Drug and Chemical Toxicology ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: http://www.tandfonline.com/loi/idct20 Gastroprotective effects of oleuropein and thymol on indomethacin-induced gastric ulcer in SpragueDawley rats Kubra Koc, Salim Cerig, Serap Ucar, Suat Colak, Murat Bakir, Huseyin Serkan Erol, Serkan Yildirim, Mirkhalil Hosseinigouzdagani, Nihal Simsek Ozek, Ferhunde Aysin, Enver Fehim Kocpinar, Harun Budak & Fatime Geyikoglu To cite this article: Kubra Koc, Salim Cerig, Serap Ucar, Suat Colak, Murat Bakir, Huseyin Serkan Erol, Serkan Yildirim, Mirkhalil Hosseinigouzdagani, Nihal Simsek Ozek, Ferhunde Aysin, Enver Fehim Kocpinar, Harun Budak & Fatime Geyikoglu (2018): Gastroprotective effects of oleuropein and thymol on indomethacin-induced gastric ulcer in Sprague-Dawley rats, Drug and Chemical Toxicology, DOI: 10.1080/01480545.2018.1530261 To link to this article: https://doi.org/10.1080/01480545.2018.1530261 Published online: 14 Nov 2018. Submit your article to this journal Article views: 2 View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idct20 DRUG AND CHEMICAL TOXICOLOGY https://doi.org/10.1080/01480545.2018.1530261 RESEARCH ARTICLE Gastroprotective effects of oleuropein and thymol on indomethacin-induced gastric ulcer in Sprague-Dawley rats Kubra Koca , Salim Ceriga, Serap Ucara, Suat Colakb, Murat Bakira, Huseyin Serkan Erolc, Serkan Yildirimd, Mirkhalil Hosseinigouzdagania, Nihal Simsek Ozeka, Ferhunde Aysina,e, Enver Fehim Kocpinarf, Harun Budakg and Fatime Geyikoglua a Department of Biology, Faculty of Science, Ataturk University, Erzurum, Turkey; bDepartment of Biology, Erzincan University, Erzincan, Turkey; cDepartment of Biochemistry, Faculty of Veterinary, Ataturk University, Erzurum, Turkey; dDepartment of Pathology, Faculty of Veterinary, Ataturk University, Erzurum, Turkey; eEast Anatolian High Technology Research and Application Center (DAYTAM), Ataturk University, Erzurum, Turkey; fDepartment of Medical Laboratory, Techniques Vocational School of Health Services, Alparslan University, Mus, Turkey; gDepartment of Molecular Biology and Genetics, Faculty of Science, Ataturk University, Erzurum, Turkey ABSTRACT ARTICLE HISTORY Ethnopharmacological studies demonstrated that thymol (Thym) and oleuropein (Ole) have therapeutic potential for gastric ulcers. The molecular mechanism underlying the gastroprotective effects of these compounds have not been elucidated yet especially for their individual and combination use at high dose. Therefore, this study was conducted to explore their gastroprotective mechanisms on indomethacin (Indo)-induced gastric ulcer model. Ole (50,100, 250, and 500 mg/kg) and Thym (50,100, 200, and 500 mg/kg) were orally administered to the rats 10 min before the induction of ulcer with Indo. The combination of 500 mg/kg doses of Ole and Thym were applied. The gastric mucosa was evaluated histopathologically. Moreover, TAC/TOS, tumor necrosis factor-alpha (TNF-a), prostaglandin E2 (PGE2), endothelial nitric oxide synthase (eNOS), and caspase-3 levels were assessed by ELISA and the caspase3 and TNF-a expressions were quantified by qRT-PCR. Indo-induced histopathological changes while Ole and Thym pretreatment prevented these effects. Unlike the 500 mg/kg dose of Ole treatment, the 500 mg/kg dose of Thym administration enhanced these damages. The decreased TAC, PGE2 levels and increased TOS, eNOS, TNF-a, caspase-3 levels were obtained in Indo group. However, these changes were reversed by Ole and Thym groups except the 500 mg/kg dose of Thym and the combination treatment groups. Similar trends were observed in the caspase-3 and TNF-a expression levels. These results demonstrated that enhanced inflammation, oxidant/antioxidant imbalance, and apoptotic activities were occurred in Indo, 500 mg/kg dose of Thym and the combination treatment groups while not in the other groups. The findings demonstrated the gastroprotective ability of Ole and low doses of Thym in gastric ulcer models. Received 7 April 2018 Revised 8 September 2018 Accepted 23 September 2018 Introduction Gastric ulcer disease is one of the most common disorders of the gastrointestinal tract with a global prevalence, affecting about 4.6 million people annually and having a mortality of one death per 10 000 cases (Oluwole 2015). Since this disease has several complications; it negatively affects the overall quality of life. It has been well known that several etiological factors like alcohol consumption, the use of nonsteroidal anti-inflammatory drugs, stress, and Helicobacter pylori infection increase the chances of gastric ulcer development (Verma and Kumar 2016). This development is related to the imbalance between mucosal aggressive and defensive factors caused by the oxidative stress and inflammation. Therefore, these factors are considered as an important contributor to the pathogenesis of gastric ulcers (Guruprasad et al. 2015). Up to now, there are many studies performed to reveal the mechanism of this disease development. However, the pathogenesis of gastric ulcer has not been clarified yet CONTACT Kubra Koc kubrakc@hotmail.com