TY - JOUR
AU1 - Mosa, Israa, F
AU2 - Abd, Haitham, H
AU3 - Abuzreda,, Abdelsalam
AU4 - Assaf,, Nadhom
AU5 - Yousif, Amenh, B
AB - Abstract Hydroxyapatite has been extensively used in tissue engineering due to its osteogenic potency, but its present toxicological facts are relatively insufficient. Here, the possible gastric toxicity of hydroxyapatite nanoparticles was evaluated biochemically to determine oxidant and antioxidant parameters in rats’ stomach tissues. At results, hydroxyapatite nanoparticles have declined stomach antioxidant enzymes and reduced glutathione level, while an induction in lipid peroxidation and nitric oxide has been observed. Furthermore, DNA oxidation was analyzed by the suppression of toll-like receptors 2, nuclear factor-kappa B and Forkhead box P3 gene expression and also 8-Oxo-2′-deoxyguanosine level as a genotoxicity indicator. Various pro-inflammatory gene products have been identified that intercede a vital role in proliferation and apoptosis suppression, among these products: tumor suppressor p53, tumor necrosis factor-α and interliukin-6. Moreover, the hydroxyapatite-treated group revealed wide histological alterations and significant elevation in the number of proliferating cell nuclear antigen-positive cells, which has been observed in the mucosal layer of the small intestine, and these alterations are an indication of small intestine injury, while the appearance of chitosan and curcumin nanoparticles in the combination group showed improvement in all the above parameters with inhibition of toxic-oxidant parameters and activation of antioxidant parameters. hydroxyapatite nanoparticles, nano-antioxidants, antioxidant enzymes, genotoxicity, oxidative DNA damage, oxidative stress, proinflammatory cytokines, gastrointestinal toxicity, histopathological architecture, immunohistochemistry, PCNA-ir cells Introduction Numerous engineered nanoparticles (ENPs) are being extensively used nowadays, and hydroxyapatite nanoparticles (HAP-NPs) have become most frequently used in biomedical and tissue engineering field for intraosseous implantation as an implant coating material or bone replacement [1]. Their distinguished properties have made them more acceptable than their microphase counterpart. The extensive utilization and production of HAP-NPs in many fields of life have raised attention about their possible toxicity, such as inflammation, cytotoxicity and the over-production of reactive oxygen species (ROS) which has increased oxidative stress are featured to be pertinent factors regarding fine particles safety, especially these down to the nano-scale [2]. Turkez has reported that particles with different sizes and morphologies, including HAP-NPs, have the potency to interact with numerous kinds of biomolecules including hemoglobin, cytochrome and DNA [3]. For this reason, nanoparticles have been used for antioxidative purposes known as nano-antioxidants, where numerous of these nanoparticles have exhibited a potential utility in nanomedicine [4, 5]. Antioxidant nanoparticles comprise a new orientation of antioxidant therapies for treatment and prevention of diseases, and they are also believed to be more efficient and functional against free radical-induced damage [6]. Furthermore, chitosan is considered as one of the most frequently used polymers in nanomedicine production, because it shows very attractive properties for drug delivery as its permeation and mucoadhesive enhancer form a protective shield for the drug [7]. Chitosan has confirmed to be more dynamic when used in a nano-size, due to its solubility in aqueous medium and cationic character, as in case of chitosan nanoparticles (CsNPs), particle size plays a vital role in penetrating the mucous layer [8]. Chitosan mucoadhesive characteristic is associated with its strong positive charge, which in turn helps in bonding with negatively charged mucus, especially in the respiratory and the gastrointestinal tract (GIT) organs. Moreover, GIT is distinguished by varying enzyme environment and pH, which with its role makes it complicated for DNA and protein/peptide drugs’ oral delivery [9]. Yadav has well defined curcumin for its anti-inflammatory, antioxidant, immunomodulation and chemo-preventive characteristics [10]. on the other hand, curcumin nanoparticles (CurNPs) were reported to have better bioavailability than their bulk counterpart, as nanotechnology is an efficient device to improve curcumin water solubility, thereby improving curcumin absorbability, dispersibility and bioavailability. Nanoparticles can easily penetrate in and pass through organisms’ cell membranes and rapidly interact with biological systems [11]. Progress in nanotechnology and insights into biomedical technologies have deducted that the administration of curcumin in nanosized form enhanced therapeutic effects of curcumin in several pre-clinical studies carried out on animal models [12]. Moreover, CsNPs and CurNPs showed a synergistic antioxidant capacity when administered together than individually reversing nephrotoxicity induced by hydroxyapatite nanoparticles at several levels [13]. Hence, in the present study, we aimed to evaluate the genotoxicity and inflammation induced in rat’s stomach in response to HAP-NP oral treatment through evaluating gastric biochemical parameters to better understand whether and how the process of toxicity could be induced. Therefore, it has been suggested that inflammation and oxidative stress are the key mechanisms accountable for ultrafine particulate adverse effects, nanoparticles. Also, analyzing the histological and immunohistochemical architecture of small intestine, and eventually exploring the anti-oxidative role of chitosan and curcumin nanoparticles against the possible gastric-induced toxicity. Materials and Methods Tested compounds and doses Hydroxyapatite was prepared using the following reagents: NaOH which was