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Original Article

Anat Cell Biol 2024; 57(1): 105-118

Published online March 31, 2024

https://doi.org/10.5115/acb.23.217

Copyright © Korean Association of ANATOMISTS.

Impact of peripheral blood mononuclear cells preconditioned by activated platelet supernatant in managing gastric mucosal damage induced by zinc oxide nanoparticles in rats

Darwish Badran1 , Ayman El-Baz El-Agroudy2 , Amira Adly Kassab2,3 , Khaled Saad El-Bayoumi2,4 , Zienab Helmy Eldken2,5 , Noha Ramadan Mohammed Elswaidy3

1Department of Anatomy and Histology, Faculty of Medicine, the University of Jordan and Ibn Sina University for Medical Sciences, Amman, 2Department of Basic Medical Sciences, Faculty of Medicine, Ibn Sina University for Medical Sciences, Amman, Jordan, 3Department of Histology and Cell Biology, Faculty of Medicine, Tanta University, Tanta, 4Department of Human Anatomy and Embryology, Faculty of Medicine, Mansoura University, Mansoura, 5Department of Physiology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Correspondence to:Amira Adly Kassab
Department of Basic Medical Sciences, Faculty of Medicine, Ibn Sina University for Medical Sciences, Amman 16197, Jordan
E-mail: amirakassab1980@gmail.com

Received: August 25, 2023; Revised: October 16, 2023; Accepted: October 31, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The world has witnessed tremendous advancements in nano-base applications. Zinc oxide nanoparticles (ZON) are widely used in food industry and medicine. Although their application is of important value, they may cause toxicity to body tissues. Peripheral blood mononuclear cells (PBMCs) proved its efficacy in tissue regeneration especially when it is preconditioned by activated platelet supernatant (APS). The aim of this study is to evaluate the effect of ZON on the gastric mucosa and the therapeutic role of the PBMCs preconditioned by APS in rats. Ten rats were donors and fifty rats were recipients. The recipients were divided into; control group, ZON group (10 mg/kg/day orally for five days) and preconditioned PBMCs group (1×107 once intravenously 24 hours after ZON). Gastric specimens were processed for histological, immunohistochemical, biochemical and quantitative real-time polymerase chain reaction studies. ZON group showed marked structural changes in the gastric mucosa. There was desquamation or deep ulceration of the epithelium. Cytoplasmic vacuoles and pyknotic nuclei were in glandular cells. Reduced proliferating cell nuclear antigen and increased tumor necrosis factor-α were in epithelial cells. There were significant elevation in malondialdahyde and reduction in glutathione, superoxide dismutase, and catalase. Enhancement in mRNA expression of nuclear factor kappa-B and cyclooxygenase-2 was detected. The preconditioned PBMCs group showed significant improvement of all parameters. So, ZON had cytotoxic effects on the gastric mucosa and the preconditioned PBMCs had a therapeutic effect on gastric mucosal damage after ZON.

Keywords: Leukocytes, mononuclear, Activated platelet supernatant, Zinc oxide nanoparticles, Gastric mucosa

Zinc oxide nanoparticles (ZON) are considered a new substitute for zinc oxide materials in industrial field, food processing, biomedicine and clinical therapy [1]. ZON have wide application in environmental control due to their antimicrobial activity [2, 3]. In medicine, ZON are commonly involved in the diagnosis, treatment and target drug delivery for cancer [4]. ZON are commonly combined with many chemotherapeutic drugs to achieve better antitumor effect [5]. ZON are synthesized by a variety of chemical, biological and physical processes from zinc oxide [6]. Due to the increased usage of ZON in food packaging or preservation, the most important route of ZON exposure in human is the oral ingestion. Also, oral exposure may occur through usage of purified water, cosmetics and toothpaste [7].

Despite their application values, they may cause toxicity to many body tissues or even induce threats to human. Cardiotoxicity, hepatotoxicity, brain damage, reproductive dysfunction and lung injury due to ZON were reported in many studies [8-13]. ZON can induce reactive oxygen species (ROS) generation, genotoxicity and apoptosis [14, 15]. The pathological mechanism of ZON toxicity may be through an inflammatory pathway which is dangerous with chronic ZON environmental exposure [16]. Other mechanisms of ZON cytotoxicity may be by particles dissolution, autophagosome accumulation and autophagic cell death [17, 18].