Apoptosis; cytokine; gastric ulcer; oleuropein; oxidative stress; thymol since it is a multifactorial disease. To elucidate the etiopathogenesis, indomethacin (Indo) and ethanol-induced gastric ulcer models in rats are commonly used as a model of differentiated-type human stomach diseases (Chen et al. 2016, Yodoi et al. 2016). These models produce a unified size of ulcer, which is very useful for the assessment of different drugs’ healing effects. The treatment strategy of the ulcer is generally based on the antisecretory therapy and proton pump inhibitors. However, these therapeutic strategies do not have a complete protective/curative effect of this disease and they have side effects. Therefore, alternative therapies based on medicinal plants are considered as better alternatives and main source of new drugs for the treatment of peptic ulcer since histological studies revealed that these medicinal plants did not show any acute toxicity. Due to their better compatibility with the human body and lesser adverse effects as compared to the current anti-ulcer agents, there is an extensive Department of Biology, Faculty of Science, Ataturk University, Erzurum, Turkey ß 2018 Informa UK Limited, trading as Taylor & Francis Group KEYWORDS 2 K. KOC ET AL. research to find out plant derived antiulcer agents (Dharmani et al. 2005, Kumar 2011). Among this agent, olive leaves, thyme oil, and their extracts are taken into consideration since they include valuable bioactive substances such as oleuropein (Ole) and thymol (Thym) (Tuck and Hayball 2002). Previous studies indicated that Ole was generally obtained from an edible food Olea europaea with various medicinal uses (El and Karakaya 2009). On the other hand, Thym is mostly derived from medicinal plants and it was extracted from Origanum species (Origanum onites and Origanum vulgare) and Thymus vulgaris (Chorianopoulos et al. 2004). Olive leaf extracts itself had a potential to prevent gastritis induced by HCl/ethanol via alleviating inflammation and oxidative imbalance (Al-Quraishy et al. 2017). Especially, the extracts of Origanum vulgare protected the intestinal mucosa from injuries (Azari et al. 2016) and it showed a possible anti-cancer activity against colon carcinogenesis (Srihari et al. 2008). Also, the extracts of T. vulgaris had potential antiulcer effects (Oliveira et al. 2014). Ole is a polyphenolic constituent of olive leaves with various biological benefits (Barbaro et al. 2014) (Figure 1(a)). The potential antioxidant activity of Ole is mainly due to the presence of hydroxyl groups in its chemical structure. These hydroxyl groups could donate hydrogen to prevent oxidation and it protects from the possible side effects of the oxidative stress (Hassanzadeh et al. 2014). In addition, Thym is a monoterpene phenolic compound found in the oils of thyme with valuable biological characteristics (Basch et al. 2004) (Figure 1(b)). The pharmacophore feature of the phenolic hydroxyl group in Thym’s chemical structure confer protection against the deleterious effects of free radicals both by absorbing or neutralizing free radicals and by augmenting endogenous antioxidants (Wojdylo et al. 2007). The therapeutic/protective efficacy of the Ole and Thym on different diseases has been indicated in traditional medicine and clinical studies (Sumbul et al. 2011, Meeran et al. 2017). Our unpublished studies on the protective effects of both Ole and Thym in Indo-induced liver and renal damages demonstrated that pretreatment with these natural compounds attenuated the adverse effects of this drug. In addition, gastroprotective effects of Thym have been demonstrated in ulcer models including Indo-induced ulcer model (Chauhan and Kang 2015, Ribeiro et al. 2016). Ribeiro et al. (2016) revealed that 100 mg/kg dose of Thym treatment reduce acute and chronic ulcers induced by ethanol, Indo, and acetic acid through the increased mucus secretion, prostaglandins, and ATP-sensitive K þ channels. In addition to Thym, the anti-ulcer activity of Ole has been indicated only in ulcer models except Indo-induced disease model (Alirezaei et al. 2012). Alirezaei et al. indicated that 12 mg/kg dose of Ole pretreatment significantly lowered gastric ulcers induced by ethanol through the elevation of glutathione peroxidase, superoxide dismutase, and catalase activities. However, in these studies, only low doses of both compounds with a range of 10–100 mg/kg were used and just biochemical and physiological parameters such as enzyme activity and mucus secretion were investigated. Up to now, there is no comprehensive study including histological, biochemical, and genetic expression examinations on the anti-ulcer activities of Ole and Thym with their low and moderate doses. Therefore, the first aim of this study was to elucidate the protective potential via histopathological, genetic, and biochemical analysis. In addition, the clinical results remained unclear for the availability and reliability of these natural compounds at higher doses. For this reason, the second aim of this study was to determine the protective efficacy of high doses