The Beneficial Effect of Nanostructured Oligochitosan against Gamma Irradiation and/or Carbon Tetrachloride-Induced Hepatic Injury in Rats

 

Enas A. Mohamed1, Ahmed M. Elbarbary2, Nashat M. M. Abd alaty1, Nashwa K. Ibrahim3, Mahmoud M. Said4*, Ahmed M. Salem4

1Nuclear Materials Authority, Cairo, Egypt.

2Polymer Chemistry Department, National Center for Radiation Research and Technology,

Atomic Energy Authority, Cairo, Egypt.

3Radiation Biology Department, National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.

4Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.

*Corresponding Author E-mail: mahmoudmsaid@sci.asu.edu.eg

 

ABSTRACT:

The current study was undertaken to investigate the hepatoprotective potential of nanostructured oligochitosan (NOC) against the synergistic toxic effects of g-irradiation exposure and carbon tetrachloride (CCl4) intoxication in male rats. Adult male rats were allocated into eight groups; control, NOC-administered, g-irradiated, CCl4-intoxicated, NOC-pretreated g-irradiated, NOC-pretreated CCl4-intoxicated, g-irradiated and CCl4-intoxicated, NOC-pretreated CCl4-intoxicated and g-irradiated. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results demonstrated that the oligochitosan prepared by exposure to gamma irradiation was in the range of nanoparticles. A synergistic hepatotoxic effect was demonstrated following the exposure of rats to g-irradiation and CCl4 intoxication, along with the induction of oxidative stress, inflammation and apoptosis. NOC was able to protect the hepatocytes from the combined toxic insults through suppressing lipid and protein oxidations, maintaining hepatic functions, downregulating the expression of some inflammatory genes, including nuclear factor kappa B (NF-kB) and interleukin 1 beta (IL-1β), as well as enhancing the expression of the antiapoptotic Bcl2 gene and suppressing the proapoptotic Bax gene expression. Histological findings of liver tissues verified the biochemical and molecular data. The study clarified some of the molecular mechanisms by which NOC protects the liver against the synergistic toxic effect of g-irradiation and CCl4.

 

KEYWORDS: Oligochitosan, nanostructures, g-irradiation, CCl4, rats, liver

 

 


INTRODUCTION:

Exposure of radiosensitive organs, such as the liver, to ionizing radiations produces excessive reactive oxygen species (ROS) that interact with biological systems and exaggerate the generation of free radicals that promote inflammatory responses, causing changes in the structure and permeability of cellular components leading to cellular damage and organ dysfunction1,2.

 

Carbon tetrachloride (CCl4), one of the most potent environmental contaminants, induces acute liver injury through various mechanisms, including inflammatory responses, oxidative stress and apoptosis3. Hepatic cytochrome P450 metabolizes CCl4 to form trichloromethyl (CCl3) free radicals that initiate the peroxidation of membrane lipids4. Secondary metabolic radicals of CCl4, such as trichloromethylperoxy radicals (Cl3COO), react with lipids and proteins and alters the permeability of the mitochondria, endoplasmic reticulum and plasma membrane, which results in cell damage5. The inflammation response is also an important event in CCl4-induced acute liver injury6. The histopathological features of the liver of CCl4-treated murines closely resemble the structural changes of chronic hepatitis and cirrhosis in humans7.

Chitin is the most abundant natural amino polysaccharide and its annual production is estimated to almost equal that of cellulose8. The principal sources of chitin are two marine crustaceans, shrimp and crab9. Chitosan, a non-toxic, biocompatible, and biodegradable polymer derived from chitin hydrolysis, is widely used in bioengineering industries and biomedical applications10. Chitosan possesses many biological properties, including hemocompatibility and antitumor11, anti‑inflammatory12, mucoadhesive, absorption enhancing and antimicrobial13, immune-stimulatory14 and antioxidant15. Furthermore, chitosan-based nanoparticles exhibit more superior activities than chitosan and are used in many applications, including non-parenteral drug delivery for the treatment of many diseases16,17.

 

Oligochitosan was prepared by several techniques, including acid hydrolysis18, ultraviolet degradation19 and gamma radiation20. In addition, ionotropic gelation, microemulsion, emulsification solvent diffusion, polyelectrolyte complex and reverse micellar methods have been employed for the synthesis of chitosan nanoparticles21.

