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Background: Fluoxetine (FLX) is one of the selective serotonin reuptake inhibitors that are widely used to treat neuropsychiatric disorders including depression, but high doses can cause several adverse effects. Fisetin (FIS), a bioactive flavonoid presents in vegetables and fruits, has antioxidant, anti-inflammatory, and anticancer effects. Aim: To evaluate the possible ameliorating effect of FIS on the hepatic alterations induced by FLX in adult male albino rats. Materials and Methods: Our study was done, for 3-weeks, on 48 rats that were divided into four groups: group I (control), Group II received FIS orally (100 mg/kg/day), Group III received FLX orally (10 mg/kg/day), and Group IV concomitantly received FLX and FIS at the same dose and manner of groups II and III. Blood and liver samples were obtained and prepared for histological, immunohistochemical, and biochemical studies. Results: FLX group revealed disturbed liver architecture, hepatocytes with vacuolated cytoplasm, inflammatory cellular infiltration, blood extravasation, and congestion of blood vessels in addition to, a significant increase in the area percentage of caspase-3, inducible nitric oxide synthase and the number of glial fibrillary acidic protein-expressing cells as well as a significant decrease in the area percentage of periodic acid–Schiff stain. Moreover, FLX significantly increased aspartate-aminotransferase and alanine-aminotransferase levels in the serum. In addition, increased malondialdehyde level and decreased superoxide dismutase, glutathione (GSH) peroxidase, and reduced GSH levels in liver tissue. The concomitant administration of FIS ameliorated these alterations. Conclusions: Administration of FIS ameliorated the histological, immunohistochemical, and biochemical alterations induced by FLX in the liver of adult male albino rats.
Keywords: Fisetin, fluoxetine, glial fibrillary acidic protein, inducible nitric oxide synthase, liver
How to cite this URL:
Shaer DF, Halim HI. The possible ameliorating role of fisetin on hepatic changes induced by fluoxetine in adult male albino rats: Histological, immunohistochemical, and biochemical study. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Mar 22]. Available from: https://www.jmau.org/preprintarticle.asp?id=369251 |
| Introduction | |  |
The most prevalent mental illness that endangers millions of people all over the world is depression.[1] It is considered the main cause of disability worldwide nowadays and greatly reduces the quality of life.[2] The selective inhibitors of serotonin reuptake are drugs that are utilized for both the short-term and long-term management of mental illnesses.[3] They are extensively metabolized by a group of enzymes called cytochrome P-450 in the liver.[4]
Fluoxetine (FLX) is a fluorine-including selective serotonin reuptake inhibitor. It is the first choice medication that is commonly prescribed for the management of numerous neuropsychiatric disorders including depression and anxiety.[5] When it is orally administered, it undergoes substantial metabolic conversion in the liver into the active metabolite norfluoxetine as well as a variety of other metabolites.[6] FLX needs 1–4 days while norfluoxetine needs 7–15 days to be eliminated from the body.[7]
Despite FLX safety has been confirmed, using excessive doses was linked to numerous health hazards including digestive troubles, nervous system disorders in addition to, sexual problems, metabolic disturbance as well as liver and kidney failure.[8],[9] In recent years, research found that both FLX and norfluoxetine have a harmful impact on rats' livers by causing oxidative stress.[9],[10]
Nowadays, flavonoids are attracting great interest owing to their powerful pharmacological and nutritional effects. They are available in dietary sources like fruits and vegetables such as strawberry, persimmon, onion, and cucumber.[11] Fisetin (FIS) is a natural flavonoid polyphenol, it has many biological activities include antioxidant,[12],[13] anti-inflammatory,[14],[15] anticancer,[16] antihyperlipidemic,[17] and neuroprotective effects.[18] FIS has strong antioxidant properties owing to its chemical structure as it can scavenge free radicals that cause oxidative stress by donating the electrons.[19] Recent studies tried to demonstrate the hepatoprotective effect of FIS on the acetaminophen-induced hepatic injury.[20],[21]
As liver diseases are of public health concern, herbal medicine has attracted researchers to find new hepatoprotective compounds that can improve and ameliorate liver injury with minor side effects.[22] Although FIS is a novel antioxidant, anti-inflammatory, and health food supplement, there is not enough data about its beneficial effects on liver injury resulted from drug intake. Hence, the current work was conducted to assess the potential role of FIS in ameliorating the hepatic alterations caused by FLX in adult male albino rats.
| Materials and Methods | | |
Drugs
FLX is commercially available in a name of philozac capsules (10 mg) which is manufactured by Amoun Pharmaceutical Company, Cairo, Egypt.
