Oxyresveratrol ameliorates ethanol-induced gastric ulcer via downregulation of IL-6, TNF-α, NF-ĸB, and COX-2 levels, and upregulation of TFF-2 levels

Oxyresveratrol ameliorates ethanol-induced gastric ulcer via downregulation of IL-6, TNF-α, NF-ĸB, and COX-2 levels, and upregulation of TFF-2 levels

Biomedicine & Pharmacotherapy 110 (2019) 554–560 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsev...

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Biomedicine & Pharmacotherapy 110 (2019) 554–560

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Oxyresveratrol ameliorates ethanol-induced gastric ulcer via downregulation of IL-6, TNF-α, NF-ĸB, and COX-2 levels, and upregulation of TFF-2 levels ⁎⁎

T



Rao Salman Aziza,1, Arfah Siddiquab,1, Muhammad Shahzada, , Arham Shabbirc, , Nadia Naseemd a

Department of Pharmacology, University of Health Sciences, Lahore, Pakistan Pharmacology section, Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan Department of Pharmacy, The University of Lahore-Gujrat campus, Gujrat, Pakistan d Department of Morbid Anatomy & Histopathology, University of Health Sciences, Lahore, Pakistan b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Artocarpus lakoocha Cytokines Cyclooxygenase Ulcer Ranitidine

Oxyresveratrol, an active ingredient of Artocarpus lakoocha, is known to possess anti-inflammatory and immunomodulatory properties. Current study investigates the immunomodulatory effect of oxyresveratrol in mouse model of ethanol-induced ulcer. Anti-ulcer effect was determined using histopathological evaluation (H& E staining) and different tests like, gastric ulcer scoring, ulcer index, total acid secretion, and gastric pH. The mRNA expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nuclear factor-kappaB (NF-ĸB), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and trefoil factor 2 (TFF-2) were evaluated using reverse transcription polymerase chain reaction (RT-PCR). The data showed marked percentage inhibition of erosion, hemorrhage, fibrinoid necrosis, inflammatory infiltrate, and ulcer in low (30 mg/kg b.w.) and high dose (50 mg/ kg b.w.) groups of oxyresveratrol. Treatment with oxyresveratrol inhibited ulcer score and ulcer index as compared with disease control group. Oxyresveratrol significantly increased gastric pH (P < 0.001) and attenuated total acid (P < 0.001) secretion. RT-PCR analysis showed significant suppression in the mRNA expression levels of IL-6 (P < 0.001), TNF-α (P < 0.01), NF-ĸB (P < 0.001), and COX-2 (P < 0.05) in oxyresveratrol treated groups, while COX-1 expression levels were found unaltered. Treatment with oxyresveratrol significantly elevated (P < 0.01) the expression levels of cytoprotective TFF-2 levels. Similar Immunomodulatory and anti-ulcer effects were found with ranitidine treatment, which was used as a reference drug. In conclusion, oxyresveratrol possess significant anti-ulcer property which might be attributed to attenuated expression levels of IL-6, TNF-α, NF-ĸB, and COX-2 and elevated expression levels of TFF-2.

1. Introduction Gastric ulcer is one of the most common diseases of gastrointestinal tract in the whole world. According to a survey, chances of ulceration is 3–4 times higher in men than women and occur mostly at older age [1]. Gastric ulcer occurs due to imbalance between constructive and destructive mechanisms of gastric system. Constructive factors include mucin and peptide secretions, prostaglandin secretion, and blood flow, while destructive factors comprise gastric acid, pepsin secretion, and

Helicobacter pylori [2,3]. Gastric ulcer can occur in all layers of stomach [1]. Injury to any layer causes disturbance in normal physiologic functioning that leads to excessive release of gastric acid, reactive oxygen radicals, and nitric oxide synthase, and lipid peroxidation [4]. NSAIDs, alcohol intake, bacterial infection, stress, and refluxed bile salts can be responsible for gastric ulceration [5]. Ethanol is mostly used to induce gastric ulcer in animal models. Ethanol ingestion causes gastric cell necrosis and vascular injury, and consequently ulceration. These effects are attributed to the generation of hydroperoxy free