Feng et al. [19] in an in vitro study showed a higher ZON solubility in a simulated human gastric fluid (pH=1.5) than in rat gastric fluid (pH=3.2). ZON are highly soluble in acidic media (2–4 pH ranges). The low pH of the gastric fluid dissolves ZON and zinc ions react with carbonate or phosphate ions forming insoluble molecules. Moreover, following its dissolution in the gastric juice the particles appear in the intestinal fluid [20]. Due to the nanometer scale of ZON, they can quickly penetrate cells and can be internalized as free nanoparticles or zinc ions in the lysosomes of mammalian cells [21]. Other data reported affection of the intestinal microflora by the orally ingested ZON [19].

These particles pass from the gastrointestinal lumen to the blood circulation to be distributed and embedded in the different body organs [22]. ZON has damaging effects on pancreas and stomach. It could induce edema, marked inflammation, mucous cell hyperplasia, vacuolar, and hyaline degeneration in the glandular stomach in addition to squamous cell hyperplasia in the non-glandular stomach. Moreover, it could induce oxidative DNA fragmentation in the gastric tissues which is initial step for apoptosis. So, ZON could impair cellular stress-defence mechanism resulting in gastric injury [23, 24].

Cell therapy either stem cell or non-stem cell based is a type of regenerative medicine that aims to enhance tissue repair or regeneration. It means treatment of some medical diseases by autologous or allogenic cellular substances. Cell therapy is involved in many therapeutic areas as cancer therapy and immunotherapy [25, 26]. Nowadays, the peripheral blood mononuclear cell (PBMC) therapy proved its effectiveness in tissue regeneration especially when they are stimulated and mobilized to peripheral blood by granulocyte colony stimulating factor (G-CSF) [27].

PBMCs are lymphocytes (T cells, B cells, natural killer cells), monocytes and dendritic cells; they are characterized by their single rounded nuclei. They can undergo proliferation and differentiation into many subsets [28]. Their safety was evaluated in many studies [29] and they proved their beneficial role in many clinical cases as bed sores, liver injury, ischemic limb and stroke [30-33]. They may play a useful role in postmenopausal osteoporosis [34]. Previous in vivo study reported their tissue regenerative ability in rats as they regulate about forty differential proteins that are involved in many signalling pathways and DNA replication [35]. Moreover, PBMC played a useful role in healing of some intestinal ulcers in experimental rat models [27].

Previous studies proved that preconditioning or priming of G-CSF mobilized PBMCs with activated platelet supernatant (APS) augments their potential in tissue regeneration. So, the APS priming may be a novel approach to enhance the efficacy of cell-based therapy [36].

From all the above-mentioned data, it is necessary to assess ZON interaction with the living tissues trying to discover key parameters involved in ZON-tissue interaction.

Aim of the study

This research aimed to study the biological effect of ZON on the gastric mucosal tissue exploring novel mechanisms involved in ZON-gastric tissue interaction. Moreover, the study evaluated the impact of the PBMCs preconditioned by APS on ZON induced gastric mucosal injury in rats.

Ethical approval

The experimental work followed guidelines of animal research approved by the Local Ethics Committee (Faculty of Medicine, Tanta University, Egypt) and it complied with the national institutes of health guide for the care and use of laboratory animals. The experiment was carried out at the animal house of the histology department (Faculty of Medicine of Tanta University, Egypt) (approval code: 36264PR210/5/23).

Chemicals

ZON (30 gm/bottle) were purchased from Nanotech Company for Photo Electronics, Cairo, Egypt. They were in the form of white powder dispersion. They were examined with the transmission electron microscope (at Tanta University, Egypt) for morphology and size. The examination revealed ZON with homogenous form and semi-circular shape. Their size ranged from 10 to 20 nm (Fig. 1).

Figure 1. Transmission electron microscopic analysis of ZON.

Granulocyte colony-stimulating factor G-CSF or Filgrastim (Neupogen) was in the form of a pre-filled syringe/solution (Amgen).

Research design

Sixty adult male albino rats (weighed 150–200 g and aged 12–16 weeks) were used in the study. Ten rats were donors and fifty rats were recipients. All animals were under the same environmental conditions in hygienic ventilated cages and had a similar balanced diet and water. For 7 days before the work, animals were acclimatized to their environment. Then, the recipients were divided into three groups:

1) Group I (control group): included three subgroups (n=ten rats/subgroup):

Subgroup IA: didn’t receive any medications and were sacrificed after 2 weeks.