of Thym and Ole with their individual and combination use on gastric ulceration. Materials and methods Animals Sixty Sprague-Dawley female rats, whose weight range 250–300 g were used in the study. Constant environmental conditions were maintained with a temperature of 22 ± 1  C, humidity of 55% and a 12-h light/dark cycle. Experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996). All experimental procedures in this study were approved by €rk University Local Ethics Committee for Animal the Atatu Experiments (No. 104, 02.06.2015). Chemicals Indo was purchased from DEVA Holding A.S. (Istanbul, Turkey). Certified chemical standard of Thym (>99.5%), Ole (HPLC grade 98%) and all the other chemicals were purchased from Sigma-Aldrich International (St. Louis, MO). Experimental design Figure 1. (a)The chemical structures of Oleopein and (b) Thymol. The design of this study related to the application of the tested agents was performed based on the previous studies about protective effects of plant-based products such as Ole and Thym (Manna et al. 2004, Sumbul et al. 2011, Alirezaei et al. 2012). In these studies, natural compounds/plant-based products were given with drugs which have tissue toxicities. One reason of this experimental design is that both compounds administered alone did not cause any biochemical and histological changes in animals (Alam et al. 1999, Kumral et al. 2015, Achour et al. 2016, El-Sayed et al. 2016). Based on DRUG AND CHEMICAL TOXICOLOGY these studies, due to ethical considerations in animal experiments especially to keep sample number at minimum, we have performed both natural products in only Indo-induced gastric ulcer models. The rats were divided into twelve groups: normal control, ulcer (25 mg/kg dose Indo only), Indo þ reference standard (50 mg/kg dose Rantidine hydrochloride [Ran]), Indo þ Ole (50, 100, 250, and 500 mg/kg doses), Indo þ Thym (50, 100, 200, and 500 mg/kg doses), Indo þ Thym (500 mg/kg dose) þ Ole (500 mg/kg dose). Doses of Ole and Thym were determined according to the previous studies (Miller et al. 2015, Halici et al. 2016) and our dose-based preliminary studies. The animals were fasted 24 h prior to administration of each of control, standard, and tested compounds. All substances used were dissolved in distilled water. First, Ole and Thym solution (1 ml/rat) were administered orally to the abovementioned groups. After 10 min of this treatment, Indo solution (0.5 ml) was given orally at the specified doses. Six hours after treatment, the rats were anesthetized using sevoflurane (3.5 vol% and 30% oxygen: 70% N2O) for 30 min using a ratadapted mask. The stomachs were dissected, opened, and rinsed with phosphate-buffered saline (PBS) to remove the gastric contents. Histopathological examinations and assessments Stomach sections were fixed in 10% formalin embedded in paraffin and then serially-sectioned (5-lm thick) by using a Leica RM2135 microtome (Leica, Berlin, Germany), mounted on glass slides and stained using with hematoxylin and eosin (H&E) solution. To localize the gastric mucus production, some slides were stained by periodic acid–Schiff (PAS) base (Sigma-Aldrich, PAS kit). The extent of mucosal fibrosis and definition of an amyloid fibril protein were detected using Masson and Congo red-staining kits, respectively. Then sections were examined under a light microscope by an experienced pathologist who was blind for the treatment (Yildirim et al. 2018). The high-resolution pictures of samples (200 and 400) were taken under bright field using an Olympus BX60 microscope. Determination of TAC/TOS, PGE2, eNOS, TNF-a levels, and caspase-3 activity All activity measurements were obtained from tissue homogenates. Fresh tissues were rinsed with ice-cold saline and immediately stored at –80  C. The tissues were weighed and homogenized in 50-mM phosphate buffered saline (PBS) at pH 7.0. Then centrifuged at 10 000 rpm at 4  C for 15 min to collect the supernatant for subsequent analysis. The automated Trolox equivalent total antioxidant capacity (TAC) and total oxidant status (TOS) assays were carried out in tissue homogenates using commercially available kits (Rel Assay DiagnosticsV, Gaziantep, Turkey). The tumor necrosis factoralpha (TNF-a), prostaglandin E2 (PGE2), endothelial nitric oxide synthase (eNOS) levels, and caspase-3 activity were acquired via the commercially available enzyme-linked immunosorbent assay (ELISA) kits. Procedures were performed according to R 3 the manufacturers’ instructions in each kit (rat TNF-a kit, Biolegend, San Diego, CA; PGE2 kit, Cayman Chemical Company, Ann Arbor, MI; eNOS kit, Korain Biotech, Junjiang Internatioanl Bldg., SH. China; caspase-3 activity kit, Beyotime Institute of Biotechnology, Haimen, China). Caspase-3 and TNF-a expressions Total RNA was isolated from frozen rat gastric tissues using Pure Link RNA Mini Kit according to manufacturer’s instruction (Thermo Fisher Scientific, ambion life technologies Cat. No. 12183018A). RNA concentrations and quality were verified by spectrophotometer (Thermo Scientific, Multiskan GO) and cDNA was synthesized using ProtoScript