Abbreviations: NSAID, non-steroidal anti-inflammatory drug; TFF-2, trefoil factor 2; OXY, oxyresveratrol; COX, cyclooxygenase; RT-PCR, reverse transcription polymerase chain reaction ⁎⁎ Corresponding author at: Department of Pharmacology, University of Health Sciences, near Khyaban e Jamia Punjab, Lahore, Pakistan. ⁎ Corresponding author at: Department of Pharmacy, The University of Lahore-Gujrat campus, near chenab toll plaza, Gujrat, Pakistan. E-mail addresses: [email protected] (M. Shahzad), [email protected] (A. Shabbir). 1 Rao Salman Aziz and Arfah Siddiqua contributed equally to this work https://doi.org/10.1016/j.biopha.2018.12.002 Received 9 October 2018; Received in revised form 27 November 2018; Accepted 2 December 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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0.2 ml/animal. Ethanol was administered one hour after the pretreatment with oxyresveratrol or ranitidine. Mice were sacrificed by cervical dislocation thirty minutes after ethanol administration [22].

radicals and superoxide anions which are produced as a result of ethanol metabolism in the body [3,4]. Ethanol administration is also associated with the imbalance of pro-inflammatory cytokines, and modulation of nitric oxide pathway [4]. TFF2 is a peptide which plays important role in gastric ulcer healing. It is usually found in the cytoplasm of epithelial cells and in the mucosal neck cells of gastrointestinal tract. TFF2 stabilizes mucin gel layer and plays its role in recovery from mucosa injury [6,7]. Interleukin-6 (IL-6) and Tumor necrosis factor α (TNF-α) are pro inflammatory cytokines which are involved in various immunological and inflammatory reactions [8]. NF-кB is a transcription factor which is stimulated in gastric ulcer tissues as inflammation occurs and further stimulates the production of other cytokines, chemokines, and growth factors [9,10]. Nowadays, the medicinal use of plant extracts and plant derived active ingredients is increasing throughout the world for the prevention and treatment of ulcer [11]. Oxyresveratrol is an active ingredient of Artocarpus lakoocha [12] and white mulberry [13]. It is known to possess anti-inflammatory, immunomodulatory, anti-asthmatic [14,15], anti-oxidant [16], tyrosinase inhibitory [17,18], and neuroprotective effects [19,20]. The current study evaluates the anti-inflammatory and immunomodulatory effects of oxyresveratrol using mouse model of ethanol-induced gastric ulcer.

2.4. Determination of gastric pH and total acid secretion in gastric juice The contents were collected from stomach after ligating the pylorus and placed for centrifugation at 3000 rpm for 10 min at 15 °C. The pH values of gastric juice acid were analyzed. Total acid secretion in the supernatant solution of the gastric juice was evaluated by titration as pH 7.0 using phenolphthalein (indicator) and a 0.01 mol−1 NaOH solution [23].

2.5. Determination of ulcer score, ulcer index, and percentage inhibition Ulcer scoring from the gastric tissues was conducted by giving 0, 1, 2, and 3 scores to no ulcer (normal mucosa), superficial ulcer (mucosal lesion limited to superficial layer), deep ulcer (mucosal lesion penetrated to deep layer), and perforation (mucosal damage with a hole), respectively. Ulcer index and percentage inhibition were determined by using formulas from a previous publication [24].

2. Methods and materials

2.6. Histopathological evaluation

2.1. Animals

The stomach was removed and fixed in 10% formalin solution. The tissues were dehydrated with ethanol, cleared in xylene, and embedded in paraffin wax. Sections of 4–5 μm thick were cut and stained with Hematoxylin and Eosin (H & E). Stomach sections were microscopically assessed for morphological changes by two histopathologists in a blind fashion. The evaluated parameters include erosions, edema, hemorrhage, fibrinoid necrosis, inflammatory infiltrate, and ulcer.

Fifty male BALB/c mice, weighing 30–35 grams were kept in experimental research laboratory at room temperature and humidity (50–65%). The animals were kept under 12 h light and dark cycles. All the experimental protocols were approved by Ethical Review Committee, University of Health Sciences, Lahore. 2.2. Experimental design

2.7. Determination of m RNA expression levels of COX-I and COX-II, IL-6, TNF-α, NF-ҡB and TFF-2

Mice were divided into 5 groups, each group containing 10 mice. Commercially purchased oxyresveratrol (Sigma-Aldrich) was used for the study.