Subgroup IB: received 0.5 ml phosphate buffered saline (PBS, a vehicle to suspend PBMC) once intravenously in the tail vein. They were sacrificed 2 weeks after PBS injection.

Subgroup IC: received 0.5 ml physiological saline solution (0.9% sodium chloride) orally once daily for five consecutive days. The physiological saline solution is used for preparing ZON suspension. They were sacrificed 24 hours after the last saline dose.

2) Group II (ZON group): ten rats received 10 mg/kg/day of ZON by oral gavage for five consecutive days. This dose was suspended in 0.5 ml physiological saline solution. Rats were sacrificed 24hours after the last ZON dose [37].

3) Group III (PBMC group): Ten rats were injected with PBMC (1×107) suspended in 0.5 ml PBS once intravenously via tail vein 24 hours after ZON administration as in group II and were sacrificed 2 weeks after PBMC injection [38].

Methods of obtaining peripheral blood mononuclear cells reconditioned by activated platelet supernatant

The procedures were done at Tissue Culture Unit of Histology Department, Tanta Faculty of Medicine (Egypt). The donor rats (10 rats) were divided into five rats for PBMC isolation and the other five rats for APS preparation.

PBMC isolation

The five donor rats were daily injected with G-CSF subcutaneously at a dose of 100 µg/kg for 3 consecutive days for mononuclear cells mobilization to the peripheral blood [36]. After alcoholic sterilization of rats’ thorax, a cardiac puncture was done to collect peripheral blood. The PBMC were isolated by density gradient centrifugation [39].

In brief, the diluted anticoagulant treated blood was layered over a separation medium with no mixing (Ficoll-Paque product) and was centrifuged for 30–50 minutes. As a result, there was formation of layers of different types of cells. Mononuclear cells and platelets were on top of the medium by their low density. Red blood corpuscles and granulocytes were at the bottom. By subsequent PBS wash, mononuclear cells were separated from platelets [40].

APS preparation

By differential centrifugation, platelet rich plasma (PRP) was prepared and activated by 10% calcium gluconate (0.1 ml/1 ml PRP) [41, 42]. After that; the platelet gel was formed from the activated PRP by incubation at 37°C for 24 hours. Then, it was centrifuged at 2,800×g for 15 minutes at 20°C, and the supernatant was obtained for PBMC priming [43, 44]. Finally, the isolated PBMC was co-cultured with the prepared APS in 5% CO2 incubator at 37°C for 6 hours [36].

Samples collection

Animals were anaesthetized by intraperitoneal injection of sodium pentobarbital at dose of 50 mg/kg [45]. Stomach was carefully dissected out, opened through the greater curvature and washed by saline. Specimens were taken from the glandular part next to the limiting ridge to be processed for histological, immunohistochemical and biochemical studies.

For light microscopy

The glandular stomach specimens were fixed in 10% neutral-buffered formalin, washed, dehydrated, cleared and finally embedded in paraffin. Then, serial sections (5 μm thickness) were stained with haematoxylin and eosin (H&E) to be examined by the light microscope [46].

For immunohistochemistry

Sections (5 μm thickness) were dewaxed, rehydrated, and washed with PBS. The sections were incubated in a humid chamber in PBS overnight at 4°C with the primary antibodies (mouse monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody, Ab-1, Clone PC10, CAT. # MS-106-R7, 1:200 dilution, Lab Vision Corporation, USA and rabbit polyclonal anti-tumor necrosis factor-α (TNF-α) antibody, 1/100 dilution, ab6671, Abcam). Thereafter, they were washed in PBS buffer and co-incubated with biotinylated secondary antibody (Dako North America, Inc.) for 1 hour at room temperature. Streptavidin peroxidase was added for 10 minutes and rinsed 3 times in PBS. The immunoreactivity was visualized using 3, 3’diaminobenzidine (DAB)-hydrogen peroxide (a chromogen). Finally, the sections were counterstained using Mayer’s haematoxylin. The negative control sections were prepared with no primary antibody [47]. Tonsils, lymph node, or small intestine were PCNA positive control. Tonsils was TNF-α positive control. For accuracy of the immuno-results, all slides were evaluated in triplicates. The epithelial cells were PCNA positive cells when they expressed brown nuclear staining. Moreover, the epithelial cells were TNF-α positive cells when they expressed brown cytoplasmic staining.

