IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-γ-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

Abstracts

Context

Hydrogen sulphide (H₂S) has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H₂S synthesis is essential to gastric protection against ethanol.

Objective

To better understanding the role of H₂S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) isoforms in gastric mucosa of mice treated with saline or 50% ethanol.

Methods

Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g). After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS.

Results

We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol.

Conclusion

Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

Gastric mucosa; Cystathionine-β-Synthase; Cystathionine-γ-Lyase; Ethanol; Immunohistochemistry; Mice

Contexto

O sulfeto de hidrogênio (H₂S) tem sido mostrado como um neuromodulador e contribuidor para a manutenção da integridade da mucosa gástrica na lesão causada por drogas antiinflamatórias não esteroidais. Previamente, demonstramos que a síntese de H₂S é essencial para a proteção da mucosa gástrica contra a administração de etanol.

Objetivo

Para compreender o papel do H₂S e a localização detalhada de sua produção no estômago normal e após lesão induzida pela administração de etanol, estudou-se a expressão das isoformas cistationina-γ-liase (CSE) e cistationina-β-sintetase (CBS) na mucosa gástrica de camundongos tratados com salina ou etanol 50%.

Métodos

Os camundongos foram tratados por gavagem com salina ou etanol 50% (0,5 mL/25 g). Após 1 hora, os camundongos foram sacrificados e os tecidos gástricos foram avaliados por análise histológica e imunoistoquímica específica para CBS e CSE.

Resultados

Foi demonstrado expressão não específica de CBS na mucosa gástrica normal e expressão de CSE ocorrendo principalmente nas células parietais dos animais tratados com etanol.

Conclusão

Assim, demonstramos que a expressão de CBS parece ser constitutiva e difusa através do epitélio gástrico, enquanto a expressão de CSE parece ser induzida nas células parietais por agentes lesivos como o etanol.

Mucosa gástrica; Cistationina-β-Sintase; Cistationina-γ-Liase; Etanol; Imunoistoquímica; Camundongos

INTRODUCTION

Ethanol ingestion causes acute gastric mucosal lesions in humans⁽⁵⁾. Ethanol-induced gastric damage is characterized by epithelial cellular loss, mucosal edema and subepithelial hemorrhage(⁶, ⁹). The molecular mechanism of ethanol-induced gastric injury has not yet been completely elucidated.

Hydrogen sulfide (H₂S) is a gaseous transmitter involved in a multitude of physiologic functions, which includes immune and inflammatory processes⁽²⁾, perception, and pain mediation, as well as control of gastric mucosal integrity, vascular tone, and the control of gastrointestinal motility⁽⁴⁾. H₂S is synthesized endogenously from L-cysteine, primarily via two enzymes: cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS)⁽⁹⁾. CBS and CSE are expressed in a different extent in neurons in the brain and in the enteric nervous system⁽⁹⁾. Recent studies suggest that H₂S may similarly contribute to mucosal defense(³, ¹⁰). The gastric mucosa expresses both CSE and CBS and has the ability to generate H₂S, but CSE seems to be the main enzyme involved in the H₂S generation by the gastric mucosa(³, ⁴).

The mechanism through which H₂S protects the stomach from damage due to ethanol has not been completely elucidated. Therefore, the aim of this study was to determine the effect of ethanol on the immunoexpression of CSE/CBS in the gastric mucosa of mice.

METHODS

Animals procedures

Male Swiss mice (25 g–30 g) were fasted 18h to 24h before the experiments. All animal treatments and surgical procedures of the present study were performed in accordance with the ethical principles of the Federal Law 11.794 (October 2008) and Decree 66689 (July 2009) dictated by the Brazilian Society of Animal Science Laboratory (SBCAL) and revised and approved by the local ethics committee (UFPI protocol 63/07).

Tissue collection and immunohistochemical assay

The present study used 10 mice and was divided into two groups: normal group (treated only saline) or 50% ethanol (0.5 mL/25 g). One hour later, the animals were sacrificed after being weighed and anesthetized with 40 mg/kg of intraperitoneal thiopental (Abbott Laboratories, Chicago, IL, USA). The stomachs were rapidly removed and a sample of the corpus region was fixed in 10% neutral buffered formalin solution, sectioned and embedded in paraffin to histological analysis.

Specific immunohistochemistry for cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) was performed according to Costa et al.⁽¹⁾. The gastric mucosal cells were identified by their specific morphology, localization and staining behavior and their total number was counted at ×250 magnification. Each microscopic slide contained three portions of the 5 cm gastric mucosa segment. A pathologist blinded to the experimental conditions used a light microscope to count all the immunopositive cells. Three full countings were performed, and the last counting results were used in the analysis. The gastric chief cell, parietal cells and mucosal cells were counted.

Statistical analysis

All values are expressed as means ± SEM. ANOVA and student test t were used to determine statistical significance of differences between groups. Differences were considered to be significant when P<0.05.

RESULTS

Figure 1 show that ethanol administration increased the CSE immunoexpression exclusively in the parietal cell and all gastric mucosal cells express CBS. In control group (saline alone) the immunostaining to CBS was especially seen in chief cells, although it was also observed some immunostaining in the mucosal cells (Figure 1 – panel B and Table 1). However, the animal treatment with ethanol 50% was accompanied by a wider parietal cells staining (Figure 1 – panel C and Table 1). Unlike CBS, there was no significant immunostaining of CSE in the control group (Figure 1 – panel D and Table 1). However, in animals treated with ethanol 50%, CSE was notably expressed in parietal cells (Figure 1 – panel E).