Total RNA was extracted from gastric tissues using standard TRIzol method. cDNA was synthesized by reverse transcription using 1 μl of oligo dt primer (10 μM), 4 μl of 5X reaction buffer, 0.5 μl of RNAse inhibitor (20 U/μL), 2 μl of dNTP mix (10 mM), 0.5 μl of M-MuLV enzyme (200 U/μL) enzyme, and nuclease free water q.s to 20 μl. 1000 ng total RNA per reaction was used as a template. The resultantly prepared cDNA was stored at -20 °C for further use as a template in PCR. Following ingredients were mixed for PCR; 2 μl of cDNA, 0.75 μl of forward and reverse primer each (10 μM), 6 μl of PCR master mix, and 2.5 μl nuclease free water. The PCR protocol include; denaturation at 95 °C for 10 s, annealing at 58–60 Co for 20 s (35 cycles), and extension at 72 °C for 30 s. The primers of TNF-α and NF-ĸB were picked from our previous publications [25,26], while primers of COX-1, COX-2, IL6, and TFF2 were designed manually (Table 1). PCR product was initially visualized using gel electrophoresis (2% agarose gel) and later semiquantified by densitometry using imageJ software.

2.2.1. Group- I (normal control group) Group-I served as normal control (NC) and normal saline was given to mice. 2.2.2. Group-II (disease control group) Group-II served as disease control (DC) and pre-treated with normal saline (10 ml/kg b.w., p.o.). 2.2.3. Group-III (low dose group) Group-III served as low dose (LD) oxyresveratrol treated group and was orally pre-treated with oxyresveratrol 30 mg/kg b.w. [21]. 2.2.4. Group-IV (high dose group) Group-IV served as high dose (HD) oxyresveratrol pre-treated group and orally dosed with 50 mg/kg b.w. of oxyresveratrol [21].

Table 1 Primer sequences.

2.2.5. Group-V (Ranitidine group) Group-V served as reference drug treated group and was administered with ranitidine orally at dose of 100 mg/kg b.w. [22].

Primers

Forward/Reverse

Sequence

COX-1

Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5'-GAAACCGCATCGCCATGGAA-3' 5'-CGCATCTCTCGGGACTCCTT-3' 5ʹ-ATCTGGCTTCGGGAGCACAA-3ʹ 5ʹ-GTGGTAACCGCTCAGGTGTT-3ʹ 5'-CCCACCAAGAACGATAGTCA-3' 5'-CTCCGACTTGTGAAGTGGTA-3' 5'-GCTGACACCCCACAACAGAA-3' 5'-GGAAGAAACACCAGGGCACT-3'

2.3. Induction of gastric ulcer in mice

COX-2

Gastric lesions were produced using absolute ethanol through intragastric administration. Mice were fasted for 15 h before the experiment, but were provided free access to water. Absolute ethanol was administered to all the groups except negative control group at a dose of

IL6 TFF2

555

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Fig. 1. Low and high doses of oxyresveratrol, and ranitidine significantly increased pH of the gastric juice as compared to disease control group (A). The total acid secretion was found significantly reduced in oxyresveratrol and ranitidine treated groups (B) as compared to disease control group. Mean ± SD is given to represent the data. **P < 0.01, ***P < 0.001.

3.5. Oxyresveratrol significantly attenuated the mRNA expression levels of IL-6

2.8. Statistical analysis The data were represented as Mean ± SD and analyzed by Graphpad prism 6. One way ANOVA was used to analyze the quantitative variables and post hoc Tukey’s test/t-test was used to compare the groups with each other.

The data exhibited amplified (P < 0.05) expression levels of IL-6 in DC group (42.65 ± 5.78) relative to NC group (31.43 ± 5.07). The treatment caused marked reduction (P < 0.01 and P < 0.001, respectively) in LD group (25.44 ± 7.56) and HD group (19.46 ± 6.86) as compared to the PC group. The standard drug ranitidine (31.57 ± 7.18) also produced significant reduction (P < 0.05) in the elevated levels of IL-6 (Fig. 3A).

3. Results 3.1. Oxyresveratrol significantly increased gastric pH

3.6. Treatment with oxyresveratrol significantly suppressed TNF-α expression levels

The data showed significantly decreased (P < 0.001) gastric pH in DC group (5.152 ± 0.062) as compared with NC group (6.743 ± 0.335). Treatment with low (5.891 ± 0.49) and high (6.585 ± 0.43) doses of oxyresveratrol significantly increased (P < 0.001) gastric pH as compared with diseased control group. Similarly, ranitidine also significantly (P < 0.001) elevated gastric pH (6.494 ± 0.225) as compared with disease control group (Fig. 1A).

