|Year : 2014 | Volume
| Issue : 1 | Page : 113-116
Effect of ovarian sex hormones on non-steroidal anti-inflammatory drug-induced gastric lesions in female rats
Tultul K Sangma, Seema Jain, Pramod K Mediratta
Department of Pharmacology, University College of Medical Sciences and Gugu Teg Bahadur Hospital, New Delhi, India
|Date of Submission||29-May-2013|
|Date of Decision||13-May-2013|
|Date of Acceptance||21-Nov-2013|
|Date of Web Publication||16-Jan-2014|
Pramod K Mediratta
Department of Pharmacology, University College of Medical Sciences and Gugu Teg Bahadur Hospital, New Delhi
Source of Support: None, Conflict of Interest: None
Objective: The objective of the following study is to investigate the effect of ovarian sex hormones on gastric ulcer in female rats.
Materials and Methods: Female rats were treated daily with estrogen (0.05 and 0.1 mg/kg), progesterone (2.0 and 5.0 mg/kg), combined estrogen (0.05 mg/kg) and progesterone (2.0 mg/kg), ranitidine (30 mg/kg) or vehicle for 7 days. Ulcers were induced with aspirin on 7 th day. Four hours later, animals were sacrificed and stomach were removed for macroscopic and biochemical examination.
Results: Estrogen in 0.05 and 0.1 doses showed 32% and 18% of ulcer inhibition, respectively, progesterone 09% and 14% inhibition in 2.0 and 5.0 mg/kg doses, respectively, whereas combined estrogen and progesterone showed 23% and ranitidine showed 60% inhibition. However, the inhibition attained and the stomach malondialdehyde and glutathione levels in sex hormone treated groups were not statistically significant when compared to control group.
Conclusion: At the tested doses, these ovarian sex hormones neither worsen nor protect against aspirin-induced gastric lesions in female rats.
Keywords: Estrogen, progesterone, ulcer, glutathione, malondialdehyde
|How to cite this article:|
Sangma TK, Jain S, Mediratta PK. Effect of ovarian sex hormones on non-steroidal anti-inflammatory drug-induced gastric lesions in female rats. Indian J Pharmacol 2014;46:113-6
|How to cite this URL:|
Sangma TK, Jain S, Mediratta PK. Effect of ovarian sex hormones on non-steroidal anti-inflammatory drug-induced gastric lesions in female rats. Indian J Pharmacol [serial online] 2014 [cited 2021 Mar 7];46:113-6. Available from: https://www.ijp-online.com/text.asp?2014/46/1/113/125191
| » Introduction|| |
Female sex steroids have been widely used as contraceptives and in hormone replacement therapy. The ulcerogenic effect of corticosteroids is well-known. However, reports available on the effect of female sex steroids on peptic ulcer are controversial. Protective role against the development of peptic ulcer by estrogen and progesterone have been reported by various workers. , Increased risk of aspirin-associated peptic ulcer in elderly female patients has been attributed to gradual decrease in serum levels of female sex hormones coupled with reduced gastric mucosal defenses.  In a study, 17 β estradiol but not progesterone was believed to stimulate duodenal mucosal bicarbonate secretion in mice via estrogen receptor dependent mechanism and this has been attributed to clinically observed lower incidence of duodenal ulcer in premenopausal women than age-matched females.  However, in another animal study, no gender difference or any influence by estrous cycle on ulcer in female rats were found. 
Other than inhibition of prostaglandin synthesis, free radicals and lipid peroxidation have also been implicated in pathogenesis of non-steroidal anti-inflammatory drug (NSAID)-induced peptic ulcer. , Estrogens, in contrast to other natural steroids, have been shown to possess antioxidant property. 
In spite of all these reports, estrogen is also supposed to cause damage of gastroduodenal mucosa probably by inactivating prostaglandins while progesterone is said to have beneficial effect.  Another study demonstrated that progesterone in early stage of pregnancy had protective effect on gastroduodenal mucosa by increasing gastric and duodenal mucus while estrogen had no effect. 
