Rhubarb water extract as a promising gastroprotective agent in Tamoxifen induced parietal cell damage in female rats: a histological study

Abstract

Tamoxifen (TAM) is one of the most used drugs in the prevention and treatment of breast cancer. A set of common side effects was recorded associating its prolonged clinical use that ranges 3–10 years. This study aimed to investigate TAM-induced parietal cells (PCs) injury in rats and the possible protective effect of rhubarb (Rh) water extract (WE). Twenty-four adult female rats were distributed as: control group, TAM-group (3 mg/kg/day TAM for 4-weeks) and TAM+Rh-group (combined 3 mg/kg/day TAM and 20 mg/kg Rh-WE for 4-weeks). Blood sample before euthanizing rats was tested for vitamin-B12. PCs in stomach fundus were examined using histological and transmission electron microscopic studies, besides immunohistochemistry for Caspase-3, proliferating cell nuclear antigen (PCNA) and hydrogen potassium (H⁺/K⁺)-ATPase. Gastric homogenates were inspected for malondialdehyde (MDA) by ELISA. TAM intake induced structural and ultrastructural alteration in rat PCs including ballooning degeneration, apoptosis, decreased canaliculi, increased tubulovesicular system and irregular-shaped mitochondria. A significant increase of Caspase-3 immunostaining and MDA expression in gastric tissue was associated with a significant decrease of PCNA and H⁺/K⁺-ATPase-immunostaining and in serum vitamin-B12 as compared to the control group. Combined oral intake of TAM and Rh-WE revealed a significant reversal of the previous findings. Conclusion: Prolonged use of oral TAM substantially affected the structure and function of gastric PCs which can be ameliorated by Rh-WE.

Introduction

Tamoxifen (TAM) has been widely used for many decades as the adjuvant treatment for patients with breast cancers (BC) [1]. TAM, a selective estrogen receptor (ER) modulator, acts as an ER antagonist in breast tissue and decreases BC mortality in female with ER-positive breast cancer. It’s also effective in primary prevention of BC in high-risk female [2]. It is associated with several physiological benefits such as the maintenance of bone density in postmenopausal female and a decrease in cardiovascular disease also with its tumoristatic action [3].

TAM commonly causes a range of side effects such as hot flashes, night sweats, gynecologic symptoms (vaginal dryness or vaginal discharge), depression, forgetfulness, sleep alterations, weight gain, and diminished sexual functioning. Occasionally, it caused more serious adverse events such endometrial hyperplasia or endometrial cancer and venous thromboembolic disease. Some side effects of TAM on the gastrointestinal system were recorded including hepatic injuries such as hepatocarcinoma, hepatic steatosis, and hepatotoxicity [4].

The gastric parietal cells (PCs) are considered one of the main controllers of the gastrointestinal tract (GIT) functions through their secretion of HCl, the main activator for digestion of food, absorption of iron, calcium, and vitamin-B12 (Vit-B12), in addition to its role in bacterial growth control [5]. Also, PCs secrete multiple growth factors like transforming growth factor (TGF)-α and epidermal growth factor that mediate the GIT mucosal cell proliferation [6]. TAM intraperitoneal injection for 3 to 7 consecutive days in mice induced damage of PCs and promoted metaplasia in chief cells of the stomach [7]. The mechanism by which TAM induces PCs atrophy is uncertain. However, Manning et al. [8] proved that TAM acts as a PCs protonophore, which can lead to back wash of acid into secreting PCs, causing their damage.

Rhubarb (Rh) is an herbaceous perennial plant with a long, fleshy stalk, commonly used for cooking and medicine. Dried Rh rhizomes were traditionally used in Chinese medicine as a natural remedy for gastrointestinal complications including diarrhea, constipation, and inflammation [9]. Two main active constituents (ethanol-soluble and water soluble) have been classified in Rh. The aqueous fraction of Rh was investigated for its potential to reduce the intestinal damage induced by the anti-metabolite chemotherapeutic drugs in rats. It was hypothesized that Rh-water extract (WE) would decrease the severity of intestinal mucositis by selectively blocking water influx into the cell, induced by a decrease in external osmotic pressure. As water efflux was unaltered by the presence of extracellular Rh-WE, the directional flow of water across the epithelial barrier, in the presence of extracellular RE, indicated that RE may alleviate water loss across the epithelial barrier and promote intestinal health in chemotherapy-induced intestinal mucositis [10].