Significantly elevated (P < 0.05) expression levels of TNF-α were found in DC group (24.91 ± 4.19) as compared to NC group (16.54 ± 2.84). Treatment with oxyresveratrol also caused significant reduction in the TNF-α expression levels in both LD (14.99 ± 4.84; P < 0.01) and HD (13.65 ± 2.62; P < 0.01) groups. Similarly, ranitidine (12.59 ± 6.11) also significantly decreased (P < 0.001) TNF-α level as compared to DC group (Fig. 3B).

3.2. Oxyresveratrol significantly decreased total gastric acid secretion Treatment with low (43.6 ± 4.4) and high doses (39.5 ± 3.5) of oxyresveratrol significantly (P < 0.05) reduced gastric acid secretion ([H+] mequiv./I/4 h) as compared with disease control (55.9 ± 1.5) group. Ranitidine also nearly normalized (36.9 ± 2.1) the gastric acid secretion (Fig. 1B).

3.7. Treatment with oxyresveratrol significantly suppressed the expression levels of NF-кB Significant up-regulated (P < 0.05) expression levels of NF-кB were found in DC group (30.01 ± 4.63) as compared to the NC group (21.33 ± 5.34). The expression of NF-кB was found significantly down-regulated in treated groups i.e. LD (P < 0.01; 18.83 ± 2.42) and HD groups (P < 0.001; 14.67 ± 3.31). When compared with DC group, ranitidine treated group (22.60 ± 3.67; P < 0.05) also showed significant reduction (Fig. 3C).

3.3. Oxyresveratrol reduced ulcer score and ulcer index Treatment with oxyresveratrol and ranitidine reduced ulcer score and ulcer index as compared with diseased control group. The maximum percentage ulcer inhibition was found (90.5%) in high dose oxyresveratrol group (Table 2).

3.8. Oxyresveratrol did not significantly inhibit COX-1 expression levels Results indicated a significant increase in mRNA expression levels of COX-1 in DC group as compared to NC group (4607 ± 1267 vs 3340 ± 843). Non-significant difference was found in LD and HD groups of oxyresveratrol as compared with DC group. Ranitidine significantly (P < 0.05) reduced the expression levels of COX-1 as compared with DC group (3653 ± 954 vs 4607 ± 1267) (Fig. 4A).

3.4. Oxyresveratrol significantly suppressed histopathological markers Treatment with high dose of oxyresveratrol caused the highest percentage inhibition of erosion, hemorrhage, fibrinoid necrosis, inflammatory infiltrate, edema, and ulcer (Table 3; Fig. 2).

3.9. Oxyresveratrol significantly inhibited COX-2 expression levels

Table 2 Oxyresveratrol reduced ulcer score and ulcer index. Groups

No. of ulcers

Ulcer score

Ulcer index

Ulcer inhibition (%)

NC DC LD HD Ranitidine

– 1.6 0.5 0.1 0.2

– 16 05 01 02

– 11.76 4.55 1.11 2.22

– – 61.3 90.5 81.1

± ± ± ±

0.4 0.1 0.02 0.05

A significant increase (P < 0.01) in the mRNA expression levels of COX-2 was found in DC group as compared to NC group (4289 ± 1434 vs 2855 ± 748). Low (3110 ± 1179), and high doses (3077 ± 1212) of oxyresveratrol showed a significant decrease (P < 0.05) in COX-2 expression levels as compared with DC group. Ranitidine treated group also showed a significant decrease (P < 0.05) in COX-2 levels as compared to DC group (3042 ± 1247 vs 4289 ± 1434) (Fig. 4B). 556

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Table 3 Effects of oxyresveratrol on histopathological parameters. Groups

Erosion score (%)

Hemorrhage score (%)

Fibrinoid necrosis score (%)

Inflammatory infiltrate score (%)

Edema score (%)

Ulcer score (%)

NC DC LD HD Ranitidine

0 (0) 10 (100) 10 (100) 03 (30) 08 (80)

0 (0) 10 (100) 01(10) 0 (0) 03 (30)

0 (0) 09 (90) 01 (10) 0 (0) 0 (0)

0 (0) 10 (100) 09 (90) 06 (60) 10 (100)

0 (0) 10(100) 07 (70) 07 (70) 10 (100)

