumin level due to improvement of synthetic function of liver tissue with insignificant reduction of serum bilirubin level which indicate the presence of the fibrous tissue around portal tracts without degradation in comparison to CPF group (B).
Histopathological evaluation of group C resulted in insignificant reduction in the fibrosis, and there were ballooning hepatocytes with vacuolar degeneration and pyknotic nuclei, decreased portal inflammatory activity and loss of architecture. Ishak scoring for this group in H& E Stain was (HAI 11, F 3) indicated moderate inflammatory reactions with moderate fibrosis. So, colchicine and silymarin were only mild improving the inflammatory reactions, with no improving to fibrosis.
This result was confirmed by Picosirius Red staining and immunohistochemical assays revealing MMP-2 and TIMP-2 expression. The percentage of the collagen proportionate area in Picosirius red stain was measured by Computed quantitative morphometry. There was no reduction in collagen deposition in group treated with Colchicine and Silymarin (group C). The content of hepatic fibrosis not significantly reduced.
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Immunohistochemical assays revealing MMP-2 and TIMP-2 expression confirmed no improving of liver fibrosis. The expression of MMP-2 was not affected by colchicine and silymarin treatment (group C). The expression of TIMP-2 was strong all over the hepatic tissue due to collagen deposition and fibrosis.
So, colchicine and silymarin treatment did not improve liver architecture and not resolved fibrous tissues with insignificant reduction of serum bilirubin level, but there was significant reduction of serum liver enzymes (ALT, AST) indicate regeneration of hepatocytes, and significant elevation of serum albumin level due to improvement of synthetic function of liver tissue suggested there is therapeutic role of colchicine and silymarin in liver degeneration only, but not in liver fibrosis and cholestatic liver disease.
Treating liver degeneration and fibrosis induced by chlorpyrifos with BM-MSCs was evaluated after four weeks of BM-MSCs transplantation (group D) to make sure homing took place. PKH26-labeled BM-MSCs in the injured liver tissue of recipient mice were detected at 30 days after transplantation by fluorescent microscope. Biochemical evaluation of BM-MSCs treatment led to significant reduction of serum liver enzymes (ALT, AST) indicate regeneration of hepatocytes, with significant reduction of serum bilirubin level which indicate improvement of the cholestatic liver disease (reduction of fibrosis of portal tracts), and significant elevation of serum albumin level due to improvement of synthetic function of liver tissue in comparison to CPF group (B). Restoration of liver
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functions suggested a possible therapeutic role of BM-MSCs in fibrotic liver injury.
BM-MSC therapy in group (D) was resulted in significant improvement of hepatic fibrosis and degeneration compared to the untreated CPF group (B). Treatment with BM-MSCs (group D) markedly reduced the extent of portal cellular infiltration and reduced congestion with improving of hepatocytes degeneration and restoration of liver architecture. There was a clear histological variability of severity within fibrosis and inflammatory activities classified by the Ishak scoring system as HAI 15, F3 group B and HAI 6, F 1 group D. According to this scoring system, ”histological improvement was observed in hepatic fibrosis after BM-MSC treatment”.
This result was confirmed by Picosirius Red staining and immunohistochemical assays revealing MMP-2 and TIMP-2 expression. The percentage of the collagen proportionate area in Picosirius red stain was measured by Computed quantitative morphometry. The content of hepatic fibrosis slightly decreased after BM-MSC treatment may due to the number of pictures taking from each group in Computed quantitative morphometric measurements must be larger (10 pictures from each animal liver organ in each group) for more detection and accuracy of collagen percentages.
Immunohistochemical assays revealing MMP-2 and TIMP-2 expression confirmed improving of liver fibrosis. The expression of MMP-2 was reduced by BM-MSCs treatment to reach weak level (group D). TIMP-2 reduced by BM-MSCs treatment reached almost
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control level. Restoration of liver architecture and degradation of fibrous tissues suggested a possible therapeutic role of BM-MSCs in fibrotic liver injury. So that, BM-MSCs therapy can ameliorates hepatic fibrosis and reverse liver degeneration with improving liver functions. These results agreed with my first hypothesis.
However, treatment with Colchicine and silymarin had insignificant antifibrotic effect on liver fibrosis and bilirubin, with significant improving of liver enzymes (ALT, AST) and synthesis (Albumin). These results do not agreed with this second hypothesis.