purchased from El-Nasr pharmaceutical chemicals Co., Egypt and Na2CO3, purchased from Riedel-de-Haën, Germany; CurNPs and CsNPs with Mw 310–375 kDa and degree of deacetylation (DD 75%) were purchased from Nanotech Egypt. HAP-NP dosage (300 mg/kg bw) was chosen and prepared according to Mosa method [13], while CsNPs dose (280 mg/kg bw) followed the same dosing as in Tang and Abdel-Wahhab study [14, 15, 16]. Apart from that, CurNPs dose (15 mg/kg bw) has followed the dosing criteria as in Yadav’s study [17]. Characterization of the used nanoparticles All the employed nanoparticles were characterized using X-ray diffractometer (XRD). X-ray diffraction analysis XRD patterns of the used nanoparticles were obtained using (Schimadzu 7000 Diffractometer, Japan), operated with Cu Kα radiation (λ = 0.15406 nm) generated at 30 mA and 40 kV with a scan rate of 5° min−1 for 2θ values between 4 and 80°. Crystallographic identification of the XRD patterns was accomplished by a comparison with standard data. Ethics statement Experimental animal and sample collection was conducted within the barrier system, and protocols were confirmed by the Institutional Animal Care and Use Committee (IACUC), (ethics approval no. 1468-104, revised 2018) and conducted in acquiescence with Alexandria University’s national ethical standards. Animal grouping and dose administration Eighty male Wistar rats with an average weight of 170–175 g were brought from the faculty of Medicine, Alexandria University. Rats were housed in cages preserved at 20–25°C with 40–70% relative humidity. Animals had ad libitum access to rodent basal diet, and sterilized tap water was consumed from water bottles, taken in consideration that food and water consumption and the gain or loss in body weights were weekly recorded. Each rat was identified by its tail tag with a unique number. Before the study was carried out, it was approved that none of these rats had any previous clinical condition. After 2 weeks of acclimation, animals were orally treated with the respective doses daily for 45 days. Rats were haphazardly divided into eight equal groups. Group 1 was marked as control; group 2 has received CsNPs; group 3 received CurNPs; group 4 received CsNPs and CurNPs; group 5 received HAP-NPs; group 6 received CsNPs and HAP-NPs; group 7 received CurNPs and HAP-NPs; and finally group 8 received CsNPs, CurNPs and HAP-NPs as it was the combination group. Blood sample collection and stomach tissue preparation At the end of the experiment, all animals were anesthetized with diethyl ether and sacrificed, and then blood samples were collected in heparin collection tubes. For plasma separation, blood was centrifuged at 860 × g for 20 minutes, and plasma was kept at −80°C until tested parameters analyses. Stomach was removed and washed with saline solution (0.9%), and then, the connective tissues and adhering fats were removed. The first part was used for DNA isolation for DNA fragmentation assessment, the second part was used for RNA isolation for gene expression analysis, the third part was immersed immediately in formalin for histological and immunohisotochemical analysis and eventually, the last part was minced and homogenized (10%, w/v) separately, in ice-cold sucrose buffer (0.25 M) in a Potter–Elvehjem-type homogenizer; then, the homogenates were centrifuged at 10 000 × g for 20 min at 4°C, to pellet the cell debris, and the supernatant was collected and stored at −80°C. Measured parameters Gene expression quantification in gastric tissues Total RNA was extracted from gastric tissues with RNeasy Extraction Kit (Qiagen®, USA). Quantitative RT-PCR assays were performed with Rotor-Gene SYBR Green RT-PCR Kit (Qiagen®, USA) on Rotor-Gene Q, (Qiagen®, Valencia, CA, USA) according to the manufacturer’s instructions. Housekeeping gene GAPDH was used as a reference gene for normalization, and primers used for rat genes are presented in Table 1. Table 1 Reverse-transcription reaction component Gene . . Primer sequence . TLR2 F 5′-CGCTTCCTGAACTTGTCC-3′ R 5′-GGTTGTCACCTGCTTCCA-3′ NF-κB F 5′-CAGGACCAGGAACAGTTCGAA-3′ R 5′-CCAGGTTCTGGAAGCTATGGAT-3′ FoxP3 F 5′-CACAACATGCGACCCCCTTTCACC-3′ R 5′-AGGTTGTGGCGGATGGCGTTCTTC-3′ GAPDH F 5′-GGGTGTGAACCACGAGAAATA-3′ R 5′-AGTTGTCATGGATGACCTTGG-3′ Gene . . Primer sequence . TLR2 F 5′-CGCTTCCTGAACTTGTCC-3′ R 5′-GGTTGTCACCTGCTTCCA-3′ NF-κB F 5′-CAGGACCAGGAACAGTTCGAA-3′ R 5′-CCAGGTTCTGGAAGCTATGGAT-3′ FoxP3 F 5′-CACAACATGCGACCCCCTTTCACC-3′ R 5′-AGGTTGTGGCGGATGGCGTTCTTC-3′ GAPDH F 5′-GGGTGTGAACCACGAGAAATA-3′ R 5′-AGTTGTCATGGATGACCTTGG-3′ Open in new tab Table 1 Reverse-transcription reaction component Gene . . Primer sequence . TLR2 F 5′-CGCTTCCTGAACTTGTCC-3′ R 5′-GGTTGTCACCTGCTTCCA-3′ NF-κB F 5′-CAGGACCAGGAACAGTTCGAA-3′ R 5′-CCAGGTTCTGGAAGCTATGGAT-3′ FoxP3 F 5′-CACAACATGCGACCCCCTTTCACC-3′ R 5′-AGGTTGTGGCGGATGGCGTTCTTC-3′ GAPDH F 5′-GGGTGTGAACCACGAGAAATA-3′ R 5′-AGTTGTCATGGATGACCTTGG-3′ Gene . . Primer sequence . TLR2 F 5′-CGCTTCCTGAACTTGTCC-3′ R 5′-GGTTGTCACCTGCTTCCA-3′ NF-κB F 5′-CAGGACCAGGAACAGTTCGAA-3′ R 5′-CCAGGTTCTGGAAGCTATGGAT-3′ FoxP3 F 5′-CACAACATGCGACCCCCTTTCACC-3′ R 5′-AGGTTGTGGCGGATGGCGTTCTTC-3′ GAPDH F 5′-GGGTGTGAACCACGAGAAATA-3′ R 5′-AGTTGTCATGGATGACCTTGG-3′ Open in new tab Oxidative stress markers and antioxidant parameters Thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO) level was evaluated according to Tappel and Zalkin and Montgomery and Dymock methods, respectively [18, 19]. Total antioxidant capacity (TAC) in stomach homogenates was measured following Koracevic method [20]. Superoxide dismutase (SOD) activity was evaluated as in Misra and Fridovich method [21]; also, the activity of glutathione peroxidase (GPx) was assayed by Chiu method [22]. In addition, the activities of glutathione S-transferase (GST) and Catalase (CAT) have been analyzed according to Habig and Luck method, respectively [23, 24]. Finally, glutathione (GSH) level was