First Strand cDNA Synthesis Kit (BioLabs, New England, Cat. No. NEB #E6300S). Quantitative real-time PCR (qPCR) was performed by using SYBR Green method. Both target and reference genes were purchased from metabion international (Martinsried, Germany). Their gene symbols were: caspase-3, TNF-a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a reference gene since it was not affected by any of the treatments. The sequences of the specific primers of rat GAPDH (NM_017008.4) were as follows: GAPDH forward primer 50 -TGGACCTCATGGCCTACATG-30 ; GAPDH reverse primer 50 -AGGGAGATGCTCAGTGTTGG-30 . Thermal cycler real-time PCR (Rotor Gene Q, Qiagen) machine was used to perform gene expression experiments. The 25 ml PCR reaction volume contained 10 ng/ml template DNA, 0.2 pmol of forward and reverse primers, 12.5 ml of SYBR Green Master Mix. The PCR conditions were as follows: 50  C for 2 min, 95  C for 10 min, 45 cycles of 95  C for 10 s, annealing, and extension at 60  C for 1 min. GAPDH expression was used for normalization and relative quantification (DDCT method). Statistical analysis Results are expressed as the mean ± SD. The differences in variance were analyzed statistically using one-way analysis of variance (ANOVA) test by Graphpad Prism version 5.0 statistics software (GraphPad, La Jolla, CA). Tukey’s test was used as a post hoc and p < 0.05 was considered as statistically significant. The superscripts of a and b were used to compare the control and Indo groups with other studied groups. Results The macroscopic examination Representative stomach of each group is shown in Figure 2. Stomach of control rats showed a normal anatomical structure with an intact corpus (Figure 2(a)), whereas the oral administration of Indo produced ulcers with well-defined hemorrhagic streaks (Figure 2(b)). Macroscopic examinations revealed that the Ran afforded the protection on the mucosa (Figure 2(c)). However, the low doses (50 and 100 mg/kg) of Ole provided a poor improvement on Indo-induced gastric ulceration (Figure 2(d,e)). On the other hand, increasing doses 4 K. KOC ET AL. Figure 2. Macroscopic findings of Ole and Thym treatments on Indo-induced gastric mucosal lesions. (a): Control (Normal), (b): Indo, (c): Indo þ Ran, (d): Indo þ50 mg/kg Ole, (e): Indo þ100 mg/kg Ole, (f): Indo þ250 mg/kg Ole, (g): Indo þ500 mg/kg Ole, (h): Indo þ50 mg/kg Thym, (i): Indo þ100 mg/kg Thym, (j): Indo þ200 mg/kg Thym, (k): Indo þ500 mg/kg Thym, (l): Indo þ500 mg/kg Thym þ500 mg/kg Ole. Abbreviations: Indo, Indomethacin; Ran, Ranitidine; Ole, Oleuropein; Thym, Thymol. (250 and 500 mg/kg) of Ole showed a remarkable protective effect in comparison with Ran-treated rats (Figure 2(f,g)). Similar effects were also detected in the Thym-treated groups (Figure 2(h–j)). However, the gastric damage in the Indo þ500 mg/kg Thym group was more profound than those of the Indo group (Figure 2(k)). Although the protective effect of Ole with 500 mg/kg was observed, this effect was found to be insufficient for the gastric injury induced by 500 mg/kg dose of Thym and Indo treatment (Figure 2(l)). Histopathological studies and assessments To evaluate the morphology of gastric mucosa, H&E, PAS, Congo Red, and Masson trichrome staining were carried out. The results of each dye are demonstrated in Figures 3–6. It was found that there was a strong correlation between the macroscopic and microscopic findings. As can be seen in Figure 3(a–h), no gastric damage was observed in control group (Figure 3(a1,a2)) whereas Indo group presented remarkably distinctive injuries to the gastric mucosa, which deeply penetrated into the gastric epithelium. Moreover, congestion, leukocyte aggregation, severe alterations in the architecture of glandular parts, hemorrhages, the depletion of the gastric mucus layer and amyloidosis were observed in Indo group (Figure 3(b1–b4)). Although, a decrease in Indoinduced gastric lesions was obtained with Ran treatment, congestion, hemorrhages, and cellular infiltration were observed in this group (Figure 3(c)). In addition, the low doses (50 and 100 mg/kg) of Ole and Thym-treatment could not return stomach tissue into its normal architecture (data not shown) (Figure 3(d–f)). This protective effect was observed only in the 200 mg/kg dose of Thmy treatment. However, the highest dose (500 mg/kg) of Thym and Indo treatment enhanced the pathological structure in the gastric DRUG AND CHEMICAL TOXICOLOGY 5 Figure 3. Rat stomach (H&E). (a): Control (a1): Normal structure of stomach (a2): Normal structure of gastric glands, (b): Indo group (b1): Focal necrosis of gastric mucosa (white arrow) (b2): Congestion (black arrow), infiltration (I), Dilatation of glands (b3): Extensive congestion in glands (black arrow) (b4): Severe hemorrhagic bands in the gastric mucosa (H), (c): Indo þ Ran group; Congestion (black arrows), hemorrhage (H), infiltration (I), (d): Indo þ250 mg/kg Ole; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym, (g1): Hemorrhage (H), (g2): Congestion (black arrow), (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole, Hemorrhage (H). For abbreviations see legend