FIS was purchased in the form of fine dark yellow powder from Sigma-Aldrich St. Louis, USA.
Animals
In this experiment, 48 adult male albino rats, weighing an average of (180–200 g) and with an average age of (2–3 months), were used. Rats were maintained in clean adequately ventilated cages and had unrestricted access to water and food ad libitum. They had been acclimatized to animal house conditions 7 days before starting any experimental procedure. The experimental procedure was approved by the local ethical committee of the Faculty of Medicine, Tanta University, Egypt (Approval number: 35482/5/22).
Four equal, randomly selected groups of rats were created as following:
Group I (Control group)
It consisted of 12 rats divided equally into three subgroups.
Subgroup (i): It consisted of 4 rats that were kept untreated during the experiment.
Subgroup (ii): It consisted of 4 rats received 2 ml of [0.5% sodium-carboxymethyl-cellulose solution (CMC-Na)], the diluting vehicle for FIS, orally by orogastric tube for 3 consecutive weeks.
Subgroup (iii): It included 4 rats that received 2 ml of physiological saline (the diluting vehicle for FLX) orally by orogastric tube for 3 consecutive weeks.
Group II (Fisetin-group)
It included 12 rats, each one received 2 ml of prepared FIS solution (FIS was diluted in a 0.5% [CMC-Na] [10 ml/kg][23]) and it was administered orally at a daily dose of 100 mg/kg by orogastric tube for 3 consecutive weeks.[24]
Group III (Fluoxetine-group)
It included 12 rats, each one received 2 ml of prepared FLX solution (FLX was dissolved in physiological saline 10 ml/kg) and it was administered orally at a daily dose of 10 mg/kg by orogastric tube for 3 consecutive weeks.[10]
Group IV (Fluoxetine and Fisetin-group)
It included 12 rats, each of them received both FLX and FIS in a similar dose and manner as previously described in Group II and Group III for 3 consecutive weeks.
The rats were euthanized at the end of the experiment with a 50 mg/kg intraperitoneal injection of sodium pentobarbital.[25]
Sampling
Blood samples
At the time of sacrifice, disodium EDTA tubes were used to collect the blood samples from each experimental animal via cardiac puncture. For serum separation, centrifugation of the samples for 20 min at 3000 rpm was done. The serum concentration of liver enzymes, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) was then measured. A clinical automated chemistry analyzer was used to perform the biochemical assay.[20]
Tissue samples
The liver of each animal was dissected; specimens from the right lobe of the liver were excised. Samples of liver tissue were used for biochemical and histological studies. The liver samples were homogenized in phosphate-buffered saline, followed by centrifugation, then collection of the supernatant for further biochemical tests. Oxidative stress biomarkers: Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reduced GSH were then estimated in the liver homogenates. These procedures were performed by kits purchased from (Bio-diagnostic Co., Egypt) following the manufacturer's guidelines and instructions.[10]
Histological methods
Light microscopic study
After fixation of the liver specimens in 10% neutral-buffered formalin, they were processed to obtain (5-μm thick) paraffin sections. Subsequently, staining of the sections with the ordinary hematoxylin and eosin (H and E) stain, to demonstrate the hepatic tissue's general histological architecture,[26] and periodic acid Schiff's (PAS) stain, for detection of the hepatocytes content of glycogen, was done.[27]
Immunohistochemical study
Some liver sections were processed to be stained with the following immunostains; caspase-3 to detect apoptotic cells,[28] glial fibrillary acidic protein (GFAP) for the detection of hepatic stellate cells (HSCs) activation,[29] and inducible nitric oxide synthase (iNOS) to assess the distribution and intensity of iNOS protein expression in hepatic tissues.[30]
