Intidhar Bkhairia*a,
Sabah Dhibibc,
Rim Nasria,
Abdelfettah Elfekib,
Najla Hfaiyedhbc,
Ibtissem Ben Amarad and
Moncef Nasria
aLaboratory of Enzyme Engineering and Microbiology, University of Sfax, National School of Engineering of Sfax (ENIS), B. P. 1173, 3038, Sfax, Tunisia. E-mail: ibkhairia@yahoo.com; Fax: +216 74 275 595; Tel: +216 96 287 128
bLaboratory of Environmental Physiopathology, Valorization of Bioactive Molecules and Mathematical Modeling, Faculty of Sciences of Sfax, Road Soukra km 3.5, PB no. 1171-14 3000, Sfax, Tunisia
cLaboratory Animal Eco Physiology, Faculty of Sciences, Sidi Ahmed Zarrouk, 2112, Gafsa, Tunisia
dHigher Institute of Biotechnology of Sfax, University of Sfax, 3000, Sfax, Tunisia
First published on 26th June 2018
This study was undertaken to examine the hepatoprotective, antioxidant, and DNA damage protective effects of protein hydrolysates from Liza aurata, against paracetamol overdose induced liver injury in Wistar rats. L. aurata protein hydrolysates (LAPHs) were mainly constituted by glutamic acid (Glu) and glutamine (Gln) and lysine (Lys). In addition, they contained high amounts of proline (Pro), leucine (Leu) and glycine (Gly). The molecular weight distribution of the hydrolysates was determined by size exclusion chromatography, which analyzed a representative hydrolysate type with a weight range of 3–20 kDa. The hepatoprotective effect of LAPHs against paracetamol liver toxicity was investigated by in vivo assay. Rats received LAPHs daily by gavage, for 45 days. Paracetamol was administrated to rats during the last five days of treatment by intraperitoneal injection. Paracetamol overdose induced marked liver damage in rats was noted by a significant increase in the activities of serum aspartate amino transferase (AST) and alanine amino transferase (ALT), and oxidative stress which was evident from decreased activity of the enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), and level of glutathione (GSH), and increased concentration of lipid peroxidation products (MDA). Furthermore, paracetamol increased the DNA damage with liver histopathological changes. LAPH pretreatment significantly attenuated paracetamol-induced hepatotoxic effects, including oxidative damage, histopathological lesions, and apoptotic changes in the liver tissue. Interestingly, LAPHs restored the activities of antioxidant enzymes and the level of GSH, ameliorated histological and molecular aspects of liver cells. The present data suggest that paracetamol high-dose plays a crucial role in the oxidative damage and genotoxicity of the liver and therefore, some antioxidants such us LAPHs might be safe as hepatoprotectors. Altogether, our studies provide consistent evidence of the beneficial effect of LAPHs on animals treated with a toxic dose of paracetamol and might encourage clinical trials.
Paracetamol (acetaminophen – APAP) is used by millions of people worldwide as a safe analgesic drug at therapeutic doses. An overdose of paracetamol results in an increase in the levels of the toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which extensively depletes hepatocellular glutathione (GSH) along with generation of reactive oxygen species (ROS).2 There are an estimated 60000 cases of paracetamol overdose annually, with most cases being intentional suicide gestures.3 Nearly 26000 overdose patients are hospitalized each year, and an estimated 1% develops severe coagulopathy or encephalopathy. The mortality attributed to acetaminophen toxicity is 500 per annum, and at least 20% of these deaths occur in patients with non-intentional paracetamol overdose.3 High dose of paracetamol is well known to be toxic to the liver.4 When acetaminophen is used in therapeutic doses, most of the drug is metabolized via glucuronidation and sulfation, a very small amount of acetaminophen is metabolized to NAPQI by the hepatic enzyme cytochrome P450 2E1 (CYP2E1).5
The antidote n-acetylcysteine (NAC) is highly effective at preventing toxicity, provided it is administered within a few hours of the overdose. Subsequently efficacy declines as the interval between the overdose and NAC administration increases.6 However, despite its efficacy in reducing mortality due to paracetamol poisoning, intravenous NAC can cause anaphylactoid reactions.7 Due to the complexity of preparation of NAC as well as its side effects, mistakes are possible.8 However, by reason of the narrow treatment and limited timing of NAC, new therapeutic interventions are necessary to be developed for the treatment of paracetamol toxicity.