Figure 2. mucosa when compared with those of the Indo group (Figure 3(g1,g2)). Moreover, the 500 mg/kg dose of Ole treatment indicated a slight protective effect on Indo þ500 mg/kg Thym and the observed pathological findings were similar to those of Indo group (Figure 3(h)). Congo Red staining findings indicated that no amyloid deposit was present in control, Indo þ250 mg/kg Ole, Indo þ500 mg/kg Ole, and Indo þ200 mg/kg Thym groups. On the contrary, this deposit was present in the Indo, Indo þ Ran, Indo þ500 mg/kg Thym, and Indo þ500 mg/kg Thym þ500 mg/kg Ole groups (Figure 4(a–h)). Based on PAS staining results (Figure 5(a–h)), control group had a gastric mucus whereas Indo induced mucus depletion. Although less mucus content was observed in Indo þ Ran treated group, an increased mucus secretion was observed with the 250, 500 mg/kg doses Ole, and 200 mg/kg dose of Thym treatment. In addition to these results, 500 mg/ kg dose of Thym treatment led to mucus depletion. The fibrotic content in stomach was evaluated based on Masson staining results, which revealed that this content was not changed control and Indo treated groups. Similarly, any fibrotic content was not observed in all of the doses of Ole and Thym (Figure 6(a–h)). 6 K. KOC ET AL. Figure 4. Rat stomach (Congo Red). (a): Stomach without amyloid in control, (b): Amyloidosis after Indo (A), (c): Indo þ Ran group; Amyloidosis (A), (d): Indo þ250 mg/kg Ole; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym; Amyloidosis (A), (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole; Amyloidosis. For abbreviations see legend Figure 2. Biochemical studies Indo treatment significantly reduced TAC and PGE2 levels while it increased TOS, eNOS, TNF-a levels, and caspase-3 activity with respect to the control group (Figure 7). On the other hand, the low doses (50 and 100 mg/kg) of Ole and Thym-treatment reversed the amount of the measured biochemical parameters induced by Indo application. However, the most profound changes in these parameters were determined in the 250 mg/kg dose of Ole and 200 mg/kg dose of Thym treated group. Ole and Thym have better protective potential against Indo-induced ulcer compared to Ran. In addition, 500 mg/kg dose of Ole provided a similar protective effect on these parameters. However, the highest dose (500 mg/kg) of Thym caused a significant increase in TOS, eNOS, TNF-a, and caspase-3 levels when compared with Indo group. However, 500 mg/kg dose of Ole treatment did not alter caspase-3 activity compared to the Indo group, implying that it could not prevent apoptotic activity induced by Indo þ500 mg/kg dose of Thym treated group. Molecular biology studies Indo administration induced a remarkable increase in mRNA levels of TNF-a and caspase-3 which were 28.5- and 5-fold higher than those levels of the control group. Both Ole and Thym treatments significantly decreased TNF-a and caspase-3 expression levels with respect to the Indo group (p and 5-fold higher than those levels of the control group. Both Ole and Thym treatments significantly de 32 and 26% of the Indo group’s values. A significant decrease in caspase-3 levels was observed which corresponds to 43 and 35% of the Indo groups’s values (p values (of the Indo group’s values. A significant decrease in caspase-3 levels was observed which co mRNA expression levels of TNF-a and caspase-3 by 35.7 and 42.4% with respect to the ulcer control group. No significant differences were also observed between Indoþ 500 mg/kg Thym þ500 mg/kg Ole and Indo-treated rats for these levels. Discussion Several studies on the pathogenesis of gastric ulcer proved that oxidative stress is the main cause of this disease. Therefore, elimination of oxidative stress by antioxidant agents, such as Ole and Thym can be used as a protective approach. The protective efficacy of these compounds with low doses between 10 and 100 mg/kg in gastric ulcer was previously demonstrated (Alirezaei et al. 2012, Chauhan and Kang 2015, Ribeiro et al. 2016). However, this ability of both molecules at moderate and high doses in gastric ulcers has not been shown yet. Acute toxicity study on Thym in rats DRUG AND CHEMICAL TOXICOLOGY 7 Figure 5. Rat stomach (PAS). (a): Gastric-mucus in controls (asterisk), (b): Indo-induced mucus depletion, (c): Indo þ Ran grup, Slight mucus content in gastric mucosa, (d): Indo þ250 mg/kg Ole; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym (g1): mucus depletion (g2): Extensive hemorrhage, (H), (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole; unregulated mucus discharge. For abbreviations see legend Figure 2. indicated that the lethal dose (LD50) of thymol was 2462.23 mg/kg (Gad 2012). Moreover, 1000 mg/kg dose of Ole was found to be nontoxic for mice (Petkov and Manolov 1972). Therefore, this study was conducted to investigate and determine for the first time, the protective mechanism of Ole and Thym at moderate and high doses in the gastric injury induced by Indo ingestion. In addition, we investigated the gastroprotective efficiency of Ole and Thym in combination especially for high dose (500 mg/kg). Indo-induced