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Effect of α-lipoic acid on fundic gastric mucosal damage induced by acetyl salicylic acid: a histological study

Yehia, Nagah M.; Abdel Salam, Nevert F.; Saleh, Hanan A.; Bayomi, Noha S.

The Egyptian Journal of Histology: June 2014 - Volume 37 - Issue 2 - p 280–291
doi: 10.1097/01.EHX.0000446586.27067.46
Original articles

Introduction α-Lipoic acid (ALA), an endogenous agent, has been shown to combat oxidative stress.

Aim of the work The aim of the study was to evaluate the protective effect of ALA on fundic gastric mucosal damage induced by acetyl salicylic acid (ASA).

Materials and methods Fifty adult male albino rats were divided into four groups: group I (the control group), group II that received ALA for 2 weeks (subgroup IIa) and for 4 weeks (subgroup IIb), group III that received ASA for 2 weeks (subgroup IIIa) and for 4 weeks (subgroup IIIb), and group IV that received ALA 30 min before ASA for 2 weeks (subgroup IVa) and for 4 weeks (subgroup IVb). At the end of the experiment, specimens from the fundus of the stomach were processed for light and electron microscopic examinations. The mean number of proliferating cell nuclear antigen (PCNA)-positive cells, parietal cells, and the mean thickness of the fundic mucosa were measured and the results were statistically analyzed.

Results Examination of sections revealed that ASA for 2 weeks induced widening of the gastric pits and focal mononuclear cellular infiltration. The mucous content of the mucosa was apparently increased and PCNA-positive cells were significantly decreased compared with the control group. ASA for 4 weeks resulted in extensive desquamation, thinning out of the mucosa, and diffuse mononuclear cellular infiltration. The collagen content of the lamina propria showed an apparent increase, whereas the mucous content showed an apparent decrease. The parietal cell count and the PCNA-positive cells were significantly decreased compared with the control group. In ultrathin sections, parietal cells showed cytoplasmic vacuoles, decreased intracellular canaliculi, and mitochondria, whereas the chief cells showed dilated rough endoplasmic reticulum and decreased secretory granules. Concomitant use of ALA showed a histological profile nearly comparable with that of the control group in both subgroups IVa and IVb.

Conclusion ALA administration prevented the structural changes of the gastric mucosa induced by ASA.

Histology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence to Hanan A. Saleh, MD, Histology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Tel: +20 100 111 1372; e-mail: dr.hanan_saleh@yahoo.com

Received February 2, 2014

Accepted March 24, 2014

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Introduction

Gastric ulcer is a multietiologic disease. Steroidal and nonsteroidal drugs have been shown to play a role in gastric ulcerogenesis 1.

NSAIDs including aspirin [acetyl salicylic acid (ASA)] are widely prescribed, despite their gastrointestinal side effects. Several regimens have been recommended to prevent serious gastrointestinal complications such as the use of safer NSAIDs and the coprescription of gastroprotective agents 2.

Reactive oxygen species (ROS) have been proved to play an important role in the pathogenesis of gastric ulcer. Accordingly, various regimens of reactive oxygen metabolite scavengers appear to be new treatment strategies for gastrointestinal diseases 3.

α-Lipoic acid (ALA) is an endogenous agent found in a variety of meats, notably kidneys, heart, and liver, as well as in spinach, broccoli, and potatoes. It has been widely used as a dietary supplement 4. It has also been shown to combat oxidative stress by quenching a variety of ROS 5,6.

Several studies have confirmed the beneficial effect of ALA in the treatment of many diseases in which ROS have been implicated, such as ischemia–reperfusion injury 7, diabetes, diabetic neuropathy 8,9, atherosclerosis, hypertension 10, neurodegeneration, disease of joints, radiation injury, and acquired immune deficiency syndrome 11.

Therefore, the aim of the present study was to evaluate the possible protective effect of ALA on the structural changes of the fundic gastric mucosa induced by administration of ASA.

