HEME OXYGENASE-1 UPREGULATED BY MELATONIN : POTENTIAL PROTECTION AGAINST BURN-INDUCED OXIDATIVE GASTRIC MUCOSAL INJURY

Melatonin is indoleamine hormone derived from Ltryptophan. Due to its lipophilic nature, it is accessible to every cell. Melatonin has immunomodulatory and antioxidant activities thus protecting tissue injury. Heat shock proteins such as HSP32 known as heme oxygenase-1 (HO-1) possesses antioxidant, anti-inflammatory, and vasodilatory properties and plays an important role in the protecting of tissues from several stresses. The aim of study is to investigate the expression of HO-1 in gastric mucosa and its connection with oxidative stress and melatonin mediated protection after thermal injury. On rats back, under anesthesia, third degree burn was applied involving 30% of total body surface area (TBSA). Melatonin (10 mg per kg body mass) was injected i.p. immediately and 12 hours after thermal skin injury. We used tissue malondialdehyde (MDA), lipid peroxidation product, as a marker of oxidative stress. Gastric mucosa histopathology were observed on light microscopy and light immunohistochemistry investigating the HO-1 too. Results: The levels of MDA in gastric mucosa were elevated (p< 0.05). The HO-1 expression was significantly increased in rats with trauma. Melatonin inhibited elevation in lipid peroxidation product and augmented the increase in expression of HO-1 in the gastric mucosa. In conclusion, our data suggest that HO-1 induction following burn injury is an adaptive response protecting gastric mucosal against further oxidative damage. Melatonin increased the antioxidant capacity and restricted burn-induced oxidative damage in gastric mucosa and thus could be used therapeutically in organ protection.


INTRODUCTION
Cutaneous burn injury causes gastric mucosal injury, alters mucosal integrity and leads to breakdown in mucosal defense -from superficial epithelial lesions to severe destructive alterations involving the underlying tissues [1,2].The underlying mechanism of the greater mucosal damage induced by thermal trauma is not clear.Some reports refer to several possible mechanisms including decreased gastric mucosal blood flow, changes in nitric oxide (NO) and pros-taglandin (PG) production and increased free radical formation [3,4].
Heme oxygenase-1 (HO-1) is a rate-limiting enzyme of heme degradation into its products carbon monoxide, free iron and biliverdin.The induction of HO-1expression is considered a primary adaptive response of the cellular defence mechanism under oxidative stress, inflammation, ischemia and reperfusion, irradiation, hypoxia, hyperoxia, heavy metal action [5].The enzyme accomplishes its antioxidant, antiinflammatory and antiapoptotic effects through its end products [5,6].HO-1 overexpression by pharmacological drug induction has been used to reduce the deleterious effect of free radicals, oxidative stress and apoptosis in various cell types and animal models.There are no data about changes in HO-1 activity after thermal injury.
Melatonin is an indole compound synthesized in the pituitary gland and many extrapineal tissues as well.It is widely disseminated in the organism and presents with pleiotropic properties, e.g.antioxidative, antiinflammatory and antiapoptotic.It has already been used as gastroprotective agent in some clinical investigations [7] and experimental models of damaged gastric mucosa [8,9].
Melatonin increases HO-1 activity and contributes to the restriction of tissue oxidative damage [10].We hypothesize that melatonin protects against reduces burn-induced oxidative gastric mucosal injury by HO-1 activation.To test this hypothesis, we examined the effect of melatonin on oxidative stress (using MDA as marker of peroxidation) HO-1 expression in gastric mucosa after experimental burns.