assayed regarding Jollow method [25]. The above-mentioned parameters were measured in compliance with Biodiagnostic Kit, Egypt manual instruction. DNA fragmentation assay 8-OH-2-deoxyguanosine (8-OH-dG) was measured according to the protocol of the manufacturer in the DNA samples, using commercial ELISA kit (ab201734, Abcam, Cambridge, UK). ELISA measurements Interleukin-6 (IL-6) was measured using ELISA kit (Kamiya Biomedical C., 12779 Gateway Drive, Seattle, WA98168). Tumor necrosis factor-alpha (TNF-α) was assayed by ELISA kits in plasma and supernatant samples (Abcam co., UK), and tumor suppressor gene (p53) was analyzed using (Active Motif co. 1914, Palomar Oaks Way, Suite 150, Carlsbad, CA 92008 USA) ELISA kits. Histopathological examination Rats’ small intestine tissues were cut and instantly placed in 10% formalin, ascendingly dehydrated using xylene and alcohol grades, then fixed for about 10 minutes in xylene and molten wax, then installed in paraffin wax and sectioned by Rotary microtome to obtain (4–6 μm) thickness and then stained with H&E following Drury method. Proliferating cell nuclear antigen immunoreactivity measurement Small intestine distribution of PCNA receptor subunits was determined in (5 μm, thickness) deparaffinized sections using an Avidin-Biotin-Peroxidase (Elite–ABC; Vector Laboratories, CA, USA) and anti-PCNA monoclonal antibody (dilution 1:100; DAKO Japan Co, Tokyo, Japan) was employed. Statistical analysis All the studied parameters were statistically analyzed using the general linear model (GLM) created by SAS [26]. Duncan’s New Multiple Range Test was applied in order to evaluate the significant difference between means [27]. Results XRD analysis has been done for the prepared nanoparticles in order to understand the skeleton structure in semi-crystalline and crystalline polymers. Hence, XRD pattern of needle-like crystal morphology of hydroxyapatite nanoparticles at different scale bars, 50–100 nm, was completely similar to the standard hydroxyapatite. Furthermore, no other impurities have been observed in the XRD pattern, indicating that the chief inorganic phase of the natural HAP-NPs is the same as HAP crystals as shown in (Fig. 1). $$ d=0.9\lambda /\beta \kern0.5em \cos \theta $$ Figure 1 Open in new tabDownload slide The XRD pattern of hydroxyapatite nanoparticles. Figure 1 Open in new tabDownload slide The XRD pattern of hydroxyapatite nanoparticles. The XRD pattern of nano-chitosan (30 nm) particle size is presented in Fig. 2. Peaks at 2θ = 9.205° and 20.05° for pure chitosan with DDA 75% confirm the semi-crystalline nature of the material. On the other hand, the XRD pattern of nano-curcumin crystals with an average particle size 40 nm exhibited a series of broadened and sharp peaks as displayed in (Fig. 3). The average crystal size (d) was calculated based on the width of the peak using Scherer’s formula, where λ is the wavelength of X-ray used, β is the full width at half maximum and θ is the Bragg’s angle of reflection. The XRD pattern peaks of CurNPs were observed at angels (17.16°, 21.05° and 23°) with an average crystallite domain size of 27 nm. Figure 2 Open in new tabDownload slide The XRD pattern of chitosan nanoparticles. Figure 2 Open in new tabDownload slide The XRD pattern of chitosan nanoparticles. Figure 3 Open in new tabDownload slide The XRD pattern of curcumin nanoparticles. Figure 3 Open in new tabDownload slide The XRD pattern of curcumin nanoparticles. Furthermore, gene expressions of toll-like receptors 2 (TLR2), nuclear factor-kappa B-p65 activity (NF-kB) and Forkhead box P3 (FoxP3) are summarized in Fig. 4 and Table 2. As HAP-NP-treated group showed a significant induction in the gene expression of TLR2, NF-kB and FoxP3, rats that received CsNPs alone or in combination with CurNPs showed non-significant high expression level of TLR2, NF-kB and FoxP3. On the other hand, the presence of CsNPs and CurNPs along with HAP-NPs in the combination group had significantly suppressed the induced expression of TLR2, NF-kB and FoxP3 and completely normalized their expression as shown in Fig. 4. Figure 4 Open in new tabDownload slide TLR2, NF-kB and FoxP3 mRNA gene expressions in gastric tissues. Values are means ± standard error (n = 8). *P < 0.05: significant differences from the control group; CsNPs; CurNPs; (Cs + Cur) NPs; HAP-NPs; (Cs + HAP) NPs; (Cur + HAP) NPs; (Cs + Cur + HAP) NPs. Figure 4 Open in new tabDownload slide TLR2, NF-kB and FoxP3 mRNA gene expressions in gastric tissues. Values are means ± standard error (n = 8). *P < 0.05: significant differences from the control group; CsNPs; CurNPs; (Cs + Cur) NPs; HAP-NPs; (Cs + HAP) NPs; (Cur + HAP) NPs; (Cs + Cur + HAP) NPs. Table 2 Mean values ± SE of gene expressions of TLR2, NF-kB and FOXP3 Experimental groups . . Parameter . TLR2 . NF-kB . Foxp3 . Control 1.0 ± 0.07e 1.00 ± 0.073d 1.00 ± 0.12c CsNPs 1.9 ± 0.16c 1.15 ± 0.064d 0.97 ± 0.10c CurNPs 1.2 ± 0.08de 1.14 ± 0.048d 1.03 ± 0.11c (Cs + Cur) NPs 1.6 ± 0.08cd 1.07 ± 0.125d 0.84 ± 0.05c HAP-NPs 5.4 ± 0.21a 4.01 ± 0.0247a 3.00 ± 0.20a (Cs + HAP) NPs 2.7 ± 0.20b 2.24 ± 0.085b 1.73 ± 0.19b (Cur + HAP) NPs 3.0 ± 0.16b 2.32 ± 0.212b 1.78 ± 0.11b (Cs + Cur + HAP) NPs 1.6 ± 0.14cd 1.73 ± 0.211c 1.27 ± 0.10c Experimental groups . . Parameter . TLR2 . NF-kB . Foxp3 . Control 1.0 ± 0.07e 1.00 ± 0.073d 1.00 ± 0.12c CsNPs 1.9 ± 0.16c 1.15 ± 0.064d 0.97 ± 0.10c CurNPs 1.2 ± 0.08de 1.14 ± 0.048d 1.03 ± 0.11c (Cs + Cur) NPs 1.6 ± 0.08cd 1.07 ± 0.125d 0.84 ± 0.05c HAP-NPs 5.4 ± 0.21a 4.01 ± 0.0247a 3.00 ± 0.20a (Cs + HAP) NPs 2.7 ± 0.20b 2.24 ± 0.085b 1.73 ± 0.19b (Cur + HAP) NPs 3.0 ± 0.16b 2.32 ± 0.212b 1.78 ± 0.11b (Cs + Cur + HAP) NPs 1.6 ± 0.14cd 1.73 ± 0.211c 1.27 ± 0.10c Open in new tab Table 2 Mean values ± SE of gene expressions of TLR2, NF-kB and FOXP3 Experimental groups . . Parameter . TLR2 . NF-kB . Foxp3 . Control 1.0 ± 0.07e 1.00 ± 0.073d 1.00 ± 0.12c CsNPs 