We aimed in the present work to investigate the potential effect of Rh-WE on TAM induced-PC injury in adult female albino rat using biochemical, histological, and immunohistochemical studies.

Materials and Methods

Material

Tamoxifen (Nolvadex) was purchased in the form of tablets (Cat. No. HY-13757A; AstraZeneca), each contained 10 mg TAM. Each tablet was dissolved in 10 ml distilled water (DW) and was given in a dose of 3 mg/kg/day orally by intra-gastric tube [11].

The stem of Rh was purchased from Harraz for Food Industry & Natural Products Company, Egypt and was authenticated by the Pharmacognosy Department, Faculty of Pharmacy, Egyptian Russian University. The Rh aqueous extract was prepared and given at a dose of 20 mg/kg/day by oral gavage tube [12].

Animals

Twenty-four adult female albino rats weighing 200–250 g were included in this study. The animals were bred in the Animal House of Faculty of Medicine, Cairo University. They were housed in hygienic stain-steel cages, kept in clean well-ventilated rooms and allowed free access to standard diet and water. The rats were kept for 48 hours in these conditions before the start of the experiment to adapt their new environment. All experimental procedures were applied according to guidelines of Institutional Animal Care and Use Committee of Cairo University with (approval number: CU III F 2 21).

Experimental design

Rhubarb water extract preparation

Rh stems (2.5 kg) were sectioned (1 cm) and boiled with absolute ethanol to remove alcohol-soluble components. Once cooled, the liquid was discarded, and the residues were further boiled with water. The Rh-WE components were retained for dehydration to obtain a concentrated powder [12].

Groups

The experimental animals were equally distributed into three groups (n=8) as follow:

Control group: rats were distributed equally into 2 subgroups:

- Subgroup Ia: 4 rats were left without any intervention.

- Subgroup Ib: 4 rats were given 0.5 ml of DW daily orally by oral gavage tube for 4 weeks.

TAM-group: each rat received 3 mg/kg/day TAM orally daily by oral gavage tube for 4 weeks [11].

TAM+Rh-group: each rat received a combined daily oral dose of 3 mg/kg TAM and 20 mg/kg Rh-WE [12] using oral gavage tube for 4 weeks.

Sample collection

Twenty-four hours before the end of the experiment (4 weeks), the rats were subjected to fasting, to ensure an empty stomach before scarification. Blood samples were taken for biochemical study just before scarification that was induced using anesthesia overdose by intraperitoneal injection of ketamine hydrochloride/xylazine (75 mg/kg and 10 mg/kg, respectively) [13]. After dissection of stomach, it was cut along the greater curvature then gastric tissue specimens were obtained from corpus region and rinsed gently with phosphate buffer saline (PBS) to remove any debris. The stomach specimens were pinned flat on solid surface by needles to extend the rugae then they were washed gently by DW [14].

The specimens were taken from corpus region of each rat were divided into three sets; the first was fixed in neutral buffered formalin (pH of 7) for 24 hours, dehydrated in graded alcohol concentration (70%, 95%, and 100%), cleared in xylene, embedded in paraffin, processed as paraffin blocks then cut at 4 μm thickness and subjected for the histological and immunohistochemical stains. The second set was fixed in glutaraldehyde and processed as resin blocks for transmission electron microscopy (TEM) evaluation. The third was preserved as homogenate at –80ºC for biochemical investigation.

Histological studies

Hematoxylin and eosin

The deparaffinized sections were subjected to hematoxylin and eosin (H&E); to assess the structural changes in the fundic gland of the stomach.