0 (0) 10 (100) 2 (20) 1 (10) 2 (20)

damage is ascribed to the recruitment of leukocytes that stimulates the inflammatory responses by increasing the levels of various pro-inflammatory cytokines [33]. In our study, we found increased levels of TNF-α, IL-6, and NF-кB expression levels in the disease control group, whereas the levels of TFF2 were significantly decreased owing to its gastro-protective activity. Previous findings showed that the inflammatory mediators played major roles in the development of gastric mucosal lesion and the levels of these markers were found significantly increased in the gastric tissue of ethanol-induced ulcer [34]. Treatment with oxyresveratrol and reference drug ranitidine improved all the altered parameters in the disease control group. IL-6 is a multifunctional cytokine and is considered as a primary regulator of both the acute and chronic inflammation, as well as, the stimulator of T and B cells [35]. It stimulates the neutrophils, macrophages, and lymphocytes at the site of inflammation to produce various noxious products, reactive oxygen radicals, and lysosomal enzymes responsible for tissue damage in gastric ulcer [36]. Previous studies have demonstrated the significantly higher mRNA levels of IL-6 in the inflamed tissues of the gastric ulcer [37]. Other studies also showed that the overproduction of IL-6 was responsible for the pathogenesis of various inflammatory diseases, and anti-IL-6 therapy produced marked therapeutic effects by blocking its signals [38]. Therefore, we intended to investigate the effect of oxyresveratrol on the mRNA expression levels of IL-6 in gastric tissues and found significant inhibition of IL-6 levels in the treatment groups. Our results are in line with inferences of previous studies showing treatment with other plant derived active ingredients of similar class, such as, Polygonum cuspidatum containing

3.10. Oxyresveratrol significantly enhanced the expression levels of TFF2 TFF2 levels were found significantly decreased (P < 0.001) in DC group (24.37 ± 4.82) as compared to NC group (44.49 ± 6.39). These reduced levels were found improved (P < 0.01) after treatment with HD group (37.53 ± 4.85). Similarly, the reference group (34.62 ± 5.81) also showed elevated (P < 0.05) levels of TFF2 in comparison with DC group (Fig. 4C).

4. Discussion Ulcer is a gastrointestinal disorder, occurs mainly in the mucosal lining of the stomach or proximal duodenum [27]. In current study, we used mouse model of ethanol-induced gastric ulcer in order to evaluate the anti-ulcer and anti-inflammatory activity of oxyresveratrol due to its resemblance with the acute gastric ulcer in humans [28]. It is the most commonly used model to induce gastric ulcer because this method is a rapid and easy way to evaluate anti-ulcer activity of any plant extract or active ingredient [29]. Ethanol ingestion produces both direct and indirect gastric damage. The direct injury is produced by rapidly entering into the gastric mucosa causing disturbance in vascular endothelium. This effect is attributed to the reduction in nitric oxide levels leading to decreased vascular permeability, which in turn causes suppression of blood flow to gastric mucosa. These events are followed by hypoxia, hemorrhagic necrosis, and reduction in gastric mucus secretion [29,30]. As a result, Na+ / k+ flow and pepsin secretion are enhanced and H+ are lost into the gastric lumen [31,32]. The indirect

Fig. 2. H & E staining of NC group at 10x magnification showing normal stomach with intact mucosa, submucosa, and muscularis mucosa layer (A). Erosion, edema, and ulcer are visible in DC group (B). The representative picture of LD group is showing erosion, edema, and inflammatory exudate but no ulcer (C). We found decreased erosion with mild edema and no ulcer in HD group (D). Similar to HD group, mild inflammatory exudate, and erosion is observed in ranitidine group with no ulcer (E). 557

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Fig. 3. Low and high doses of oxyresveratrol, and ranitidine significantly attenuated relative mRNA expression levels of IL6 (A), TNF-α (B), and NF-ĸB (C) as compared to DC group. Mean ± SD is given to represent the data. *P < 0.05, **P < 0.01, ***P < 0.001.