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Materials and methods

Fifty adult male albino rats (3-month-old) were used in this study with an average weight of 200 g. They were purchased from the Animal House of the Faculty of Medicine, Ain Shams University. They were kept in wire mesh cages under proper sanitary conditions at room temperature. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Research Center, Faculty of Medicine, Ain Shams University. The animals were fed on a balanced rat chow and water ad libitum. They were classified into four groups:

  • Group I (the control group): this group included 10 rats. They were given daily equivalent volume of distilled water (DW) by gastric gavage. Five rats were sacrificed after 2 weeks (subgroup Ia) and the other five rats were sacrificed after 4 weeks (subgroup Ib).
  • Group II: this group included 12 rats. They were subdivided into two subgroups (IIa and IIb), six animals each. They were given ALA (100 mg/kg/day) daily by gastric gavage for 2 weeks (subgroup IIa) and for 4 weeks (subgroup IIb).
  • Group III: this group included 14 rats. They were subdivided into two subgroups (IIIa and IIIb), seven animals each. They were given ASA (200 mg/kg/day) daily for 2 weeks (subgroup IIIa) and for 4 weeks (subgroup IIIb).
  • Group IV: this group included 14 rats that were subdivided into two subgroups (IVa and IVb), seven animals each. They were given ALA (100 mg/kg/day) 30 min before the administration of ASA (200 mg/kg/day) daily for 2 weeks (subgroup IVa) and 4 weeks (subgroup IVb).
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Drugs

  • Acetyl salicylic acid: a commercially available formulation of ASA was used (Rivo tablets 320 mg; Arab Pharma & Chemical Mfg Co., Egypt). Five tablets were dissolved in 40 ml DW (40 mg/ml) and administered by gastric gavage at a dose of 200 mg/kg/day 12.
  • α-Lipoic acid: a commercially available formulation of ALA was used (Thiotacid tablets 300 mg; Eva Pharma, Cairo, Egypt). Four tablets were dissolved in 60 ml DW (20 mg/ml) and administered by gastric gavage at a dose of 100 mg/kg/day 3.

At the end of the experiment, the animals were sacrificed by cervical dislocation. Their stomachs were dissected out, opened along their greater curvature, and washed gently with saline. Specimens from the fundus were excised. They were fixed in 10% formalin and then processed for formation of paraffin sections (5-μm-thick). Sections were stained by H&E, Masson’s trichrome stain for collagen 13, and by the combined Alcian blue–periodic acid–Schiff (PAS) technique for acid and neutral mucins 14.

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Immunohistochemical staining

Avidin–biotin peroxidase technique for proliferating cell nuclear antigen (PCNA) using monoclonal antibody was performed (dilution 1:2000; MP Biomedicals LLC, Germany) 13.

Specimens from the fundic mucosa of the control group, subgroups IIIb, and IVb were fixed in chilled 3% formol glutaraldehyde. They were processed and ultrathin sections were cut and examined with Philips 400 transmission electron microscope at the Electron Microscopy Unit, Faculty of Science, Ain Shams University.

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Morphometric and statistical study

The following parameters were measured using the image analyzer Leica Q500 MC program: (i) the mean thickness of the gastric mucosa; (ii) the mean number of PCNA-positive cells/high-power field in the isthmus region of the fundic gland; and (iii) the mean number of parietal cells/high-power field in the neck region of the fundic glands.

All parameters were measured in five fields per slide. Five slides were used for each group. The mean for each animal was calculated (n=5). Analysis of variance tests were used to compare the different parameters between the groups. Calculations were performed using SPSS (version 17; SPSS Inc., Chicago, Illinois, USA). P value was considered significant if less than 0.05.

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Results

Group I (the control group: Ia and Ib)

H&E-stained sections of the fundic mucosa of the stomach showed that it was formed of surface columnar epithelial cells with underlying lamina propria and muscularis mucosa. The lamina propria consisted of loose connective tissue and was occupied by straight tubular fundic glands having short narrow pits and lying perpendicular to the surface. The fundic glands could be differentiated into three parts: innermost isthmus, middle neck, and outer base. The muscularis mucosa was formed of smooth muscle fibers arranged as inner circular and outer longitudinal fibers (Fig. 1).

Figure 1

Figure 1

The pits and isthmus appeared to be lined by surface columnar mucous-secreting cells having basal rod-shaped nuclei and apical vacuolated cytoplasm (Fig. 2a). The necks of the glands were lined by groups of mucous neck cells having flattened basal nuclei and vacuolated cytoplasm and parietal cells (Fig. 2b).

Figure 2

Figure 2

Parietal cells were present all over the glands resting on the basement but not reaching the lumen. They appeared large polyhedral with acidophilic cytoplasm and central rounded vesicular nuclei (Fig. 2b).

The bases of the glands were lined by chief cells and parietal cells. The chief cells appeared with basal rounded vesicular nuclei and deeply stained basal basophilic cytoplasm with apical acidophilic zymogen granules (Fig. 2c).

Few collagenous fibers were seen within the lamina propria, mostly between the bases of the fundic glands (Fig. 3).

Figure 3

Figure 3

In combined Alcian blue–PAS reaction, the surface columnar mucous-secreting cells showed PAS-positive reaction, whereas the mucous neck cells showed Alcian blue-positive reaction. A mixed coat of both types appeared covering the surface of the mucosa (Fig. 4).