Liver sections were dewaxed and rehydrated. To reduce the nonspecific background staining and inhibit the activity of endogenous peroxidase; 3% hydrogen peroxide was added to sections for 10 min. After that, preheated citrate buffer was added to sections for antigen retrieval. Incubation of sections was done, in a humidified chamber overnight, with the subsequent primary antibodies; Caspase-3 (PP229AA polyclonal antibody, obtained from Biocare Medical Company, USA), iNOS (RB-9242-R7 polyclonal antibody, obtained from Thermo Fisher Scientific, USA), and GFAP (ab7260 polyclonal antibody, Abcam Medical Company, UK). The secondary biotinylated goat antirabbit antibody was added on the slides after their washing. Subsequently, the slides were stained with diaminobenzidine, a chromogenic substrate, and incubated until the desired reaction was achieved. Finally, the slides were counterstained using Mayer's hematoxylin stain.[31]
Positive results of activated caspase-3 appeared as cytoplasmic and/or nuclear reaction,[32] while for GFAP and iNOS appeared cytoplasmic.[33],[34] Positive control for caspase-3 was from the placenta and for GFAP was from the brain (cerebellum, striatum), whereas for iNOS was from the lung. Negative control was run automatically without adding the primary antibody.[31] Finally, light microscope with built-in camera from Leica was used to examine and photograph all the specimens of the current study.
Morphometric study
Ten different, random, nonoverlapped fields were selected from each slide in each group to be quantified, by ImageJ application software (National Institute of Health, Bethesda, Maryland, USA), for the underlying parameters:
- The mean area percentage of glycogen, in sections stained with PAS (×400).
- The mean area percentage of positive caspase-3 immunoreaction (×400).
- The mean area percentage of positive iNOS immunoreaction (×400).
- The mean number of GFAP +ve cells (×400).
Statistical analysis
The measured morphometric parameters, liver enzymes, and liver oxidative stress biomarkers were statistically analyzed to evaluate the differences between the different studied groups using the SPSS program for Microsoft Windows (IBM Corp, Version 25.0. Armonk, NY, USA). The calculated numbers were considered for comparison and statistical analysis using the one-way analysis of variance test followed by Tukey's post hoc test to compare all groups. All statistical values were recorded in the form of mean ± standard deviation. If P ≤ 0.05, significant differences were considered.[35]
| Results | | |
No deaths of the experimental animals were noted during the study period. Moreover, the three subgroups (i, ii, iii) in the control group revealed nonstatistical differences in the histological, immunohistochemical, and biochemical results; hence, all of them were presented as the control group.
Histological results
H and E stain
Both control and FIS groups exhibited the normal hepatic architecture with tightly packed hepatic cords extended outward from the central vein (CV) to the portal regions and separated by sinusoids [Figure 1]a. The hepatic cords consisted of polygonal hepatocytes that had granular eosinophilic cytoplasm and centrally placed rounded nuclei. Some hepatocytes were binucleated. Sinusoidal capillaries lined by endothelial cells appeared in between the hepatic cords [Figure 1]b. Portal tracts (also known as the portal triads) were seen at hepatic lobules' corners and each tract contained branches from the hepatic artery and portal vein as well as a small bile duct [Figure 1]c. | Figure 1: (a) A photomicrograph of a liver section from control group showing closely packed cords of normal hepatocytes (arrow) radiating from a CV to the portal areas (P) and separated by sinusoids (S) (H and E, ×200, Scale bar = 50 μm). (b) A photomicrograph of a liver section from control group showing cords of normal hepatocytes (arrows), having granular acidophilic cytoplasm and vesicular nuclei, radiating from a CV and separated by sinusoids (S). Binucleated hepatocytes are also observed (arrowheads) (H and E, ×400, Scale bar = 50 μm). (c) A photomicrograph of a liver section from control group showing the portal tract that contains branches from the PV and HA as well as a small BD (H and E, ×400, Scale bar = 50 μm). (d) A photomicrograph of a liver section from FLX-group showing hepatocytes having vacuolated cytoplasm either with ill-defined fragmented nuclei (arrowheads) or absent nuclei (arrows). Notice inflammatory cellular infiltration (bifid arrows) (H and E, ×400, Scale bar = 50 μm). (e) A