In recent years, a great deal of interest has been expressed regarding the productions, characterizations, and applications of protein hydrolysates and food-derived biopeptides due to their numerous beneficial health effects. Peptides with biological activities have shown promise as pharmaceutics with the potential to treat a wide variety of diseases. Indeed, increasing demands for functional foods for health care and disease risk reduction are prevalent throughout the world.9 The past decade has also witnessed intense interest in functional foods or dietary photochemical which can influence the pharmacological activity of drugs and their toxicities by modifying metabolism system, including drug-metabolizing enzymes and transporters.10
In the pharmaceutical industry, liver, a vital organ in the body responsible for metabolizing and detoxification of substances,11 is one of the routinely assessed organs during preclinical safety evaluations. Indeed several biological compounds with antioxidant properties proved effective in protecting the liver against deleterious effects of paracetamol overdose. In addition, protein hydrolysates from fish are considered as reservoirs of structurally diverse bioactive materials with numerous biological effects for human's body affects either directly or indirectly in maintaining good health.12 Depending on the composition and the sequence of amino acids, these protein hydrolysates, which contain a complex mixture of peptides, can exhibit diverse activities.
In fact, experimental data showed the antihypertensive,13 antioxidative14,15 and anti-diabetic16 properties from marine protein hydrolysates.
In a previous study, we demonstrated that LAPHs obtained from L. aurata proteins using a commercial, endogenous and microbial proteases, exhibited a variable extend antioxidant activities.15 In addition, the RP-HPLC analysis demonstrates that the bioactivities of LAPHs were directly related to the hydrophobic properties of peptides. However, the protective role of biopeptides from L. aurata against paracetamol induced liver injury has not been investigated. Hence, we proposed to scrutinize hepatoprotective effect of LAPHs against paracetamol-induced acute liver injury. In addition, the feasible molecular mechanisms underlying this hepatoprotective effect are discussed, involving antioxidant activities.
Golden grey mullet (L. aurata) is one of the mullet species which is widely distributed in the Mediterranean Sea and Black Sea, Atlantic coasts from the Azores and Madeira northward to the British Isles, and the southern coasts of Norway and Sweden.17 L. aurata is relatively important in the fish catches of Tunisia, and is utilized for human consumption.
The aim of the present study was to evaluate, for the first way, the hepatoprotective potential and antioxidative effects of LAPHs in subacute toxicity induced by paracetamol overdose.
Nutritional properties (%) | |
Moisture (maximal) | 14 |
Fibers (maximal) | 5 |
Proteins (minimal) | 18 |
Fat (maximal) | 3 |
Ash (maximal) | 13.5 |
Carbohydrate | 46.5 |
Calorific value (kcal kg−1) | 2846 |
Amino acid (%) | |
Methionine | 0.36 |
Cysteine | 0.26 |
Threonine | 0.62 |
Tryptophan | 0.2 |
Mineral mix (mg kg−1) | |
Manganese | 80 |
Fer | 48 |
Cuivre | 18 |
Zinc | 64 |
Selenium | 0.28 |
Cobalt | 0.2 |
Iode | 2 |
Vitamin and antioxidant (mg kg−1) | |
Vitamine A | 11200 |
Vitamine D3 | 2800 |
Vitamine H | 25 |
Antioxidant (BHA–BHT) | 100 |
Group 1: normal rats (NR) fed with the standard diet and water, Group 2: rats treated with 325 mg of paracetamol/kg body weight (bw), by intraperitoneal injection, during the last five days of treatment period. This dose provoked toxicity without lethality. Groups 3, 4, 5, 6 and 7: rats received 350 mg kg−1 bw of PH-LA, PH-ES, PH-TR, PH-A2, and PH-A26, daily by gavage, during 45 days. They were treated intraperitoneally with paracetamol for the last five days of treatment period.
At the end of the experimental period, animals of the different groups were sacrificed by cervical decapitation to avoid stress.
Trunk blood samples were collected into EDTA tubes. Some of them were immediately used for the determination of hematological parameters. The sera samples were collected after centrifugation (2200 × g, 15 min, 4 °C), and the liver of each rat was carefully excised.