1.9 ± 0.16c 1.15 ± 0.064d 0.97 ± 0.10c CurNPs 1.2 ± 0.08de 1.14 ± 0.048d 1.03 ± 0.11c (Cs + Cur) NPs 1.6 ± 0.08cd 1.07 ± 0.125d 0.84 ± 0.05c HAP-NPs 5.4 ± 0.21a 4.01 ± 0.0247a 3.00 ± 0.20a (Cs + HAP) NPs 2.7 ± 0.20b 2.24 ± 0.085b 1.73 ± 0.19b (Cur + HAP) NPs 3.0 ± 0.16b 2.32 ± 0.212b 1.78 ± 0.11b (Cs + Cur + HAP) NPs 1.6 ± 0.14cd 1.73 ± 0.211c 1.27 ± 0.10c Experimental groups . . Parameter . TLR2 . NF-kB . Foxp3 . Control 1.0 ± 0.07e 1.00 ± 0.073d 1.00 ± 0.12c CsNPs 1.9 ± 0.16c 1.15 ± 0.064d 0.97 ± 0.10c CurNPs 1.2 ± 0.08de 1.14 ± 0.048d 1.03 ± 0.11c (Cs + Cur) NPs 1.6 ± 0.08cd 1.07 ± 0.125d 0.84 ± 0.05c HAP-NPs 5.4 ± 0.21a 4.01 ± 0.0247a 3.00 ± 0.20a (Cs + HAP) NPs 2.7 ± 0.20b 2.24 ± 0.085b 1.73 ± 0.19b (Cur + HAP) NPs 3.0 ± 0.16b 2.32 ± 0.212b 1.78 ± 0.11b (Cs + Cur + HAP) NPs 1.6 ± 0.14cd 1.73 ± 0.211c 1.27 ± 0.10c Open in new tab Furthermore, the treatment with HAP-NPs alone resulted in a significant induction in 8-Oxo-2′-deoxyguanosine (8-OH-dG) by about 5-fold compared to the control, also a significant (P < 0.05) increase in the levels of TBARS and NO, while the treatment with CsNPs and CurNPs alone or in combination with HAP-NPs has significantly corrected and completely normalized the expression of 8-OH-dG and decreased TBARS and NO levels Fig. 5 and Table 3. Figure 5 Open in new tabDownload slide Gastric tissue content of 8-OH-2-deoxygunaine (8-OH-dG), thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO). Figure 5 Open in new tabDownload slide Gastric tissue content of 8-OH-2-deoxygunaine (8-OH-dG), thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO). Table 3 Mean values ± SE of gastric content of 8-Oxo-2′-deoxyguanosine, thiobarbituric acid-reactive substances and nitric oxide Experimental groups . Parameter . 8-OH-dG* . TBARS* (mU/mg protein) . NO* (mU/mg protein) . Control 2.11 ± 0.20d 5.7 ± 0.30d 37.1 ± 1.61c CsNPs 1.97 ± 0.08d 5.2 ± 0.28d 32.6 ± 1.61cd CurNPs 2.00 ± 0.09d 4.9 ± 0.34d 32.3 ± 1.24cd (Cs + Cur) NPs 1.88 ± 0.12d 4.6 ± 0.37d 28.3 ± 0.98d HAP-NPs 11.39 ± 0.58a 16.5 ± 0.26a 56.8 ± 1.68a (Cs + HAP) NPs 4.89 ± 0.38b 15.4 ± 0.98ab 46.9 ± 0.82b (Cur + HAP) NPs 5.32 ± 0.38b 14.2 ± 0.77b 49.8 ± 0.99b (Cs + Cur + HAP) NPs 3.78 ± 0.52c 10.4 ± 0.30c 44.9 ± 2.66b Experimental groups . Parameter . 8-OH-dG* . TBARS* (mU/mg protein) . NO* (mU/mg protein) . Control 2.11 ± 0.20d 5.7 ± 0.30d 37.1 ± 1.61c CsNPs 1.97 ± 0.08d 5.2 ± 0.28d 32.6 ± 1.61cd CurNPs 2.00 ± 0.09d 4.9 ± 0.34d 32.3 ± 1.24cd (Cs + Cur) NPs 1.88 ± 0.12d 4.6 ± 0.37d 28.3 ± 0.98d HAP-NPs 11.39 ± 0.58a 16.5 ± 0.26a 56.8 ± 1.68a (Cs + HAP) NPs 4.89 ± 0.38b 15.4 ± 0.98ab 46.9 ± 0.82b (Cur + HAP) NPs 5.32 ± 0.38b 14.2 ± 0.77b 49.8 ± 0.99b (Cs + Cur + HAP) NPs 3.78 ± 0.52c 10.4 ± 0.30c 44.9 ± 2.66b *Mean values within a column not sharing a common superscript letter (a, b, c and d) were significantly different, P < 0.05. Gastric tissue content of 8-OH-2-deoxygunaine (8-OH-dG), thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO). Open in new tab Table 3 Mean values ± SE of gastric content of 8-Oxo-2′-deoxyguanosine, thiobarbituric acid-reactive substances and nitric oxide Experimental groups . Parameter . 8-OH-dG* . TBARS* (mU/mg protein) . NO* (mU/mg protein) . Control 2.11 ± 0.20d 5.7 ± 0.30d 37.1 ± 1.61c CsNPs 1.97 ± 0.08d 5.2 ± 0.28d 32.6 ± 1.61cd CurNPs 2.00 ± 0.09d 4.9 ± 0.34d 32.3 ± 1.24cd (Cs + Cur) NPs 1.88 ± 0.12d 4.6 ± 0.37d 28.3 ± 0.98d HAP-NPs 11.39 ± 0.58a 16.5 ± 0.26a 56.8 ± 1.68a (Cs + HAP) NPs 4.89 ± 0.38b 15.4 ± 0.98ab 46.9 ± 0.82b (Cur + HAP) NPs 5.32 ± 0.38b 14.2 ± 0.77b 49.8 ± 0.99b (Cs + Cur + HAP) NPs 3.78 ± 0.52c 10.4 ± 0.30c 44.9 ± 2.66b Experimental groups . Parameter . 8-OH-dG* . TBARS* (mU/mg protein) . NO* (mU/mg protein) . Control 2.11 ± 0.20d 5.7 ± 0.30d 37.1 ± 1.61c CsNPs 1.97 ± 0.08d 5.2 ± 0.28d 32.6 ± 1.61cd CurNPs 2.00 ± 0.09d 4.9 ± 0.34d 32.3 ± 1.24cd (Cs + Cur) NPs 1.88 ± 0.12d 4.6 ± 0.37d 28.3 ± 0.98d HAP-NPs 11.39 ± 0.58a 16.5 ± 0.26a 56.8 ± 1.68a (Cs + HAP) NPs 4.89 ± 0.38b 15.4 ± 0.98ab 46.9 ± 0.82b (Cur + HAP) NPs 5.32 ± 0.38b 14.2 ± 0.77b 49.8 ± 0.99b (Cs + Cur + HAP) NPs 3.78 ± 0.52c 10.4 ± 0.30c 44.9 ± 2.66b *Mean values within a column not sharing a common superscript letter (a, b, c and d) were significantly different, P < 0.05. Gastric tissue content of 8-OH-2-deoxygunaine (8-OH-dG), thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO). Open in new tab As shown in Fig. 6, the activities of antioxidant enzymes (GPx, GST, CAT, SOD, GSH and TAC) have been significantly (P < 0.05) declined in HAP-NP-treated group, while, the presence of CsNPs and/or CurNPs in the combination group was capable to normalize the activities of these enzymes as addressed in (Table 4). Figure 6 Open in new tabDownload slide The levels of GPx, GST, CAT, SOD, GSH and TAC in rats’ stomach. Figure 6 Open in new tabDownload slide The levels of GPx, GST, CAT, SOD, GSH and TAC in rats’ stomach. Table 4 Mean values ± SE of stomach glutathione peroxidase, glutathione S-transferase, catalase, superoxide dismutase, glutathione and total antioxidant capacity Experimental groups . Parameter . GPx* (mU/mg protein) . GST* (mU/mg protein) . CAT* (mU/mg protein) . SOD* (mU/mg protein) . GSH* (mU/mg protein) . TAC* (mU/mg protein) . Control 36.0 ± 1.90c 32.6 ± 1.45bc 29.4 ± 1.66b 41.7 ± 1.91b 30.6 ± 1.12b 7.5 ± 0.54b CsNPs 43.4 ± 1.38b 36.2 ± 1.68ab 31.5 ± 1.12b 45.4 ± 2.77ab 36.0 ± 0.46a 8.7 ± 0.17a CurNPs 41.7 ± 1.83b 36.1 ± 2.04ab 31.6 ± 1.27b 40.7 ± 1.73b 36.3 ± 1.88a 9.1 ± 0.21a (Cs + Cur) NPs 49.6 ± 2.43a 38.1 ± 1.80a 35.1 ± 0.79a 49.2 ± 2.41a 37.4 ± 1.02a 9.5 ± 0.39a HAPNPs 14.7 ± 1.52e 12.0 ± 1.42f 8.2 ± 0.32f 14.8 ± 0.82e 11.4 ± 0.73e 4.7 ± 0.21d (Cs + HAP) NPs 24.9 ± 0.75d 22.3 ± 1.13e 19.0 ± 0.66e 27.6 ± 1.27cd 20.4 ± 0.89d 5.6 ± 0.41cd (Cur + HAP) NPs 22.5 ± 0.85d 26.7 ± 1.21de 22.1 ± 0.74d 23.7 ± 1.37d 21.3 ± 0.89d 5.8 ± 0.26c (Cs + Cur + HAP) NPs 32.8 ± 1.20c 28.1 ± 1.46cd 25.8 ± 0.92c 32.3 ± 1.81c 26.5 ± 1.09c 