Immunohistochemistry

The deparaffinized sections were immunohistochemically stained in Histology Department, Kasr Al-Ainy Faculty of Medicine-Cairo University, for caspase-3 (Cas-3) as a marker of apoptosis [15], proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation [16], and for H⁺/K⁺-ATPase as a marker for the activity of gastric PCs [17]. The sections were put on positive-charged slides, deparaffinized in xylene and rehydrated in descending graded alcohol. Retrieval was done by boiling in 10 ml citrate buffer (pH 6), then washing in PBS. Quenching of endogenous peroxidase by hydrogen peroxide was followed by washing in PBS the incubation of the primary antibodies (Ab) in humidity champers. The anti-PCNA Ab (Cat. No. DPABH-0371; Creative Diagnostics), anti-Cas-3 Ab (Cat. No. E-AB-63510; Elabscience), and anti-H⁺/K⁺-ATPase Ab were applied at a dilution of 1:100. After washing with PBS, sections were incubated with the secondary Ab Mouse/Rabbit Poly Detector Plus DAB HRP Brown Detection System (Cat. No. BSB 0261; Bio SB Inc.) for 30 min then diaminobenzidine was used for color development. The negative control was performed by all steps omitting adding the primary Ab.

Histological scoring system

The morphological variables of the gastric affection were graded according to our GAP histological score. We evaluated “G” for glandular epithelial damage in the corpus (regarding H&E results), “A” for cellular apoptosis (regarding Cas-3 immunostaining) and “P” for PCs loss (regarding H⁺/K⁺ ATPase immunostaining) to directly assess and score the degree of gastric damage and PCs loss in the gastric mucosa of the corpus (Table 1). The total score for the was calculated by summing the scores from each parameter then the mean±SD was calculated for total scores.

Table 1.

Histological scoring system

Parameter	Grade 0 (none)	Grade 1 (mild)	Grade 2 (moderate)	Grade 3 (severe)
Gland epithelial damage	No damage	Occasional loss	Up to 50% loss	>50% loss
Cellular apoptosis (Cas-3 immunostaining)	No apoptotic cells	<5% of total cells	5%–15% of total cells	>15% of total cells
PCs loss (H+/K+-ATPase immunostaining)	No loss (strong immunostaining)	Occasional loss (<33% reduction in stained cells)	33%–50% reduction in PCs (scattered, weakly stained cells)	>50% reduction in PCs (minimal to no staining)

PCs, parietal cells; Cas-3, caspase-3.

TEM

The tissue specimens were post-fixed in osmium tetroxide (1%) for 4h at 4°C, dehydrated in ascending graded ethanol then in propylene oxide and embedded in resin. The semithin sections were obtained and examined by the light microscopy to confirm the precise region that illustrated PCs. Ultrathin (60 nm) sections were obtained and stained with uranyl acetate/lead citrate and examined at different magnifications by TEM JEOL (JEM-1400 TEM; JEOL) in Electron Microscopy Unit, Faculty of Agriculture, Cairo University, Egypt.

Biochemical investigations

- Malondialdehyde (MDA) in tissue samples obtained from the stomach of each rat, as a marker for oxidative stress [18].

- Vit-B12 in blood from samples taken from the tail vein of each rat, as a marker for PCs function [19].

Morphometric measures

The data were obtained using “Leica Qwin-C 500” image analyser computer system (Leica Imaging System Ltd.) in Histology Department, Kasr Al-Ainy, Cairo University. Images were captured live onto the screen from sections using a digital video camera (Panasonic colour CCTV camera; Matsushita Communication Industrial Co. Ltd.) connected to Olympus light microscope (Olympus BX-40). Six non-overlapping fields from each slide/group were analysed to estimate:

- The area percent (%) of Cas-3 positive immune-stained cells at magnification 200×.

- The number of PCNA positive immune-stained cells at magnification 200×.

- The optical density of H⁺/K⁺-ATPase positive immuno-stained PCs at magnification 400×.

Statistical analysis

The measurements obtained were analyzed using Statistical Package for Social Science (SPSS) software version 16 (SPSS Inc.). Comparison between different groups was made by using one-way analysis of-variance (ANOVA) followed by post-hoc Tukey test. The results were expressed as mean±SD. The differences were considered statistically significant when probability P-value was <0.05 [20].

Results

Morphological alteration in the gastric fundus

Histological examination of the control subgroups was similar revealing normal architecture. The gastric mucosa illustrated long straight closely packed glands with numerous PCs mainly in the isthmus/neck region and in the upper part of the gland with acidophilic granular cytoplasm and vesicular nuclei. The chief cells were detected mainly in the lower part of the gland displaying basophilic cytoplasm. The TAM-group closely packed tubular glands with numerous PCs showing pyknotic nuclei and either deeply acidophilic or ballooned vacuolated cytoplasm. The obvious encroachment of many basophilic chief cells in middle and basal part of the gland was noted. The TAM+Rh-group displayed gastric mucosa with straight closely packed tubular glands harboring many PCs with acidophilic cytoplasm and rounded vesicular nuclei. However, a few PCs illustrated pyknotic shrunken nuclei and either darkly stained acidophilic or vacuolated cytoplasm (Fig. 1).