Estimation of the levels of the oxidative stress markers malondialdahyde, glutathione, catalase, and superoxide dismutase in gastric homogenate

Stomach has been excised and quickly washed with icy cold saline with concentration (0.9%). Part of glandular stomach has been taken for determination of oxidative stress markers. Gastric homogenates were prepared during which one gram of the stomach was homogenized in 5 ml of PBS. The homogenized sample was then centrifuged at 3,000 rpm in a bench centrifuge for 15 minutes. The supernatants from gastric homogenate were used for determination of tissues content of the lipid peroxidation marker, malondialdehyde (MDA), the antioxidant reduced glutathione (GSH) as well as the activity of the antioxidant enzyme catalase (CAT), and superoxide dismutase (SOD) by colorimetric method using commercially available kits (Bio-Diagnostics) according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

Assessment of the nuclear factor kappa-B (NF-κB) and cyclooxygenase-2 (COX-2) mRNA expression levels was done by extraction of the total RNA from the frozen glandular gastric tissue (ten specimens per group) using RNA Mini kit and the manufacturer protocol (Qiagen RNeasy). Nanodrop spectrophotometer was used for RNA quantification in each sample. Synthesis of cDNA was done from 1,000 ng total RNA for each sample by reverse transcription. NF-κB and COX-2 mRNA expression levels were measured and analysed by Stratagene, MX3000P QPCR System (Agilent Technologies) and MxPro QPCR Software (Agilent Technologies). The annealing temperatures were: 62°C for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 60°C for NF-ĸB and 58°C for COX-2. The sequenced primers were used according to Kim et al. [48] (Table 1). GAPDH was a housekeeping gene for comparing relative level of the target genes. NF-κB, COX-2, and GAPDH GenBank accession was #, M61909.1, NM011198.4, and GU214026.1 respectively.

Table 1 . Real-time polymerase chain reaction primer sequences for NF-κB and COX-2 mRNA expression

GeneForward primer (5’-3’)Reverse primer (5’-3’)
GAPDH5’-CATCTTCCAGGAGCGAGACC-3’5’-TCCACCACCCTGTTGCTGT-3’
NF-κB5’-CAATGGCTACACAGGACCA-3’5’-CACTGTCACCTGGAAGCAGA-3’
COX-25’-CACTACATCCTGACCCACTT-3’5’-ATGCTCCTGCTTGAGTATGT-3’

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NF-κB, nuclear factor kappa-B; COX-2, cyclooxygenase-2.



Morphometric study

All photos were taken by LEICA DM 750 Microscope with ICC50E Camera Module (Leica Microsystems AG) at Basic Medical Sciences Department, Faculty of Medicine, Ibn Sina University for Medical Sciences, Amman, Jordan. Morphometrical evaluation of the photos was done by an image analysis image J program (revamped by US software developer Wayne Rasband at the National Institutes of Health, Public Domain, BSD-2). Area percentage (area %) of PCNA and TNF-α positive reaction was measured (at a magnification of 400 in Ten non-overlapping fields of each DAB-stained slide).

Statistical analysis

Statistical package for social sciences statistical analysis version 11.5 (SPSS Inc.) software was utilized for analysis of the obtained data. The data were subjected to one-way analysis of variance (ANOVA) as well as Tukey’s procedure. The mean values as well as their standard deviation (mean±SD) for all parameters were calculated. P<0.05 or >0.05 were significant or nonsignificant respectively [49].

No animal death occurred during the experiment.

Histological results (H&E staining)

Group I (control): No observed structural differences were found in all subgroups. The mucosa of the fundus of the glandular stomach appeared with normal histological findings. Thick intact mucosa was seen which was formed of surface epithelium, lamina propria and muscularis mucosa. The lamina propria contained densely packed simple tubular glands, blood vessels, lymphoid cells and smooth muscle cells. These glands were long straight that extended through the full thickness of the lamina propria (Fig. 2A). The glands arose from the surface epithelium by a pit and were composed of isthmus, neck and base. The surface and the pits were lined with simple columnar cells (mucous secreting) with elongated nuclei and pale acidophilic cytoplasm. The acidophilic parietal cells with central rounded nucleus predominated in the superficial part of the glands but the darker chief cells were mainly found in the basal part (Fig. 2B, C).