Figure 4

Figure 4

On immunohistochemical staining for PCNA, the sections showed strong positive reaction for PCNA in the cells of the isthmus region of the fundic glands (Fig. 5).

Figure 5

Figure 5

On electron microscopy, the parietal cells appeared with rounded euchromatic nuclei. The cytoplasm contained numerous mitochondria and the characteristic intracellular canaliculi with many microvilli (Fig. 6). Chief cells showed basal euchromatic nuclei. Apical moderately electron-dense secretory granules and numerous cisternae of rough endoplasmic reticulum (RER) were detected (Fig. 7).

Figure 6

Figure 6

Figure 7

Figure 7

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Group II

On examination of sections of the different stains and techniques, the fundus of the stomach after 2 and 4 weeks of ALA administration showed a picture similar to that of the control group.

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Group III

Subgroup IIIa

Examination of the sections showed that the thickness of the mucosa was apparently comparable with that in the control group with preservation of the regular arrangement of the fundic glands (Fig. 8). Focal lesions of the fundic mucosa were detected. Partial desquamation and sloughing of the surface epithelium and widening of the gastric pits were seen (Fig. 8). Within the affected areas of the fundic mucosa, the surface columnar mucous-secreting cells showed deeply stained pyknotic nuclei (Fig. 9a).

Figure 8

Figure 8

Figure 9

Figure 9

Many parietal cells appeared ballooned and vacuolated with eccentric pyknotic nuclei (Fig. 9b). The bases of the glands were disorganized and most of the chief cells had deeply stained pyknotic nuclei (Fig. 9c).

The lamina propria showed focal areas of mononuclear cellular infiltration (Figs 8 and 9a). The collagen content was similar to that seen in sections of the control group.

On Alcian blue–PAS stain, the surface mucous coat, pit regions, isthmus, necks, and the bases of the glands showed an apparent increase in Alcian blue-positive reaction in comparison with the control group (Fig. 10).

Figure 10

Figure 10

Immunohistochemical staining for PCNA showed an apparent decrease in PCNA-positive reaction in the isthmus region of the fundic glands as compared with that in the control group (Fig. 11).

Figure 11

Figure 11

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Subgroup IIIb

H&E-stained sections revealed extensive affection of the gastric mucosa. The gastric pits were wide, and an apparent decrease in the thickness of the mucosa was detected in comparison with the control group (Fig. 12).

Figure 12

Figure 12

The surface columnar mucous-secreting cells were desquamated in many areas. They appeared flattened with deeply stained pyknotic nuclei (Fig. 13a). The parietal cells showed apparent decrease in their number and size with deeply stained pyknotic nuclei (Fig. 13b). Most of the chief cells in the bases of the fundic glands were disorganized and showed pyknotic nuclei (Fig. 13c).

Figure 13

Figure 13

In certain areas, there was desquamation and shedding of the epithelium. Extensive diffuse mononuclear cellular infiltration was detected in the lamina propria (Fig. 12). Congested blood vessels were also detected (Figs 12 and 13c).

The collagen fiber content in the basal parts of the mucosa was apparently increased in comparison with the control group (Fig. 14).

Figure 14

Figure 14

Most of the specimens revealed marked reduction in both Alcian blue and PAS-positive reactions (Fig. 15a). In some areas, an apparent Alcian blue-positive reaction was seen in the bases of the glands (Fig. 15b).

Figure 15

Figure 15

Immunohistochemical staining for PCNA showed an apparent depletion in PCNA-positive reaction in the isthmus region of the fundic glands in comparison with the control group (Fig. 16).

Figure 16

Figure 16

On electron microscopy, the parietal cells showed irregular nuclei with perinuclear space. Numerous cytoplasmic vacuoles were detected and mitochondria were hardly seen. The intracellular canaliculi and microvilli were unapparent (Fig. 17).

Figure 17

Figure 17

The chief cells appeared with irregular flattened nuclei. The cytoplasm showed few irregular dilated cisternae of RER (Fig. 18).

Figure 18

Figure 18

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Group IV

Subgroups IVa and IVb

Examination of H&E-stained sections of group IV (IVa and IVb) showed that the structure and thickness of the fundic mucosa appeared comparable with that in the control group. The mucosa appeared intact with short narrow pits of regularly arranged fundic glands (Fig. 19).

