HISTOLOGICAL AND BIOCHEMICAL EVALUATION OF DIFFERENT TREATMENT MODALITIES OF LIVER CIRRHOSIS IN ALBINO RATS
Einas Ahmed Kassem Kassem;
Abstract
Summary and Conclusion
I
n Egypt, liver cirrhosis represents a major health crisis in view of the high prevalence rate of hepatitis virus C (HCV) infection. Liver cirrhosis is characterized by massive deposition of fibrous tissue and the conversion of normal liver architecture into structurally well defined abnormal nodules. These lobules are surrounded by thickened fibrous septa that are infiltrated by inflammatory cells. Portal hypertension resulting from an increased intra hepatic resistance is the earliest and most important consequence of cirrhosis. It is eventually complicated by splenomegaly and esophageal varices. Liver cell failure goes hand in hand with the cirrhotic process leading to ascites and encephalopathy.
The cirrhotic changes are conducted mainly by activated stellate cells (HSCs) located in the spaces of Disse and to lesser extent by portal fibroblasts and bone marrow-derived collagen producing cells. Activation of stellate cells takes place in response to oxidative stress, profibrogenic cytokines, apoptotic bodies or inflammatory signal transduction pathways. Hepatic stellate cells activation is followed by mitosis and production of increasing amount of extracellular matrix. Activated HSCs also show enhanced production of metalloproteinase inhibitors which inhibit the naturally occurring collagenolytic enzymes that cause collagen degradation and remodeling. HSCs activation has been recently linked to stellate cell autophagy and the role of autophagy inhibitors in the management of liver cirrhosis has been emphasized.
Proper understanding of the cirrhotic mechanism pointed out to the notion that cirrhosis is not only a controllable process but also can be a reversible one. Elimination of the trigger of cirrhosis removes the survival factor necessary for the activation of HSCs. This would lead to rapid decline in metalloproteinase inhibitors expression with consequent enhancement of the collagenolytic activity, extracellular matrix (ECM) degradation and HSCs apoptosis.
The main aim of the present study was to assess the potentials of chloroquine phosphate and α-chymotrypsin in resolving liver cirrhosis induced in albino rats by CCl4/ethanol treatment. These two medications were selected on basis of their previously documented biological actions. These biological actions seemed to have the potential to interrupt the viscous circle of HSCs activation, collagen production, metalloproteinase inhibitor secretion, inhibition of HSCs apoptosis with consequent over deposition of ECM and inhibition of collagenolysis. It also aimed at reproducing the earlier data documenting the efficacy of two other compounds, green tea extract and ciprofloxacin, in the amelioration of liver cirrhosis and compare with the other two drugs.
Fifty four albino rats have been used in this study. They were divided into six equal groups as follows:
Group 1: (Healthy Control group) receiving no treatment.
Group 2:(CCl4-Ethanol group) receiving intraperitoneal injection of 1.0 ml/kg body weight of 10% CCl4 in olive oil three times a week for 12 weeks with no other treatment for two more weeks.
Group 3: (Green Tea group) treated as in group 2 and provided with 1.5% green tea extracts as a sole source of drinking water for two weeks.
Group 4: (Ciprofloxacine group) treated as in group 2 and were given a daily dose of Ciprofloxacine (antibiotic) 7.2 mg dissolved in 0.5 ml distilled water by gastric gavage for two more weeks.
Group 5: (Chloroquine group) treated as in group 2 and were orally supplied with (Chloroquine phosphate) at a dose of (10 mg per kg) for two weeks by gastric tube.
Group 6: (α-Chymotrypsin group) treated as in group 2 and received intraperitoneal (I.P) doses (15mg) of α –Chymotrypsin per kg body weight thrice per week for two weeks.
At the end of the experiment, all rats were sacrificed by decapitation, blood was collected, and livers were excised and kept in buffered formalin for histological examination. EDTA blood was used for determination of the complete blood count (CBC). Sera were separated for the determination of the liver function parameters and serum α-fetoprotein level. The liver function parameters included the determination of serum total bilirubin, direct bilirubin, serum alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP) activities together with serum total proteins, albumin and globulin.