Figure 2. Representative photomicrographs of H&E stained section of group I showing: (A) The fundic mucosa with smooth epithelial surface (arrows), lamina propria (*) and muscularis mucosa (M). Notice the simple tubular glands (G) and lymphoid cells (L) in the lamina propria. (B) The surface and pits lined by simple columnar cells with oval nuclei and pale acidophilic cytoplasm (arrows). The underlying lamina propria contains blood vessels and smooth muscle cells (*). Notice acidophilic parietal cells (arrow heads) predominating in the upper part of the glands. (C) The darker chief cells (arrow heads) predominating in the basal part of the glands. Notice the blood vessels, lymphoid cells and smooth muscle cells of the lamina propria (*). (A) Magnification ×100, (B, C) ×400.

Group II (ZON group): The fundic mucosa showed sloughing and desquamation of the surface epithelium or even deep ulceration extending to muscularis mucosa (Fig. 3A, B). The parietal cells revealed reduced acidophilia and vacuolation of the cytoplasm in addition to deeply stained pyknotic nuclei of other cells (Fig. 3C). Cells in the basal part of the glands were shrunken with deeply stained pyknotic nuclei in addition to dilated congested blood vessels (Fig. 3D).

Figure 3. Representative photomicrographs of H&E stained section of group II showing: (A) Sloughing and desquamation of the surface epithelium (arrows). (B) Deep ulceration of the epithelium extending to muscularis mucosa (arrow). Notice reduced cytoplasmic acidophilia of some parietal cells (arrow heads). (C) Reduced acidophilia, cytoplasmic vacuolation (arrows) and deeply stained pyknotic nuclei (arrow heads) of the parietal cells. (D) Shrunken cells in the basal part of the glands with deeply stained pyknotic nuclei (arrow heads). Notice markedly dilated congested blood vessels (*). (A) Magnification ×100, (B–D) ×400.

Group III (PBMC group): Improved histological appearance of the fundic mucosa was noticed in this group. The mucosa showed more or less the same fundus structure as that of the control one (Fig. 4A). The surface and the pits showed simple columnar epithelium. Closely packed glands with nearly intact lining cells were also seen. A few dilated blood vessels were observed in focal areas. However, a few vacuolated or shrunken glandular cells were found in focal areas (Fig. 4B, C).

Figure 4. Representative photomicrographs of H&E stained section of group III showing: (A) The fundic mucosa with smooth epithelial surface (arrows). Notice closely packed simple tubular glands (G) in the lamina propria. (B) The surface and pits lined by simple columnar mucous cells with oval nuclei (arrows). (C) Closely packed glands with nearly intact lining cells (G) and a few dilated blood vessels were observed in the lamina propria (*). Notice a few vacuolated or shrunken glandular cells (arrow heads). (A) Magnification ×100, (B, C) ×400.

Immunostaining results of the proliferating cell nuclear antigen

Immunostaining showed that PCNA was characteristically expressed in the epithelial cells lining the neck of the fundic glands giving brown nuclear reaction. In control group I, intense PCNA reaction was observed in many epithelial cells. In group II, reduced number of PCNA positive cells with very weak PCNA expression in most cells was noticed. Group III showed moderate PCNA expression in many cells (Fig. 5).

Figure 5. Representative photomicrographs of proliferating cell nuclear antigen (PCNA) immunostained sections; (A) Group I shows intense PCNA reaction in the nuclei of many epithelial cells in the neck of the fundic glands (arrows). (B) Group II shows reduced PCNA positive cells with very weak PCNA nuclear expression in most epithelial cells (arrows). (C) Group III shows moderate PCNA nuclear expression in many epithelial cells (arrows). (A–C) Magnification ×400.

Immunostaining results of tumour necrosis factor-α

The study revealed that TNF-α was expressed as brown colour of the cytoplasm of the epithelial cells of the fundic glands. Group I expressed a very weak TNF-α immunoreaction in a few epithelial cells. Group II had strong TNF-α expression in many epithelial cells. Group III showed moderate TNF-α expression in some epithelial cells (Fig. 6).

