Does Dactyloctenium aegyptium ethanolic extract protect against hormonal, histological and immunohistochemical alterations induced by sodium fluoride in rat adrenal gland?

ABSTRACT This work aimed to perform a biochemical, histological, and immunohistochemical assessment of the influence of sodium fluoride on the adrenal gland of rats and the possible ameliorating role of Dactyloctenium aegyptium (D. aegyptium). Thirty-five male albino rats were divided into five equal groups: group I served as the control group, group II received sodium fluoride (5 mg/kg), group III received D. aegyptium (200 mg/kg), and groups IV and V received sodium fluoride and D. aegyptium (100 and 200 mg/kg respectively) simultaneously for 28 days. Aldosterone, corticosterone, nitric oxide, sodium, potassium, and chloride were measured in serum samples. Adrenal gland specimens were processed for histological, histochemical, and immunohistochemical studies. The sodium fluoride group recorded a significant reduction in serum levels of aldosterone, corticosterone, potassium, and chloride (p ≤ 0.0001) and a significant increase in nitric oxide (p ≤ 0.0001) and serum sodium (p = 0.0076) when compared with the control group. Histological changes in the sodium fluoride treated group showed cytoplasmic vacuolation, scattered apoptotic cells, and hemorrhage. The sodium fluoride treatment significantly decreased glycogen (p = 0.0002), total protein) p = 0.0007), and DNA (p ≤ 0.0001) and increased inducible nitric oxide synthase (p = 0.0001). Co-treated groups with D. aegyptium showed almost average values for most parameters and a nearly standard architecture. The present data answers the question of our research that D. aegyptium ethanolic extract could protect against sodium fluoride’s adverse effects in rat adrenal glands on the physiological and histological levels.


Introduction
The adrenal gland is found at the apex of each kidney.It secretes various hormones, including cortisol, aldosterone, adrenaline, and a small number of androgens, which are sex hormones.The adrenal gland hormonestdslevel serve a variety of crucial roles.They help to regulate blood pressure, digest protein and fat, balance blood sugar, and respond to stress.Some hormones, such as cortisol, can cause adrenal gland diseases [1,2].
Harmful chemicals can disrupt the endocrine system, which controls hormones, metabolism, and transportation [1].
Among the elements in the planet's crust, fluoride is the thirteenth most prevalent.About 200 million people throughout the world consume water with a fluorine concentration of 1.5 mg/L or more, according to the World Health Organization (WHO).Consumption of fluoridated water, food, and other fluoride-containing products can lead to a condition known as fluorosis [3].Many studies demonstrated the hazardous effects of fluoride on various organs, such as the brain, gastrointestinal tract, kidney, and liver [4].When consumed in excess, fluoride can induce dental and skeletal fluorosis, which disrupt average enamel growth and bone homeostasis, respectively.Extreme fluoride exposure also leads to pathological and physiological alterations in the adrenal gland, steroid hormones, and reproductive hormones in rats [5].
Researchers have tried to identify a wide variety of naturally occurring compounds that not only have significant biological activity but also have an adequate response against the damage caused by toxicants [6].
Dactyloctenium aegyptium (D. aegyptium) is a Gramineae (Poaceae) plant native to tropical and subtropical regions.Its medicinal properties make it a valuable herb.The D. aegyptium plant is beneficial for the digestive system, and pharmacological tests have also found that it has antioxidant, anti-inflammatory, anticancer, and antipyretic properties [7].Our previous investigation showed that this plant's ethanolic extract is rich in flavonoids, phenolic acids, and derivatives.Eight phenolic acids, two phenolic derivatives, and four flavonoids were found [8].To the best of our knowledge, there are no scientific records on the biological effects of D. aegyptium in the adrenal glands of experimental animals.The study aimed to investigate the potential protective impact of D. aegyptium on sodium fluoride (NaF)induced hormonal, histological, histochemical, and immunohistochemical alterations in the adrenal gland.

Chemicals
The NaF was purchased from LOBA Chemie Pvt. Ltd. in Mumbai, India.NaF was provided as a white powder and dissolved in distilled water.