Fig. 1.

H&E-stained sections in the gastric mucosa of the experimental rats. (A, B) Control group: straight closely packed tubular gastric glands show isthmus/neck (Is/N) and base (b) regions occupying the whole thickness of the lamina propria with minimal connective tissue in-between. Numerous parietal cells (PCs) illustrate acidophilic cytoplasm and vesicular nucleus (arrows) in the (Is/N) region and in the upper part of the gland. The chief cells (arrowheads) display basophilic cytoplasm and are located mainly in the lower part of the gland. The underlying muscularis mucosa (mm) is noted. (C, D) Tamoxifen (TAM)-group: straight closely packed tubular glands perpendicular to the surface formed of (Is/N) and base (b) and occupy the whole thickness of the lamina propria. Numerus PCs show pyknotic nuclei (bifid arrows) and either vacuolated cytoplasm (wavy arrows) or deep acidophilic cytoplasm (curved arrows). Apparent increase in the basophilic chief cells (arrowheads) encroaching the lower half of the gland is noted. (E, F) TAM+rhubarb-group: straight closely packed tubular glands displaying (Is/N) and base (b) and many PCs with acidophilic cytoplasm and rounded vesicular nuclei (arrows). Few PCs illustrate pyknotic shrunken nuclei (bifid arrows). Magnifications: (A, C, and E) 100×; (B, D, and F) 200×.

Immunohistochemistry results

Few cells with positive Cas-3 immunostaining were detected within the mucosa of the control rats. However, TAM-group displayed numerous PCs with strong cytoplasmic Cas-3 immunostaining with a significant increase in the mean area % of Cas-3 as compared to the control rats. The gastric mucosa of the TAM+Rh-group showed some cells displaying mild/moderate Cas-3 immunostaining with a significant decrease of the mean area % of Cas-3 as compared to TAM-group (Fig. 2).

Fig. 2.

Immunohistochemistry of caspase-3 (Cas-3) in the gastric mucosa of the corpus region. (A) Negative control: negative immunostaining. (B) Control: few cells with mild cytoplasmic Cas-3 immunostaining scattered in the glands. (C) Tamoxifen (TAM)-group: numerous cells with strong cytoplasmic Cas-3 immunostaining throughout the glands. (D) TAM+rhubarb (Rh)-group: many cells with mild-to-moderate cytoplasmic Cas-3 immunostaining. Magnifications: 200×; Inset, 1,000×. (E) Histogram representing quantification of the mean area % of Cas-3 immunostaining. *Significant (P<0.05) compared to the control group. ^#Significant compared to TAM-group.

The control corpus tissue demonstrated many PCNA immune-positive cells, mainly in the isthmus/neck region and few scattered in the upper part of the gland region. Meanwhile, TAM-group illustrated many PCNA immuno-positive cells in the isthmus/neck region. The mean number of PCNA immuno-positive cells in the gastric gland in TAM-group was substantially decreased as compared to the control group. The upper part of the mucosa of the TAM+Rh-group showed numerous strongly stained PCNA immuno-positive cells in the isthmus/neck region and scattered in the upper part of the gland region. The mean number of PCNA immuno-positive cells was significantly increased in the upper part of the mucosa as compared to the control and TAM-group (Fig. 3).

Fig. 3.

Immunohistochemistry of proliferating cell nuclear antigen (PCNA) in the gastric mucosa of the corpus region. (A) Control: many PCNA immune-positive cells with strong nuclear immunostaining (arrows), mainly in the isthmus/neck region and few scattered in the upper part of the gland. (B) Tamoxifen (TAM)-group: many PCNA immune-positive cells with strong nuclear immunostaining (arrow) were noted in the isthmus/neck region. (C) TAM+rhubarb (Rh)-group: numerous PCNA immune-positive cells with strong nuclear immunostaining (arrows) throughout the gland. Magnifications: 200×; Inset, 1,000×. (D) Histogram representing quantification of the mean number of PCNA immune-positive cells. *Significant (P<0.05) compared to the control group. ^#Significant compared to TAM-group.