Some liver samples were immediately removed, rinsed in ice-cold physiological saline solution, fixed in 10% buffered formalin solution and embedded in paraffin for histological studies. The other ones were homogenized in Tris-buffer-saline (pH 7.4) and then centrifuged (3500 × g, 20 min) at 4 °C. The supernatants were frozen and stored for further use in subsequent enzymatic assays. All samples were stored at −80 °C until further use.
The focus of recent research has been on different protein hydrolysates which were prepared at the same enzyme/substrate ratio (E/S = 3 U mg−1) and after incubation for 6 hours, PH-LA showed the highest degree of hydrolysis (DH = 13.05%), followed by PH-TR (DH = 12.67%), PH-ES (DH = 12.5%), PH-A26 (DH = 9.25%), and PH-A2 (DH = 8.0%).
Treatment of protein with different enzymes produced a mixture of bioactive peptides with a different degree of hydrolysis (DH) which also could be responsible for the different range of antioxidant capacity.15
The molecular mass distribution of the LAPHs was carried out using HPLC-SEC analysis. The SEC spectra of the molecular weight (MW) peptides are shown in Fig. 1(A). The profiles of SEC spectra revealed the differences in the molecular mass distribution depending on the proteases used and consequently on the degree of hydrolysis (DH). Further, the elution profile of PH-TR and PH-LA gave the highest intensities for the last eluting peptides, characterized by their low MW. This result is in accordance with the high DH obtained for this hydrolysate. Indeed, more the DH value increased more the low MW peptides (<10 kDa) content increased. As shown in Table 2, the levels of peptides below 3 kDa obtained in PH-LA, PH-TR, PH-ES, PH-A26, and PH-A2, were 34.09%, 29.92%, 37.9%, 22.3% and 15.18%, respectively, which is in accordance with their DHs.
SEC distribution | ||||||
---|---|---|---|---|---|---|
a SEC method was used to calculate the MW distribution of the LAPHs. | ||||||
Mass (kDa) | >20 | 20–10 | 10–5 | 5–3 | <5 | <3 |
PH-LA | 9.16 | 12.61 | 20.53 | 23.61 | 57.7 | 34.09 |
PH-TR | 9.27 | 12.05 | 37.1 | 11.66 | 41.58 | 29.92 |
PH-ES | 6.7 | 16.43 | 27.64 | 11.52 | 49.23 | 37.9 |
PH-A26 | 11.13 | 18.14 | 28.67 | 19.76 | 42.06 | 22.3 |
PH-A2 | 16.29 | 20.48 | 27.92 | 20.13 | 35.31 | 15.18 |
Protein hydrolysates obtained after proteins hydrolysis are composed of free amino acids and short chain peptides, and exhibit many advantages as nutraceuticals or functional foods because of their amino acid profile.
The amino acids composition of any food protein has a significant role in various physiological activities of the human body and affects either directly or indirectly the maintenance of good health.27 Table 3 shows the amino acid composition of the LAPHs. It is obviously shown that the LAPHs contained almost all the essential and non-essential amino acids with dominance of Glu, Gln, and Lys. Pro, Leu and Gly were also present in relatively high amounts. However, the contents of His, Met, Phe and Ser were very low, as demonstrated by our results. Eight key amino acids were observed in the hydrolysate products, namely leucine, isoleucine, valine, lysine, methionine, tyrosine and phenylalanine. These amino acids are essential daily food intakes to assure normal human growth. Furthermore, amino acid compositions may also be important to antioxidant activity. Himaya et al.28 argue that hydrophobic amino acids facilitated interactions with hydrophobic targets, such as the cell membrane, and thereby, enhanced the bioavailability. Additionally, aromatic amino acids increased the antioxidant activities of peptides and protein hydrolysates because they easily donate protons to electron-deficient radicals and maintain their stabilities via resonance structures and enhance radical scavenging activities.29 Therefore, the antioxidant activities of LAPHs could be related to the high hydrophobic and aromatic amino acid contents.