Data from the present study showed that CCl4/ethanol treated rats experienced significant decrease in hemoglobin (Hb), hematocrite (HCT), total leucocytic count (TLC) and platelet count. They also showed that these animals experienced marked deterioration of the liver function manifested by significant rise in ALT, AST, GGT, total and direct bilirubin and significant decrease in serum total protein and albumin. Data also showed significant rise in α-fetoprotein in response to CCl4/ethanol treatment. Green tea, ciprofloxacine and α-chymotrypsin treatment resulted in the restoration of Hb and HCT of CCl4/ethanol treated animals. All compounds corrected the decrease of TLC and platelets counts. Improvement of liver function parameters were detected in response to all therapeutic modalities except the ALT activity in green tea supplemented animals. CCl4/ethanol treatment induced profound disruption of the liver architecture consistent with liver cirrhosis. Marginal resolution of cirrhosis was reported in animals receiving no treatment for two weeks after completion of CCl4/ethanol administration. Treatment with chloroquine phosphate and green tea showed signs of amelioration of the feature of cirrhosis in most animals. Examination of liver sections from both groups revealed more or less normal appearance of the liver architecture where hepatic lobules appeared intact with normal contour of central veins. Portal areas showed fewer number of inflammatory cells and minimal signs of necrosis. Treatment with ciprofloxacine and α-chymotrypsin induced lesser degree of amelioration in the cirrhosic features caused by CCl4/ethanol treatment. This was manifested by the presence of different degrees of damage in the liver tissue. In most of the sections of the two groups portal areas showed infiltration with leucocytes and activation of Kupffer cells. Areas of fibroblatic activity or fibrosis were seen in the portal area.
I
n Egypt, liver cirrhosis represents a major health crisis in view of the high prevalence rate of hepatitis virus C (HCV) infection. Liver cirrhosis is characterized by massive deposition of fibrous tissue and the conversion of normal liver architecture into structurally well defined abnormal nodules. These lobules are surrounded by thickened fibrous septa that are infiltrated by inflammatory cells. Portal hypertension resulting from an increased intra hepatic resistance is the earliest and most important consequence of cirrhosis. It is eventually complicated by splenomegaly and esophageal varices. Liver cell failure goes hand in hand with the cirrhotic process leading to ascites and encephalopathy.
The cirrhotic changes are conducted mainly by activated stellate cells (HSCs) located in the spaces of Disse and to lesser extent by portal fibroblasts and bone marrow-derived collagen producing cells. Activation of stellate cells takes place in response to oxidative stress, profibrogenic cytokines, apoptotic bodies or inflammatory signal transduction pathways. Hepatic stellate cells activation is followed by mitosis and production of increasing amount of extracellular matrix. Activated HSCs also show enhanced production of metalloproteinase inhibitors which inhibit the naturally occurring collagenolytic enzymes that cause collagen degradation and remodeling. HSCs activation has been recently linked to stellate cell autophagy and the role of autophagy inhibitors in the management of liver cirrhosis has been emphasized.
Proper understanding of the cirrhotic mechanism pointed out to the notion that cirrhosis is not only a controllable process but also can be a reversible one. Elimination of the trigger of cirrhosis removes the survival factor necessary for the activation of HSCs. This would lead to rapid decline in metalloproteinase inhibitors expression with consequent enhancement of the collagenolytic activity, extracellular matrix (ECM) degradation and HSCs apoptosis.