Figure 6. Representative photomicrographs of tumor necrosis factor-α (TNF-α) immunostained sections; (A) Group I shows weak TNF-α immunoreaction in the cytoplasm of a few epithelial cells of the fundic glands (arrows). (B) Group II shows strong TNF-α cytoplasmic immunoreaction in many epithelial cells (arrows). (C) Group III shows moderate TNF-α cytoplasmic immunoreaction in some epithelial cells (arrows). (A–C) Magnification ×400.

Morphometric & statistical results of PCNA and TNF-α expression

The mean area % of PCNA expression showed a statistically significant decrease in group II (29.63±2.67) compared to group I (38.25±1.53). Moreover, group III showed non-significant decrease (37.42±1.57) compared to group I (Fig. 7).

Figure 7. Morphometrical and statistical analysis of proliferating cell nuclear antigen (PCNA) mean area %.

The statistical analysis revealed a significant increase in the mean area % of TNF-α expression in group II (47.92±1.89) as compared to group I (33.75±1.01). Whereas, group III showed non-significant increase in TNF-α mean area % (34.20±1.10) compared to group I (Fig. 8).

Figure 8. Morphometrical and statistical analysis of tumor necrosis factor-α (TNF-α) mean area %.

Oxidative stress assessment

There was a significant elevation in the concentration of MDA and a significant reduction in GSH, SOD, and CAT in group II compared to group I. On the other hand, group III showed a significant elevation in GSH, SOD, and CAT and significant decrease in MDA compared to group II. No significant difference was observed when group III was compared with group I regarding the mentioned parameters (Table 2).

Table 2 . Mean±SD for laboratory parameters

ParametersGroup I (control)Group II (ZON group)Group III (PBMC group)
GSH (U/mg protein)85.333±0.57266.97±3.2a)84.70±0.525b)
SOD (U/mg protein)24.65±0.78919.55±0.68a)23.833±0.28b)
CAT (U/mg protein)54.533±0.66238.117±0.845a)53.533±0.882b)
MDA (nmol/mg protein)0.533±0.1631.450±0.409a)0.867±0.314b)

ZON, zinc oxide nanoparticles; PBMC, peripheral blood mononuclear cell; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde.

a)Significant vs. group I, b)significant vs. group II.



Quantitative real-time PCR (RT-qPCR)

Our results revealed enhancement in the mRNA expression levels of NF-ĸB, and COX-2 genes (5.333±0.683 and 6.47±1.04, respectively) in group II as compared to group I (GAPDH) (1.033±0.528 and 1.217±0.649, respectively). In addition, our data demonstrated significant improvement in the mRNA expression of the above genes in group III as compared to group II. Also, mRNA expression of NF-ĸB and COX-2 genes in group III (1.6±0.54 and 1.9±0.434, respectively) displayed no significant difference compared to group I (GAPDH) (Fig. 9).

Figure 9. mRNA expression of nuclear factor kappa-B (NF-ĸB) and cyclooxygenase-2 (COX-2).

ZON proved their efficacy in many industrial and medical fields [1]. ZON are commonly involved in cancer diagnosis and treatment [4, 5]. Despite their values, they have in vivo and in vitro cytotoxic effects [8-10]. PBMC therapy is effective in tissue regeneration as reported in many studies [31-33]. Priming or preconditioning of the PBMC with APS has been known to enhance their tissue regenerative capacity and angiogenesis [36]. So, our study was designed to demonstrate the biological effect of ZON administration on the gastric mucosal tissue trying to discover the key parameters involved in ZON induced tissue injury. Moreover, the study evaluated the possible curative role of the PBMCs preconditioned by APS on ZON induced gastric mucosal injury in rats.

Our results showed that ZON administration to rats (group II) induced marked structural changes in the gastric mucosa. There was sloughing and desquamation of the surface epithelium or even deep ulceration. Cytoplasmic vacuoles and pyknotic nuclei appeared in most glandular cells. Reduced PCNA expression and increased TNF-α expression were recognized in most epithelial cells. There was also a significant elevation in the gastric tissue concentration of MDA and a significant reduction in GSH, SOD, and CAT. Enhancement in the mRNA expression of NF-ĸB, and COX-2 genes was also detected. The morphometric and statistical analysis confirmed our immunohistochemical and biochemical findings. Treatment with the preconditioned PBMC (group III) caused improvement of all mentioned histological, immunohistochemical and biochemical parameters compared to ZON group (group II).