photomicrograph of a liver section from FLX-group showing some hepatocytes are fused together with loss of their boundaries and exhibit highly eosinophilic cytoplasm and deeply stained nuclei (circle). Notice a congested central vein (wavy arrow) (H and E, ×400, Scale bar = 50 μm). (f) A photomicrograph of a liver section from FLX-group showing markedly dilated and congested blood sinusoids (wavy arrow). Notice hepatocytes with vacuolated cytoplasm and deeply stained nuclei (arrows) (H and E, ×400, Scale bar = 50 μm). (g) A photomicrograph of a liver section from FLX-group showing congestion of the portal vein (wavy arrow) and inflammatory cells (bifid arrows) infiltrate the portal area (H and E, ×400, Scale bar = 50 μm). (h) A photomicrograph of a liver section from FLX-group showing disturbed architecture of the hepatic tissue with areas of eosinophilic homogenization (stars) and blood extravasation (wavy arrow) (H and E, ×400, Scale bar = 50 μm). (i) A photomicrograph of a liver section from FLX and FIS-group showing cords of nearly normal hepatocytes (arrow) separated by sinusoids (S), with some hepatocytes appear binucleated (arrowhead). Few shrunken darkly stained nuclei (curved arrows) and mild congested blood sinusoids (wavy arrows) are noticed (H and E, ×400, Scale bar = 50 μm). (j) A photomicrograph of a liver section from FLX and FIS-group showing more or less normal portal tract components with focal inflammatory cellular infiltration (bifid arrows) in the portal area (H and E, ×400, Scale bar = 50 μm). CV: Central vein, S: Sinusoids, P: Portal, PV: Portal vein, HA: Hepatic artery, BD: Bile duct, FLX: Fluoxetine, FIS: Fisetin
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The FLX-group exhibited obvious alterations in the liver structure such as hepatocytes with vacuolated cytoplasm that showed either ill-defined fragmented nuclei or absent nuclei. Inflammatory cellular infiltration was also noticed [Figure 1]d. Some hepatocytes appeared coalesced and exhibited highly eosinophilic cytoplasm, deeply stained nuclei, and lost cellular boundaries, in addition to congestion of CVs [Figure 1]e. Moreover, there were hepatocytes with vacuolated cytoplasm and deeply stained nuclei, in addition to markedly dilated congested blood sinusoids [Figure 1]f. Congested portal veins with inflammatory cells infiltrating the portal area were observed [Figure 1]g. Furthermore, disturbed architecture of the hepatic tissue with areas of eosinophilic homogenization and blood extravasation was also noticed [Figure 1]h.
However, FLX and FIS-group revealed almost normal liver structure, although there were few darkly stained nuclei, mild congested blood sinusoids [Figure 1]ixs, and focal cellular infiltration around the portal area [Figure 1]j.
Periodic acid Schiff stain
PAS-stained liver tissues of both the control group and FIS-group revealed high glycogen content of most of the hepatocytes [Figure 2]a. In the FLX-group, there was marked depletion in the glycogen content in most of the hepatocytes. Few hepatocytes displayed moderate to high glycogen content [Figure 2]b. However, FLX and FIS-group showed more glycogen content in hepatocytes than the FLX-group [Figure 2]c. | Figure 2: (a) A photomicrograph of a liver section from control group showing strong PAS positive reaction in the hepatocytes (arrows) (PAS, ×400, Scale bar = 50 μm). (b) A photomicrograph of a liver section from FLX-group showing weak PAS positive reaction in most hepatocytes (arrows). Few hepatocytes display moderate to strong PAS positive reaction (arrowheads) (PAS, ×400, Scale bar = 50 μm). (c) A photomicrograph of a liver section from FLX and FIS-group showing moderate to strong PAS positive reaction in the hepatocytes (arrows) (PAS, ×400, Scale bar = 50 μm). FLX: Fluoxetine, FIS: Fisetin, PAS: Periodic acid Schiff's
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Immunohistochemical results
Caspase-3 immunostaining