The gastric mucosa of the control group harbored numerous active PCs with moderate/strong H⁺/K⁺-ATPase immunostaining scattered throughout the gland, indicating various stages of activity. The TAM-group showed some active PCs with mild H⁺/K⁺-ATPase immunostaining. The mean optical density of H⁺/K⁺-ATPase immuno-positive cells in TAM-group exhibited a significant decrease as compared to the control group. The gastric mucosa of the TAM+Rh-group showed many active PCs with moderate immunostaining displaying a significant increase of the mean optical density of H⁺/K⁺-ATPase immuno-positive cells as compared to TAM-group (Fig. 4).

Fig. 4.

Immunohistochemistry of H⁺/K⁺-ATPase in the gastric mucosa of the corpus region. (A) Control: numerous parietal cells (PCs) with strong H⁺/K⁺-ATPase immunostaining diffusely in the cytoplasm (arrows) and in the apical cell membrane (arrowheads). (B) Tamoxifen (TAM)-group: few PCs scattered throughout the gland with mild cytoplasmic (arrow) and membranous (arrowhead) H⁺/K⁺-ATPase immunostaining. (C) TAM+ rhubarb (Rh)-group: many PCs with moderate cytoplasmic (arrows) and membranous (arrowhead) H⁺/K⁺-ATPase immunostaining. Magnifications: 200×; Inset, 1,000×. (D) Histogram representing quantification of the optical density of H⁺/K⁺ATPase immuno-positive cells. *Significant (P<0.05) compared to the control group. ^#Significant compared to TAM-group.

Histological scoring by glandular/apoptosis/parietal cell loss (GAP) system

A significant increase of the histological scoring in TAM-group (7.50±0.50) was detected as compared to the control group (1.87±0.90), indicating elevated gastric mucosal damage. Meanwhile, TAM+Rh-group scoring (3.87±0.60) revealed a significant decrease as compared to TAM-group scoring.

Ultrastructure changes of gastric PCs

Ultrastructure examination of the control PCs showed a pear-shape with rounded euchromatic nucleus, numerous microvilli within numerous intracellular canaliculi, abundant uniform mitochondria and few tubulovesicular system. Examination of TAM-group showed PCs with heterochromatic nucleus, numerous tubulovesicular structures, few intracellular canaliculi, mitochondria illustrated bizarre shapes with different sizes and crystolysis with apparent reduction of the mitochondrial numbers. TAM+Rh-group illustrated PCs with rounded euchromatic nucleus, numerous intracellular canaliculi with microvilli and many oval mitochondria (Fig. 5).

Fig. 5.

Ultrastructure images of parietal cells (PCs) from the rat corpus region. (A) Control: a pear-shaped PC with central rounded euchromatic nucleus (N), numerous microvilli (arrow) projecting into the lumina of numerous intracellular canaliculi (c). The cytoplasm contains abundant mitochondria (mi) and a few tubulovesicular (T) systems. (B) Tamoxifen (TAM)-group: PC with heterochromatic nucleus (N). The cytoplasm shows numerous tubulovesicular (T) structures, bizarre-shaped mitochondria (mi) illustrating different sizes, distorted cristae (crystolysis) (arrowhead) and areas of cytoplasmic rarefaction (asterisk). (C) TAM+rhubarb (Rh)-group: PC with rounded euchromatic nucleus (N), numerous intracellular canaliculi (c) with numerous microvilli (arrow). The cytoplasm shows many oval mitochondria (mi). Magnification: 8,000×.

Biochemical results

The mean value of gastric tissue MDA (Table 2) was significantly elevated in TAM-group as compared to the control group. Administration of Rh-WE significantly lowered the mean value of MDA in the gastric tissue as compared to TAM-group.

Table 2.

The tissue gastric malondialdehyde and serum Vit-B12 in the experimental groups

	Control	TAM-group	TAM+Rh-group
Tissue MDA (nmol/g)	48.0±3.87	113.4±3.78*	67.9±6.36*, **
Serum Vit-B12 (pmol/L)	811.3±107.86	288.3±75.21*	641.7±66.46*, **

Values are presented as mean±SD. Vit-B12, vitamin-B12; MDA, malondialdehyde; TAM, Tamoxifen; Rh, rhubarb. *Significant (P<0.05) compared to the control group; **Significant compared to TAM-group.