gastric ulcer model was preferred since this drug often causes gastrointestinal side-effects in patients and is widely used to induce gastrointestinal ulcers in animal models (Park et al. 2014, Yodoi et al. 2016). A number of studies suggested that Indo had common severe effects on the gastric mucosa, such as ulceration, deep erosions with shedding of the superficial epithelial cell, and cellular infiltration (Xie et al. 2013, Farzaei et al. 2015). Therefore, to assess the severity of gastric damage induced by Indo, a detailed microscopic analysis was performed on H&E stained sections. In agreement with the previous studies, we detected a disarrangement of epithelial barrier integrity and serious congestion of gastric vessels with Indo treatment. On the contrary, the Ole (250 and 500 mg/kg) and Thym (200 mg/kg) pretreatment prevented the formation of these damages. It has been known that any disruption of the epithelial barrier causes gastric ulceration (Saeki et al. 2000). Therefore, Indo-induced gastric ulcer may be arised from the disruption of mucosal barrier. In addition, this drug-induced congestion may be due to the constriction of the small arteries and venules and the restriction of mucosal blood flow (Freudenthaler et al. 2016). Based on these findings it can be inferred that, the curative potential of Ole and Thym may be stem from the strengthened mucosal barrier, maintenance of mucosal integrity, and accurate blood flow (Chauhan and Kang 2015). In consistence with our findings, the protective of Ole and Thym pretreatments in gastric ulcer were also demonstrated in the histological structure of stomach in other studies (Dekanski et al. 2009, Chauhan and Kang 2015). In addition to H&E staining, to better understand the architectural alterations in the gastric mucosa cells, we carried out Congo red, PAS, and Masson staining. Congo red staining results indicated that an interstitial amyloid deposit was found with Indo administration. However, these deposits were not seen in the 250, 500 mg/kg doses of Ole and 200 mg/kg dose of Thym treated group. This deposit formation may be due to Indo-enhanced oxidative stress (Alirezaei et al. 2014). However, the elimination of these amyloid 8 K. KOC ET AL. Figure 6. Rat stomach (Masson). (a): Control stomach, (b): Unchanged fibrotic content after Indo (A), (c): Indo þ Ran grup; Unchanged fibrotic content, (d): Indo þ250 Ole mg/kg; normalized appearances of stomach, (e): Indo þ500 mg/kg Ole; normalized appearances of stomach, (f): The healed ulcer in Indo þ200 mg/kg Thym, (g): The damaged gastric tract in Indo þ500 mg/kg Thym; unchanged fibrotic content, (h): Ulcer in Indo þ500 mg/kg Thym þ500 mg/kg Ole; Normal fibrotic content. For abbreviations see legend Figure 2. deposits may be associated the reduced oxidative stress by Ole and Thym treatments. A previous study stated that this deposit was responsible for the formation of hemorrhages. Therefore, Indo-induced gastric hemorrhages may be arisen from the formation of amyloid deposits (Miller et al. 2015). It has been reported that mucus secretion had an important role in the gastroprotection since the mucus lipids and glycoproteins had an antioxidant property and protected the mucus against oxygen radicals (Gong et al. 1990, Tarique et al. 2016). Therefore to evaluate the mucus content, PAS staining was performed, indicating a decrease in the mucus secretion by Indo treatment. This decrease may be due to the Indo-enhanced free radical production (Naito and Yoshikawa 2016). However, enhanced mucus secretion was observed in the 250, 500 mg/kg doses of Ole and 200 mg/kg dose of Thym treated group, which may be arisen from the decreased free radical amount by these compounds. In consistence with our results, the enhanced effect of Thym on the mucus secretion was previously demonstrated in Indoinduced gastric ulcer rat model (Ribeiro et al. 2016). In addition to histopathological examinations, the determination of biochemical changes is an important factor for revealing tissue damage caused by oxidative stress. Numerous antioxidant enzymes are involved in the protection of gastric mucosa from oxidative stress. The imbalance between oxidant status and antioxidant capacity is a substantial determinant for demonstration of the oxidative damage. Therefore, TAC and TOS levels were determined in Indo, as well as in Ole and Thym pretreated tissue homogenates to assess alterations in oxidative stress levels of stomach. According to our study, Indo treatment inhibited the production of antioxidants, whereas Ole (p According to our study, Indo treatment inhibited the production of antioxidants, whereas Ole (hym nd Thym d to standard drug (ranitidine). Moreover, it has been reported that the use of antioxidants as dietary supplements in combination with conventional treatment could play a pivotal part in the management of gastric and duodenal ulcers (Lee et al. 2016). Besides, TOS level was significantly lower in the control group (p  Besides, TOS level was significantly lower in thIndo group. TOS levels were higher in Ole and Thymtreated groups than in Indo