The main aim of the present study was to assess the potentials of chloroquine phosphate and α-chymotrypsin in resolving liver cirrhosis induced in albino rats by CCl4/ethanol treatment. These two medications were selected on basis of their previously documented biological actions. These biological actions seemed to have the potential to interrupt the viscous circle of HSCs activation, collagen production, metalloproteinase inhibitor secretion, inhibition of HSCs apoptosis with consequent over deposition of ECM and inhibition of collagenolysis. It also aimed at reproducing the earlier data documenting the efficacy of two other compounds, green tea extract and ciprofloxacin, in the amelioration of liver cirrhosis and compare with the other two drugs.
Fifty four albino rats have been used in this study. They were divided into six equal groups as follows:
Group 1: (Healthy Control group) receiving no treatment.
Group 2:(CCl4-Ethanol group) receiving intraperitoneal injection of 1.0 ml/kg body weight of 10% CCl4 in olive oil three times a week for 12 weeks with no other treatment for two more weeks.
Group 3: (Green Tea group) treated as in group 2 and provided with 1.5% green tea extracts as a sole source of drinking water for two weeks.
Group 4: (Ciprofloxacine group) treated as in group 2 and were given a daily dose of Ciprofloxacine (antibiotic) 7.2 mg dissolved in 0.5 ml distilled water by gastric gavage for two more weeks.
Group 5: (Chloroquine group) treated as in group 2 and were orally supplied with (Chloroquine phosphate) at a dose of (10 mg per kg) for two weeks by gastric tube.
Group 6: (α-Chymotrypsin group) treated as in group 2 and received intraperitoneal (I.P) doses (15mg) of α –Chymotrypsin per kg body weight thrice per week for two weeks.
At the end of the experiment, all rats were sacrificed by decapitation, blood was collected, and livers were excised and kept in buffered formalin for histological examination. EDTA blood was used for determination of the complete blood count (CBC). Sera were separated for the determination of the liver function parameters and serum α-fetoprotein level. The liver function parameters included the determination of serum total bilirubin, direct bilirubin, serum alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP) activities together with serum total proteins, albumin and globulin.
Data from the present study showed that CCl4/ethanol treated rats experienced significant decrease in hemoglobin (Hb), hematocrite (HCT), total leucocytic count (TLC) and platelet count. They also showed that these animals experienced marked deterioration of the liver function manifested by significant rise in ALT, AST, GGT, total and direct bilirubin and significant decrease in serum total protein and albumin. Data also showed significant rise in α-fetoprotein in response to CCl4/ethanol treatment. Green tea, ciprofloxacine and α-chymotrypsin treatment resulted in the restoration of Hb and HCT of CCl4/ethanol treated animals. All compounds corrected the decrease of TLC and platelets counts. Improvement of liver function parameters were detected in response to all therapeutic modalities except the ALT activity in green tea supplemented animals. CCl4/ethanol treatment induced profound disruption of the liver architecture consistent with liver cirrhosis. Marginal resolution of cirrhosis was reported in animals receiving no treatment for two weeks after completion of CCl4/ethanol administration. Treatment with chloroquine phosphate and green tea showed signs of amelioration of the feature of cirrhosis in most animals. Examination of liver sections from both groups revealed more or less normal appearance of the liver architecture where hepatic lobules appeared intact with normal contour of central veins. Portal areas showed fewer number of inflammatory cells and minimal signs of necrosis. Treatment with ciprofloxacine and α-chymotrypsin induced lesser degree of amelioration in the cirrhosic features caused by CCl4/ethanol treatment. This was manifested by the presence of different degrees of damage in the liver tissue. In most of the sections of the two groups portal areas showed infiltration with leucocytes and activation of Kupffer cells. Areas of fibroblatic activity or fibrosis were seen in the portal area.
Other data
| Title | HISTOLOGICAL AND BIOCHEMICAL EVALUATION OF DIFFERENT TREATMENT MODALITIES OF LIVER CIRRHOSIS IN ALBINO RATS | Other Titles | التقييم الهستولوجى والبيوكيميائى لأنماط علاجية مختلفة للتشمع الكبدى فى الجرذان البيضاء | Authors | Einas Ahmed Kassem Kassem | Issue Date | 2015 |
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