Plant materials and extraction process
The D. aegyptium's aerial parts were gathered from Kafr-Saad, Damietta, Egypt, in March-April 2020 during flowering, followed by air-drying.Mansoura University Herbarium in Egypt received a voucher specimen (DA-X317-015-M1921).Air-dried powdered plant aerial parts (500 g) were extracted with 70% ethanol, filtered, and dried under vacuum at 40°C to produce 21.0 g of dark black gum.

Animals
Thirty-five adult male albino rats weighing 120-130 g were used in this investigation and were purchased from the Animal House at the National Research Centre in Giza, Egypt.All rats were kept in metal cages with constant temperature, humidity, a light/dark cycle, and excellent ventilation with unrestricted food and water.The Ethics Committee of Suez University, Suez, Egypt, approved the experimental procedure (approval number 13323b).

Experimental design
For 28 days, five groups of seven rats each received the following interventions: Group I: Rats received drinking water.
Group II: Rats received 5 mg/kg NaF dissolved in distilled water through a stomach tube daily [9].
Group III: Rats received D. aegyptium extract (200 mg/kg) orally dissolved in distilled water.The dose of the plant extract was evaluated and calculated in our previous study [8].

Histological and histochemical studies
At the end of the experiment, the animals were sacrificed and carefully dissected, then the adrenal glands were removed.After being fixed in 10% formal saline for 24 hours, the adrenal glands were washed, dehydrated, cleared in xylene, and finally embedded in paraffin.Sections of 5 μm thickness were cut and processed for hematoxylin and eosin (H&E) stain for histological evaluation [15], Periodic Acid Schiff (PAS) stain for glycogen detection [16], Mercury Bromophenol Blue stain for total protein detection [17] and Feulgen stain for DNA content demonstration [18].

Immunohistochemical study
Labeled streptavidin-biotin immunoperoxidase staining with an inducible nitric oxide synthase (iNOS) antibody revealed iNOS in tissue sections.The sections were treated with 0.01% trypsin at 37°C for 10 minutes and rinsed for 5 minutes after xylene deparaffinization and rehydration in 0.1 mol/l PBS.Body peroxidases were suppressed by 0.5% H 2 O 2 treatment for 5 minutes.Cytoplasmic iNOS was identified by polyclonal rabbit anti-iNOS immunoperoxidase dye.Sections were treated with a 1:100 primary antibody at 4°C for 60 minutes.The secondary antibody was added after two PBS washes.Biotinylated secondary antibodies were in allpurpose kits.Slides were mounted with D.P.X and counterstained with Mayer's hematoxylin.Normal rat serum (×100 diluted) replaced the iNOS-specific primary antibody in negative controls [19].

Histomorphometric evaluations and image analysis
The Leica Qwin 500 Image Analyzer (England) analyzed five randomly chosen fields at 400× from each slide to acquire histomorphometry data.After examining all digital images with the semi-quantitative scoring system (Image J software, a Java-based image assessment application) in the Pathology Department of the National Research Centre in Giza, Egypt, the average was calculated: (1) Measurement of mean thickness of the whole adrenal cortex (μm) in H&E-stained sections.(2) The mean percentage (%) of PAS-stained area.

Statistical analysis
GraphPad Prism 9 was utilized for the estimation of statistics.The data was presented as the mean ± SEM.Mean differences across the various groups were calculated using a one-way analysis of variance (ANOVA) and then subjected to Tukey's test for multiple comparisons.At P < 0.05, the results appeared to be significant.The data was found to be regularly distributed when the Shapiro-Wilk test was used to determine normality.

Serum aldosterone and corticosterone
Compared to the control group, serum aldosterone and corticosterone levels were significantly decreased at P < 0.05 in the Na-F treatment group.D. aegyptium extract treated group showed no significant difference in aldosterone and corticosterone levels compared to the control group.Serum aldosterone and corticosterone levels in rats treated with NaF that also received D. aegyptium extract (100 or 200 mg/kg) were significantly increased at P < 0.05 compared to the NaF group by 40.45% and 59.54% and 47.36% and 61.44% respectively (Figure 1(a,b)).

Serum NO
Serum levels of NO were significantly increased at P < 0.05 in the NaF treatment group compared to the control group.NO levels showed insignificant differences between D. aegyptium extract treated and control groups.The animals treated with NaF and D. aegyptium extract (100 or 200 mg/kg) showed a significant decrease in NO at P < 0.05 (Figure 2).