group. Our results were in accordance with those of Chu et al. (2016) that evaluated the oxidative stress using Indo. Along with the determination of TAC and TOS levels, eNOS, TNF-a, PGE2, and Caspase-3 amounts were also detected as bioimportant markers of oxidative stress. eNOS causes loss of membrane fluidity, impairment of ion transport and membrane integrity, and ultimately failure in cellular function (Bindu et al. 2013). eNOS generated at the inflammatory site contributed to the onset and progression of gastric ulcer (El-Ashmawy et al. 2016). Previous studies stated that the first physiologic defense DRUG AND CHEMICAL TOXICOLOGY 9 Figure 7. The effects of Thym and Ole on stomach (a) TAS (b) PGE2 (c) TOS (d) eNOS (e) TNF-a (f) Caspase-3 levels after treated with Indo. Control: TAS, PGE2, TOS, eNOS, TNF-a, and Caspase-3 levels in normal rats. Indo: Indo induced a significant increase in TOS, eNOS, TNF-a levels and caspase-3 activity, while decrease TAC and PGE2 levels in comparison to the control. Indo þ Ran: Reversal of TAS, PGE2, TOS, eNOS, TNF-a, and Caspase-3 levels in Ran treated rats in comparison to Indo. Indo þ50 mg/kg Ole, Indo þ100 mg/kg Ole, Indo þ50 mg/kg Thym, Indo þ100 mg/kg Thym: Dose-dependent improvement of Ole and Thym treatment on the levels of biochemical parameters against Indo-induced ulcer. Indo þ200 mg/kg Thym, Indo þ250 mg/kg Ole, Indo þ500 mg/kg Ole: Ole and Thym treatment most profoundly improved the levels of biochemical parameters against Indo-induced ulcer. Indo þ500 mg/kg Thym: Thym caused a significant increase in TOS, eNOS, TNF-a and caspase-3 levels when compared with Indo group. Indo þ500 mg/kg Thym þ500 mg/kg Ole: Worsening effects of Thym and Ole treatment on the levels of biochemical parameters against Indo-induced ulcer. Data are presented as mean ± SD (n ¼ 5). a denotes significant differences between other studied groups and control (a1:p < 0.05, a2:p < 0.01, a3:p < 0.001, a4:p < 0.0001), b denotes significant differences between other studied groups and Indo group (b1:p < 0.05, b2:p < 0.01, b3:p < .0.001, b4:p < 0.0001) by Tukey’s multiple range tests. For abbreviations see legend Figure 2. against cellular damage in any organ was the inflammation emerging with rapid vascular changes (i.e., increased permeability and blood flow), followed by cellular alteration (e.g., infiltration by acute and chronic inflammatory cells). Indoinduced eNOS can cause serious early vascular injury resulting in blood flow stasis and necrosis of peripheral epithelial and mesenchymal cells (Antonisamy et al. 2014). In order to further confirmation of the NO production, TNF-a was measured as an indicator of NO production in endothelial cells (Boss et al. 2016). Our data support a similar situation in ulcerated animals where inducible TNF-a production enhanced inflammation-associated cytokines such as eNOS (Jagla and Pechanova 2015). In our experiments, we observed that high doses of Ole (250 and 500 mg/kg) lead to the changes in microcirculation modulated through eNOS and anti-inflammatory activity. Olive polyphenols have been found to play an important protective role in inflammationrelated diseases (Wedler et al. 2016). The polyphenols are 10 K. KOC ET AL. Figure 8. The effects of Thym and OLE on stomach relative mRNA expression of (a) TNF-a (b) Caspase 3 after treated with Indo. Data are presented as mean ± SD (n ¼ 5). a denotes significant differences between other studied groups and control (a4:p < 0.0001), b denotes significant differences between other studied groups and Indo group (b4:p < 0.0001) by Tukey’s multiple range tests. For abbreviations see legend Figure 1. For abbreviations see legend Figure 2. vasoprotective and the protection mechanisms may include the upregulation of eNOS expression and NO production (Bertin et al. 2016), reduction in TNF-a and superoxide anion generation (Sinnett et al. 2016). In this study, however, antiinflammatory function of Ole may be due to its direct effect on neutrophils, in which inhibited the production of proinflammatory cytokines, such as TNF-a and eNOS. The inflammation is aggravated by the recruitment of inflammatory cells, such as neutrophils and mononuclear cells (Murch et al. 1991). A number of studies indicated that Indo induced the infiltration of neutrophils in gastric mucosa, which directly correlated with the genesis of ulcerative lesions with the amount of migrant neutrophils and the initiation of acute inflammation process. In innate immune response, neutrophils represent the first step of basic defense. On the other hand, there was also alternative ways of free radical generation and other forms of ROS, such as superoxide anions (Wallace and Granger 1996). Finally, the inflammation of gastric mucosa induced the release of cytokines (Wallace et al. 2004). Our first explanation for this is that the Ole treatment may prevent the inflammatory process and suppress inflammatory responses of inflammatory cells in Indo-induced inflammation. Prostaglandins play a key role in the defense mechanisms of the gastric mucosa, and their production seems to be dependent on the capacity