Amino acids | PH-A2 | PH-A26 | PH-LA | PH-TR | PH-ES |
---|---|---|---|---|---|
a Essential amino acids. HAA: hydrophobic amino acid. Values are given as mean SD from triplicate determinations (n = 3), a,b,c,d,e in the same line indicate significant differences (p < 0.01). | |||||
Hydrophilic amino acid | |||||
Aspartic acid and asparagine (Asp and Asn) | 9.84 ± 0.10b | 10.02 ± 0.80a | 10.06 ± 0.06a | 9.92 ± 0.80a | 10.13 ± 1.10a |
Glutamic acid and glutamine (Glu and Gln) | 15.75 ± 1.00d | 16.80 ± 0.10a | 16.18 ± 0.20b | 15.2 ± 0.35e | 16.00 ± 0.71c |
Serine (Ser) | 3.93 ± 0.01d | 4.39 ± 0.10a | 4.01 ± 0.19c | 3.83 ± 0.21e | 4.19 ± 0.30b |
Glycine (Gly) | 7.41 ± 0.02d | 8.33 ± 0.11a | 8.09 ± 0.20b | 6.63 ± 0.01e | 7.95 ± 0.42c |
Histidinea (His) | 2.04 ± 0.02c | 2.15 ± 0.12b | 2.20 ± 0.27a | 1.84 ± 0.02d | 2.26 ± 0.61a |
Arginine (Arg) | 5.01 ± 0.01a | 4.02 ± 0.13b | 2.18 ± 0.31e | 4.21 ± 0.10c | 3.95 ± 0.10d |
Threoninea (Thr) | 4.48 ± 0.20d | 5.00 ± 0.14b | 5.40 ± 0.35a | 4.10 ± 0.32e | 4.89 ± 0.30c |
Tyrosine (Tyr) | 2.48 ± 0.52b | 2.42 ± 0.15b | 2.60 ± 0.39a | 2.40 ± 0.02b | 2.63 ± 0.95a |
Lysinea (Lys) | 11.09 ± 0.03b | 8.02 ± 0.16e | 10.45 ± 0.43c | 16.67 ± 1.23a | 8.91 ± 0.10d |
Hydrophobic amino acid | |||||
Proline (Pro) | 8.81 ± 0.00b | 8.73 ± 0.17c | 8.26 ± 0.47d | 8.27 ± 0.10d | 8.94 ± 0.14a |
Alanine (Ala) | 7.04 ± 0.00b | 7.02 ± 0.17b | 7.08 ± 0.51b | 6.41 ± 0.10c | 7.87 ± 0.00a |
Valinea (Val) | 4.56 ± 0.00b | 4.53 ± 0.18b | 4.86 ± 0.55a | 4.23 ± 0.21c | 4.51 ± 0.1b |
Methioninea (Met) | 2.81 ± 0.00c | 2.97 ± 0.19b | 3.04 ± 0.59a | 2.51 ± 0.15d | 3.03 ± 0.14ab |
Isoleucinea (Ile) | 4.18 ± 0.05c | 4.47 ± 0.20b | 4.65 ± 0.63a | 4.56 ± 0.61a | 4.42 ± 0.16b |
Leucinea (Leu) | 7.31 ± 0.10d | 7.58 ± 0.21c | 7.87 ± 0.68b | 6.28 ± 0.13a | 7.44 ± 0.94c |
Phenylalaninea (Phe) | 3.26 ± 0.10b | 3.50 ± 0.72a | 3.01 ± 0.72c | 2.90 ± 0.75d | 2.85 ± 0.27d |
Total | 100 | 100 | 100 | 100 | 100 |
Antioxidative activity of LAPHs, using protein oxidation with Cu2+/H2O2 assay, was objectified, in this study, by a decrease in the BSA degradation induced by Cu2+/H2O2. Densitometric analysis of the gel electrophoretic samples is presented in Fig. 1(C). The Cu2+/H2O2 system is a metal-catalyzed system and can produce ROS and altered spectroscopic properties of albumin, increased protein carbonyl content and resulted in several conformational changes. Interestingly, LAPHs reduced significantly protein damage and protein band intensity was restored to control levels. PH-A26 (3 mg ml−1), was a better protector against the metal catalyzed protein oxidative damage. The incubation of BSA with PH-A26, PH-TR, PH-LA, PH-ES, and PH-A2, increased significantly the band intensities by 86.20%, 84.0%, 81.0%, 78.0%, and 75.5%, respectively, compared to the BSA incubated only with Cu2+/H2O2 (Fig. 1(C)). The inhibitory effect of LAPHs might be explained by the scavenging of hydroxyl radical generated in Cu2+/H2O2 system. The results obtained above are in agreement with our previously findings,15 with regards to the scavenging effects of LAPHs.