Serum Na + , K + and Cl −
Compared to the control group, the serum Na + level was significantly increased.In contrast, serum K + and Cl − levels were significantly decreased at P > 0.05 in the Na-F group compared to the control group.D. aegyptium extract treated group showed no significant difference in Na + , K + , and Cl − levels compared to the control group.The serum Na + levels of NaF-treated rats given 100 or 200 mg/kg D. aegyptium extract were significantly decreased by −11.23% and −13.79%, respectively, while K + and Cl − levels were significantly increased compared to the NaF group by 4.1% and 8.21%and 26.57% and 33.35%, respectively (Figures 3(a-c)).

Histological and histomorphometric results
H&E-stained adrenal gland sections of the control group exhibited the normal histoarchitecture of the adrenal gland (Figure 4(a,b)).
Adrenal gland sections of NaF-treated rats revealed disruption of the histoarchitecture of the adrenal gland.The zona glomerulosa showed dispersed cells with vacuoles in their cytoplasm.In contrast, the zona fasciculata showed apoptotic cells separated by average blood sinusoids (Figure 4(c)).The zona reticularis showed scattered apoptotic cells.In contrast, the medulla showed hemorrhage, apoptosis, and vacuolated cytoplasm (Figure 4(d)).Cortical thickness was significantly decreased at p < 0.05 compared to the control group (Figure 4(h)).D. aegyptium extracttreated rats revealed normal adrenal gland histoarchitecture.D. aegyptium extract group exhibited similar cortical thickness to the control group (Figures 4(e,h)).Histopathological examination of adrenal gland sections of NaFtreated rats given D. aegyptium extract (100 or 200 mg/kg) showed normal zona glomerulosa, zona fasciculata, and intervening blood capillaries with a significant increase at p < 0.05 in cortical thickness (Figures 4(f,g,h)).

PAS reaction
The adrenal gland sections of the control animals showed dense magenta-stained PAS glycogen in the cells of both the zona glomerulosa and zona fasciculata (Figure 5(a)).The adrenal gland sections of NaF-treated rats revealed weak PAS reactivity in most of the zona glomerulosa and zona fasciculata cells (Figure 5(b)).Digital image analysis showed a significant decrease in PASpositive materials (%) at P > 0.05 (Figure 5(f)).The D. aegyptium-treated rats' adrenal glands showed a strong PAS reaction in the cells of both the zona glomerulosa and zona fasciculata (Figure 5(c)).The adrenal gland sections from NaF-treated rats given 100 or 200 mg/kg D. aegyptium extract showed dense magentastained positive glycogen for PAS in zona glomerulosa and zona fasciculata compared to the NaF group (Figures 5(d,e,f)).

Total protein
Examination of the adrenal glands sections of the control rats showed deeply stained fine blue granules of total protein in most of the zona glomerulosa and most of the zona fasciculata cells (Figure 6(a)).Sections of adrenal glands from NaFtreated rats revealed faint staining in zona glomerulosa cells and most zona fasciculata cells (Figure 6(b)).Most of the zona glomerulosa and zona fasciculata cells in D. aegyptium-treated rats showed heavily stained blue granules (Figures 6(c,  f)).Adrenal gland sections of NaF-treated rats given D. aegyptium extract (100 or 200 mg/kg) showed dark blue granules in most zona glomerulosa and zona fasciculata cells as compared to the NaF-treated group (Figures 6(d,e,f)).

DNA content
Microscopic examination of sections of the adrenal glands of control rats exhibited a regular DNA distribution in nuclei of zona glomerulosa cells and zona fasciculata cells (Figure 7(a)).Sections of adrenal glands of NaFtreated rats revealed weak positivity for Feulgen in most of the zona glomerulosa cell nuclei and most of the zona fasciculata cells nuclei.DNA (%) stained area was significantly decreased at P < 0.05 in the NaF group compared to the control group (Figures 7(b,f)).Adrenal gland sections of D. aegyptium-treated rats exhibited high positivity for Feulgen in most of the zona glomerulosa cell nuclei and in most zona fasciculata cell nuclei (Figure 7(c)).Examination of sections of adrenal glands of NaF-treated rats that were given D. aegyptium extract (100 or 200 mg/kg) exhibited a strong Feulgen reaction in most of the zona glomerulosa cell nuclei and most of the zona fasciculata cell nuclei as compared to the NaF-treated group (Figures 7(d,e)).