Considering these interesting yet confusing findings, the present study was planned to investigate the effect of these ovarian sex hormones on aspirin-induced gastric ulcers in female rats.
| » Materials and Methods|| |
Female Wister albino rats weighing between 150 and 200 g were procured from Central Animal House, University College of Medical Sciences (UCMS) and Guru Teg Bahadur Hospital, Delhi. The animals were housed in polypropylene cages under standard laboratory conditions and were acclimatized for 4-5 days before the experiments in animal house facility of the Department of Pharmacology, UCMS. Animals were fed with standard pellet diet and potable water ad libitum. Animals were maintained on natural light-dark cycle; temperature: 22 ± 1°C and humidity: 50 ± 2%. Permission was obtained from the Institutional Animal Ethics Committee and the care of animals was done as per "Committee for the Purpose of Control and Supervision of Experiments on Animals: Guidelines for laboratory animal facilities".
Drug Treatment and Ulcer Induction
Rats were divided into seven groups (n = 6) and received following treatments once a day for 7 days per orally (p.o.).
- Group 1: Control (distilled water),
- Group 2: Conjugated estrogen (0.05 mg/kg),
- Group 3: Conjugated estrogen (0.1 mg/kg),
- Group 4: Medroxyprogesterone (2.0 mg/kg),
- Group 5: Medroxyprogesterone (5.0 mg/kg),
- Group 6: Medroxyprogesterone (2.0 mg/kg) + Conjugated estrogen (0.05 mg/kg),
- Group 7: Ranitidine (30.0 mg/kg).
Animals were given food and water ad libitum for 5 days of the experimental period. On 6 th day, food was withdrawn and on 7 th day water was stopped soon after administration of the drugs. One hour later, aspirin (an NSAID) in the dose of 400 mg/kg body weight, p.o. was administered. Animals were killed 4 h after aspirin dosing, and stomachs were dissected out. The stomachs were cut opened along greater curvature, washed with normal saline and assessed for mucosal damage.
Gastric mucosal damage was observed by magnifying glass and assessed by scoring technique as follows:
- 0: Apparently normal,
- 0.5: Redness,
- 1.0: Spot ulcers,
- 1.5: Hemorrhagic streaks,
- 2.0: Ulcers >3 but ≤5 ulcers,
- 3.0: Ulcers >5. 
Mean ulcer score in each stomach was taken as ulcer index (UI) for the individual rat and inhibition percentage was calculated by the following formula: ([UI control mean−UI treated mean]/UI control mean) × 100.
| » Assessment of Oxidative Stress|| |
After macroscopic evaluation, gastric mucosa of each stomach was scraped with scalpel and stored in −80°C for future use. At the time of biochemical test, stored tissues were weighed and homogenized with 10 times ice cold 0.1 M Na 2 HPO 4 buffer (pH 7.4). Homogenates were centrifuged at 4000 rpm for 15 min and supernatants were used for assay of lipid peroxidation product-malondialdehyde (MDA) and antioxidant non-protein thiol-reduced glutathione (GSH) levels. Double beam ultraviolet-visible spectroscopy spectrophotometer (UV5704SS, Electronics Configuration of India Limited) was used for estimation of samples.
MDA was estimated by thiobarbituric acid reactive substances assay as described by Ohkawa et al.  The results were evaluated from the standard curve (0.01, 0.02, 0.04 and 0.08 nmol/ml) and expressed as nmol/g of tissue.
Determination of GSH was carried out according to the method of Ellman.  Results were evaluated from the standard curve (12, 24, 36, 48, 60 and 72 μg/ml) and expressed as nmol/mg of tissue.
| » Statistical Analysis|| |
All data were expressed as mean ± standard error of the mean with n = 6 in each group. One-way analysis of variance followed by Tukey-Kramer test was used. A P value < 0.05 was considered to be significant.
| » Results|| |
All rats survived 7 days of treatment. Animals in all groups developed mucosal damage except two animals in ranitidine group whose mucosa apparently looked normal. In the present study, initial trial was carried out with single dose of 200 mg/kg, p.o. of aspirin in three rats of distilled water group. There was no apparent gastric mucosal damage with this dose, so the findings were not included in the data. In the next trial, aspirin was administered in a dose of 400 mg/kg, p.o. to same number of animals. At this dose, all animals showed gastric erosion. Thus, this dose of aspirin was used for induction of ulcer.
