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CCl4 and TAA were purchased from Sigma-Aldrich, SL, USA via a local distributor. TIBC colorimetric reagent set was obtained from Pointe Scientific® Inc., MI, USA via a local distributor. Rat ferritin ELISA kit was acquired from Genway Biotec®, Inc., CA, USA, via a local distributor. RT-PCR Trizol reagent, Oligo (dT) primer, Superscript Ⅱ reverse transcriptase, Taq DNA polymerase, hepcidin, IL-6, and β-actin primers were supplied by Invitrogen, Carlsbad, CA. All other chemicals were of analytical grade and available from local distributors.
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Thirty male albino rats aged 6 weeks and weighing 180-200 g were acclimatized under hygienic condition and given free access to clean water and balanced diet. The rats were then randomly separated into three groups (10 each group) in separate cages and subjected to different treatments. Subchronic liver injury/fibrosis was induced in groups 2 and 3 by repeated intraperitoneal injections of CCL4 (100 μL/100 g rat weight diluted as 1:4 in corn oil) or TTA (10 mg/100 g rat weight dissolved in isosaline) twice a week for 8 weeks (modified after Scholten et al.[11] and Wallace et al.[12], respectively). Control group (C) was injected with vehicles (corn oil and isosaline) at the same time. On the day after the last administration, blood samples were collected from the medial canthus venous plexus under light ether anesthesia. Two types of blood samples were saved. The first sample was received into EDTA tube for blood examination. The second sample was received into a plain sampling tube, left to coagulate, and centrifuged. Serum was harvested for determination of transaminases, iron, UIBC, and ferretin. After blood collection, the rats were sacrificed. The livers were removed, and pieces (100 mg) were submerged in Trizol reagent for RNA isolation and PCR. All procedures had ethical approval. The animals received daily human care, and the study protocols complied with our institutional guidelines. The study conformed to ARRIVE guidelines (http://www.nc3rs.org.uk/arrive guidelines) developed by the National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs).
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An auto-hematology analyzer (Mindray®, Model BC-2800Vet, Shenzhen, China) was used to determine erythrocytic parameters, including red blood cell (RBC) count, packed cell volume (PCV), hemoglobin concentration (HGB), mean cell volume (MCV), mean cell hemoglobin, and mean cell hemoglobin concentration; and leukocytic parameters, including total white blood cell count, lymphocyte, granulocyte, monocytes, and platelets (PLT) counts[13].
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Serum biomarkers (ALAT and ASAT) in the liver were measured spectrophotometrically using diagnostic kits from Analyticon® (Lichtenfels, Germany) following the instructions of the manufacturer.
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Serum Iron was determined based on the ferrozine principle described by Stookey[14] by using a diagnostic kit. Transferrin-bound iron in the sample (500 μL) was released at an acidic pH (2.5 mL of 220 mmol/L hydroxylamine hydrochloride in acetate buffer, pH 4.5) and reduced from ferric to ferrous. These ions reacted with ferrozine (50 μL, 16.7 mmol/L) to form a violet colored complex, which was measured spectrophotometrically at 560 nm wavelength.
TIBC was determined by calculation as the algebraic sum of the 'serum iron concentration' plus the 'UIBC'[15]. UIBC was determined first by ferrozine reaction after adding a known concentration (500 μL of the standard 500 μg/dL) of ferrous ions to the serum sample (500 μL) at an alkaline pH (Tris 500 mmol/L, pH 8.1). The ferrous ions will bind to transferrin at the unsaturated iron binding sites. The additional unbound ferrous ions were measured using the ferrozine reaction (adding 50 μL of the iron color reagent, namely, 16.7 mmol/L ferrozine and measuring the intensity of violet color at 560 nm). UIBC is the difference between the concentration of ferrous ions added and the unbound serum ions measured.
Transferrin saturation% was calculated as: [Serum iron (μg/dL)/TIBC (μg/dL)] × 100[16].