6.5 ± 0.18c Experimental groups . Parameter . GPx* (mU/mg protein) . GST* (mU/mg protein) . CAT* (mU/mg protein) . SOD* (mU/mg protein) . GSH* (mU/mg protein) . TAC* (mU/mg protein) . Control 36.0 ± 1.90c 32.6 ± 1.45bc 29.4 ± 1.66b 41.7 ± 1.91b 30.6 ± 1.12b 7.5 ± 0.54b CsNPs 43.4 ± 1.38b 36.2 ± 1.68ab 31.5 ± 1.12b 45.4 ± 2.77ab 36.0 ± 0.46a 8.7 ± 0.17a CurNPs 41.7 ± 1.83b 36.1 ± 2.04ab 31.6 ± 1.27b 40.7 ± 1.73b 36.3 ± 1.88a 9.1 ± 0.21a (Cs + Cur) NPs 49.6 ± 2.43a 38.1 ± 1.80a 35.1 ± 0.79a 49.2 ± 2.41a 37.4 ± 1.02a 9.5 ± 0.39a HAPNPs 14.7 ± 1.52e 12.0 ± 1.42f 8.2 ± 0.32f 14.8 ± 0.82e 11.4 ± 0.73e 4.7 ± 0.21d (Cs + HAP) NPs 24.9 ± 0.75d 22.3 ± 1.13e 19.0 ± 0.66e 27.6 ± 1.27cd 20.4 ± 0.89d 5.6 ± 0.41cd (Cur + HAP) NPs 22.5 ± 0.85d 26.7 ± 1.21de 22.1 ± 0.74d 23.7 ± 1.37d 21.3 ± 0.89d 5.8 ± 0.26c (Cs + Cur + HAP) NPs 32.8 ± 1.20c 28.1 ± 1.46cd 25.8 ± 0.92c 32.3 ± 1.81c 26.5 ± 1.09c 6.5 ± 0.18c *Mean values within a column not sharing a common superscript letter (a, b, c, d, e and f) were significantly different, P < 0.05. (GPx) stomach glutathione peroxidase, (GST) glutathione S-transferase, (CAT) catalase, (SOD) superoxide dismutase, (GSH) glutathione and (TAC) total antioxidant capacity. Open in new tab Table 4 Mean values ± SE of stomach glutathione peroxidase, glutathione S-transferase, catalase, superoxide dismutase, glutathione and total antioxidant capacity Experimental groups . Parameter . GPx* (mU/mg protein) . GST* (mU/mg protein) . CAT* (mU/mg protein) . SOD* (mU/mg protein) . GSH* (mU/mg protein) . TAC* (mU/mg protein) . Control 36.0 ± 1.90c 32.6 ± 1.45bc 29.4 ± 1.66b 41.7 ± 1.91b 30.6 ± 1.12b 7.5 ± 0.54b CsNPs 43.4 ± 1.38b 36.2 ± 1.68ab 31.5 ± 1.12b 45.4 ± 2.77ab 36.0 ± 0.46a 8.7 ± 0.17a CurNPs 41.7 ± 1.83b 36.1 ± 2.04ab 31.6 ± 1.27b 40.7 ± 1.73b 36.3 ± 1.88a 9.1 ± 0.21a (Cs + Cur) NPs 49.6 ± 2.43a 38.1 ± 1.80a 35.1 ± 0.79a 49.2 ± 2.41a 37.4 ± 1.02a 9.5 ± 0.39a HAPNPs 14.7 ± 1.52e 12.0 ± 1.42f 8.2 ± 0.32f 14.8 ± 0.82e 11.4 ± 0.73e 4.7 ± 0.21d (Cs + HAP) NPs 24.9 ± 0.75d 22.3 ± 1.13e 19.0 ± 0.66e 27.6 ± 1.27cd 20.4 ± 0.89d 5.6 ± 0.41cd (Cur + HAP) NPs 22.5 ± 0.85d 26.7 ± 1.21de 22.1 ± 0.74d 23.7 ± 1.37d 21.3 ± 0.89d 5.8 ± 0.26c (Cs + Cur + HAP) NPs 32.8 ± 1.20c 28.1 ± 1.46cd 25.8 ± 0.92c 32.3 ± 1.81c 26.5 ± 1.09c 6.5 ± 0.18c Experimental groups . Parameter . GPx* (mU/mg protein) . GST* (mU/mg protein) . CAT* (mU/mg protein) . SOD* (mU/mg protein) . GSH* (mU/mg protein) . TAC* (mU/mg protein) . Control 36.0 ± 1.90c 32.6 ± 1.45bc 29.4 ± 1.66b 41.7 ± 1.91b 30.6 ± 1.12b 7.5 ± 0.54b CsNPs 43.4 ± 1.38b 36.2 ± 1.68ab 31.5 ± 1.12b 45.4 ± 2.77ab 36.0 ± 0.46a 8.7 ± 0.17a CurNPs 41.7 ± 1.83b 36.1 ± 2.04ab 31.6 ± 1.27b 40.7 ± 1.73b 36.3 ± 1.88a 9.1 ± 0.21a (Cs + Cur) NPs 49.6 ± 2.43a 38.1 ± 1.80a 35.1 ± 0.79a 49.2 ± 2.41a 37.4 ± 1.02a 9.5 ± 0.39a HAPNPs 14.7 ± 1.52e 12.0 ± 1.42f 8.2 ± 0.32f 14.8 ± 0.82e 11.4 ± 0.73e 4.7 ± 0.21d (Cs + HAP) NPs 24.9 ± 0.75d 22.3 ± 1.13e 19.0 ± 0.66e 27.6 ± 1.27cd 20.4 ± 0.89d 5.6 ± 0.41cd (Cur + HAP) NPs 22.5 ± 0.85d 26.7 ± 1.21de 22.1 ± 0.74d 23.7 ± 1.37d 21.3 ± 0.89d 5.8 ± 0.26c (Cs + Cur + HAP) NPs 32.8 ± 1.20c 28.1 ± 1.46cd 25.8 ± 0.92c 32.3 ± 1.81c 26.5 ± 1.09c 6.5 ± 0.18c *Mean values within a column not sharing a common superscript letter (a, b, c, d, e and f) were significantly different, P < 0.05. (GPx) stomach glutathione peroxidase, (GST) glutathione S-transferase, (CAT) catalase, (SOD) superoxide dismutase, (GSH) glutathione and (TAC) total antioxidant capacity. Open in new tab Treatment with HAP-NPs alone caused a significant (P < 0.05) increase in the level of tumor suppressor p53, tumor necrosis factor-α (TNF-α) and interliukin-6 (IL-6), while in the combination group, the presence of CsNPs and CurNPs has significantly (P < 0.05) decreased the levels of p53, TNF-α and IL-6 as shown in Fig. 7 and confirmed in Table 5. Figure 7 Open in new tabDownload slide The levels of P53, TNF-α and IL-6 in gastric tissues. Figure 7 Open in new tabDownload slide The levels of P53, TNF-α and IL-6 in gastric tissues. Table 5 Mean values ± SE of tumor suppressor P53, tumor necrosis factor-α and interliukin-6 in gastric tissues Experimental groups . . Parameter . P53* (pg/ml protein) . TNF-α* (pg/ml tissue) . IL-6* (pg/ml tissue) . Control 9.7 ± 0.54e 52 ± 1.12e 148 ± 3.3d CsNPs 8.1 ± 0.58e 43 ± 2.05ef 126 ± 5.1ef CurNPs 9.4 ± 0.44e 47 ± 2.37ef 143 ± 4.6de (Cs + Cur) NPs 7.9 ± 0.22e 40 ± 2.02f 117 ± 5.6f HAPNPs 37.0 ± 0.82a 162 ± 2.22a 297 ± 8.2a (Cs + HAP) NPs 23.5 ± 0.58b 122 ± 4.07b 227 ± 9.7bc (Cur + HAP) NPs 25.3 ± 0.99b 105 ± 3.21c 245 ± 7.2b (Cs + Cur + HAP) NPs 17.1 ± 0.78c 83 ± 3.12d 214 ± 3.1c Experimental groups . . Parameter . P53* (pg/ml protein) . TNF-α* (pg/ml tissue) . IL-6* (pg/ml tissue) . Control 9.7 ± 0.54e 52 ± 1.12e 148 ± 3.3d CsNPs 8.1 ± 0.58e 43 ± 2.05ef 126 ± 5.1ef CurNPs 9.4 ± 0.44e 47 ± 2.37ef 143 ± 4.6de (Cs + Cur) NPs 7.9 ± 0.22e 40 ± 2.02f 117 ± 5.6f HAPNPs 37.0 ± 0.82a 162 ± 2.22a 297 ± 8.2a (Cs + HAP) NPs 23.5 ± 0.58b 122 ± 4.07b 227 ± 9.7bc (Cur + HAP) NPs 25.3 ± 0.99b 105 ± 3.21c 245 ± 7.2b (Cs + Cur + HAP) NPs 17.1 ± 0.78c 83 ± 3.12d 214 ± 3.1c *Mean values within a column not sharing a common superscript letter (a, b, c, d, e and f) were significantly different, P < 0.05, tumor suppressor P53 (P53) , tumor necrosis factor (TNF-a)- and interliukin-6 (IL-6). Open in new tab Table 5 Mean values ± SE of tumor suppressor P53, tumor necrosis factor-α and interliukin-6 in gastric tissues Experimental groups . . Parameter . P53* (pg/ml protein) . TNF-α* (pg/ml tissue) . IL-6* (pg/ml tissue) . Control 9.7 ± 0.54e 52 ± 1.12e 148 ± 3.3d CsNPs 8.1 ± 0.58e 43 ± 2.05ef 126 ± 5.1ef CurNPs 9.4 ± 0.44e 47 ± 2.37ef 143 ± 4.6de (Cs + Cur) NPs 7.9 ± 0.22e 40 ± 2.02f 117 ± 5.6f HAPNPs 37.0 ± 0.82a 162 ± 2.22a 297 ± 8.2a (Cs + HAP) NPs 23.5 ± 0.58b 122 ± 4.07b 227 ± 9.7bc (Cur + HAP) NPs 25.3 ± 0.99b 105 ± 3.21c 245 ± 7.2b (Cs + Cur + HAP) NPs 17.1 ± 0.78c 83 ± 3.12d 214 ± 3.1c