Immunohistochemical demonstration of iNOS
The adrenal gland sections of control rats showed negative iNOS reactivity in both of the zona glomerulosa and zona fasciculata (Figure 8(a)).Sections of the adrenal glands of NaF-treated rats showed a strong iNOS immunoreactivity in zona glomerulosa and zona fasciculata, with a significant increase in iNOS (%) stained area at P < 0.05 compared to the control group (Figures 8(b,f)).Sections of adrenal glands from rats treated with D. aegyptium showed negative iNOS reactivity in zona glomerulosa and zona fasciculata cells (Figures 8(c,  f)).Adrenal gland sections from NaF-treated rats given D. aegyptium (100 or 200 mg/kg) showed decreased iNOS reactivity in the zona glomerulosa and negative reactivity in the zona fasciculata as compared to the NaF-treated group (Figures 8(d,e,f)).

Discussion
The current study aimed to determine whether D. aegyptium extract, when combined with NaF, offers any protection.The plant was discovered to possess antibacterial, diuretic, antipyretic, anti-proliferative, and antioxidant properties [7].
Our previous study indicated through HPLC results that the ethanolic extract of this plant is extremely rich in flavonoids, phenolic acids, and phenolic derivatives.It was demonstrated that the primary chemicals include quercetin, catechin, gallic acid, naringenin, and chlorogenic acid, whereas the minor compound is caffeic acid [8].
According to the current biochemical findings, NaF-treated rats exhibited a significant decrease in aldosterone and corticosterone serum levels compared to the control group.Since the zona fasciculata oversees the production and secretion of glucocorticoids, adrenal cortex damage is a significant factor in the disruption of steroidogenesis.According to Elshennawy and Aboelwafa [20], this may occur because of cytochrome P450 enzyme disruption, further inhibiting cholesterol manufacturing.
A similar reduction in corticosterone levels was seen in the tramadol-treated group compared to the control group [21].Shashi and Tikka [22] presented similar findings, stating that significant decreases in aldosterone and corticosterone were observed after fluoride exposure in rats.These findings suggest that zona fasciculata cells were damaged by NaF intoxication, leading to compromised function and, ultimately, reduced synthesis and secretion of corticosterone hormone.
The levels of Na + were significantly increased, whereas the serum levels of both K + and Cl − were significantly decreased in the NaF group compared to the control group.These findings align with Rosol et al. [23], who found that zona glomerulosa-secreted aldosterone stimulates renal potassium excretion and enhances salt reabsorption.There is an electrolyte imbalance when there are disturbances in this area.
According to the current findings, administration of D. aegyptium extract with NaF significantly increased serum aldosterone and corticosterone secretion to levels that resumed normal levels.Antioxidants, a class of phenolic chemicals with the potential to enhance adrenal hormones, are abundant in the plant extract.These findings are supported by Shashi and Tikka [22], who found that curcumin prevented the adverse effects of NaF consumption by restoring normal aldosterone and mg/kg) showing average capsule (thick arrow), average zona glomerulosa (ZG), and zona fasciculata with average cells (ZF) and average intervening blood sinusoids (thin arrow).(h) Shows changes in cortical thickness following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/group.# P <0.05 compared to the control group.*P <0.05 compared to the NaF-treated group.corticosterone levels, leading to electrolyte balance.This may be because it has antiinflammatory and antioxidant effects.
Regarding histological findings, cortex and medulla alterations and a significant reduction in cortical thickness were found upon histological and histomorphometry assessment of adrenal tissues in the current investigation following exposure to NaF.Apoptotic cells were found in the zona fasciculata, while dispersed cells with vacuoles in their cytoplasm were found in the zona glomerulosa, which was separated by average-sized blood sinusoids.The zona reticularis included a few scattered apoptotic  cells, while the medulla contained a zone of hemorrhage, apoptotic cells, and cells with vacuolated cytoplasm.These results confirmed the present biochemical results.Similar findings agree with Abdel-Aziz [24], who found that nicotine treatment caused the zona fasciculata cells to form lipid droplets and display cytoplasmic vacuolation, which is a defect in the production and secretion of glucocorticoids.
Similar results were also observed by Shalaby et al. [25], who found that tramadol therapy led to severe histopathological alterations in the adrenal cortex, including disrupted architecture and decreased cell size accompanied by pyknotic nuclei.Adrenal medulla hemorrhage was also discovered.A possible cause is adrenal gland inflammation, which leads to vascular restriction and congestion.Such a result could be attributed to low corticosterone levels [26].