for free-radical-neutralization and the antioxidant potential following the damage by Indo (da Silva Junior et al. 2016). Especially, PGE2 increases the resistance of microvascular endothelial cells and prevents thrombin-induced deterioration of endothelial junctions (Alirezaei et al. 2014). Therefore, the deficiency in prostaglandin levels may play a substantial role in the pathogenesis of the formation of ulcers induced by Indo, and this may be due to the presence of oxidative damage (Boss et al. 2016). Thereby, drugs that have an antioxidant effect can protect the gastric mucosa against damage caused by ulcerogenic agents (Bouaziz et al. 2005). Our second explanation encompasses the antioxidant activity of agents like Ole. In this study, oral administration of Ole with indicated an antioxidant activity significantly prevents gastric injuries induced by Indo (Otani et al. 2016). It has been reported that these agents contain a phenolic hydroxyl group, which allows them to perform antioxidant activity by scavenging free radicals (Lin et al. 2016). Thus, it can be said that Ole may be beneficial for the prevention of oxidative stress in the stomach and hemorrhage can be controlled with PGE2 regulation. Apoptosis is another important pathophysiological pathway activated during the formation of Indo-induced ulcer (Yin et al. 2016). There are two major apoptotic pathways (extrinsic and intrinsic). Caspase-3 is a key factor particularly associated with the DRUG AND CHEMICAL TOXICOLOGY intrinsic pathway and its activation may take place either following the activation of the mitochondrial-mediated pathway or the death receptor pathway (Bakkali et al. 2008). In this study, the high doses of Ole (250 and 500 mg/kg) provided a significant reduction in pro-apoptotic marker (Caspase-3) in Indo-induced ulceration. However, the therapeutic potential of 500 mg/kg Ole was inadequate against apoptotic behavior of high dose Thym (500 mg/kg) and Indo in which they used together in a treatment. There have been no documented reports on antiulcerogenic activity of increasing doses of Thym. As a phenolic compound, Thym has a high potential to scavenge oxidative radicals by donating protons from its phenolic hydroxyl groups (Michiels et al. 2010). According to a previous study, lower doses of Thym (30 and 100 mg/kg) displayed gastroprotective actions on the acute ulcer model (10 mg/kg Indo) through mechanisms involving the increase in mucus secretion, prostaglandin levels, and activity of ATP-sensitive K þ channels (Slamenova et al. 2013, Halici et al. 2016). A diet supplemented with high doses of Thym (500 and 2000 mg/kg) in piglets could improve stomach and gut health as antimicrobial (Gao et al. 2016). However, our in vivo study revealed a negative effect of the highest dose of Thym (500 mg/kg) on Indo-induced gastric ulcer in rats. This may be due to the irreversible cell membrane damaging activity of Thym associated with its antifungal activity (Thakre-Nighot and Blikslager 2016). Hence the Ole could not prevent these damages in Indo þ Thym group. Additionally, gene expression analysis of caspase-3 and TNF-a supported the findings of biochemical assays (Figure 8). TOS, eNOS, and TNF-a measurements indicated that they may have a potential to trigger the apoptotic pathways, whereas PGE2 was able to inhibit apoptosis and regulate the gene expressions of caspase-3 and TNF-a. Of 500 mg/kg dose of Thym-treatment caused a significant increase in caspase-3 and TNF-a expression as well as inflammatory mediators and oxidative stress determinants. Conclusion The findings of this study showed that high doses (250 and 500 mg/kg) of Ole and 200 mg/kg dose of Thym have therapeutic potential against gastric mucosal damage caused by Indo via anti-inflammatory, anti-oxidative, and anti-apoptotic mechanisms. On the other hand, the high dose of Thym treatment (500 mg/kg), showed serious side effects. The administration of 500 mg/kg dose combination of Ole and Thym revealed that this dose of Ole treatment is inadequate for the prevention of apoptosis and gastric damages induced by high dose of Thym administration. Since the current antiulcer agents as synthetic drugs are expensive and are likely to produce more side effects when compared to herbal medicines, Ole and Thym may be more effective and less toxic therapeutic potential for the treatment of peptic ulcers as comparison to the synthetic anti-ulcer agents. However, to able to be proved as anti-ulcer agents, comprehensive studies on high doses of these agents are required. 11 Disclosure statement The authors declare that there are no conflicts of interest. Funding This work was supported by BAP (No. 2015/344) from Atat€ urk € Universitesi. ORCID Kubra Koc http://orcid.org/0000-0001-6208-165X References Achour, I., Arel-Dubeau, A.M., and Renaud, J., 2016. Oleuropein prevents neuronal death, mitigates mitochondrial superoxide production and modulates autophagy in a dopaminergic cellular model. International Journal of Molecular Sciences, 17 (8), 1293–1310. Alam, K., et al., 1999. 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