Experimental groups . . Parameter . P53* (pg/ml protein) . TNF-α* (pg/ml tissue) . IL-6* (pg/ml tissue) . Control 9.7 ± 0.54e 52 ± 1.12e 148 ± 3.3d CsNPs 8.1 ± 0.58e 43 ± 2.05ef 126 ± 5.1ef CurNPs 9.4 ± 0.44e 47 ± 2.37ef 143 ± 4.6de (Cs + Cur) NPs 7.9 ± 0.22e 40 ± 2.02f 117 ± 5.6f HAPNPs 37.0 ± 0.82a 162 ± 2.22a 297 ± 8.2a (Cs + HAP) NPs 23.5 ± 0.58b 122 ± 4.07b 227 ± 9.7bc (Cur + HAP) NPs 25.3 ± 0.99b 105 ± 3.21c 245 ± 7.2b (Cs + Cur + HAP) NPs 17.1 ± 0.78c 83 ± 3.12d 214 ± 3.1c *Mean values within a column not sharing a common superscript letter (a, b, c, d, e and f) were significantly different, P < 0.05, tumor suppressor P53 (P53) , tumor necrosis factor (TNF-a)- and interliukin-6 (IL-6). Open in new tab Figure 8A–D represent the histological alterations of rats’ small intestine in different experimental groups stained with Hematoxylin & Eosin. The following groups, starting with control as in G1, CsNP-treated group as in G2, CurNP-treated group as in G3 and both CsNP- and CurNP-treated group as in G4, revealed no histological alterations, well-preserved mucosal integrity with well-arranged finger–like villi and their epithelial linings. Normal features of sub mucosa and other duodenal layers were observed, and also few inflammatory cells were seen to infiltrate into the villous stroma and sub mucosa. Small intestine of rats treated with HAP-NPs has showed abnormalities such as, villus shortening and fusion with variable degrees of epithelial atrophy, crypt damage, in addition to increased leukocyte infiltration in the lamina propria and marked increased goblet cell number in both villi and crypts as in Fig. 8E. HAP-NP- and CsNP-treated group has revealed more or less normal mucosal structure with few inflammatory cells and mild increased in goblet cell number in villi as shown in Fig. 8F. Small intestine sections of group treated with HAP-NPs and CsNPs have revealed moderate villus damage as shortening, fusion and epithelial atrophy, mild crypt hyperplasia, moderate increased goblet cell number and an increased leukocyte infiltration in the lamina propria as in Fig. 8G. On the other hand, small intestine sections of rats in the combination group that received HAP-NPs, CsNPs and CurNPs revealed mild villus shortening and fusion, epithelial atrophy and mild crypt hyperplasia as in Fig. 8H. Figures 8 Open in new tabDownload slide (A–H) Photomicrographs of rats’ small intestine sections in the different experimental groups stained with Haematoxylin & Eosin. Figures 8 Open in new tabDownload slide (A–H) Photomicrographs of rats’ small intestine sections in the different experimental groups stained with Haematoxylin & Eosin. The detection and distribution in PCNA immunoreactivity (PCNA-ir) in small intestine sections in the different experimental groups are represented in Fig. 9A–H. Small intestine section in control G1, CsNP-treated group as in G2, CurNP-treated group as in G3, and both CsNP- and CurNP-treated group as in G4 showed faint to mild positive reaction for PCNA-ir (grade 1) in mucosa layer as shown in Fig. 9A–D. In contrast, strong positive reactions were detected for PCNA-ir (grade 5) in group treated with HAP-NPs as shown in Fig. 9E as the intensity of PCNA-ir in HAP-NP-treated group showed a significant increase when compared to the control group. However, moderate positive reactions for PCNA-ir (grade 3) were observed in the group treated with HAP-NPs and CsNPs, while strong positive reactions (grade 5) for PCNA-ir were detected in group treated with HAP-NPs and CurNPs as shown in Fig. 9F and G, respectively. In the combination group, treatment with HAP-NPs, CsNPs and CurNPs revealed faint to mild positive reactions for PCNA-ir (grade 1) in mucosal layer as in Fig. 9H. Figures 9 Open in new tabDownload slide (A–H) Photomicrographs of rats’ small intestine sections in the different experimental groups stained with PCNA-ir. Figures 9 Open in new tabDownload slide (A–H) Photomicrographs of rats’ small intestine sections in the different experimental groups stained with PCNA-ir. Discussion Gastrointestinal tract is a key factor of the immune system, as well as a fundamental route of exposure for macromolecules to get in the body. The GIT epithelium is in direct contact with the ingested matter, which is absorbed by the villi. To date, studies on absorption, exposure and bioavailability are fundamentally concerned with the dermal and inhalation routes, and few are concerned with toxicity following oral uptake, especially in relation to nanoparticle ingestion [28]. Previous studies have reported that gastric exposure is one of the important nanoparticle (NP) absorption pathways. Several articles have revealed that multiple NPs can be absorbed from the GIT into the bloodstream and deposited in secondary organs. Interaction with the GIT mucosa ends up with physical adverse effects on motility luminal components, as they have crucial part in normal gut metabolic, physiologic and immune function [29, 30, 31]. However, further information is definitely required in such cases where nanoparticles are persistent and might induce prolonged oxidative stress, inflammation or migrate to various organs [32]. Although HAP-NPs are among various crystalline nanoparticles that have attracted researchers’ attention recently and variously have been used as a carrier for different cargoes such as DNA [33], siRNA [34] and anticancer drugs [35] and employed in scaffold production for tissue engineering [36], they might lead to one or more endpoints, e.g. inflammatory response inducement, cytotoxicity, genotoxicity and ROS generation. After HAP-NPs step inside the body via oral uptake, they undergo conditions that are completely different from those experienced in other routes of exposure [37]. Perhaps, the intestine high ionic strength and the low pH of stomach will condemnatorily affect nanomaterial properties, potentially yielding products