The mean value of the serum Vit-B12 (Table 2) was significantly decreased in TAM-group as compared to the control group. However, TAM+Rh-group displayed a significant increase of the mean value of the serum Vit-B12 as compared to TAM-group.

Discussion

TAM is a cornerstone in the adjuvant therapy of ER-positive BC by reducing the recurrence and increasing the survival of BC patients [21]. It is usually prescribed orally for long use duration; from 3–10 years [22]. However, the recommended long use of TAM is a challenge due to a wide range of associated side effects [23]. This study aimed to investigate the TAM induced PCs’ injury in adult female albino rat and the potential protective effect of Rh-WE.

In our work, adult female rats were used to investigate the effect of TAM on the gastric PCs by a dose of 3 mg/kg/day for 4-weeks. It was demonstrated that the essential stomach morphology and digestive function in the rat are similar to human, particularly at the microscopic level [24]. For adjusting the dose of TAM in our study, the clinically used human dose (20 to 40 mg/day) [25] was converted into rat dose according to Nair and Jacob [26] using the following formula: “rat dose (mg/kg)=human dose (mg/kg)×0.162.” The average rat dose of TAM was 3 mg/day to be equivalent to human dose with an average weight.

The lipophilicity of TAM and its extensive binding to serum proteins prolonged TAM action in target tissues [27]. For novelty of our work, we used oral TAM for a long experimental period by the same method and route of administration that mimic human usage, and not in acute onset as previous studies [28, 29]. TAM was given to the rats for 4-weeks that were equivalent to about 3 years in human life [30] to simulate the chronic use of TAM.

Histological examination of the gastric glands in corpus tissue after daily oral intake of TAM induced cellular damage of the PCs including apoptotic nuclear changes, cytoplasmic ballooning and bizarre-shaped mitochondria with crystolysis. These findings were accompanied by a significant increase in Cas-3 immunostaining and MDA expression in the gastric tissue, indicating enhanced apoptosis and oxidative stress. TAM hydrophobic properties facilitate its rapid accumulation in the phospholipid bilayers of membranes. It stimulates apoptosis and oxidative stress by elevation of mitochondria nitric oxide synthase, suppression of mitochondrial respiration and decrease of cytochrome C that stimulates mitochondrial lipid peroxidation [31]. In a study by Keeley et al. [32], gastric toxicity resulting from PCs death was detected in mice after intraperitoneal administration of 100–200 mg/kg TAM for 3 days. Moreover, the effect of TAM on in-vitro isolated mitochondria from rat liver and bovine heart revealed an inhibitory effect of both ATP synthase and respiratory complex I and suppressed the mitochondrial permeability transition causing mitochondrial dysfunction [33]. Cytoplasmic entry of mitochondrial contents, specifically cytochrome C, promote downstream activation of caspase-cascade [34].

The MDA is a widely known marker for oxidative stress and activation of programmed cell death [35]. TAM was accused to inhibits fatty acid β-oxidation and synthesis of dicarboxylic acid leading to mitochondria damage which causes excessive release of ROS and lipid peroxidation of the fatty acids [36]. The raised tissue MDA levels is associated with increased generation of ROS which affects the antioxidant enzyme activities and spurs oxidative stress in cells, impairment of mitochondrial outer membrane permeability and triggering of apoptosis [31, 37]. ROS serve as a crucial link between caspase-dependent and caspase-independent apoptosis because they are implicated in both processes [38].