MATERIALS AND METHODS Experimental thermal skin injury
All procedures were conducted in compliance with the national laws and policies, in conformity with the international guidelines.
Age-matched male rats weighing between 220 and 250 g fasted for 12 h were allowed free access to water before injury.Animals were housed in a 20º C and offered rat chow and water ad libitum.They were kept in dark/light cycles (12:12 h) in individual wire-bottomed cages.Thus, lights were turned off at 8:00 p.m. and turned on at 8:00 a.m. for achieving the satisfactory photoperiod.After light ether inhalation, general anesthesia was intraperitoneally http://dx.doi.org/10.5272/jimab.2015212.779performed using thiopental (30 mg/kg).In order to accomplish 30% of third degree burn hot boiling water (98º C) was applied on the back of the animals during a period of 10 sec.For those rats which were subjected to burn injury, 4 mL of physiological saline was intraperitoneally applied for immediate resuscitation after the trauma.No animals died within the first 24 h of post-burn period.
All the animals were intraperitoneally given buprenorphine (0.3 mg/kg b.w.) twice daily for pain control post-burn.They were re-anesthetized with thiopental and sacrificed 24 h after burns as stomach was Histopathological analysis Tissue specimens were fixed in 10% buffered formalin (pH 7.2), dehydrated in ascending series of ethyl alcohol (70%-100%), cleared in methyl benzoate and embedded in paraffin wax.Tissue sections of 5 µm were stained with hematoxylin and eosin (H&E) and examined using light microscope (Olympus BH-2, Tokyo, Japan).Histopathological changes were examined at a magnification of 400x (high power field).

Biochemical analysis
Membrane lipid peroxidation was assayed by Malone dialdehyde (MDA) measured by its thiobarbituric acid (TBA) reactivity of stomach homogenate using the method of Porter et al. [11].Results were expressed as nmol MDA/ g tissue.They were determined using the extinction coefficient of MDA-TBA complex at 532 nm = 1.56 x 105 cm -1 M -1 solution.
Immunohistochemistry Rat stomach specimens were fixed in 10% neutral buffered formalin and embedded in paraffin.The deparaffinized and dehydrated sections (5 µm thick) were treated with 1% hydrogen peroxide for peroxidase activity inhibition for 5 min.Then they were rinsed in 0.1 M phosphate buffered saline (PBS) (pH 7.4) and treated in normal goat serum for 20 min.Subsequently, the sections were incubated with polyclonal primary antibody for 24 h at room temperature.Rabbit anti-HO-1 antibody (Santa Cruz, USA) was used.After rinsing with PBS, the sections were incubated for 20 min in goat anti-rabbit immunoglobulins at room temperature.Then they were rinsed in PBS again, treated with rabbit peroxidase-antiperoxidase complex for 20 min at room temperature and then rinsed in PBS.Finally, peroxidase activity was estimated by the diaminobenzydine-tetrachloride H2O2-method.Negative controls were incubated with non-immune sera instead of primary antibody.

Statistical analysis
Using GraphPadInStat software, the data were statistically analysed using a one-way analysis of variance (ANOVA) and expressed as mean ±SEM.The level of significance was determined by unpaired Student's t test.A value of p<0.05 was considered statistically significant.

Histological results
In control group the surface and glandular epithelium of gastric mucosa was intact (Fig. 1A).In the burned group were degeneration of the surface and glandular epithelium of gastric mucosa with loss of surface epithelial cells.Hyperemia (vascular congestion) and leukocyte infiltration was found (Fig. 1B).The gastric mucosa of melatonintreated rats was almost intact.The congestion and leukocyte infiltration were minimal (Fig. 1C).

MDA levels in gastric mucosa
Gastric mucosal MDA levels in the burned group were significantly higher (by 43%, p<0.05) than those of the control one (Fig. 2).In the melatonin-treated group, tissue levels were diminished, remaining close to the control values.
HO-1 expression in gastric mucosa HO-1 expression was low and was localized in endothelial cells in the upper part of the mucosa in the control group (Fig. 3A).In the burned group, HO-1 content of the endothelial cells was moderate and mainly in endothelial cells in the upper part of the mucosa (Fig. 3B).HO-1 expression in the melatonin-treated group was moderate to high and was found in both endothelial cells and epithelial cells (Fig. 3C).