All histological alterations in the adrenal gland caused by fluoride were reversed after treatment with D. aegyptium extract, and gland functioning was restored.There is speculation that the phenolic compounds it contains are responsible for its antioxidant and anti-inflammatory effects.Comparable findings were reported by Shashi and Tikka [22], who stated that zona glomerulosa, zona fasciculata, and zona reticularis were present in the adrenal cortex and adrenal medulla of rats treated with 200 mg/kg b.w./day of curcumin for 20 days, just as they were in the control group.Such findings are in agreement with Bahaa El-Din [27], who found that resveratrol's potent antioxidant action can reverse histopathological changes, alleviating fluoride toxicity.
Like the protective effect of quercetin against fluoride toxicity in the brain, Nabavi et al. [28] found that quercetin's high antioxidant activity abrogates the fluoride-induced reactive oxygen species (ROS) in various organs.Prunella vulgaris (1.575 g of crude drug) reduced fluoride-induced oxidative damage in rat kidneys in a recent study by Li et al. [29].This finding demonstrated that natural antioxidants can reduce the harmful effects of hazardous elements on several bodily systems.
According to histochemical data, NaF treatment decreased glycogen, total protein, and DNA in zona glomerulosa and zona fasciculata cell nuclei.Such results agree with Hanafy [30], who reported that carbimazole treatment reveals a considerable depletion of general carbs in zona glomerulosa and zona fasciculata and is absent in zona reticularis.In rats with high levels of the oxidative stress biomarker malondialdehyde MDA, we previously demonstrated that fluoride ingestion decreased antioxidant biomarkers (SOD and GSH enzymatic activity) [8].These findings add significance to the growing body of evidence that identifies oxidative stress damage as a critical mechanism of fluoride-induced harm.When reactive oxygen species interact with cell membranes, they produce malondialdehyde, which is often regarded as the most reliable biomarker of this sort of lipid peroxidation.High quantities of H 2 O 2 produced in cells via regulated mechanisms may contribute to increased lipid peroxidation owing to fluoride toxicity.Extremely high levels of H 2 O 2 are toxic to cells because they lead to the oxidation of protein, lipid, and DNA [31].
As shown in the results, co-administration with D. aegyptium extract revealed a significant increase in glycogen, total protein, and DNA in the zona glomerulosa and zona fasciculata cells.The results found by Girsang et al. [32] were similar, and they concluded that chlorogenic acid may serve as a protective agent by reducing levels of ROS.Ferulic acid inhibits free radical production enzymes and enhances scavenger enzyme activity, according to Zduska et al. [33].To be effective as an antioxidant, ferulic acid must be able to react with free radicals to produce stable phenoxyl radicals.This substance could also donate hydrogen to the radicals, allowing them to grow their own atoms.This is especially crucial in preventing the lipid acids in cell membranes from being damaged by undesirable autoxidation processes.Thus, the oxidation of biological targets like proteins, lipids, and DNA is prevented.
Immunohistochemical results showed that NaF-treated sections stained with anti-iNOS antibodies significantly increased the stained iNOS mean area (%) compared to the control group.This finding is consistent with the findings of Luo et al. [34], who confirmed that NaF treatment induces inflammatory mediators like iNOS, producing reactive nitrogen species like NO.The increased production of free radicals induced by NaF may be to blame.Such results are consistent with those of Hamza et al. [35], who discovered that increased lipid peroxidation and free radical production are suggested to mediate the harmful effects of fluoride on soft tissues.
In the current study, iNOS immunoreactivity was significantly decreased by D. aegyptium extract.The extract's bioactive components may reduce NO production.The antioxidant and free radical-scavenging properties of D. aegyptium derivatives, particularly syringic and ferulic acids, may be to blame.Accordingly, Kim et al. [36] found four hydroxycinnamic acid derivatives, two free cinnamic acids, p-coumaric acid, and ferulic acid in maize bran (80%) ethanolic extract.The antioxidant response of syringic acid in asthmatic groups was demonstrated by the discovery of increased levels of SOD and GSH in the Syringic acid treatment group compared to the asthmatic control group.Interestingly, Syringic acid reduced levels of ROS, NO, and MDA in mice [37].These findings are also consistent with those of Ghasemi et al. [38], who discovered that ferulic acid and other derivatives of the Ferula plant can inhibit the activity of pro-inflammatory cytokines and inhibit cell death.Superoxide dismutase, glutathione peroxidase, catalase activity, and glutathione level were all shown to rise.At the same time, oxidative stress markers, including malondialdehyde, reactive oxygen species, and nitric oxide, were reduced when these plants and their constituents were present.
Our results confirmed that 200 mg/kg D. aegyptium extract decreased pathological changes better than 100 mg/kg in a doseresponse manner.Moreover, the study found that D. aegyptium extract normalizes adrenal hormones and decreases oxidative stress.Chlorogenic, syringic, and ferulic acids enhance D. aegyptium antioxidant properties [8,39].