Under normal condition, the gastric mucosal cells are continuously undergoing apoptosis and rapidly replaced by newly proliferating cells present in isthmus/neck region for maintaining integrity of the gastric mucosa [28]. In our work, an obvious encroachment of many chief cells beyond the base of the gland was associating PCs damage in TAM-group. Interestingly, PCNA positive cells were restricted to the isthmus/neck region when examining the upper part of gastric mucosa for PCNA immune staining which is a polypeptide essential for DNA replication and repair. Damage of gastric PCs is a critical precursor of spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach, in which chief cells are reprogrammed to re-enter the cell cycle and transdifferentiate into metaplastic mucus secreting SPEM-cells [39]. Gastric metaplasia consistently appears in the deep part of fundic gland associating diffuse PCs damage as in autoimmune gastritis, Helicobacter pylori-induced atrophic gastritis and animal models of acute injury [40]. SPEM is a multifactorial process that involves several mechanisms including PCs atrophy and expression of several metaplasia-promoting immune response and release of metaplasia-promoting factors [41]. A single high dose (3 mg/20 g body weight) of TAM in mice agitated apoptosis of more than 90% of all PCs and induced chief cells’ metaplasia with increased proliferation of stem/progenitor cells within 3 days [42]. In addition, TAM boosted expression of the pathognomonic markers of SPEM such as CD44 variant isoform 9, neck cell marker trefoil factor 2 and secreted SPEM marker Clusterin 10 [43].

In this work, PCs in TAM-group showed heterochromatic nucleus and numerous tubulovesicular structures with significant decrease of the mean optical density of H⁺/K⁺-ATPase immuno-staining as compared to the control group indicating significant decrease of PCs activity. Changes in PCs structure coups with is acid secretion activity. Non-secreting PCs display distinctive invagination of the apical membrane deep into the cytoplasm, forming numerous tubulovesicles [44]. The H⁺/K⁺-ATPase is the major protein constituent of the secretory membrane system of the PCs. Acid secretion is controlled by neurohormonal pathway that stimulates translocation of the membrane-bound H⁺/K⁺-ATPase enzyme from the cytoplasmic tubulovesicular membranes to the intracellular canaliculi of the stimulated PCs [5]. High-dose TAM functions as PCs protonophore that causes back wash of acid into the secreting PCs with subsequent cell death. This is facilitated by its hydrophobic criteria [8].

Prolonged administration of TAM revealed a significant decrease in the mean value of serum Vit-B12 level in comparison to the control group. Vit-B12 absorption requires intrinsic factor (IF) released by PCs in the stomach to be transported to the distal ileum. Destruction of PCs leads to inability to absorb Vit-B12 [45]. Chemotherapeutic drugs and conditions affecting PCs as autoimmune gastritis or H-pylori infection cause loss of PCs with subsequent IF deficiency and long-term deficiency of Vit-B12 [46].

In the present work, Rh-WE from the stalk revealed ameliorative effect against TAM-induced PCs damage. Gastric mucosa of TAM-Rh-group illustrated many viable PCs with acidophilic cytoplasm and vesicular nuclei. The mean area % of Cas-3 immunostaining and MDA expression in the gastric tissue were statistically decreased as compared to TAM-group. Rheum palmatum L. is an ancient natural herb belonging to the Polygonaceae family and widely distributed in temperate climates. It has pleiotropic effects such as anti-inflammatory, antibacterial and anti-fibrotic roles due to high levels of polyphenols and antioxidants components [47]. The plant leaves are usually not recommended owing to high content of several organic acids as oxalic acid that results in kidney stone formation [48, 49]. However, its root and stalk are frequently used in food and in pharmaceuticals due to their myriad valuable biologically active molecules they have [50]. Pharmacologically, there are two main active constituents that have been classified in Rh stalks and widely affected by the extraction solvent, alcohol-soluble and water-soluble extracts [51, 52]. Rh alcoholic extract is enriched in lipophilic compounds, such as free anthraquinones (emodin, physcion, chrysophanol), dianthrones (sennosides), and stilbenes (rhapontigenin), which are often associated with laxative and cytotoxic properties [53]. In contrast, Rh aqueous extract contains more polar constituents, such as anthraquinone glycosides, phenolic acids (gallic acid), tannins, acylglucosides, condensed tannins, and related flavonoids [54]. Although emodin and rhein are traditionally classified as poorly water-soluble, several studies have confirmed their presence in aqueous Rh extracts in measurable concentrations. Zhu et al. [55] and Zhumashova et al. [56] reported the identification of Rh bioactive materials in water-based preparations using HPLC and LC-MS/MS techniques. Study focusing on phytochemical profiling of Rh-WE are using LC-MS/MS to quantify anthraquinones and correlate their concentration with the observed gastric protective effects is recommended in the future work.