Agarose gel electrophoresis showed undetectable DNA laddering in hepatic tissues of the control rats (Fig. 2, lane 1). However administration of paracetamol (325 mg kg−1 bw) caused DNA damage resulted in DNA shearing with DNA ladder pattern (a hallmark of necrosis) (Fig. 2(A), lane 2). According to Scott et al.33 free radical generation, following xenobiotic exposure, may lead to an extensive DNA damage giving rise to mutations and/or cell death. Oxidative stress induced by oxygen-derived radicals can produce numerous modifications in DNA including base and sugar lesions, strand breaks, DNA–protein cross-links and base-free sites. Flaks and Flaks34 reported that paracetamol overdose could increase the mutation rates through oxidative damage. DNA gel electrophoresis results supported the view that LAPHs protected liver cells from necrosis and/or apoptotic death induced by paracetamol overdose. Interestingly, LAPHs prevented DNA fragmentation as evidenced by the absence of DNA laddering patter (Fig. 2(A), lane 3–7).
The protection of the DNA could be caused by the DNA repair enzyme(s) such as OGG1, stimulated by LAPHs, which must be present in the nucleus to reduce the 8-oxo-deoxyguanosine (8-oxodG) incision activity.
The AST, ALT and LDH activities in the serum of the paracetamol treated group increased by 37%, 71.5% and 55.5%, respectively, compared to the control (Table 4). A significant increase in the ALP (71.8%) was also noted in paracetamol treated group. Similar results were reported by Venkatachalam and Muthukrishnan.35 An elevation in transaminases ALP and LDH activities are attributed to the liver injury. When the liver cell plasma membrane is alterated, a variety of enzymes usually located in the cytosol are relegated into blood stream. The increased production of serum enzymes in blood stream was associated with central submassive necrosis of liver which causes severe hepatic injury.36
Groups | AST (U L−1) | ALT (U L−1) | Glucose (mmol L−1) | LDH (U L−1) | ALP (U L−1) |
---|---|---|---|---|---|
a AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; glucose; ALP, alkaline phosphatase; PH-LA, PH-TR, PH-ES, PH-A2 and PH-A26: protein hydrolysate obtained with crude enzyme from L. aurata, trypsin, esperase, Pseudomonas aeruginosa A2 and Bacillus subtilis A26 respectively. Data expressed as mean ± SD in each group (n = 6). a,b,c,d,e,f The means with no common superscripts differ significantly (p < 0.01). | |||||
Control | 196 ± 13.3b | 88.32 ± 3.21d | 5.41 ± 0.39de | 1006 ± 24ef | 341 ± 27ef |
Paracetamol (P) | 269.15 ± 27.32a | 151.3 ± 6.35a | 9.45 ± 0.41a | 1563 ± 38a | 586 ± 27a |
PH-LA/(P) | 177.96 ± 15.32c | 111.32 ± 7.32bc | 6.34 ± 0.47c | 1241 ± 17b | 407 ± 8.0bc |
PH-TR/(P) | 145.32 ± 18.32e | 120.96 ± 6.18b | 7.21 ± 0.12b | 1205 ± 21bc | 413 ± 15b |
PH-ES/(P) | 156.89 ± 11.66de | 117.69 ± 5.32b | 6.89 ± 0.61bc | 1102 ± 33d | 397 ± 19c |
PH-A2/(P) | 166.32 ± 14.32d | 99.64 ± 4.62c | 5.21 ± 0.14e | 1086 ± 27de | 388 ± 10cd |
PH-A26/(P) | 198.25 ± 15.26b | 89.34 ± 2.94d | 5.91 ± 0.36cd | 1074 ± 35ef | 361 ± 22e |
A significant increase in the serum glucose (74.67%) levels was also detected. Hinson et al.37 reported hyperglycemia (500 mg kg−1 bw) and glycosuria in an acetaminophen-overdosed patients. The data presented in Table 4 show, compared to the paracetamol treated group, a significant decrease in all parameters cited above in all protein hydrolysates treated groups, and values obtained were similar or slightly higher than those of the normal rats (p < 0.01).