Serum Transferrin concentration was calculated from its correlation with TIBC, where one molecule of transferrin can bind to two molecules of iron at two high-affinity binding sites, by using the following equation according to previous works[17-18]:
$$ \text{TIBC }\left( \text{ }\!\!\mu\!\!\text{ mol/L} \right)\text{ = 25}\text{.1 }\!\!\times\!\!\text{ transferrin }\left( \text{g/L} \right) $$ (1) where (μg/dL) = 0.179 (μmol/L) according to SI conversion.
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Ferritin, the iron storage protein, was determined by ELISA kit according to the instructions of the manufacturer. Ferritin in the serum samples reacted with anti-ferritin antibodies, which were adsorbed onto the surface of the polystyrene microtiter plate. After the removal of unbound proteins by washing, anti-ferritin antibodies conjugated with horseradish peroxidase (HRP) were added. These enzyme-labeled antibodies formed complexes with the previously bound ferritin. After another washing step, the enzyme bound to the immunosorbent was assayed by adding a chromogenic substrate, 3, 3′, 5, 5′-tetramethylbenzidine (TMB). The quantity of the bound enzyme varied directly with the concentration of ferritin in the sample tested. Absorbance was recorded using a microplate reader at 450 nm to measure the concentration of ferritin in the test sample.
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Total cellular RNA was extracted from the hepatic specimens by Trizol reagent according to Chomczynski and Sacchi[19]. In brief, 100 mg of each liver tissue specimen was homogenized in 1 mL of Trizol reagent by using a power homogenizer (IKA Labortechnik Staufen, Staufen, Germany). The homogenized samples were transferred to 1.5 mL Eppendorf tubes and added with 0.2 mL of chloroform for phase separation. After vigorous shaking for 15 s, the samples were centrifuged at 12, 000 rpm and 4 ℃ for 15 min. The mixture separated into a lower red, phenol-chloroform phase, an inter-phase, and a colorless upper aqueous phase. RNA remained in the aqueous phase. For RNA precipitation, the aqueous phases were transferred to fresh tubes and added with 0.5 mL of isopropanol. The tubes were incubated at room temperature for 15 min and centrifuged again at 12, 000 rpm for 15 min. The RNA precipitate, which is often invisible before centrifugation, formed a gel-like pellet on the side and bottom of the tube. The pellets were washed in 75% ethanol after removal of the supernatant and recentrifuged. The washing alcohol solution was removed, and the pellets were left at room temperature for 10 min to dry up. The dried pellets were redissolved in RNase-free water according to the size of the pellet. RNA concentration was determined by recording their absorbance (A) at 260 and 280 nm with a spectrophotometer (the A260/280 values were less than 1.6 in all samples assuming no protein contamination). RNA samples were subjected directly to reverse transcription (RT).
RT was performed with RNA random primers by using RT-PCR kit (Takara Bio INC., Shiga, Japan) according to the instructions of the manufacturer. Aliquots of 2.5 μg of the isolated RNA in a suitable volume of RNase-free water (1-5 μL according to the concentration of RNA measured in the sample; preferred to be added lastly to avoid trans-contamination) were used for RT. The obtained first-strand complementary DNAs (cDNAs) were kept at -20 ℃ until PCR was performed.
PCR was performed using the same kit with rat hepcidin, IL-6, and β-actin primers prepared by nucleic acid synthesis system (Rikaken, Nagoya, Japan). The sense (forward) and antisense (reverse) sequences were 5′-GCT GCC TGT CTC CTG CTT-3′ and 5′-TTA CAG CAT TTA CAG CAG AAG AGG-3′ for hepcidin; 5′-TGA TGG ATG CTT CCA AAC TG-3′ and 5'-GAG CAT TGG AAG TTG GGG TA -3′ for IL-6; and 5′-TGT CAC CAA CTG GGA CGA TA-3′ and 5′-AAC ACA GCC TGG ATG GCT AC-3′ for β-actin.