 

The present study aims at evaluating the probable protective potential of the synthesized nanostructured oligochitosan (NOC) against γ-irradiation and/or CCl4-induced hepatic injury in male rats and to explore some of the underlying molecular mechanisms that may contribute to its beneficial role.

 

MATERIALS AND METHODS:

Chemicals:

Chitosan (Mw 100,000-300,000 and degree of deacetylation [DD] ≤ 85%) was supplied from Acros Organics (Belgium). Thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (TEP) and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) were purchased from Sigma-Aldrich (Germany). Carbon tetrachloride (CCl4) and lactic acid were purchased from El-Gomhoreya Company (Cairo, Egypt).

 

Radiation-induced degradation of chitosan polymers:

Radiation-induced degradation of chitosan polymers was done in our laboratory according to the previously reported methods20,22. Briefly, a weight of one-gram chitosan powder was dissolved in 100 ml of lactic acid solution (1% v/v in distilled water) at room temperature with overnight stirring. The solution was then filtered to remove any impurities or undissolved polymers. The chitosan solution was subjected to g-irradiation at ascending doses (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 kGy) using the 60Co Canadian Irradiator (Atomic Energy of Canada Ltd, Ontario, Canada) at a dose rate of 1.41 kGy/hr.

 

Analysis and characterization of oligochitosan:

Determination of the relative viscosity of oligochitosan:

The Ubbelohde viscometer was used to investigate the abnormal viscosity behavior of oligochitosan solutions23. The flow time of the pure solvent in seconds, namely (to), was measured by a thoroughly cleaned viscometer, then after drying the viscometer, the flow time of the oligochitosan solution with different concentrations, namely (t) was measured. Having (t) and (to), the relative viscosity (ηrel) of irradiated oligochitosan solutions was calculated as follows: (ηrel) = (t) / (to)

 

Determination of free radical scavenging activity of oligochitosan:

The ability of oligochitosan to scavenge DPPH radicals was colorimetrically assayed24. The radical scavenging activity (%) was calculated from the decrease in absorbance value at 517 nm of oligochitosan solution (0.1 mM in 95% ethanol) or ascorbic acid solution (as standard) in comparison with a negative control (lactic acid 1% v/v) according to the following equation: (Acontrol - A sample or standard/A control) × 100

 

Characterization of oligochitosan:

Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy (Bruker Optik GmbH, Ettlingen, Germany) was used to investigate the chemical structure of oligochitosan in the range of 4000-400 cm-1. The UV absorbance of oligochitosan was measured in a UV spectrophotometer (JASCO V-560, Japan) in the range of 190-400 nm. Similarly, the size distribution of oligochitosan was determined using dynamic light scattering (DLS-ZP/Particle SizerNicomp 380 ZLS, USA). For DLS measurements, 1 ml of oligochitosan solution was dispersed in 20 ml distilled water, stirred in an ultrasonic water bath for 30 min to reach homogeneity, filtered and the supernatant was then used for subsequent measurements. The mean diameter of oligochitosan particles was investigated using Transmission Electron Microscopy (TEM) (JEOL JSM-100 CX, Japan) with an acceleration voltage of 80 kv. For TEM observations, the samples were prepared by making a suspension from oligochitosan solutions in acetone in an ultrasonic water bath, centrifuged to separate the polymer matrix and collimate the large size particles, then a drop of the suspension was placed into the carbon grid and left to dry at room temperature.

 

Animals:

Adult male outbred Sprague Dawley rats were obtained from the Nile Company for Pharmaceuticals and Chemical Industries (Cairo, Egypt). Animals were housed in plastic cages and maintained under standard conditions of temperature, humidity and 12/12 hr light/dark cycles along the experimental period. Animals were provided with a pelleted diet containing all the necessary nutritive elements. The animals had free access to pelleted diet and tap water and left for a one-week period before the start of the experiment as an acclimatization period. All animal procedures were in accordance with the General Guidelines of Animal Care and Recommendations of the Canadian Council for Animal Care25, and all applicable institutional guidelines were followed.