The degree of apoptosis was detected by caspase-3 immunostaining. The positive caspase-3 immunoexpression appeared as brown cytoplasmic coloration. There was weak and localized positive caspase-3 immunoexpression in the cytoplasm of few hepatocytes in the control group and FIS group [Figure 3]a. However, in the FLX-treated group, diffuse strong positive caspase-3 immunoexpression was noticed in many hepatocytes [Figure 3]b. FLX and FIS-group exhibited localized moderate positive caspase-3 immunoexpression in some hepatocytes [Figure 3]c. | Figure 3: (a): A photomicrograph of a liver section from control group showing localized weak positive expression of caspase-3 (arrows) in the cytoplasm of few hepatocytes (Caspase-3, ×400, Scale bar = 50 μm). (b) A photomicrograph of a liver section from FLX-group showing diffuse strong positive caspase-3 expression (arrows) in most hepatocytes (Caspase-3, ×400, Scale bar = 50 μm). (c): A photomicrograph of a liver section from FLX and FIS-group showing localized moderate positive caspase-3 expression (arrows) in the cytoplasm of some hepatocytes (Caspase-3, ×400, Scale bar = 50 μm). FLX: Fluoxetine, FIS: Fisetin
Click here to view |
Inducible nitric oxide synthase immunostaining
Liver sections were stained against iNOS to assess the distribution and intensity of iNOS protein expression in hepatic tissues in all groups. The brownish-yellow cytoplasmic coloration of the hepatocytes was regarded as a positive immunoreaction. Both control and FIS groups revealed localized weak positive iNOS-expression on the walls of the hepatic sinusoids [Figure 4]a. In the FLX-group, there was strong diffuse positive iNOS expression in the hepatic tissues [Figure 4]b. Focal positive iNOS expression appeared in FLX and FIS-group compared to FLX-group [Figure 4]c. | Figure 4: (a) A photomicrograph of a liver section from control group showing weak localized positive iNOS-expression on walls of the hepatic sinusoids (arrows) (iNOS, ×400, Scale bar = 50 μm). (b) A photomicrograph of a liver section from FLX-group showing diffuse strong positive iNOS-expression in the hepatic tissues (arrows) (iNOS, ×400, Scale bar = 50 μm). (c) A photomicrograph of a liver section from FLX and FIS-group showing focal areas of positive iNOS-expression in the hepatic tissues (arrows) (iNOS, × 400, Scale bar = 50 μm). FLX: Fluoxetine, FIS: Fisetin, iNOS: Inducible nitric oxide synthase
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Glial fibrillary acidic protein immunostaining
To detect the GFAP within the HSCs, liver tissues from all studied groups were stained with GFAP immunostaining. Both control and FIS groups [Figure 5]a showed mild positive GFAP immunoreaction (brown coloration) in few HSCs in between the hepatocytes. In the FLX-group, an obvious increment in the count of GFAP-positive HSCs with apparent strong positive immunoreaction was noticed [Figure 5]b. However, FLX and FIS-group revealed a moderate count of the GFAP positive cells with moderate positive immunoreaction [Figure 5]c. | Figure 5: (a) A photomicrograph of a liver section from control group showing few GFAP positive hepatic stellate cells with mild positive GFAP immunoreaction (arrows) (GFAP, ×400, Scale bar = 50 μm). (b) A photomicrograph of a liver section from FLX-group showing numerous GFAP positive hepatic stellate cells with apparent strong positive immunoreaction (arrows) (GFAP, ×400, Scale bar = 50 μm). (c) A photomicrograph of a liver section from FLX and FIS-group showing some GFAP positive hepatic stellate cells with moderate positive immunoreaction (arrows) (GFAP, ×400, Scale bar = 50 μm). FLX: Fluoxetine, FIS: Fisetin, GFAP: Glial fibrillary acidic protein
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Morphometric results
The morphometric study revealed nonsignificant statistical differences in the morphometric results between Group I and Group II. Otherwise, FLX group revealed a high-significant decrement in the area percent of PAS and a high-significant increment in the area percent of caspase-3 and iNOS as well as in the number of GFAP-positive cells in comparison to the control group. However, the FLX and FIS-group exhibited a high-significant increment in the area percent of PAS and a high-significant decrement in the area percent of caspase-3 and iNOS as well as in the number of GFAP-positive cells versus that in the FLX-group. Furthermore, FLX and FIS-group showed nonsignificant statistical differences in the morphometric results versus the control group, except for PAS as there was a significant difference [Histogram 1].