FIGURE 1.
Immunohistochemical features of cystathionine γ-lyase (CBS) and cystathionine β–synthetase (CSE) in the gastric mucosa of normal mice and ethanol-induced gastric damage. (A) Negative control (immune-negative cells); (B-C) Immunostaining for CBS in the stomach of an animal treated only with saline (panel B) or treated with ethanol 50% (panel C) with arrows marking indicating the chief cells and arrowheads indicate labeling in mucosal cells (panel B) or parietal cells (panel C). (D-E) There was no significant immunostaining of CSE in the control group (panel D). However, in animals treated with ethanol 50%, was observed to mark CSE in parietal cells (panel E) and some mucosal cells. (F) Counting of CBS and CSE positive immunostained cells (five animals from each group). The results are presented as means ± S.E.M. * < 0.05 vs saline + CSE group

TABLE 1.
Ethanol effect on the immunohistochemical expression of CBS and CSE in gastric mucosa cells of mice

Experimental group	Enzyme	Number of chief cells	Number of parietal cells	Number of mucosal cells
Saline	CBS	19.3 ± 2.1	8.9 ± 1.7	24.5 ± 4.3
Saline	CSE	2.2 ± 1.0	3.1 ± 1.2	3.2 ± 2.3
Ethanol	CBS	23.8 ± 5.2	19.2 ± 4.2a	9.6 ± 2.3a
Ethanol	CSE	8.8 ± 3.3	21.9 ± 2.2b	12.5 ± 1.0b

• Results are mean ± SEM of 5-6 mice.
• ^a
  P<0.05, when compared with CBS in saline group;
• ^b
  P<0.05, when compared with CSE in saline group

DISCUSSION

It was demonstrated that H₂S contributes to the maintenance of gastric mucosal integrity in damage caused by nonsteroidal anti-inflammatory drugs⁽¹⁰⁾ and ethanol⁽³⁾. In the present study, we evaluated the ethanol effect on the immunohistochemical expression of CBS and CSE in gastric mucosa of mice. Cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) can use L-cysteine as an alternative substrate to form hydrogen sulfide (H₂S). The gastric mucosa expresses both CSE and CBS and has the ability to generate H₂S⁽¹⁰⁾, and CSE seems to be the main enzyme involved in the H₂S generation by the gastric mucosa(⁹, ¹⁰).

Detailed localization of CSE and CBS in stomach has not yet been evaluated and general agressors has ability to modulate CSE expression, but they do not affect CBS expression. Our results clearly showed that administration of ethanol resulted in an increased immunostaining of CSE but not of CBS in the gastric wall. We results demonstrated that the control group (saline) showed only immunostailing of CBS, that was expression mainly in chief and mucosal cells. However, after ethanol administration, there was also expression in parietal cells. Likewise, the ethanol promotes expressions of CSE in parietal cells. Our findings show that ethanol promotes an increase in coexpression of CSE especially in the parietal cells, suggesting that these cells may be mainly responsible for the synthesis of H₂S involved stomach gastroprotection. In fact, we previously demonstrated that H₂S donor and H₂S precursor (L-cysteine) decreased ethanol-induced gastropathy⁽¹⁰⁾. Thus, this increase expression of CSE may be a mechanism of defense against the ethanol-induced gastric damage.

Little is known about the cellular consequences of an elevated CSE expression or about the associated increase in endogenously produced H₂S. CSE-derived H₂S inhibits cell proliferation(⁸, ¹⁴) and induces cell death predominantly by an apoptotic mechanism in polymorphonuclear cells(¹¹, ¹³). Deficiency of CSE activity in humans is presumed to cause cystathioninemia (cystathioninuria), an autosomal recessive inborn error probably with no consistent clinical consequence(¹², ¹⁴).

The CSE transcript, protein and enzymatic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli⁽⁷⁾. These results suggest important physiological roles for CSE in mice. We demonstrated that, after ethanol administration, almost all gastric mucosal cells express CBS while the expression of CSE occurs almost exclusively in the parietal cell. These results showed that the gastric epithelium therefore could be one source of H₂S in the gastric wall. Furthermore, the coexpression of CSE and CBS in gastric mucosal might be an indication that they are equally involved in the production of H₂S(⁴, ⁷).

Our data supports that CSE expression increased during the gastric damage induced by ethanol and it suggests the involvement of H₂S during this event. Because the CSE/H₂S pathway is also involved in regulating gastrophaty induced by ethanol these observations raise the possibility of H₂S-modifying agents being used to improve resistance to gastric mucosa injury. Overall, our incomplete understanding of the role and mechanisms of action of H₂S in many aspects of physiology and pathophysiology of the gastrintestinal tract provides a field for extensive research in the future.

CONCLUSIONS

We conclude that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol. Thus, future studies need to address the relevance of CBS and CSE produced H₂S for the signaling and gastroprotect effects.

We appreciate the excellent technical support of Maria Silvandira. The authors gratefully acknowledge the financial support from Research Foundation for the State of Piauí (FAPEPI) and National Council of Technological and Scientific Development (CNPq) (Brazil).

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Publication Dates

• Publication in this collection
  April-June 2013

History

• Received
  15 Feb 2013
• Accepted
  12 Mar 2013