arachidonic acid pathway and are inhibited by NSAIDs. COX-1 is considered as “housekeeping” enzyme and is constitutively expressed in gastrointestinal tract, while COX-2 dominates at the site of inflammation. However, some studies suggest that both COX-1 and COX-2 are involved in hemostasis and function as inflammatory modulator as well [48–50]. We found significant inhibition in the expression levels of COX-2 isoenzyme which could be one of the reasons behind anti-inflammatory effect of oxyresveratrol. TFF2 is present mainly in the stomach, and is known for its role in immune system and protection of gastrointestinal epithelia upon injury [51]. It is known to increase the viscosity of gastric mucin and stabilize the gel network [52]. It promotes mucosal healing through the stimulation of proliferation and reduction of gastric acid secretion from parietal cells. TFF2 deficiency has also been shown to aggravate inflammatory responses after gastric injury. Earlier reports showed that mice deficient in TFF2 had increased number of activated parietal cells, enhanced gastric acid secretion, and were more susceptible to ulceration [6,53]. In our study, the levels of TFF2 decreased markedly in disease control group and increased levels were observed after treatment with oxyresveratrol. These findings are supported by previous reports which showed that treatment with recombinant TFF2 promoted cell migration and protected against ethanol-induced gastric ulceration [54,55].

resveratrol caused reduction in IL-6 levels using different inflammatory models [39]. TNF-α is another important pro-inflammatory cytokine having pleoitropic functions [40]. It plays a central role in the development of gastric ulcer by initiating the acute inflammatory response, accompanied by neutrophil infiltration into gastric mucosa [41]. It modulates apoptotic cell death of gastric mucosa which occurs through activation of caspase-3 pathway [42,43]. It is also known to suppress gastric microcirculation, cell proliferation, and angiogenesis at the ulcer margin, thus delays ulcer healing [33,44]. Therefore, it may be perceived that reduction in TNF-α level might facilitate the ulcer healing. In this context, TNF-α expression levels were examined in our study, and results revealed elevated levels in disease control group which were found significantly attenuated in oxyresveratrol treated groups. Our results are simultaneous with the previous study of Du et al. [45], which showed amelioration of gastric ulcer after treatment with Veronicastrum axillare and anti-ulcer activity was attributed to the reduction in the elevated TNF-α levels. In gastric epithelial cells, NF-кB plays a key role in the regulation of genes that govern the onset of acute inflammatory reaction [46]. In an inactivated state, NF-кB is retained within the cytoplasm and bound to IкB protein [9]. Upon exposure to variety of inflammatory stimuli, the enzyme IкB kinase causes phosphorylation of IкB protein and later degradation of the IкB complex. These events lead towards activation of NF-кB which then translocates into the nucleus and causes the transcriptional activation of other pro-inflammatory cytokines like TNF-α and IL-6 [34,47]. Several studies have previously demonstrated that down-regulation of NF-кB signaling pathway alleviates ethanol-induced gastric ulcer [33]. Current study also showed that ingestion of ethanol significantly enhanced the expression level of NF-кB and administration of oxyresveratrol effectively inhibited the raised NF-кB levels. Thus, it may be suggested that down-regulation of NF-кB after treatment with oxyresveratrol led to the alleviation in the levels of pro-inflammatory cytokines and subsequently resulted in the amelioration of ethanol-induced gastric ulcer. COX isoenzymes are known to produce prostaglandins from

5. Conclusion Current study suggests that oxyresveratrol possesses significant antiulcer, anti-inflammatory, and immunomodulatory properties in mouse model of ethanol-induced gastric ulcer. This inference is attributed to the down-regulated TNF-α, IL-6, NF-ҡB, and COX-2 expression levels and up-regulated TFF-2 expression levels after treatment with oxyresveratrol. Similarly, ranitidine also significantly attenuated pro-inflammatory markers and elevated expression levels of gastro-protective TFF-2. The combined use of oxyresveratrol and ranitidine could have strong anti-ulcer effect together. Further studies are suggested for this purpose. To what level, oxyresveratrol clinically alters the levels of

Fig. 4. Low and high doses of oxyresveratrol, and ranitidine significantly decreased relative mRNA expression levels of COX-2 (B), and TFF2 (C) as compared to DC group. However, only ranitidine significantly suppressed COX-1 expression levels (A). Mean ± SD is given to represent the data. *P < 0.05, **P < 0.01, ***P < 0.001. 558

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inflammatory and immunomodulatory markers also require further studies. [22]

Conflict of interest The authors declare no conflict of interest

[23]

Acknowledgement

[24]

The authors wish to pay thanks to Department of Immunology, Department of Hematology, Department of Biochemistry, and resource lab, University of Health Sciences, Lahore for providing technical facilities.

[25]

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