DISCUSSION
Our results showed a significant MDA increase as a marker of lipid peroxidation in stomach including with histopathological alterations such as degenerative changes in gastric mucosa in the rat model of burns.The present findings showed that administration of melatonin decrease oxidative gastric mucosal injury by upregulation of HO-1 expression.
Burn induced gastro-intestinal mucosal injury [12,13].Oxidative stress is crucial in pathogenesis of these changes.Ischaemia due to vasospasm in the splanchnic area and inadequate perfusion cause overproduction of oxygenderived free radicals [14,15].Our data demonstrated microcirculatory disorders with congestion and leukocyte infiltration as well as degeneration of the surface and glandular epithelium.It could be assumed that activation of leukocytes and xanthine/xanthine oxidase system under condition of ischemia and microcirculatory disorders are potential source of free oxygen radicals in the stomach.The excessive free radicals and cytokines, e.g.TNF-α, interleukin 6 (IL-6) generated from inflammatory cells and the activation of lipid peroxidation may cause cell membrane destruction and degenerative changes in gastric mucosa.Oxidative stress is recognized as an important mechanism of apoptosis of epithelial cells in gastric mucosal injury [16].
Melatonin treatment limits congestion and leukocyte infiltration and degeneration of the surface and glandular epithelium and loss of surface epithelial cells of gastric mucosa as well.As a scavenger of both oxygen-and nitrogenbased reactants, it inhibits the recruitment of inflammatory   cells, the production of pro-inflammatory cytokines and acute stress proteins, which may also contribute to the restriction of burn-induced tissue-oxidative damage, including that of the gastric mucosa [17,12].
Melatonin prevents oxidative stress induced by ischemia / reperfusion (I/R) in gastric mucosa [18].The protective effect of melatonin against gastric mucosal -oxidative damage is associated with the restriction of tissue lipid peroxidation and glutathione depletion after burns [12].Moreover, melatonin acts to stabilize cell membranes, thereby making them more resistant to oxidative attacks [19].Melatonin can protect against free-radical damage throughout the cell and limits membrane lipid peroxidation and proteins in the cytosol and oxidation of DNA in the nucleus [20].
Oxidative stress could be counterbalanced by induction of ROS detoxifying enzymes such as HO-1, SOD, glutathione S-transferase (GTS) [5,21].Because HO-1 is induced by oxidative stress, it could be speculated that the increased HO-1 expression in gastric mucosa is likely related to overproduction of free radicals after burns.A growing body of evidence indicates that HO-1 activation may play an important protective role in acute gastric and colonic damages.Induction of HO-1 protein has been reported to protect against a variety of stress conditions such as ischemia reperfusion, ethanol and NSAID [22,23,24].
Our data show that melatonin augments the increased HO-1 expression in burned rats.Both endogenous HO-1 and melatonin-induced HO-1 are responsible for degrading the prooxidative heme and produce equimolar quantities of biliverdin, carbon oxide (CO) and free iron [5,6].Biliverdin is subsequently converted to endogenous antioxidative bilirubin via the action of biliverdin reductase.Free iron is promptly sequestered into ferritin, resulting in lowering the prooxidant state of the cell.CO improves the microcirculation by vasodilation and inhibition of platelet aggregation [5].Ryter et al. demonstrate that CO can inhibit the production of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and macrophage inflammatory protein-1β] and stimulate the synthesis of the antiinflammatory cytokine IL-10.CO has antiapoptotic effect [5].Therefore, HO-1 up-regulation by melatonin may provide cytoprotection against oxidative injury, gastric mucosal injury and apoptosis [25,26].Other studies also suggest that HO-1 metabolites also show protective effects against the gastrointestinal lesions [22, 23,24].

CONCLUSION
Melatonin increases HO-1 activity acting as a natural inducer antioxidant protection and protects gastric mucosa against oxidative injury after thermal trauma.This is the first report showing the protective role of HO-1 against burn-induced gastric mucosal lesions.

Fig. 2 .
Fig. 2. Effect of melatonin on MDA levels in gastric mucosa after burns.Results are given as mean + SEM.*p < 0.05 for control animals; •p < 0.05 for burned, non-treated rats.C, Controls; B, burned rats; B + M, burned rats, treated with melatonin.