 

Radiation facilities:

Rats were exposed to whole body gamma irradiation using a Gammacell 40 Cesium Irradiator (Atomic Energy of Canada Ltd, Ontario, Canada) installed in the National Center for Radiation Research and Technology (NCRRT) of the Egyptian Atomic Energy Authority (EAEA, Cairo, Egypt). Animals were first restrained, placed in a well-ventilated canister and then exposed to a single acute sublethal dose of 5Gy g-irradiation (inside a cylindrical stainless-steel double capsule) at a dose rate of 0.43 Gy/min2.

 

Study design:

According to our DLS and TEM results, the synthesized 30 kGy nanostructured oligochitosan (NOC) was selected for oral administration in the current study. A total of 64 adult male rats were allocated into eight groups as follows: Group I (Control); Group II (NOC): Rats were orally administered NOC (140 mg/kg bw)26 via intragastric tube five times per week for 3 consecutive weeks; Group III (R): Rats were exposed to a single whole body 5Gy g-irradiation dose; Group IV (NOC+R): Rats were orally administered NOC, then exposed to a single whole body 5Gy g-irradiation dose; Group V (CCl4): Rats were subcutaneously (s.c.) injected with CCl4 (2 ml /kg bw; 50% v/v in olive oil)27 twice per week for two consecutive weeks; Group VI (NOC+CCl4): Rats were orally administered NOC along with s.c. injections of CCl4 starting one week later after NOC administration; Group VII (CCl4+R): Rats were s.c. injected with CCl4, then exposed to a single whole body 5Gy g-irradiation dose; Group VIII (NOC+CCl4+R): Rats were orally administered NOC along with s.c. injections of CCl4 then exposed to a single whole body 5Gy g-irradiation dose.

 

Sample preparation:

At the end of the experiment, rats were anesthetized and blood was collected by heart puncture into a plain tube, left to coagulate at 37ºC for 15 min, centrifuged and the separated serum was stored at -20ºC until analysis. The liver was excised at autopsy, rinsed in ice cold isotonic sterile saline, blotted dry with a filter paper and then dissected into 3 portions. The first part was preserved at -20ºC for further oxidative stress markers analysis. The second part of the liver was preserved at -80ºC for molecular investigation, whereas the last part was kept in 10% neutral formalin for histopathological analysis.

 

Preparation of tissue homogenate:

Whole liver homogenate (10%) was prepared on ice by homogenizing one part of liver in 10 volumes of ice-cold isotonic saline in an electric homogenizer (Glas-col., Terre Haut, USA), and was used for the assay of lipid and protein peroxidation.

 

Biochemical assays:

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities, total and direct bilirubin levels, total protein and albumin concentrations were determined using commercial assay kits purchased from BioMed Diagnostics (Cairo, Egypt). In the liver tissue homogenate, the concentration of malondialdehyde (MDA), the end product of lipid peroxidation, was determined colorimetrically28 and the protein carbonyl content (PCC) level, a marker for protein peroxidation, was assayed29.

 

RNA extraction and real-time quantitative polymerase chain reaction:

Total cellular RNA was extracted from frozen liver using RNeasy® Mini kit (Qiagen, Hilden, Germany). The concentration and purity of total RNA were assessed by measuring absorbance at 260 and 280 nm, respectively, in a spectrophotometer (Nano Drop 2000, Thermo Fisher Scientific, USA). First-strand complementary DNA (cDNA) was synthesized using Thermo ScientificTM RevertAidTM First-Strand (cDNA) synthesis Kit (Fermentus, Thermo Fisher Scientific Inc, Runcorn, UK). Real-time polymerase chain reaction (PCR) amplification and analysis were performed in an optical 96-well plate in ABI PRISM 7500 Fast Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using Power SYBR® Green PCR Master Mix (Applied Biosystems). The amplification protocol consisted of 40 cycles (denaturation at 95°C for 15 sec, annealing at 55°C for 20 sec and extension at 72°C for 20 sec). Primers used were 5′-CATTGAGGTGTATTTCACGG-3′ (forward primer) and 5′-GGCAAGTGGCCATTGTGTTC-3′ (reverse primer) for nuclear factor kappa B (NF-kB), 5′-TGATGTCCCATTAGACAGC-3′ (forward primer) and 5′-GAG GTG CTG ATG TACCAG TT-3′ (reverse primer) for interleukin 1 beta (IL-1β), 5′-CCCTGTGCACTAAAGTGCCCC-3′ (forward primer) and 5′-TTCTTCACGATGGTGAGCG-3′ (reverse primer) for Bax, 5′-CTACGAGTGGGATGCTGGAGG-3′ (forward primer) and 5′-GTCAGATGGACA-CATGGTG-3′ (reverse primer) for B-cell lymphoma 2 (Bcl2), 5′-CTCAACTACATGGTCTACATG-3′ (forward primer) and 5′-CCATTCTCGGCCTTGA-CTGT-3′ (reverse primer) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; the housekeeping gene) . The relative expression of selected genes was determined by the ΔΔCT method30.