Water Rh extract showed variable activities including gastroprotective [40], neuroprotective [57], anticancer, antimicrobial, hepatoprotective and anti-inflammatory [58]. Emodin, the active ingredient of Rh, substantially suppressed the inflammatory, oxidative stress and apoptotic factors by alleviating the MDA, superoxide dismutase, interleukin (IL)-6, tumor necrosis factor (TNF)-α and Cas3 activity in diabetic nephropathy rat model [59]. The antioxidant/anti-inflammatory effect of rhein was documented in the transgenic zebrafish line inflammation model through their ability to decrease proinflammatory cytokines (such as IL-6, IL-1β, and TNF-α) and oxidative stress which in turn, reduces cell death [60]. Moreover, Rh components ameliorated inflammation and apoptosis in pancreatic acinar cell by downregulating phosphorylated-AKT (protein kinase B) and nuclear factor-kappa beta (NF-kB), in pancreatic tissue, both in-vivo and ex-vivo [61].

The upper part of the gastric mucosa of the control and TAM+Rh-group displayed a significant increase of the mean number of proliferating PCNA-positive cells in isthmus/neck region and the upper part of the gland region as compared to TAM-group; suggesting enhanced proliferative activity to renew the mucosa by the gastric stem cells. In our ultrastructural study, the morphology and number of the mitochondria in the PCs with Rh-WE administration was obviously improved. PCNA is a key protein involved in DNA replication and cell proliferation. Previous studies have illustrated that Rh-WE can affect PCNA expression with potentially increasing diethylnitrosamine-induced PCNA expression in liver tissues [62]. Rh extracts can influence various proteins involved in cell renewal and signaling pathways, including TGF-β1 and Wnt/β-catenin signaling pathway [63]. Moreover, they have displayed potential in perpetuation of gastric mucosa homeostasis and regulating the cell cycle and division. Previous works documented the beneficial effect of Rh extract on the gut microbiome and mucus production, impacting the overall health of gut permeability and integrity and the gastric environment [64]. Rh ingredients promoted regenerative capacity of pancreas and maintained mitochondrial function with acute pancreatitis of rat. They enhanced expression of regeneration parameters as TGF-β1and ki-67 and modulated the activity of PI3K/AKT/mTOR signaling pathway that maintains the differentiating state of cells [61, 65].

The gastric mucosa of the TAM+Rh-group illustrated many active PCs with euchromatic nuclei, numerous intracellular canaliculi. These findings were associated with a significant increase of the mean optical density of H⁺/K⁺-ATPase immuno-staining and the mean values of serum Vit-B12 in TAM+Rh-group as compared to TAM-group, indicating regained PCs activity. Previous studies revealed the protective effects of the GIT mucosa. In rat model with obstructive jaundice, Rh alleviated the damage of bile duct and colonic mucosa and hepatocytes. It ameliorated the cell–cell junction inclination and mitochondrial function, decreased inflammation, necrosis and oedema [66]. The key active ingredient of Rh, rhein, diminished the gastric mucosal injury in chronic atrophic gastritis mice models. It exerted anti-inflammatory and anti-oxidative stress roles in gastric mucosa by inhibiting TNF-α, IL-1β, and IL-6 in mitogen-activated protein kinase (MAPK) pathway and erythroid 2 (NFE2)-like bZIP transcription factor 2 (Nrf2) [67]. Furthermore, both MAPK and Nrf2 pathways stimulation was documented in inducing gastric cell damage [68].

In conclusion, our study indicated that prolonged TAM administration significantly compromised the structure and function of the PCs in the gastric mucosa of rats, as evidenced by histological and ultrastructural alterations, increased apoptosis, and reduced levels of Vit-B12. However, concurrent administration of Rh-WE demonstrated a protective effect and effectively reversed the detrimental changes induced by TAM. This suggests that Rh-WE may serve as a beneficial adjuvant therapy in mitigating the gastric side effects associated with long-term TAM use. Further investigations are warranted to elucidate clinical implications of these findings.

Limitations and future direction

While our work illustrated comprehensive morphological analysis with structural correlation, we did not biochemical inflammation markers or signaling pathways. Further future studies should investigate these parameters to provide a more comprehensive understanding of the gastroprotective mechanism of Rh extract.