with different toxicity impacts [38]. Furthermore, pH changes in the mucus, small intestine and resident GIT lumen microbiota add to the environmental complexity induced by nanoparticles and affect their prospected toxicity within the GIT [39]. Moreover, previous studies have proposed that lipid peroxidation and oxidative stress regulate nanoparticle-induced cell membrane disruption, DNA damage and cell apoptosis [40, 41]. Nevertheless, ROS, in turn, regulate intra-cellular calcium concentrations, induce cytokine production, activate transcription factors [42] and lead to inflammation [43]. Furthermore, the studies on HAP-NP cytotoxicity are adequately informative, and also, facts and ideas are worthy of mention. Generally, NP toxicity may have a detrimental effect on tissues and the whole cell through changing its architecture by inducting toxic impacts on cell component. However, at the molecular level, nanoparticle toxicity is ranging from direct effects on protein function and structure by either inhibition or activation to effects on the gene expressions. Some studies have provided spacious evidence on the cytotoxicity induced by HAP-NPs, as it might be due to cellular oxidative stress induction through excessive generation ROS and free radicals [44]. However, oxidative stress is associated with lipid and protein oxidation, leading to an intensive change in mitochondrial function. Any alteration in the permeability of mitochondrial membrane is recognized as an early stage of apoptosis [45]. A recent simulating study implemented by Mohamed has showed that the oral administration of titanium dioxide nanoparticles at different doses has resulted in persistent oxidative stress induction as revealed by significant elevations in malondialdehyde (MDA) and NO levels and a reduction in GSH level and CAT activity in gastric tissues of mice, which is in agreement with the present results, in which HAP-NPs caused ROS generation and induced oxidative stress with elevated levels of hydrogen peroxide and MDA, leading to a depletion in different expressions of gastric antioxidant enzymes [46]. Mainly, the impairment in stomach antioxidant defense system is connected to NO and ROS hyper-production, which in turn triggers oxidative stress in stomach tissues upon HAP-NP exposure. In addition, the inhibited activities of GSH-dependent enzymes (GST and GPx) are due to GSH depletion. The decrease in SOD and GPx level and the increase in MDA levels might lead to oxidative DNA damage, while the main route of GSH depletion is the protection against oxidative stress [47]. Subsequently, a previously published study indicated that new nano-structural endodontic cement based on calcium silicate with hydroxyapatite, exhibited genotoxic risk on human lymphocytes [48]. Also, concerns about HAP-NPs induced-nephrotoxicity has been raised through DNA damage via forming oxidative stress with high concentrations, that in turn caused a significant elevation in 8-OH-dG level, which is considered as a sensitive oxidative DNA damage marker, also an increase in micronuclei, sister chromatid exchange, and chromosome aberration rates as compared to untreated culture [49]. Accordingly, these findings have come in harmony with the present results, as genotoxicity hazards induced by HAP-NPs have been denoted by 8-OH-dG formation. Toll-like receptors (TLRs) are a membrane protein family that plays a vital role in host defenses against microbial pathogens by arising instinctive immune response and by inducing signals that initiate an adaptive immune response [50]. TLR mRNAs are expressed at higher levels in cells and tissues such as the gastrointestinal tract, lungs, spleen, colon [51], small intestinal epithelial and gastric pit cells and fatal intestinal cells [52]. Furthermore, numerous anti- and pro-inflammatory cytokines are produced as a result of bacterial ligands via activation of TLRs, for example, IL-6, IL-8, IL-10 and TNF-α [53, 54, 55]. In addition, TLRs act in cell to cell interactions and during cell development [56]. Extensive investigations have demonstrated that some TLRs can inhibit or promote cellular responsiveness to activating ligands [57]. TLR2 and TLR4 which exist on cell surface have been found to interpose immune responses to pathogens. They predominantly recognize viral proteins emitted into extracellular milieu due to their cellular location, examples are macrophages, monocytes, dendritic cells, T and B cells including Tregs [58]. Other cell types expressing TLR2 are hepatocytes, microglia, endothelial and epithelial cells [59]. Hereinafter, among the Foxp transcriptional factor family, which includes Foxp1, Foxp2 and Foxp3, only Foxp3 is capable to inhibit cytokines production and regulate T-cell cytokine gene expression, because Foxp3 advocates its effect by repressing transcriptional activators activity, which is vital for cytokine gene expression. Additionally, Foxp3 physically associates with nuclear factor-kappa B (NF-κB) and stops its ability to induce NF-κB-dependent gene expression, as Foxp3 is inducted to cytokine gene promoters by NF-κB and nuclear factor of activated T-cells (NFAT), because its recruitment to the promoter would stop any transcriptional activity, where NF-κB and NFAT are two key transcription factors substantial for many cytokine gene expressions, and an extravagant activation of NF-κB would lead to immediate inflammation, premature death and cachexia [60]. A study carried out by Williams has reported that oral exposure to silver nanoparticles even at low doses has increased the expressions of vital immunomodulating genes, for example, TLR2, GRP43, FoxP3 and TLR4, modulating the intestinal tract homeostasis and modifying the immune response of the gut [61]. FoxP3 and TLR2 gene expression dynamics might be due to immune tolerance intermediated by T cells [62]. In line with the present results, the observed