 

Histological examination:

After fixation, liver specimens were further processed and embedded in paraffin. Serial 4 μm-thick sections were cut, stained with hematoxylin and eosin (H&E) and examined under a light microscope.

 

Statistical analysis:

The Shapiro-Wilks test for normality showed that all data were normally distributed (p>0.05). Statistical analysis of difference between means was carried out using one-way analysis of variance (ANOVA). In case of a significant F-ratio, post hoc Duncan's test for multiple comparisons was used to evaluate the statistical significance between treatment groups at p<0.05 level of significance. All the statistical analysis was done using Statistical Package for Social Science (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA).

 

RESULTS:

The relative viscosity of oligochitosan:

The relative viscosity of oligochitosan solution decreased with increasing the irradiation dose and the decrease in the viscosity was very fast up to 30 kGy (Figure 1A).

 

The scavenging activity of oligochitosan:

DPPH has a characteristic absorption band at λmax 517 nm, which decreases significantly upon exposure to proton radical scavengers31. The scavenging activity (%) of oligochitosan on DPPH radicals was enhanced by γ-irradiation. The DPPH scavenging activity (%) of oligochitosan irradiated at 10, 20, 30, 40, and 50 kGy was 48, 58, 81, 83, and 84%, respectively, compared to non-irradiated chitosan (22%) (Figure 1B). This result shows that the antioxidant activity of oligochitosan was enhanced by γ-irradiation.

 

Characterization of Oligochitosan:

FTIR spectroscopy:

The FTIR spectrum of non-irradiated chitosan (Figure 1C, curve I) shows absorption bands at 3410 cm-1 υ (O-H and N-H stretch), 2918-2869 cm-1 attributed to υ (C-H) stretching, 1652 cm-1 corresponding to the stretching of amide υ (C=O) of acetyl groups in chitosan, 1598 cm-1 υ (N-H bond), 1381 cm-1 (amide due to combination of NH deformation and the C-N stretching vibration), 1154 cm-1 (asymmetric bridge-O-stretch) and 1087 cm-1 (skeletal vibration involving the C-O stretch). On the other hand, FTIR spectra of oligochitosan (Figure 1C, curves II-V) show the same characteristic absorption peaks of non-irradiated chitosan, indicating that the main polysaccharide structure of the resulting oligomers is retained. Also, it was observed that by increasing the irradiation dose, there is an increase in intensities of the absorption peaks at 1385, 1598 and 1638 cm-1 corresponding to C-O stretching, N-H bending and C=O of amide group, respectively. This may be due to the decrease in inter- and/or intra-molecular hydrogen bonding formed between the -OH and -NH2 groups. The peak at 1100 cm-1 that corresponds to the ether bond in the pyranose ring has no significant change indicating the structural stability of β-glycosidic bonds in the molecular chains of oligochitosan after irradiation.

 

UV-Vis spectroscopy:

An absorption peak at 272 nm was observed for oligochitosan, caused by the n→σ* transition for the amino groups and may also be assigned to the n→π* transition for the carbonyl or carboxylic groups of remaining acetylated group in the chitosan chain (32) (Figure 1 D). Also, this peak can be ascribed to carbon-oxygen double bonds (-C=O group) formed after the main chain scission of chitosan and/or formation of hydroxyl group by hydrogen abstraction reaction. The intensity of this peak increased with increasing the irradiation dose. Furthermore, the aqueous chitosan solution had a pale yellowish color that changed to brown by irradiation confirming the formation of the unsaturated bonds (-C=O group). As the exposure dose increases, the brown color intensity changes to deep ones. These findings are consistent with that previously reported by many authors20,33.