Lesions in various numbers, sizes and forms were seen on the surface of the stomach and were more evident in control group (mean score: 1.83 ± 0.25). Macroscopic scoring done to assess severity of gastric damage did not show significant reduction of gastric lesions in different groups pretreated with female sex steroid hormones [Table 1]. However, in ranitidine treated group a significant reduction of gastric damage (P < 0.05 when compared with control) was found.
|Table 1: Effect of female sex hormones and ranitidine on macroscopic gastric lesion caused by aspirin in rats|
Click here to view
Assuming the percentage of inhibition by control group as 0.00%, maximum percentage inhibition was seen in ranitidine treated group (68.0%) followed by estrogen (0.05 mg/kg) treated group (32.0%). In groups 3, 4, 5 and 6 the observed percentage inhibitions were 18.0%, 9.0%, 14.0% and 18.0%, respectively.
Assessment of Oxidative Stress
There was no significant change in gastric MDA and GSH levels in the groups pretreated with female sex steroid hormones when compared to control group [Table 2]. However, the group pretreated with ranitidine showed significant decrease in MDA content (*P < 0.01) and increase in GSH content (**P < 0.05) when compared to control group.
|Table 2: Effects of female sex steroid hormones and ranitidine on the MDA and reduced GSH levels in stomach of aspirin-induced gastric lesion|
Click here to view
| » Discussion|| |
In the present study, the effects of female sex steroid hormones were observed on aspirin-induced gastric ulcer in rats. Peptic ulcer is a common disease, which could be related to stress, smoking, use of NSAIDs, alcohol usage and gender differences. Sex-dependent differences of peptic ulcer could influence the incidence and severity of ulcer as risk of ulcer is more frequent in men than in women. However, after age of 70 years the increased incidence of ulcer has also been observed in postmenopausal women, thus sex dependent effect become less noticeable.  In males, more number of parietal cells have been found as compared to females and thus, are more sensitive to the formation of ulcer.  In our study, only female rats were used, and this may be the reason for the low sensitivity of the animals to aggressive factor at lower dose of aspirin.
In addition, the role of oxidative stress has been reported in pathogenesis of ulcer in many experimental and clinical studies. Various free radicals are involved in mucosal damage caused by ingestion of alcohol, indomethacin, aspirin etc. These free radicals lead to lipid peroxidation, deoxyribonucleic acid damage and change in metabolism of cells and are responsible for damage to cell membranes. 
Various findings also indicate involvement of female sex steroids in pathophysiological mechanisms of ulcers. Presence of both estrogen and progesterone receptors have been reported in gastric tissues of human. However, it is not clear whether these receptors are regulated by the exogenous treatment of sex hormones. Several investigators had studied effects of estrogen and progesterone, alone or in combination, in experimental models of gastric ulcer. However, these experimental and clinical studies have shown contradictory results about the effects of these hormones. It was shown that estrogen protected duodenal mucosa from cysteamine in rats.  In contrast, another study demonstrated that 17 β-estradiol has pro-ulcerogenic effect and exacerbates cysteamine-induced gastroduodenal ulcers in rats while progesterone was shown to decrease it.  In addition, both progesterone and estrogen alone and in combination significantly reduced the area of acute gastric lesions in aspirin and indomethacin-induced ulcer models. ,, It has been suggested that effect of exogenous administration of estrogen and progesterone on gastric ulceration is dose and time dependent. In a study, it was found that medroxyprogesterone at high dose caused more reduction in severity of gastroduodenal damage and increase in mucus level as compared to high dose of estradiol.  In another study, it was observed that ethinylestradiol (0.01 mg/kg) offered greater protection during long-term than short term therapy. This protection was more with progesterone (1.0 mg/kg) when compared to estrogen during long term therapy. However, the combination of estrogen (0.006 mg/kg) and progesterone (0.06 mg/kg) for long term provided no such protection against gastric lesions. 