downregulation of T-cell activators and microbial recognition receptor-associated genes suggests that exposure to HAP-NPs may result in gastrointestinal immune function depression. In fact, HAP-NP-mediated changes in TLR2 and FoxP3 might be due to downstream modulation of inflammation-associated genes. On the other hand, treatment with HAP-NPs has induced preliminary inflammation in rats which was indicated by elevated levels of IL-6 and TNF-α. In comparison with the present results, Mosa indicated that HAP-NPs secreted cytokines and chemokines that contribute to leukocyte chemotaxis, and emphasize an inflammation in host tissues, specifically in kidneys [13]. Hence, our results showed that the pro-inflammatory status induced by HAP-NPs, led to pro-inflammatory mediators’ over-production such as IL-6 and TNF-α and ROS-mediated activation of NF-κB. It is well documented that TNF-α and NO are produced by macrophages and they play a vital role in tumor conditions, as TNF-α is an essential factor in tumor promotion and regulation of other cytokines production which is involved in tumor development and chronic inflammation, through the NF-kB pathway [63, 64]. Furthermore, P53 is an ideal indictor for evaluating HAP-NPs biological safety [65]. Previous studies have demonstrated that HAP-NPs have activated p53 and induced intracellular ROS production, which might be responsible for cell apoptosis and DNA damage [66], p53 caused apoptosis using a downstream mediator as ROS which is a key role in apoptosis induction [67, 68]. Additionally, in our blot results, it is confirmed that HAP-NPs are accompanied by an increase in p53 expression that can be induced by hypoxia and DNA damage as many reports have shown that p53 plays a crucial role in apoptosis especially after HAP-NPs treatment and down-regulate Bcl-2 family expression [69]. On the other hand, the administration of CsNPs and/or CurNPs along with HAP-NPs has significantly decreased the level of TNF-α and IL-6 in gastric tissues, which is in line with the previous findings of Rogers reporting that CurNPs have anti-inflammatory activity in IL-1β and TNF-α down-regulating through impairing NF-κB activation [70]. Also, Anand, Abdolahi and Trivedi confirmed that CurNPs were capable to reduce the levels of many pro-inflammatory cytokines, improve phagocytosis extent and killer cells natural activity, because it has enormous pharmacological activities such as anti-inflammatory-, anticarcinogenic- and antimicrobial-activity [71, 72]. Also, Yadav found that CurNPs were capable to ameliorate fluoride and arsenic detrimental effect in rat blood and tissues through ROS levels downregulation and also restoring the blood GSH level [17]. Furthermore, El-Denshary concluded that treatment with CsNPs has ameliorated body antioxidant capacity, reduced oxidative stress by antioxidant enzyme induction and reduced MDA levels [73]., in line with the present results where CsNPs and CurNPs are able to work synergistically in order to scavenge gastric toxicity induced by HAP-NPs. The most frequent histological sites of hydroxyapatite crystals deposition are the kidneys, lungs, heart and the GIT, deposition of hydroxyapatite crystals is sometimes spotted in the stomach fundic glands [74]. The preferential deposition in the stomach is partially illustrated by the alkaline environment due to free hydrogen ions secretion, which promotes HAP-NPs precipitation. Hydroxyapatite crystals are heavily deposited in the smooth muscle cells, as intestinal calcinosis is accompanied by calcium deposition in the proper duodenum, oesophagus muscular layer, large intestine, and stomach [75]. In compliance with our results, HAP-NPs have induced many histological abnormalities in the rat small intestine, such as villus shortening and fusion with variable degrees of epithelial atrophy, crypt damage, in addition to increased leukocyte infiltration in the lamina propria and marked increased goblet cell number in both villi and crypts. In addition to the histological study, proliferating cell nuclear antigen (PCNA) were substantial to give another aspect into HAP-NPs toxicity mechanisms. The present results showed that oral administration of HAP-NPs caused highly statistically significant increase (P < 0.05) in PCNA immunoreactivity and the proliferative capacity of PCNA stained cells compared to untreated ones. These elevations might be as a result of ROS over production due to the interaction of these nano-particulates with DNA molecules leading to variations in genes expression connected with cell proliferation. PCNA overexpression increase confirms the histopathological alterations in small intestine. As, previous studies showed that PCNA is fundamental in repair and replication of DNA [76, 77]. Therefore, molecular bases are essential to gastric pathogenesis following HAP-NPs administration. It increases mRNA and cytokines levels and inflammation related-proteins including IL-6, p53 and TNF-α. PCNA induction is required for both DNA repair and replication followed a similar temporal and spatial pattern to p53 which reflects p53 gene tumor-suppression function [78]. Conclusion Based on the present findings, orally treated hydroxyapatite nanoparticles appeared to induce severe toxic effects on functional gastrointestinal biochemical parameters with respect to oxidative stress, immune function and other physiological aspects. 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TI - Bio-evaluation of the role of chitosan and curcumin nanoparticles in ameliorating genotoxicity and inflammatory responses in rats’ gastric tissue followed hydroxyapatite nanoparticles’ oral uptake
JF - Toxicology Research
DO - 10.1093/toxres/tfaa054
DA - 2020-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/bio-evaluation-of-the-role-of-chitosan-and-curcumin-nanoparticles-in-6UVlHlTlQd
SP - 493
EP - 508
VL - 9
IS - 4
DP - DeepDyve
ER -