 

Dynamic light scattering:

The average particle size of non-irradiated chitosan and oligochitosan was determined by the dynamic light scattering (DLS) as shown in Figure 2 (A-D). It was observed that the size of oligochitosan was decreased with increasing the irradiation dose, recording 479.6 nm for non-irradiated chitosan, 360.7, 289.1 and 272.3 nm for oligochitosan irradiated at 20, 30 and 40 kGy, respectively.

 

Transmission electron microscope:

The transmission electron microscope (TEM) images show that oligochitosan has nanostructures (NOC). The mean diameter of NOC particles was under 100 nm at 20-40 kGy irradiation doses and most of them have individual spherical shape with good distribution and without aggregation. In addition, the size of NOC was    decreased by increasing the irradiation dose (Figure 3 A-C).


 

Figure 1: The effect of g-irradiation on the viscosity of oligochitosan solutions (A). The DPPH scavenging activity of non-irradiated chitosan and oligochitosan (B). FTIR spectra of non-irradiated chitosan (I) and oligochitosan (II, 10 kGy; III, 20 kGy; IV, 30 kGy; and V, 40 kGy) (C). UV-Vis spectra of non-irradiated chitosan and oligochitosan (D).

 


 

Effect of different treatments on serum enzymes level:

Figure 4 demonstrates that exposure of male rats to a single 5 Gy dose whole body g-irradiation, CCl4 intoxication or combined treatments caused severe hepatic injury as demonstrated by the significant increase the activity of serum liver enzymes, mainly AST, ALT, ALP, GGT and LDH (250, 230 and 330%; 250, 200 and 300%; 66.08, 59.91 and 92.95%; 54.64, 48.2 and 90.21% and 167.49, 161.08 and 218.47%, respectively), compared to the untreated control group. By contrast, pretreatment of rats with NOC followed by exposure to either g-irradiation, CCl4 intoxication, or both treatments ameliorated the induced hepatoxicity, as evidenced by the significant decrease in serum AST, ALT, ALP, GGT and LDH activities (42.86, 48.48 and 44.19%; 46.43, 41.67 and 37.5%; 23.87, 22.87 and 30.37%; 14.5, 15.13 and 23.71%; 38.40, 41.98 and 44.47%, respectively), compared to non-NOC-pretreated respective treatments.


 

Figure 2: Particle size distribution using DLS measurements of non-irradiated chitosan (A) and oligochitosan irradiated at 20 kGy (B), 30 kGy (C) and 40 kGy (D).

 


 

Effect of different treatments on serum proteins and bilirubin levels:

Data shown in Figure 5 demonstrate that exposure of male rats to a single 5 Gy dose whole body g-irradiation, CCl4 intoxication or combined treatments disrupted the hepatic function as demonstrated by the significant decrease in the level of serum proteins, mainly total protein, albumin and globulin concentrations (44.36, 38.18 and 48.22%; 42.13, 31.47 and 35.47%; 47.23, 47.23 and 65.68%, respectively), and by contrast total and direct bilirubin levels were significantly increased (53.85, 50.55 and 87.91%; 140.74, 133.33 and 188.89%, respectively), whereas serum albumin/globulin (A/G) ratio was significantly increased (92.75 %) in CCl4-intoxicated g-irradiated rats, compared to untreated control animals. Pretreatment of rats with NOC followed by exposure to either g-irradiation, CCl4 intoxication or both treatments ameliorated the aforementioned parameters, as evidenced by the significant increase in serum total protein, albumin and globulin concentrations (31.11, 22 and 26.27%; 35.02, 25.68 and 10.74%; 24.48, 15.38 and 66.67%, respectively), whereas total and direct bilirubin levels were significantly increased (14.29, 13.14 and 26.32%; 27.69, 30.16 and 30.77%, respectively), compared to non-NOC-administered respective groups. On the other hand, serum albumin/globulin (A/G) ratio in NOC-pretreated CCl4-intoxicated and g-irradiated rats was significantly decreased (32.71%), compared to non-NOC-pretreated respective groups.