Our study evaluated the ability of ZON to induce inflammation and oxidative stress. The results showed increased TNF-α expression, a significant elevation in the concentration of MDA, a significant reduction in GSH, SOD, and CAT and enhancement in the mRNA expression of NF-ĸB, and COX-2 genes in the gastric tissue of ZON group. TNF-α, NF-ĸB, and COX-2 are markers for inflammation. MDA, GSH, SOD, and CAT are indicators for oxidative stress. Our results coincided with Aboulhoda et al. [50] who reported decreased antioxidative enzyme activity and increased MDA with oral ZON. The mechanism of ZON tissue injury may be by increased ROS generation when ZON penetrate into the cytoplasm of mammalian cells. ROS can disrupt the mitochondrial functions, lipids, proteins and DNA molecules. This leads to endoplasmic reticulum oxidative stress, autophagy and cell death. ROS lead to decreased mitochondrial membrane potential, downregulation of Bcl-2 protein and upregulation of Bax protein with subsequent mitochondrial induced apoptosis. Moreover, ROS caused lipid peroxidation triggering tumor suppressor P53 apoptotic mechanism by nuclear DNA damage [21]. Also, ROS activate the inflammatory genes triggering tissue inflammation [51].

TNF-α is an early inflammatory marker and can be produced by many cells as epithelial and endothelial cells causing recruitment of inflammatory cells. So, increased TNF-α by ZON is responsible for the acute inflammatory reaction in the gastric tissue. Also, ZON may activate the immune cells to secrete inflammatory cytokines. The increased TNF-α expression by ZON may be mediated by ROS or the toll-like receptors (TLRs) pathways. Moreover, TLR6 mediates mitogen-activated protein kinase signalling leading to upregulation of the TNF-α [52, 53].

NF-ĸB is a regulatory factor of gene transcription for more than 100 proinflammatory genes involved in the immunity and inflammation. NF-ĸB pathway could increase TNF-α and COX-2 expression leading to inflammatory reaction. The increased expression of NF-ĸB in this study is attributed to ROS generation by ZON. So, ZON can induce tissue damage by ROS/NF-ĸB signalling pathway [54-56].

COX-2 is an inducible enzyme for pro-inflammatory cytokines. NF-ĸB is the major up regulator for COX-2 leading to acute tissue injury. In addition, COX-2 activation induces caspase 3/7 mediated apoptosis due to ZON induced mitochondrial dysfunction [57-59].

So, the increased TNF-α,NF-ĸB, andCOX-2 expressions by ZON result in upregulation of several genes that are involved in the gastric mucosal inflammation. This inflammation damages the epithelial barrier and gastric glands of the lamina propria.

In this study, reduced PCNA immune-expression in the gastric glands of ZON group was noticed. PCNA is a polypeptide essential for DNA replication and repair. The reduced PCNA positive cells is an indicator for disruption of the cell proliferation due to the damaging effect of ZON on nuclear DNA by oxidative stress and lipid peroxidation. ROS reacts directly with DNA damaging pyrimidine and purine bases. The genotoxic effect of ZON was proved in many studies [60, 61].

This study revealed severe structural changes in the gastric mucosa. The cytoplasmic vacuolation of the glandular epithelial cells was attributed to changes in the cell membrane permeability with accumulation of excess intracellular fluid in the form of vacuoles. Moreover, ZON could interact with cellular proteins resulting in cellular degenerative effects. The study revealed also deeply stained pyknotic nuclei which is attributed to oxidative stress mediated apoptosis [62]. This cytotoxic effect of ZON may be due to the intracellular ZON particles dissolution with subsequent increase in the cytosolic ionic zinc. ZON enter the cells by endocytotic vesicles, fuse with endosomes with low p H medium which is suitable for particles dissolution. Then, the ionic zinc leaks to the cytosol. The increased ionic zinc may be the main cause of the cell membrane injury and DNA damage [17, 63]. In a trial to keep cytoplasm homeostasis, mitochondria try to sequester ionic zinc which in turn damage the mitochondrial membrane permeability triggering apoptosis [64, 65].