The findings of the present study indicated that, the PH-TR treated rats showed the highest decrease in AST activity (46%), followed by PH-ES (41.7%), PH-A2 (38.25%), PH-LA (33.8%), and PH-A26 (26.35%), whereas the PH-S treated rats showed the highest decrease in ALT, LDH, and ALP activities (41%, 31.25%, and 38.40%, respectively) (p < 0.01).
The significant decrease in ALT and AST activities in the LAPHs pretreated rats demonstrated their hepatoprotective effects against paracetamol damage. Similar result was reported by Galal et al.38 The possible hepatoprotective mechanisms of LAPHs may be due to preventing the process of lipid peroxidation, inhibiting the cytochrome P-450 activity and stabilizing the hepatocellular membrane.
The mechanism of hepatoprotection by protein hydrolysates generally exerts multiple effects. Although they show hepatoprotection due to antioxidant effect, there are other effects like immunomodulatory39 and anti-inflammatory.40
The results presented in Fig. 2(B) show that the level of MDA in liver tissue increased in the paracetamol-treated rats by 147%, compared to the control group, which indicate the oxidative effect of paracetamol. Several authors have also reported an increase in lipid peroxydation following administration of high doses of paracetamol in rats.41 Increased lipid peroxidation in paracetamol group, as evidenced by the elevated level of MDA in hepatic tissues, could be expected owing to the depletion in GSH stores and reduced GPx activity.
In our study LAPHs have shown to have a protective effect against damage caused by oxidative stress which is provoked by liver toxicity induced by overdose of paracetamol. We have seen that PH-LA, PH-TR, PH-ES, PH-A2, and PH-A26 were able to reduce the concentration of hepatic MDA by 57.77%, 61.50%, 70.20%, 62.75%, and 70.81%, respectively, compared to the paracetamol treated group. Hepatic MDA contents of PH-ES and PH-A26 were even lower than that of the control group.
The obtained results indicate that the administration of LAPHs effectively inhibited lipid peroxidation induced by paracetamol and demonstrate their beneficial effects.
The activities of antioxidant enzymes in liver of experimental rats are shown in Fig. 2(C). Our results revealed that hepatotoxicity induced by paracetamol overdose, significantly decreased the activities of SOD, GPx, and CAT by 59.5%, 48.8%, and 59.6%, respectively, as compared to the control group (p < 0.01). This might lead to decreased antioxidant defense and increased oxidative stress and thereby the tissue injury occurs. Similar results have been reported by Athira et al.,43 paracetamol has been reported to decrease the antioxidant enzymes activities in hepatic cells.
Pretreatment with LAPHs before paracetamol administration prevented the reduction of antioxidant enzyme activities. This might reflect the antioxidant potency of LAPHs. Our results are in agreement with reports of other workers which demonstrated that the administration of goby and zebra blenny to rats fed high fat-high fructose diet44 or alloxan induced diabetic rats27 increased SOD, CAT and GPx activities. The increase of antioxidant enzyme activities demonstrates the existence of the antioxidant peptides with potent free radical-scavenging activities in LAPHs.
These findings were attributed to small peptides that preventing the generation of free radicals. The improvement in the expression of these antioxidant enzymes in rats treated with LAPHs suggest that this hepatic antioxidant defense is reactivated by peptides with a resulting increase in the capacity of detoxification through enhanced scavenging of reactive oxygen radicals.
The measurement of non-enzymatic antioxidant content is a great biomarker for paracetamol intoxication. As shown in Fig. 2(D), the level of GSH in hepatic tissue of the paracetamol treated group, was markedly reduced by 49% as compared to the control group (p < 0.01). The decrease in the concentration of GSH in paracetamol-treated group is also an indicator of oxidative damage. The excess of NAPQI first depletes the GSH level, and then covalently binds to thiol groups of intracellular proteins, thus generating reactive oxygen species (ROS) that triggers hepatocellular necrosis.45 However, the levels of GSH, in rats pretreated with different LAPHs, were significantly restored by 90.5%, 70.05%, 84.7%, 95.26%, and 68.84%, respectively after PH-LA, PH-TR, PH-ES, PH-A2, and PH-A26 treatment, compared to the paracetamol group.