 

Figure 3: TEM images of nanostructured oligochitosan (NOC) irradiated at 20 kGy (A, 26.1-74.1 nm), 30 kGy (B, 16.1-37.3 nm) and 40 kGy (C, 4.38-15.50 nm).

 


 

Figure 4: Change in serum aspartate aminotransferase (AST, A), alanine aminotransferase (ALT, B), alkaline phosphatase (ALP, C), g-glutamyl transferase (GGT, D) and lactate dehydrogenase (LDH, E) activities in different groups.

C: Control; NOC: Nanostructured Oligochitosan; R: g-Irradiation; CCl4: Carbon Tetrachloride.

 

Figure 5: Change in serum total protein (T. PRO, A), albumin (ALB, B), globulin (GLO, C) concentrations, as well as albumin/globulin (A/G) ratio (D), total bilirubin (T. BIL, E) and direct bilirubin (D. BIL, F) levels in different groups.

C: Control; NOC: Nanostructured Oligochitosan; R: g-Irradiation; CCl4: Carbon Tetrachloride.

 


 

Effect of different treatments on oxidative stress parameters:

Exposure of male rats to a single 5 Gy dose whole body g-irradiation caused severe oxidative stress in the liver as revealed by the significant increase in hepatic MDA (86.06%, Figure 6A) and PCC (120.51%, Figure 6B) levels, compared to untreated control animals. Similarly, treatment of rats with CCl4, either alone or combined with g-irradiation exposure, significantly elevated hepatic MDA and PCC levels (77.35 and 121.88%; 106.82 and 138.63%, respectively). By contrast, pretreatment of rats with NOC followed by exposure to either g-radiation, CCl4 intoxication or both treatments, ameliorated the induced oxidative stress, as evidenced by the significant decrease in MDA and PCC levels (24.41, 25.14 and 30.99%; 35.29, 31.70 and 31.14%, respectively), compared to non-NOC-pretreated respective groups.


 

Figure 6:  Change in hepatic malondialdehyde (MDA, A) level, protein carbonyl content (PCC, B), gene expression of nuclear factor kappa beta (NF-kb, C), interleukin 1 beta (IL-1β, D), Bax (E), B-cell lymphoma 2 (Bcl2, F) levels in different groups.

C: Control; NOC: Nanostructured Oligochitosan; R: g-Irradiation; CCl4: Carbon Tetrachloride.

 


Effect of different treatments on inflammation and apoptosis markers:

Data shown in Figure 6 (C-F) 4 illustrate that exposure of male rats to a single 5 Gy dose whole body                 g-irradiation, CCl4 intoxication or both treatments markedly induced hepatic inflammation and altered apoptotic homeostasis, as revealed by the significant sharp upregulation in the mRNA expression of the transcription factor NF-kB, the proinflammatory cytokine IL-1β and the proapoptotic Bax genes (469.31, 276.24 and 664.36%; 586.14, 421.78 and 795.05%; 692.47, 545.16 and 913.98%, respectively), and by contrast a significant down-regulation was observed in the antiapoptotic Bcl2 mRNA gene expression (72.28, 63.37 and 83.17 %, respectively), compared to the untreated control group. On the other hand, pretreatment of rats with NOC followed by either exposure to g-irradiation, CCl4 intoxication or both treatments reduced hepatic inflammation and counterbalanced altered apoptosis, as demonstrated by the significant downregulation in NF-kb, IL-1β and Bax mRNA genes expression (42.09, 44.74 and 45.60%; 39.39, 40.61 and 45.46%; 35.82, 39.17 and 49.42%, respectively), along with a significant upregulation in Bcl2 mRNA expression (110.71, 70.27 and 370.59%, respectively), non-NOC-pretreated respective groups.