The PCR products were size fractionated by 1.5% agarose gel electrophoresis starting with 70 V for 20 min and continuing with 120 V until aliquots reached the desired separation levels. The DNA bands were visualized with an ultraviolet trans-illuminator (UVP, CA, USA). Quantification of hepcidin, IL-6, and β-actin amplified DNAs was carried out by densitometric analysis using Scion Image 4.02 software (Scion Corporation, Maryland, USA), setting the control density as the unity and calculating hepatotoxins as folds.
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Data are expressed as mean ± standard error of the mean of 10 (control) or 9 (groups 2 and 3) separate observations. Observations were compared using ANOVA followed by LSD as post-hoc test at P level of 0.05. All procedures of statistics and graphing were completed using the computer program GraphPad Prism® version 6 (GraphPad Inc., CA, USA).
Chemicals and Reagents
Animal Models and Study Design
Hemogram
Transaminase Assay
Iron/UIBC/TIBC Assay
Ferritin Assay
RT-PCR of Hepcidin and IL-6 in Hepatocytes
Data Presentation and Analysis
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Nine of 10 rats in groups 2 and 3 survived until the end of the experimental course. The subchronic TAA and CCL4 treatments induced significant elevations of the liver enzymes, namely, AST and ALT, which indicated damage to liver cells. The values recorded after CCl4 were lower than those recorded after TAA (Table 1).
Parameter Weeks Control TAA CCl4 ALT (U/L) 8 23.87 ± 1.92 275.0 ± 14.3* 155.0 ± 11.3* AST (U/L) 8 29.53 ± 2.05 308.1 ± 18.9* 216.3 ± 12.6* Note. *Means significantly (P < 0.05) different from corresponding control; TAA: thioactamide, 10 mg/100 g rat weight, dissolved in isosaline; CCl4: carbon tetrachloride, 100 μL/100 g rat weight, diluted as 1:4 in corn oil; ALT: alanine aminotransferase; AST: aspartate aminotransferase. Mean ± SE, n = 9 for treated groups (twice weekly, for 8 weeks) and 10 for the control (isosaline/corn oil). Table 1. Transaminases as Liver Serum Biomarkers after Repeated i.p Injections of TAA and CCl4 to Rats Compared to Those of Normal Control
The erythrocyte and leukocyte parameters and those of platelets were significantly (P > 0.05) altered after repeated subchronic hepatotoxin injections compared with those in the control group (Tables 2, 3, and 4). Table 2 shows the diminished erythrocyte count, Hb concentration, hematocrit value, and erythrocytic indices toward anemic condition in the CCl4 model but not in the TAA model. Table 3 shows the data of leukogram, indicating significant leukocytosis with significant granulocytosis and insignificant lymphocytopenia. The altered leukogram was evident in both models. Table 4 shows that the platelets exhibited thrombocytopenia and decreased plateletcrit with insignificantly altered MPV index after subchronic treatment with both hepatotoxins and increased the PDW index only in the case of TTA. Thromocytopenia was more drastic in the TAA model than in the CCl4 model.