Figure 19

Figure 19

Surface columnar mucous-secreting cells appeared intact with rod-shaped nuclei and apical vacuolated cytoplasm (Fig. 20a). Parietal cells appeared with central rounded vesicular nuclei and acidophilic cytoplasm among groups of mucous neck cells having flattened basal nuclei and pale vacuolated cytoplasm (Fig. 20b). Chief cells appeared lining the bases of the glands with basal rounded nuclei. They had basal basophilic cytoplasm with apical acidophilic zymogen granules (Fig. 20c).

Figure 20

Figure 20

On Masson’s trichrome stain, few collagenous fibers appeared between the bases of the fundic glands comparable with that in the control group (Fig. 21).

Figure 21

Figure 21

On Alcian blue–PAS stain, the surface columnar mucous-secreting cells showed a PAS-positive reaction, whereas the mucous neck cells showed Alcian blue-positive reaction. A mixed coat of both types appeared covering the surface of the mucosa (Fig. 22).

Figure 22

Figure 22

Immunohistochemical staining for PCNA showed positive reaction located at the isthmus of the glands comparable with that in the control group (Fig. 23).

Figure 23

Figure 23

On electron microscopy, subgroup IVb showed that the parietal cells appeared with rounded euchromatic nuclei and numerous mitochondria. The intracellular canaliculi showed many microvilli (Fig. 24).

Figure 24

Figure 24

The chief cells showed basally located euchromatic nuclei, apical moderately electron-dense secretory granules, and abundant RER (Fig. 25).

Figure 25

Figure 25

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Statistical and morphometric results

The mean thickness of the gastric mucosa and the mean number of parietal cells showed significant reduction in subgroup IIIb as compared with that in the control group. The other groups showed a nonsignificant change compared with the control group. There was also a significant increase in subgroup IVb as compared with subgroup IIIb in these two parameters (Table 1).

Table 1

Table 1

The mean number of PCNA-positive cells showed a significant reduction in subgroups IIIa and IIIb as compared with that in the control group, whereas the other groups showed a nonsignificant difference. In contrast, there was a significant increase in subgroup IVa as compared with subgroup IIIa and also in subgroup IVb as compared with subgroup IIIb (Table 1).

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Discussion

The present study was carried out to evaluate the protective effects of ALA on fundic mucosal damage induced by ASA (aspirin).

In this study, administration of aspirin for 2 weeks induced focal damage to the fundic mucosa in the form of widening of the gastric pits and desquamation and sloughing of mucosal cells into the lumen. Moreover, the prolonged period of aspirin administration for 4 weeks resulted in more extensive lesions, including thinning of the mucosa, sloughing, and congestion of blood vessels.

These findings were in accordance with those of previous researchers who attributed these alterations to decreased antioxidant enzymes such as glutathione, glutathione peroxidase, catalase, and superoxide dismutase with aspirin 15. In contrast, aspirin administration was found to increase the volume of gastric juice secretion and total acidity leading to sloughing ulceration of the mucosa 12.

There was a significant reduction in the thickness of the gastric mucosa after 4 weeks of aspirin administration as compared with that in the control group. Similarly, in a clinical study, 38% of NSAIDs-treated patients showed chronic gastritis with thinning out of the gastric mucosa 16. Furthermore, in mice, aspirin resulted in gastric inflammation and decreased mucosal thickness 17.

In this study, parietal and chief cells of aspirin-treated rats appeared with pyknotic nuclei, and a significant reduction in parietal cell count was detected as compared with the control group. These changes were attributed to the generation of ROS resulting in oxidative damage in the cellular membrane and cell lysis 18 or to increased mitochondrial permeability, mitochondrial failure, and translocation of the intramitochondrial protein apoptosis-inducing factor 19.

In addition, the current study revealed congestion of the blood vessels in the lamina propria of the fundus of aspirin-treated rats. This was in accordance with a previous study in which a single oral dose of 200 mg/kg of aspirin produced hemorrhagic linear erosions with dilatation and congestion of the nearby blood vessels 20.

The cellular infiltration detected in the present study could be explained by the loss of integrity of the intercellular junctions caused by aspirin 16. This exposes the mucosa to the effect of acid and enzymes, with subsequent bacterial invasion. Bacteria will then secrete chemotactic factors for neutrophils, lymphocytes, and macrophages 16.

Consequently, many growth factors become elaborated by the inflammatory mononuclear cells attracted to the site of injury. Among these growth factors was the basic fibroblastic growth factor, which induced fibroblast proliferation and collagen deposition at the site of inflammation 21. This could explain the increased collagen content detected in this study after 4 weeks of aspirin administration.