The results of present study demonstrated that in aspirin-induced gastric ulcer test, estrogen in dose of 0.05 mg/kg caused more inhibition of ulcer formation when compared to higher dose (0.1 mg/kg) of estrogen while progesterone showed protective effect at higher dose (5.0 mg/kg). Combined administration of estrogen and progesterone showed more inhibition of ulcer formation when compared to progesterone treated groups. However, estrogen, progesterone and combined estrogen and progesterone in the current dose ranges used per se did not show any significant effect on aspirin-induced gastric ulcer when compared to control group. Probably these results, at the cellular level, may be due to the low sensitivity of gastric parietal cells to the current doses of these hormones. This could also be due to small number of animals used in the study. The levels of MDA and GSH were also evaluated and it was found that the female sex hormones at tested doses did not change MDA and GSH levels significantly when compared to control group [Table 2]. These findings are in line with the macroscopic evaluation, and suggests that these hormones did not elicit antioxidant effect at tested doses.
| » Conclusion|| |
This study suggests that ovarian sex hormones at tested dose levels for period of 7 days were found neither to worsen nor protect the ulcerogenic effect of aspirin. Further studies using other ulcer producing models with wider range of doses for are required to determine the effect of sex hormones on gastric secretion, gastrin production, generation of prostaglandins and gastric mucosal blood flow.
| » References|| |
|1.||Kurt D, Saruhan BG, Kanay Z, Yokus B, Kanay BE, Unver O, et al. Effect of ovariectomy and female sex hormones administration upon gastric ulceration induced by cold and immobility restraint stress. Saudi Med J 2007;28:1021-7. |
|2.||Machowska A, Szlachcic A, Pawlik M, Brzozowski T, Konturek SJ, Pawlik WW. The role of female and male sex hormones in the healing process of preexisting lingual and gastric ulcerations. J Physiol Pharmacol 2004;55 Suppl 2:91-104. |
|3.||Okada K, Inamori M, Imajyo K, Chiba H, Nonaka T, Shiba T, et al. Gender differences of low-dose aspirin-associated gastroduodenal ulcer in Japanese patients. World J Gastroenterol 2010;16:1896-900. |
|4.||Smith A, Contreras C, Ko KH, Chow J, Dong X, Tuo B, et al. Gender-specific protection of estrogen against gastric acid-induced duodenal injury: Stimulation of duodenal mucosal bicarbonate secretion. Endocrinology 2008;149:4554-66. |
|5.||Uslu E, Aydin S, Carkman S, Uzun H, Altinli E, Apaydin BB, et al. Effects of gender on stress ulcer formation in rats. Tohoku J Exp Med 2002;197:17-26. |
|6.||Biswas K, Bandyopadhyay U, Chattopadhyay I, Varadaraj A, Ali E, Banerjee RK. A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical. J Biol Chem 2003;278:10993-1001. |
|7.||Dengiz GO, Odabasoglu F, Halici Z, Suleyman H, Cadirci E, Bayir Y. Gastroprotective and antioxidant effects of amiodarone on indomethacin-induced gastric ulcers in rats. Arch Pharm Res 2007;30:1426-34. |
|8.||Behl C, Skutella T, Lezoualc'h F, Post A, Widmann M, Newton CJ, et al. Neuroprotection against oxidative stress by estrogens: Structure-activity relationship. Mol Pharmacol 1997;51:535-41. |
|9.||Drago F, Montoneri C, Varga C, Làszlò F. Dual effect of female sex steroids on drug-induced gastroduodenal ulcers in the rat. Life Sci 1999;64:2341-50. |
|10.||Montoneri C, Drago F. Effects of pregnancy in rats on cysteamine-induced peptic ulcers: Role of progesterone. Dig Dis Sci 1997;42:2572-5. |
|11.||Amir M, Kumar S. Synthesis and evaluation of anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation properties of ibuprofen derivatives. Acta Pharm 2007;57:31-45. |
|12.||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. |
|13.||Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7. |
|14.||Odabasoglu F, Cakir A, Suleyman H, Aslan A, Bayir Y, Halici M, et al. Gastroprotective and antioxidant effects of usnic acid on indomethacin-induced gastric ulcer in rats. J Ethnopharmacol 2006;103:59-65. |
|15.||Ashokan KV, Kurane MM, Pillai MM. Effect of overiectomy and of estrogen administration upon duodenal ulceration induced by cysteamine. IUFS J Biol 2010;69:7-16. |
|16.||Aguwa CN. Effects of exogenous administration of female sex hormones on gastric secretion and ulcer formation in the rat. Eur J Pharmacol 1984;104:79-84. |
|17.||Chakravarty K, Mukherjee BP. Study of the effect of oral contraceptive steroids on experimentally induced ulcers in rat stomach. Indian J Pharmacol 1991;23:214-8. |
[Table 1], [Table 2]