In summary, the present findings demonstrate the capability of LAPHs in promoting natural defense against ROS produced by paracetamol induced hepatotoxicity.
In fact, we obtained in the present study an increase in the platelet number after paracetamol administration (Table 5). The hematological profile of the control and treated groups are presented in Table 5. Treatment with paracetamol resulted in a significant decrease in the levels of RBC (red blood cell), Hb (hemoglobin) and Ht (hematocrit) suggesting an anemia installation for rats treated with paracetamol. The diminution of blood cell count is proved by the decrease of Ht level. As known, RBCs are responsible for carrying oxygen to the body' tissues thus, changes in the numbers and/or morphology of the RBCs may indicate abnormalities or some hematological conditions.
Groups | RBC (106 μl−1) | Ht (%) | Hb (g dl−1) | CMV (fentolitre) | WBC (103 μl−1) | Plt (103 mm−3) |
---|---|---|---|---|---|---|
a RBC, red blood cell; WBC, white blood cell; Ht, hematocrit; Hb, hemoglobin; CMV, mean cell volume; Plt, platelets. Data are expressed as mean ± SD in each group (n = 6). a,b,c,d,e,f,g The means with no common superscripts differ significantly (p < 0.01) | ||||||
Control | 8.65 ± 0.37a | 40.7 ± 0.86a | 13.7 ± 0.95a | 55.1 ± 0.85g | 10.32 ± 0.5g | 645 ± 22.3e |
Paracetamol (P) | 5.23 ± 0.47e | 36.6 ± 0.8d | 9.6 ± 0.9e | 22.1 ± 0.97f | 13.56 ± 0.13a | 1234 ± 31.6a |
PH-LA/(P) | 6.98 ± 0.44d | 38.6 ± 0.57b | 10.32 ± 0.81d | 38.9 ± 0,67e | 11.62 ± 0.48c | 820 ± 43.6g |
PH-TR (P−1) | 7.31 ± 0.63c | 40.5 ± 0.35a | 10.97 ± 0.68c | 40.1 ± 0.69c | 11.32 ± 0.72d | 836 ± 63.1f |
PH-ES (P−1) | 8.07 ± 0.42b | 37.6 ± 0.97c | 12.95 ± 0.67a | 47.32 ± 0,37a | 10.67 ± 0.35f | 969 ± 38.6c |
PH-A2/(P) | 8.32 ± 0.19a | 39.6 ± 0.81ab | 10.99 ± 0.63c | 39.6 ± 0,77d | 12.36 ± 0.29b | 987 ± 55.32b |
PH-A26/(P) | 8.04 ± 0.72ab | 38.1 ± 0.44bc | 11.27 ± 0.52b | 45.12 ± 0.15b | 10.93 ± 0.77e | 852 ± 34.33d |
Generally, paracetamol overdose induced hematotoxicity which was marked by several abnormalities such as leukopenia, granulocytosis and neutropenia, thrombocytopenia, and pancytopenia in rats. The recorded hematotoxicity could be secondary to the deleterious effect of paracetamol on organs of hematopoiesis in the body which include liver.46
Interestingly, this study shows that the LAPHs could contain candidate bioactive peptides reversing the hematotoxic effect of paracetamol, with ensuing improvement of hematopoiesis.
However, the rat groups pre-treated with PH-LA, PH-A2 and PH-A26 exhibited significant liver protection against paracetamol-induced liver damage, as indicated by the presence of normal hepatic cells and absence of necrosis (Fig. 3(C), (F) and (G)). These results suggest the protective effect of LAPHs against chemical toxicity induced in rats. It was evidenced from the histopathological observation; the ability of LAPHs to reverse the hepatic lesions. The livers of animals treated with showed marked improvement in hepatocyte architecture in different areas around the central veins and portal tracts. This finding was consistent with the levels of the enzyme markers. Sigala et al.50 reported that the presence of mitotic cells in hepatocytes was assessed as an index of liver proliferative capacity in response to toxin-induced injury.
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