Conclusion
The present data confirms our question research about the protective effect of D. aegyptium extract against fluoride-induced adrenal hormone profile stress.D. aegyptium treatment normalized blood aldosterone and corticosterone levels to nearly control values.It abolished NaF poisoning-induced histological changes, which could contribute to the utilization of this important herb as a secure protective and intervention for adrenal disorders.

Figure 2 .
Figure 2. Shows changes in nitric oxide concentrations following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/group.# P <0.05 compared to the control group.*P <0.05 compared to the NaF-treated group.

Figure 4 .
Figure 4. Light microscope photomicrographs showing histological alterations in the adrenal gland of different experimental groups (H&E stain, scale bar: 20 μm).(a) Section from a control rat showing average capsule (thick arrow), average zona glomerulosa (ZG), average zona fasciculata (ZF) with average intervening blood capillaries (thin arrow) (b) Section from a control rat showing zona fasciculata, zona reticularis (ZR) with average blood sinusoids (S), and medulla with average cells (white arrow) and average blood sinusoids (black arrow).(c) Section from a NaF-treated rat showing zona glomerulosa with scattered cells with cytoplasmic vacuoles (thick arrow), and zona fasciculata showing scattered apoptotic cells (thin arrow) with average intervening blood sinusoids (white arrow) (d) Section from another NaF-treated rat showing zona reticularis with scattered apoptotic cells (thin arrows), and medulla showing area of hemorrhage (arrow head), scattered cells with apoptosis (white arrow), and others with vacuolated cytoplasm (thick arrow) (e) Section from D. aegyptium-treated rat showing average capsule (thick arrow), average zona glomerulosa (ZG), and zona fasciculata with average cells (ZF) and average intervening blood sinusoids (thin arrow).(f) Section from of NaF-treated rat that received D. aegyptium (100 mg/kg) showing average zona glomerulosa (ZG), zona fasciculata with average cells (ZF) and average intervening blood sinusoids (black arrow).(g) Section from of NaF-treated rat that received D. aegyptium (200

Figure 5 .
Figure 5.Light microscope photomicrographs showing histochemical alterations in the adrenal gland tissues of different experimental groups (PAS stain, scale bar: 20 μm).(a) Section from control group showing dark, magenta-stained positive glycogen for PAS in zona glomerulosa cells (thick arrow), and in zona fasciculata cells (thin arrow).(b) Section from NaF-treated group showing weak reaction for PAS in zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow).(c) Section from D. aegyptium-treated group showing deep magenta-stained positive glycogen for PAS in zona glomerulosa cells (thick arrow), and in zona fasciculata cells (thin arrow).(d) Section from of NaF-treated rat that received D. aegyptium (100 mg/kg) showing increase in positivity for PAS in zona glomerulosa cells (thick arrow), and in zona fasciculata cells (thin arrow).(e) Section from of NaF-treated rat that received D. aegyptium (200 mg/kg) showing strong positivity for PAS in zona glomerulosa cells (thick arrow), and in zona fasciculata cells (thin arrow).(f) Shows changes in PAS-positive materials-stained area (%) and following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/group.# P <0.05 compared to the control group.*P <0.05 compared to the NaF-treated group.