Histological findings:

Histological examination of the liver sections from control (Figure 7A) and NOC-treated animals (Figure 7B) shows normal histological structures of hepatocytes. On the other hand, clear damaging effects were demonstrated in the liver of rats either exposed to g-irradiation (Figure 7C), intoxicated with CCl4 (Figure 7E) or both treatments (Figure 7G), as represented by inflammatory cells infiltration in the portal area with ballooning degeneration and fibrosis, as well as congestion in the portal vein associated with apoptosis. By contrast, a notable protection was recorded in the liver of NOC-pretreated rats prior to exposure to g-irradiation (Figure 7D), CCl4 intoxication (Figure 7F) or both treatments (Figure 7H).

 

DISCUSSION:

Chitosan is a biopolymer that is degraded by ionizing radiation through the β-(1-4) glycosidic bond cleavage, with a subsequent reduction in its molecular weight34. From the current FTIR and UV results, the proposed mechanism of degradation of chitosan irradiated in the liquid is illustrated (Figure 8). Our TEM results show that irradiation of chitosan yielded oligochitosan in the nanostructure range with a very narrow size distribution. Furthermore, the size of NOC decreases with increasing the irradiation dose. The reason for a higher NOC diameter in DLS analysis is due to the hydrodynamic radii of nanoparticles in aqueous media or due to the association and aggregation of oligochitosan molecules in H2O causing an increase in particles diameter, leading to agglomeration enhancement and size increase35.

 

Impaired liver functions in the current study and the induced oxidative stress following exposure to                g-irradiation and/or CCl4 intoxication were previously reported by many authors36,37. Exposure to ionizing radiation results into water hydrolysis, producing excess hydroxyl (OH) radicals38. Also, bioactivation of CCl4, one of the hepatotoxin xenobiotics, by cytochrome P450-2E1 produces hazardous radicals that interact with polyunsaturated fatty acids (PUSFAs) in the phospholipids portion of hepatocytes, with the overproduction of ROS and enhancement of lipid peroxidation as well as oxidative protein damage39. In response to the induced oxidative stress in the liver, the integrity of cellular membranes was disturbed, increasing therefore membrane fluidity and facilitating leakage of intracellular hepatic marker enzymes into the blood stream40.

 

Inflammation is a hepatic wound-healing response to injury induced by several stimuli. It may be beneficial in the short term; however, chronic inflammation and the related regenerative wound-healing response are associated with the development of fibrosis, cirrhosis and hepatocellular carcinoma (HCC)41. NF-κB is a transcription factor that modulates genes involved in numerous biological processes, including cell growth, inflammation, cell survival and cell differentiation42. It induces the expression of various proinflammatory cytokines such as TNF-α, IL-1β and IL-643. Oxidative stress and ROS trigger the activation of NF-κB, which displays a crucial role in the inflammatory cascade by initiating the gene transcription of many proinflammatory cytokines44. In agreement with our results, Alkhalf and Khalifa45 demonstrated a significant upregulation in hepatic NF-κB mRNA expression in g-irradiated rats, suggesting an inflammatory response. Similarly, Al-Rasheed et al.46 revealed that the intoxication of male rats with CCl4 produces liver damage, accompanied with a significant increase in hepatic NF-κB, Smad2 and TGF-β mRNA expressions.

 

The mitochondrial pathway of apoptosis is chiefly controlled via the proteins of the Bcl2 family. Bax, a pro-apoptotic member of the Bcl2 family, translocates to the mitochondria, increases the permeability of the mitochondrial membrane causing the release of cytochrome c, which leads to the induction of the mitochondrial apoptotic pathway47. On the other hand, the function of Bcl2 in the apoptosis process is inhibiting the conductivity of ion channels in mitochondrial membrane, preventing therefore mitochondrial disruption and cytochrome c release48.

 

Exposure of rats to g-irradiation and/or CCl4 intoxication in the current study altered apoptotic homeostasis in the liver, where a marked significant elevation in Bax gene expression occurred, accompanied with a sharp decrease in Bcl2 gene expression. Similarly, Abu-Khudir et al.49 reported that g-irradiation-induced hepatic injury in male rats was associated with the upregulation of hepatic Bax expression, whereas Bcl2 expression was repressed. Similarly, hepatic injury induced by CCl4 intoxication in rats was associated with severe apoptosis and necrosis46. The authors reported a significant decrease in Bcl2 content (which functions to prevent cell death), accompanied with a significant increase in Bax mRNA expression (a cell death signal accelerator).

