الفهرس 
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Abstract
The current study was carried out to evaluate the impact of galectin-3 (Gal-3) inhibition on liver hepatocyte nuclear factors HNF4α and HNF1α gene expression after acetaminophen (APAP) induced hepatotoxicity in Wistar rats. Furthermore the study investigated the changes in hepatotoxicity severity after Gal-3 inhibition. In this study acetaminophen induced toxicity performed through single high toxic dose or long-term administration of relative high dose. This study may present a new protective way against acetaminophen induced hepatotoxicity through inhibiting galectin-3 functionality.
Materials and Methods
Experimental animals
The present study was conducted on eighty eight adult Wistar rats weighing from 100 to 120 gm. The animals were maintained on standard laboratory diet and water ad libitum, housed in stainless steel cages under standard environmental conditions and according to NRC ethical committee guidelines.
Chemicals
Acetaminophen (APAP) was purchased from Sigma Aldrich Co., St. Louis, Missouri, United States. It was suspended in 20% Tween-80 shortly before administration.
Modified citrus pectin (MCP) was purchased from EcoNugenics Inc., Santa Rosa, CA, USA. It was obtained as powder and dissolved in rats’ drinking water.
Experimental design
The rats were divided into 4 groups; three of them divided into subgroups. group A: normal control. Groups B1 and B2: Rats received MCP (1% wt.) dissolved in drinking water daily for a period of 3 days and 4 weeks respectively. Groups C1, C2 and C3: rats received orally single dose of APAP (3.5 g/Kg b.wt) and were sacrificed at 24 hours (h), 48 h and 72 h after APAP administration respectively. group C4: rats received oral APAP dose (1 g/kg b.wt) daily for 4 weeks. Groups D1, D2 and D3: Rats received oral single APAP dose (3.5 g/kg b.wt) and were supplied with MCP (1% wt.) dissolved in drinking water, then were sacrificed at 24 h, 48 h and 72 h after APAP single dose administration respectively. group D4: rats were treated by MCP as in group B2 along with receiving daily an oral APAP dose (1 g/kg b.wt) for 4 weeks.
Collection of blood and tissue samples
At the end of the experiment, blood samples were collected and serum was separated for biochemical analyses. The rats were sacrificed and liver samples were collected for biochemical and histopathological examination.
Biochemical analyses results
Administration of APAP either through single toxic dose or subtoxic dose long-term course resulted in hepatic damage represented by significant (P < 0.05) increases in serum activities of alanine and aspartate aminotransferase (ALT and AST), alkaline phosphatase (ALP) enzymes and level of total bilirubin, along with APAP toxic effect on kidney which caused elevation in serum urea and creatinine levels in comparison with normal control.
This toxic effect was strongly obvious after the long-term subtoxic dose course (group C4) and also after 24 h of APAP single toxic dose administration (group C1) compared to normal control group, then it diminished gradually after 48h (group C2) but still significant in most parameter levels and almost disappeared after 72h (group C3) which showed no significant difference compared to normal control group.
Meanwhile administration of modified citrus pectin (MCP) along with APAP improved the parameters levels towards normal control values. That occurred especially on long-term APAP subtoxic dose administration (group D4) and also after 24h of APAP single toxic dose administration (group D1) which showed significant change (P < 0.05) in all the parameters and 48h (group D2) with significant change (P < 0.05) in most of parameters when compared with their time correspondent groups (C4, C1 and C2), which received APAP only.
In comparison with normal control group, APAP administration led to significant elevation (P < 0.05) in liver levels of Gal-3 and tumor necrosis factor-alpha (TNF-α), and significant depression (P<0.05) in cytochrome P450 (CYP2E1), reduced glutathione (GSH) levels and glutathione reductase (GR) and glutathione peroxidase (GPx) activities.
All these changes occurred after both long-term APAP sutoxic dose and also at 24h after APAP single toxic dose administration, then withdrew gradually after 48h and almost disappeared after 72h which showed insignificant difference versus normal control group except for higher TNF-α and CYP2E1 levels.
Modified citrus pectin administartion reduced APAP induced toxic effect and also improved the previous parameters levels towards normal control values. Again this was most obvious after long-term APAP subtoxic dose administration (group D4) and also after 24h of APAP single toxic dose administration (group D1) which showed significant change (P < 0.05) in all the parameters, and 48h (group D2) with significant change (P < 0.05) in some of the parameters compared with their time correspondent groups (C4, C1 and C2) that received APAP only.
Quantitative gene expression levels of HNF4α and HNF1α
Acitaminophen administration led to significant depression (P < 0.05) in expression level of liver hepatocyte nuclear factor HNF4α after 24 and 48 h of single toxic dose administration and also after subtoxic long-term course in comparison with normal control group.
Meanwhile there was significant depression (P < 0.05) in expression level of liver HNF1α after 24 h and notable decrease after 48 h of APAP single toxic dose administration, compared to normal control group.
Administration of MCP induced significant increase in both liver HNF4α and HNF1α expression levels after both APAP subtoxic dose long-term course and also after 24 h of APAP single toxic dose administration, compared to their correspondent groups without MCP treatment (groups C4 and C1).
Meanwhile there was almost no significant difference in all previous markers among control group, MCP only treated groups and the groups treated with only APAP (except for TNF-α and CYP2E1 levels) or accompanied by MCP and were sacrificed after 72h of APAP administration and the group received APAP and MCP for long-term period (groups A, B1, B2, C3, D3 and D4).
Histopathological results
Administration of APAP either through subtoxic dose for long-term period or single toxic dose resulted in disturbance of the structure of hepatic lobule, appearance of focal necrosis of hepatocytes and hydropic degeneration. This was obvious clearly in the long-term period group and the groups received the single toxic dose after 24 h, stilled present after 48 h and decreased relatively after 72 h. On the other hand, liver sections of rats received MCP with APAP showed reduction in number and areas of necrotic regions compared to their correspondent groups that received only APAP.
Quantitative evaluation of liver damaged area showed significant (P<0.05) increase due to APAP administration through subtoxic dose for long-term period or single toxic dose. The damaged areas were the greatest after 24 h of APAP single toxic dose administration then decreased gradually after 48 h and 72 h. however the groups received MCP showed significant (P < 0.05) reduction of hepatic damaged areas when compared with their time correspondent groups without MCP treatment.
In conclusion, APAP administration either through single toxic dose or long-term subtoxic dose exposure caused reduction in HNF4α and HNF1α genes expression levels, which may be through the elevation in levels of inflammatory cytokines that inhibits their expression. By inhibiting bioactivity of Gal-3, which is an important mediator of inflammation, by using MCP; an improvement observed in HNF4α and HNF1α gene expression levels in addition to other hepatorenal function and APAP detoxification pathway parameters. So Inhibition of Gal-3 using MCP could be helpful against APAP induced acute and accumulative liver toxicity.