Biochemical results
Liver function tests
Regarding the liver function tests, comparing Group II with Group I exhibited a nonsignificant statistical difference. In contrast, FLX-group revealed a high significant increment in the liver enzymes; ALT and AST as compared to the control group. Whereas in the FLX and FIS-group, the enzyme levels were highly-significantly decreased compared to the FLX-group. Additionally, the levels of ALT and AST in the FLX and FIS-group were not significantly different compared to their levels in the control group [Histogram 2].
Liver oxidative stress biomarkers
Concerning the liver oxidative stress biomarkers, the statistical analysis demonstrated a nonsignificant statistical difference in-between Group I and Group II. Nevertheless, the administration of FLX in Group III caused a high significant increment in the hepatic MDA and a high significant decrement in the hepatic SOD, GSH-Px, and GSH compared to the control group. However, when FIS was given simultaneously with FLX in group IV, this resulted in a high significant decrement in MDA and a high significant increment in SOD, GSH-Px and GSH as compared to FLX-group. Furthermore, there was a nonsignificant difference in the tissue levels of MDA, SOD, GSH-Px and GSH between the FLX and FIS-group and the control group [Histogram 3].
| Discussion | | |
FLX is a drug that is frequently utilized for the management of major-depressive and panic disorders. Unfortunately, disturbed liver function was recorded in 0.5%–3% of patients who received antidepressant medications including FLX. FLX administration could induce liver injury by different mechanisms including induction of inflammation, oxidative stress, and apoptosis.[10] FIS is a naturally safe flavonoid that is available in many different fruits and vegetables, and it has anti-inflammatory and antioxidant effects.[12],[13] This study was performed to evaluate the possible ameliorating role of FIS on the hepatic alterations caused by FLX.
The current work revealed that FLX caused histological alterations and disturbed architecture of the hepatic tissue. These alterations included hepatocytes with vacuolated cytoplasm and fragmented nuclei, coalesced hepatocytes with deeply stained nuclei and lost cellular boundaries, in addition to congested blood sinusoids, central and portal veins together with inflammatory cellular infiltration, areas of eosinophilic homogenization and blood extravasation. These findings were consistent with those of Yılmaz et al.,[36] Elgebaly et al.,[37] Zlatković et al.,[38] and Ganguly et al.[39] who studied the influence of FLX on the hepatic architecture in the albino rats. They observed hepatocyte necrosis, cytoplasmic vacuolation, inflammatory cellular infiltration, and hydropic degeneration in the livers of FLX-treated rats. Zidan and Alazouny[40] as well noticed irregular cellular outlines with darkly stained nuclei, cytoplasmic vacuolation, and cellular infiltration in the parotid gland of rats that received FLX.
These histological alterations could be attributed according to Karimi-Khouzani et al.[41] and Abo-Ouf[42] to the inflammation and oxidative stress induced by FLX. They stated that FLX administration increased the pro-inflammatory cytokines (Tumor Necrosis Factor alpha [TNF-α] and Interleukin [IL-1] β levels) and decreased the level of antioxidants in the hepatic tissue of albino rats. Moreover, the irregularity in the hepatic architecture and coalesced hepatocytes as well as cytoplasmic and nuclear alterations were attributed by other previous studies to FLX-induced free radicals release that could induce oxidative damage of the hepatocellular proteins and fatty acids of hepatocyte's, membrane as well as organelles' membranes.[43],[44] This effect on the organelles, membranes could increase their permeability with subsequent disorders of the cytoplasmic and cell organelles' ions concentration which in turn caused vacuolation as reported by Ali et al.[45] Furthermore, the dilation and congestion of blood sinusoids, central and portal veins were considered by Elhelaly et al.[46] as an inflammatory response against reactive-oxygen-species (ROS) to improve blood circulation to the injured area.