Erythrocytic Parameters Groups Control TAA CCl4 RBC (1012/L) 8.02 ± 0.31 7.12 ± 0.39 5.11 ± 0.34* PCV (%) 46.81 ± 2.63 39.37 ± 3.95 26.02 ± 2.14* HGB (g/dL) 15.21 ± 1.88 12.73 ± 0.82 8.21 ± 0.75* MCV (fL) 58.35 ± 1.73 55.29 ± 3.11 50.91 ± 1.52* MCH (pg) 18.97 ± 1.55 17.84 ± 1.15 16.07 ± 0.97 MCHC (g/dL) 32.51 ± 3.01 32.26 ± 2.25 31.56 ± 1.77 Note. *Mean significantly (P < 0.05) different from it; RBC: red blood count; PCV: packed cell volume; HGB: hemoglobin concentration; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; TAA: 10 mg/100 g rat weight dissolved in isosaline; CCl4: 100 μL/100 g rat weight diluted as 1:4 in corn oil. Mean ± SE, n = 9 for treated groups (twice weekly, for 8 weeks) and 10 for the control (isosaline/corn oil). Table 2. Erythrogram after Repeated i.p. Injections of TAA and CCl4 to Rats, Compared to that of Normal Control
Leukocytic Parameters Groups Control TAA CCl4 WBC (109/L) 13.53 ± 2.43 21.13 ± 2.13* 26.33 ± 2.30* Lymph. (%) 71.83 ± 3.53 67.41 ± 3.25 68.06 ± 3.19 Mid-sized (%) 2.44 ± 0.52 2.29 ± 0.23 3.07 ± 0.30 Gran. (%) 24.23 ± 0.75 30.38 ± 1.19* 29.41 ± 1.55* Note. *Mean significantly (P < 0.05) different from it. WBC: white blood cell; Lymph: lymphocytes; Mid-sized: mid-sized cells; Gran: granulocytes; TAA: 10 mg/100 g rat weight dissolved in isosaline; CCl4: 100 μL/100 g rat weight diluted as 1:4 in corn oil. Mean ± SE, n = 9 for treated groups (twice weekly, for 8 weeks) and 10 for the control (isosaline/corn oil). Table 3. Leukogram after Repeated i.p. Injections of TAA and CCl4 to Rats, Compared to that of Normal Control
Platelet Parameters Groups Control TAA CCl4 PLT (109/L) 655.6 ± 17.44 388.8 ± 11.2* 442.6 ± 14.2* PCT (%) 0.53 ± 0.02 0.36 ± 0.05* 0.45 ± 0.03* MPV (fL) 8.13 ± 0.41 9.26 ± 0.26 10.17 ± 0.28 PDW (%) 16.38 ± 1.02 22.48 ± 1.06* 19.12 ± 1.11 Note. *Mean significantly (P < 0.05) different from it; PLT: platelet; PCT: procalcitonin; MPV: mean platelet volume; PDW: platelet distribution width; TAA: 10 mg/100 g rat weight dissolved in isosaline; CCl4: 100 μL/100 g rat weight diluted as 1:4 in corn oil; Mean ± SE, n = 9 for treated groups (twice weekly, for 8 weeks) and 10 for the control (isosaline/corn oil). Table 4. Platelet Count and Indices after Repeated i.p. Injections of TAA and CCl4 to Rats, Compared to that of Normal Control
Serum analytical assays revealed significantly increased erythropoietin (EPO) concentration in the CCL4 group compared with that in the control and TAA group. The levels of serum iron, UIBC/TIBC, transferrin, and transferrin saturation% significantly decreased, and that of ferritin increased in the CCL4 group compared with those in the control rats. In the TAA model, the serum iron and iron indices changed toward iron overload, although this change was insignificant (Figure 1A-F).
Figure 1. Changes in erythropoietin (A), serum iron (B), ferritin (C), UIBC/TIBC (D), transferrin saturation% (E), and serum transferrin concentration (F) in rats after repeated (twice weekly, for 8 weeks) i.p. injection of TAA (10 mg/100 g rat weight dissolved in isosaline) and CCl4 (100 μL/100 g rat weight diluted as 1:4 in corn oil) in comparison with normal rats (isosaline/corn oil); Mean ± SE, n = 9 for treated groups and 10 for the control. *P < 0.05.
RT-PCR analysis of the hepatic specimen revealed increased expression of hepcidin mRNA associated with increased expression of IL-6 mRNA in the CCL4 group. The TTA group showed unexpected significant decrease in hepcidin mRNA expression, and that of IL-6 did not significantly change (Figure 2A-B).
Figure 2. RT-PCR products and fold analysis of liver hepcidin (A) and IL-6 (B) mRNA expression in rats after repeated (twice weekly, for 8 weeks) i.p. injections of TAA (10 mg/100 g rat weight dissolved in isosaline) and CCl4 (100 μL/100 g rat weight diluted as 1:4 in corn oil) in comparison with normal rats (isosaline/corn oil); Mean ± SE of 3 observations; control value was set as the unit and those of hepatotoxins were set as folds. **P < 0.01.