The findings of the present study revealed that, after 2 weeks of aspirin administration, the Alcian blue binding capacity was much increased at the surface mucous coat, pit regions as well as in the mucous neck cells. These results were in agreement with those of previous researchers who observed that, in low-dose aspirin takers, the collected gastric juice showed a significant increase in the gastric mucous secretion as compared with the control group, suggesting a functional adaptive response 22.

In contrast, after 4 weeks of aspirin intake, depletion in both PAS and Alcian blue reaction was observed. These findings were in accordance with previous reports, which reported that, during long-term administration of aspirin, there was inhibition of glycoprotein biosynthesis by the epithelial cells, indicating failure of gastric adaptation against the injurious effects of aspirin 23–25.

In certain areas, the bases of some fundic glands of the mucosa were positive for Alcian blue staining. Chief cells could store mucous in stressful circumstances to increase the cytoprotective potential of the gastric mucosa 26. Accordingly, the basal cells detected with Alcian blue stain in group IIIb could be chief cells, which started to synthesize mucous in addition to their serous secretion in order to contribute to the gastric defensive mechanisms against aspirin.

In the present study, a significant decrease in the labeled nuclei for PCNA was detected after 2 and 4 weeks of aspirin intake. This denoted a decrease in the proliferating capacity of stem cells. This was similar to previous reports 27,28. In view of this, Wallace 29 explained that aspirin reduced the binding capacity of the epithelial growth factor to its receptor and inhibited the epithelial growth factor-signaling pathways. He added that there was an increase in the synthesis of antiproliferative products of arachidonic acid metabolism 29.

The different mechanisms by which aspirin induced mucosal damage included a direct cytotoxic mechanism through osmotic lysis 29, suppression of cyclo-oxygenase enzyme and inhibition of prostaglandin synthesis 30, reduced blood flow 31, and accumulation of oxygen free radicals 32.

In the present study, ALA administration before aspirin preserved the structure of the gastric mucosa from its injurious effects. Previous studies revealed a protective effect of ALA before indomethacin 33 and ethanol intake 3 as detected by reduced ulcer index and decreased congestion of blood vessels.

ALA is a dithiol antioxidant and is an important cofactor in pyruvate dehydrogenase and ketoglutarate dehydrogenase enzymes in the mitochondria 3. It preserves the membrane-bound ATPase enzyme 4. ALA exerts antioxidant effects on the biological systems through ROS quenching and also through an action on transition metal chelation 34. It was also capable of quenching reactive nitrogen species, thus preventing DNA damage and reduced lipid peroxidation 3. Furthermore, ALA markedly improved cysteine availability within the cell, resulting in accelerated glutathione synthesis compensating for its depletion during oxidative stress 35.

In the present study, ALA administration revealed preservation of the cells lining the fundic glands as detected by light and electron microscopy. In a previous study, DNA fragmentation, apoptosis, and cell death were significantly reduced when ALA was concomitantly administered with acetic acid 4.

The collagen fiber content was preserved with ALA administration in this study. According to previous reports, ALA reduced the transforming growth factors involved in fibroblast differentiation and collagen production 36.

Furthermore, in the current study, ALA prevented mononuclear cellular infiltration and the congestion of blood vessels. It was proved in a previous study that ALA suppressed neutrophil accumulation in the intestine, and consequently the release of toxic metabolites and proteases 5. Moreover, ALA showed potent anti-inflammatory effects through inhibition of vascular adhesion molecule and intercellular adhesion molecule production. It also inhibited chemoattractant protein secretion and natural killer cell activation and cytotoxicity 37.

The present study revealed preservation of mucous production by the glandular epithelium in groups IVa and IVb as compared with that in the control group. This was in agreement with the study by Gutierrez-Cabano 38 who found that ALA protection was related to an increase in both the fluid volume and the mucous volume retained in the gastric lumen and to the regeneration of exogenous and endogenous antioxidants.

In the present study, preservation of the proliferative cells was detected by PCNA technique in rats receiving ALA before aspirin. This indicated normal cellular proliferation, which maintained mucosal integrity. In view of this point, previous studies stated that ALA could have a dual action. It might act as a growth factor or stimulate other growth factors or receptors in normal cells. However, it might act as an antiproliferative agent in cancer cells through arrest of the cell cycle 39,40.

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Conclusion

From the previous results, it could be concluded that ALA protected the structure of the fundic mucosa against the injurious effects of aspirin.

Clinical studies are recommended to detect the benefit of ALA when concomitantly used with aspirin.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table

Table

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Keywords:

acetyl salicylic acid; electron microscopy; gastric mucosa; α-lipoic acid; proliferating cell nuclear antigen

© 2014 The Egyptian Journal of Histology