Interestingly, the concomitant use of FIS with FLX in the current study preserved most of the normal histological structure of the hepatic tissue. These results could be related to the anti-inflammatory and antioxidant properties of FIS as reported by Hussein et al.,[47] Naeimi and Alizadeh,[48] and Hu et al.[49] They explained that FIS could exert its anti-inflammatory effect through reducing the release of the pro-inflammatory cytokines as TNF-a and IL-6, while its antioxidant effect could be achieved by several mechanisms including enhancement of the anti-oxidants inside the cells, inhibition of enzymes that produce ROS, scavenging metal ions' chelating transition and being a substrate for oxidoreductase activity.
Using PAS stain in the current study revealed obvious depletion of glycogen granules in the hepatocytes of liver sections of FLX-treated rats. This was verified by the statistical-analysis of the morphometric results that exhibited a highly significant decrement in the area percentage of PAS in FLX-group versus the control group. This finding agreed with Dorelle et al.[50] who observed a 50% decrement in glycogen content in the liver of fish that received FLX. That depletion of glycogen was explained by El Kalawy et al.[51] as the oxidative stress associated with FLX suppresses cytochrome oxidase and dehydrogenase enzymes which in turn increases liver glycogenolysis and glycolysis. On the other hand, there was a high-significant increment in the area percentage of PAS in FLX and FIS-group versus the FLX-group. This is in accordance with the findings of a study was conducted by Constantin et al.[52] who demonstrated that FIS could decrease glycogen breakdown.
The above-discussed histopathological changes were supported by the results of the immunohistochemical study. Regarding caspase-3 immunostaining, FLX-group revealed an increase in caspase-3 immunoreactivity which was confirmed by the morphometric study followed by the statistical analysis that demonstrated a high-significant increment in the caspase-3 area percentage in FLX-group versus the control group. The same finding was previously noticed by Mohamed Kamel[10] who reported that FLX administration caused a significant increment in caspase-3 immunoreactivity in the hepatic tissue by two and a half times versus the control group. Sakr et al.[53] and Elsedawi et al.[54] explained the increment in the immunoexpression of caspase-3 by the FLX-induced oxidative stress. On the contrary, a previous study reported that FLX enhanced cellular proliferation and prevented dentate gyrus apoptosis in rats.[55]
Furthermore, a high significant decrement in the area percent of caspase-3 was recorded in FLX and FIS-group as compared to FLX-group. This indicated that the coadministration of FIS with FLX ameliorated the FLX-induced apoptosis in the hepatic tissues. This finding coincided with Sun et al.[56] who found that FIS protected hepatocytes from apoptosis and oxidative stress caused by alcohol consumption. Another study revealed that FIS could protect cardiomyocytes from cell death by suppressing ROS release and caspase activity.[57] They explained that the anti-apoptotic impact of FIS was due to the suppression of caspase-9 expression. The latter is the initiator-caspase in the intrinsic pathway of apoptosis triggered by oxidative stress.[56],[57]
In the present study, iNOS immunostaining was utilized as an oxidative stress marker. The obtained liver sections from FLX-group exhibited widely distributed strong iNOS immunoreaction in the hepatic tissue. Moreover, the statistical study showed a high-significant increment in the area percent of iNOS expression in the FLX-group versus the control group. This agreed with Issa and El-Sherif[58] who found a significant up-regulation of iNOS expression in their study about anthracene harmful effects on the tissues of liver and lung in albino rats and they reported that the immunoexpression of iNOS was utilized as an indicator for oxidative stress.
It is noteworthy that hepatocyte injury seen in H and E stained sections of FLX-group was correlated to the overexpression of iNOS that was detected by immunohistochemistry. This could be explained by increased iNOS expression in hepatocytes upon exposure to a variety of stress conditions such as the use of toxic substances, inflammation, and ischemia-reperfusion injury.[59],[60],[61] Moreover, iNOS generates a large amount of NO which is one of the reactive radicles that is involved in hepatic injury.[62],[63]
However, FIS administration in FLX and FIS-group caused a high-significant decrement in iNOS expression that appeared in focal areas of the hepatic tissues. Our finding matched with that of Elmehy et al.[24] who noticed a marked reduction in iNOS level in the hepatic tissues of mice that received FIS. They reported that FIS had a remarkable anti-inflammatory and antioxidant effect on the liver tissue. Another study done by Hada et al.[64] showed that FIS could suppress NO production through decreasing mRNA expression of the iNOS, so they suggested that FIS could reduce oxidative damage and attenuate oxidative stress.