Figure 6 .
Figure 6.Light microscope photomicrographs showing histochemical alterations in adrenal gland tissues of different experimental groups (Bromophenol blue stain, scale bar: 20 μm).(a) Section from control group showing average distribution in most of zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow) (b) Section from NaF-treated group showing weak blue granules in most of zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow) (c) Section from D. aegyptium-treated group showing intensely stained fine blue granules in most of zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow) (d) Section from of NaF-treated rat that received D. aegyptium (100 mg/kg) showing restoration of total protein in most of zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow) (e) Section from of NaF-treated rat that received D. aegyptium (200 mg/kg) showing strong staining in most of zona glomerulosa cells (thick arrow), and in most of zona fasciculata cells (thin arrow).(f) Shows changes total protein-stained area (%) and following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/group.# P <0.05 compared to the control group.*P <0.05 compared to the NaF-treated group.

Figure 7 .
Figure 7.Light microscope photomicrographs showing histochemical alterations in the adrenal gland tissues of different experimental groups (Feulgen stain, scale bar: 20 μm).(a) Section from control group showing strong positive staining for Feulgen in the majority of zona glomerulosa cell nuclei (thick arrow), and in most of zona fasciculata cell nuclei (thin arrow) (b) Section from NaF-treated group showing marked decrease in DNA in most of zona glomerulosa cell nuclei (thick arrow), and in most of zona fasciculata cell nuclei (thin arrow) (c) Section from D. aegyptium treated group showing strong Feulgen staining in the majority of in most of zona glomerulosa cell nuclei (thick arrow), and in most of zona fasciculata cell nuclei (thin arrow).(d) Section from of NaF-treated rat that received D. aegyptium (100 mg/kg) showing restoration of DNA with highly significant positivity for Feulgen throughout the zona glomerulosa cell nuclei (thick arrow), and moderate in most of zona fasciculata cell nuclei (thin arrow).(e) Section from of NaF-treated rat that received D. aegyptium (200 mg/kg) showing increase in DNA content with a high reactivity for Feulgen in most of zona glomerulosa cell nuclei (thick arrow), and in most of zona fasciculata cell nuclei (thin arrow).(f) Shows changes DNA stained area (%) and following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/ group.# P <0.05 compared to control group.*P <0.05 compared to NaF-treated group.

Figure 8 .
Figure 8.Light microscope photomicrographs showing immunohistochemical alterations in the adrenal gland tissues of different experimental groups (iNOS immunostaining, scale bar: 20 μm).(a) Section from control group showing negative cytoplasmic reactivity for iNOS in zona glomerulosa (thick arrow) and in zona fasciculata (thin arrow).(b) Section from NaF-treated group showing strong iNOS positive immunoreaction in zona glomerulosa (thick arrow) and in zona fasciculata (thin arrow).(c) Section from D. aegyptium-treated group showing no iNOS expression in zona glomerulosa (thick arrow) and in zona fasciculata (thin arrow).(d) Section from of NaF-treated rat that received D. aegyptium (100 mg/kg) showing weak iNOS reactivity in zona glomerulosa (thick arrow), and no reactivity in zona fasciculata (thin arrows).(e) Section from of NaF-treated rat that received D. aegyptium (200 mg/kg) showing decrease in cytoplasmic reactivity for iNOS in zona glomerulosa (thick arrow) and negative reaction in zona fasciculata (thin arrow).(f) Shows changes in iNOS stained area (%) following NaF (5 mg/kg) and D. aegyptium (100 and 200 mg/kg) treatment for 28 days.The results are expressed as mean ± SEM, n = 7 rats/ group.# P <0.05 compared to the control group.*P <0.05 compared to the NaF-treated group.