 


 

Figure 7:  Photomicrographs of liver sections from rats in the control (A) and NOC-treated group (B) showing the normal histological structure of the central vein (CV) and surrounding hepatocytes in the parenchyma. The g-irradiated group (C) showed hepatic fibrosis (f) and inflammatory cells infiltration (m) in the portal area with ballooning degeneration (b) of the hepatocytes. The NOC-pretreated g-irradiated group (D) showing diffuse ballooning degeneration (b) in the hepatocytes. The CCl4 group (E) showing ballooning degeneration of hepatocytes (b), few inflammatory cells infiltration (m) and fibrosis (f) in the portal area, which was extended in the hepatic parenchyma between the degenerated hepatocytes. The NOC-pretreated CCl4-intoxicated group (F) showing ballooning degeneration (b) in a diffuse manner all over the hepatocytes in the parenchyma with centro-lobular necrosis (n) around the central vein (CV). The CCl4-intoxicated g-irradiated group (G) showing congestion in the portal vein (PV) associated with apoptosis (arrow) of the hepatocytes in the adjacent surrounding parenchyma in the portal area. The NOC-pretreated CCl4-intoxicated g-irradiated group (H) showing the hepatocytes in the centro-lobular zone with mild necrosis (n) and ballooning degeneration (b) of some hepatocytes (H&E, ×40).

 

Figure 8: A proposed mechanism of g-irradiation-induced degradation of chitosan.

 


 

The antioxidant potential of chitosan is attributed to the capability of hydroxyl and amine groups to trap free radicals and subsequent formation of stable macromolecule radicals50. In addition, the characterization of the synthesized NOC in the present study revealed that they have a low molecular weight and a high degree of de-acetylation, which results in the increase of positive charges due to free amino groups that facilitates the coupling process with other molecules. Consequently, NOC can exhibit more superior activities than chitosan per se due to their small size and quantum size effects26. Therefore, NOC stabilized the cell membrane of hepatocytes against the synergistic damaging effect of g-irradiation and CCl4 intoxication as well as ROS production by virtue of its radical scavenging activity, chelating ability and inhibition of free radicals formation, which enhances the antioxidant defense system that ultimately suppressed g-irradiation- and CCl4-induced lipid and protein oxidations, preventing the leakage of intracellular enzymes into the blood and maintaining hepatic functions.

 

Previously, Ma et al.51 demonstrated that chitosan nanoparticles significantly downregulated the gene expression of NF-κB in lipopolysaccharide-stimulated RAW 264.7 macrophages through the blockade of the degradation of inhibitory kappa B alpha (IκB-α) and subsequent translocation of NF-κB from cytoplasm into the nucleus. Also, Tu et al.52 reported that chitosan nanoparticles suppressed lipopolysaccharide-induced inflammatory response in Caco-2 cells by reducing cytoplasmic IκB-α degradation and nuclear NF-κB p65 levels and decreasing the secretion of the pro-inflammatory cytokines, including TNF-α, MIF, IL-8 and MCP-1. Furthermore, the anti-inflammatory and antiapoptotic potentials of chitosan nanoparticles were verified in a hepatocellular carcinoma model in rats via modulating the signaling pathways of GSK-3, c-FOS, NF-κB and TNF-α53. Taken altogether, NOC counteracts the toxic synergistic effects of g-irradiation and CCl4 through the suppression of inflammation, as evidenced by the downregulation of NF-κB and IL-1β genes expression, as well as protecting the hepatocytes from cell death, by enhancing Bcl2 gene expression and downregulating Bax expression.

 

In conclusion, NOC protects the hepatocytes from the toxic synergistic effects of g-irradiation exposure and CCl4 intoxication in rats, through its radical scavenging and antioxidant activities, anti-inflammatory potential and antiapoptotic effects.

 

DISCLOSURE STATEMENT:

No potential conflict of interest was reported by the authors.

 

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Received on 26.04.2020            Modified on 25.06.2020

Accepted on 28.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):2243-2257.

DOI: 10.52711/0974-360X.2021.00398