In the current study, GFAP was used as a marker for HSCs activation.[65] There was a high-significant increment in the count of GFAP +ve cells in FLX-group in comparison with the control. This finding coincided with that of Bayomy et al.[29] and Morini et al.[65] who demonstrated that GFAP-expressing stellate cells were increased in acute hepatic injury compared to those in the quiescent state.
On the other hand, the coadministration of FIS in the FLX and FIS-group caused a high-significant decrement in the count of GFAP +ve cells compared with that in the FLX-group. El-Fadaly et al.[66] reported that FIS could prevent HSCs from activating and proliferating by different mechanisms including inhibition of multiple profibrogenic factors and blocking of the pathway of (Wnt\β-catenin) which modulates cell's proliferation, survival, behavior, and fate.[67]
In FLX-treated rats, a high significant increment in the serum concentrations of liver enzymes (ALT and AST) was noticed. Such an increase suggested damage to hepatocytes and breaking down of their membranes.[36],[68],[69] These results matched those of a previous study done by Zlatković et al.[38] who reported an increment in serum ALT and AST that was mostly caused by FLX-induced cell membrane damage. Furthermore, Yılmaz et al.[36] and Inkielewicz-Stępniak[70] noticed elevated serum AST and ALT levels upon exposure to FLX in high doses. Ganguly et al.[39] as well reported elevated ALT and AST in FLX-treated rats and they attributed this increment to ROS-induced hepatocyte disintegration and necrosis.
We also noted that the serum concentration of AST and ALT was highly significantly decreased when FIS was co-administered with FLX versus their serum levels while administering FLX alone. As stated by Zhao et al.,[20] FIS could reduce the leakage of liver enzymes into the bloodstream owing to its potent antioxidant properties. Moreover, FIS could normalize the level of liver enzymes in acetaminophen-intoxicated mice and in thioacetamide-treated rats as reported by other studies.[21],[66],[71]
Regarding the oxidative stress biomarkers, there was a high significant increment in hepatic MDA and a high significant decrement in hepatic SOD, GSH-Px and GSH levels in the FLX group compared to the control group. These results agreed with an earlier study by Elgebaly et al.[37] who found that FLX treatment enhanced the oxidative stress index and caused a reduction in the overall antioxidant capacity in rats' livers. Previous research demonstrated that hepatotoxicity caused by FLX was related to lowered antioxidant defenses (SOD, GSH-Px and GSH) and increased MDA (the final product of lipid-peroxidation). Furthermore, ROS overproduction induced by FLX could be harmful to the macromolecules inside cells such as DNA and lipids as well as proteins.[10],[37],[39],[41]
Interestingly, our result showed that administering of FIS attenuated oxidative stress brought on by FLX. This was verified by the high significant decrement in MDA and increment in SOD, GSH-Px and GSH in the hepatic tissue of the FLXandFIS-group compared to the FLX-group. Numerous studies confirmed that FIS possesses potent antioxidant properties.[20],[21],[46]
Thus, these findings denoted that FLX could induce oxidative stress by free radicals' formation during its biotransformation in the liver with subsequent liver inflammation and damage. FIS co-administration with FLX attenuated the hepatic alterations induced by FLX through the regulation of both oxidative stress and inflammation.
| Conclusions and Recommendation | | |
Based on this study, we concluded that the administration of FIS had ameliorating effects regarding the histological, immunohistochemical, and biochemical alterations caused by FLX in the liver of adult male albino rats. This hepatoprotective influence of FIS could be related to its antioxidant capability. We recommend careful monitoring of liver functions in FLX-treated patients for the early detection of liver injury and to conduct more research for the better comprehension of the molecular pathways that clarify how FIS prevented liver damage brought on by FLX.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Hend Ibrahim Abd El Halim,
Department of Histology and Cell Biology, Faculty of Medicine, Tanta University, Tanta, 31527
Egypt
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/jmau.jmau_84_22
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] |
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