In this study, we evaluated the possible therapeutic effect of the PBMCs preconditioned by APS on ZON induced gastric mucosal injury (group III). The results revealed marked improvement in all tested histological, immunohistochemical and biochemical parameters which were confirmed by statistical analysis. Our findings supported previous studies on the PBMC and their therapeutic effects may be attributed to promotion of angiogenesis and tissue repair ability [36]. PBMC possesses cytoprotective potential through their anti-inflammatory, anti-oxidant and anti-apoptotic role. Moreover, PBMC can increase the cellular proliferation. PBMC could be attracted to the injured tissue by chemotactic factors secreted from the tissue injury [66, 67]. In the injured tissue, PBMC have the potential to differentiate into functional mature cells as they include multipotent progenitors. These multipotent progenitors could be differentiated into epithelial cells. In addition, PBMC contain angioblasts (endothelial progenitor cells) which migrate to the injured tissue to differentiate into endothelial cell promoting angiogenesis [68-70]. The differentiation of these endothelial progenitor cells plays a basic role in healing of the gastric ulcers [71].

The therapeutic efficacy of the PBMC in ZON-induced gastric injury may be also attributed to the anti-inflammatory and immunomodulatory action of their biological paracrine secretomes such as non-oxidized lipids, proteins and extracellular vesicles. These secretomes can suppress the elevated TNF-α, NF-ĸB, and COX-2 alleviating gastric mucosal inflammation and promoting mucosal regeneration [72-74]. Also, PBMC could modulate the anti-oxidant markers, diminish apoptosis and increase epithelial cell proliferation helping in gastric mucosal healing [66]. The secretomes of PBMC could elevate the pro-survival and anti-apoptotic proteins resulting in cytoprotective effects [75].

Before obtaining PBMC samples from the donor rats, G-CSF was used for 3 days to induce progenitor cells mobilization from their bone marrow to peripheral blood [68]. Also, preconditioning of PBMC with the APS enhanced their tissue regenerative efficacy by increasing the level of certain cytokines as transforming growth factor-β and interleukin (IL)-1β which help in increasing the reparatory macrophage M2 and decreasing the proinflammatory macrophage M1. M2 macrophage play anti-inflammatory role and induce angiogenesis resulting in tissue repair. In addition, the preconditioned PBMC cause recruitment an appropriate host cells (by paracrine action) promoting tissue regeneration. Furthermore, utilization of autologous platelets to obtain APS prevent immunogenicity. Moreover, we use the supernatant without platelets because platelets can form thrombus in the injured tissue [36, 41]. APS contain growth factors that help in differentiation of the stem cells, promote angiogenesis, increase collagen synthesis resulting in tissue repair. Also, APS could supress the proinflammatory mediators as TNF-α and increase the anti-inflammatory ones as IL-10 and IL-4 mediating extracellular matrix synthesis and tissue regeneration [76, 77].

Moreover, the preconditioned PBMC caused a significant increase in PCNA expression in the gastric glands. This may be as a result of their therapeutic role in improving the mucosal regeneration and re-epithelization. The preconditioned PBMC could home to the injured tissue by chemotactic factors and then differentiate into epithelial cells [66]. This finding was in accordance with another study which proved the regenerative ability of PBMC on the endometrium which revealed normal histology in the PBMC-recipient mice with appearance of the injected PBMC in the uterine tissue [78].

From the present results, we could conclude that ZON induced marked structural alterations in the rat gastric mucosa. It induced inflammation and oxidative stress in the gastric mucosal tissue with reduced epithelial cell proliferation. The study suggested a possible mechanism involved in ZON induced gastric mucosal damage which is mediated by ROS/NF-ĸB/TNF-α and COX-2 signalling pathways. Moreover, the preconditioned PBMC with APS promoted gastric mucosal tissue regeneration which is attributed to their anti-inflammatory, anti-oxidant and anti-apoptotic roles. Accordingly, the preconditioned PBMC with APS may be useful as a potential therapeutic agent in managing ZON induced gastric mucosal injury. Further clinical studies should be done for evaluation of the therapeutic efficacy of the preconditioned PBMC in patient receiving ZON. Moreover, great effort should be done to develop a novel strategy to protect or treat such damaged tissues due to ZON interaction.

Conceptualization: DB. Data acquisition: NRME. Data analysis or interpretation: AAK, KSEB. Drafting of the manuscript: AAK, ZHE. Critical revision of the manuscript: DB, AEBEA. Approval of the final revision of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

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