Modulation of the oscillatory mechanics of lung tissue and the oxidative stress response induced by arginase inhibition in a chronic allergic inflammation model

Abstract

Background: The importance of the lung parenchyma in the pathophysiology of asthma has previously been demonstrated. Considering that nitric oxide synthases (NOS) and arginases compete for the same substrate, it is worthwhile to elucidate the effects of complex NOS-arginase dysfunction in the pathophysiology of asthma, particularly, related to distal lung tissue. We evaluated the effects of arginase and iNOS inhibition on distal lung mechanics and oxidative stress pathway activation in a model of chronic pulmonary allergic inflammation in guinea pigs.

Methods: Guinea pigs were exposed to repeated ovalbumin inhalations (twice a week for 4 weeks). The animals received 1400 W (an iNOS-specific inhibitor) for 4 days beginning at the last inhalation. Afterwards, the animals were anesthetized and exsanguinated; then, a slice of the distal lung was evaluated by oscillatory mechanics, and an arginase inhibitor (nor-NOHA) or vehicle was infused in a Krebs solution bath. Tissue resistance (Rt) and elastance (Et) were assessed before and after ovalbumin challenge (0.1%), and lung strips were submitted to histopathological studies.

Results: Ovalbumin-exposed animals presented an increase in the maximal Rt and Et responses after antigen challenge (p<0.001), in the number of iNOS positive cells (p<0.001) and in the expression of arginase 2, 8-isoprostane and NF-kB (p<0.001) in distal lung tissue. The 1400 W administration reduced all these responses (p<0.001) in alveolar septa. Ovalbumin-exposed animals that received nor-NOHA had a reduction of Rt, Et after antigen challenge, iNOS positive cells and 8-isoprostane and NF-kB (p<0.001) in lung tissue. The activity of arginase 2 was reduced only in the groups treated with nor-NOHA (p <0.05). There was a reduction of 8-isoprostane expression in OVA-NOR-W compared to OVA-NOR (p<0.001).

Conclusions: In this experimental model, increased arginase content and iNOS-positive cells were associated with the constriction of distal lung parenchyma. This functional alteration may be due to a high expression of 8-isoprostane, which had a procontractile effect. The mechanism involved in this response is likely related to the modulation of NF-kB expression, which contributed to the activation of the arginase and iNOS pathways. The association of both inhibitors potentiated the reduction of 8-isoprostane expression in this animal model.

Figure 1

Timeline of the experimental protocol. The guinea pigs underwent 7 inhalations (2 per week with 2- to 3-day intervals over 4 weeks) with aerosols of normal saline or ovalbumin solution and increasing doses of antigen. For the 1^st through the 4^th inhalations, the dose used was 1 mg/mL of ovalbumin (2 weeks). In the 5^th and 6^th inhalations (3^rd week), the animals inhaled 2.5 mg/mL of ovalbumin, and for the 7^th inhalation (beginning in the 4^th week) 5 mg/mL of antigen was used. Treatment with 1400W started 30 minutes before the 7^th inhalation and was given daily until the oscillatory mechanics assessment. The solution of ovalbumin or normal saline was continuously aerosolized for 15 minutes or until respiratory distress occurred. Seventy-two hours after the 7^th inhalation, the animals were anesthetized, exsanguinated, and their lungs were removed for oscillatory mechanics measurement. Nor-NOHA (10 μM) was infused in the bath during the evaluation of oscillatory mechanics.

Figure 2

“Box-plots” of the percentage of increase in tissue resistance (Rt%) (A) and elastance (Et%) of the five experimental groups (B). *p<0.001 compared to SAL, OVA-NOR, OVA-W, and OVA-NOR-W groups.

Figure 3

“Box-plots” of arginase 2 expression (%) in the alveolar septa of the five experimental groups. *p<0.001 compared to SAL, OVA-NOR, OVA-W, and OVA-NOR-W groups. **p<0.001 compared to OVA-NOR-W group.

Figure 4

“Box-plots” of arginase 2 activity in the alveolar septa of the five experimental groups. *p<0.05 compared to SAL, OVA-NOR and OVA-NOR-W groups. There were no differences between OVA and OVA-W groups.

Figure 5

“Box-plots” of iNOS-positive cells in the alveolar septa of the five experimental groups. *p<0.001 compared to the SAL, OVA-NOR, and OVA-NOR-W groups. **p<0.001 compared to OVA-NOR-W group.

Figure 6

“Box-plots” of NF-kB expression (%) in the alveolar septa of the five experimental groups. *p<0.05 compared to SAL, OVA-NOR, OVA-W and OVA-NOR-W groups. **p<0.001 compared to SAL, OVA-NOR and OVA-NOR-W groups.

Figure 7

“Box-plots” of PGF2α expression (%) in the alveolar septa of the five experimental groups. *p<0.001 compared to SAL, OVA-NOR, OVA-W and OVA-NOR-W groups. **p<0.001 compared to OVA-NOR group. ^#p<0.001 compared to SAL and OVA-NOR.

Figure 8

Guinea pig lung tissue samples obtained from controls (panels A, B, C and D), OVA-exposed and vehicle-treated animals (panels E, F, G and H), OVA-exposed and nor-NOHA treated animals (panels I, J, K and L), OVA-exposed and 1400 W treated animals (panels M, N, O and P), and OVA-exposed and nor-NOHA and 1400 W treated animals (panels Q, R, S and T). Immunohistochemistry analysis for arginase (panels A, E, I, M and Q - ×400), iNOS positive cells (panels B, F, J, N and R - ×1000), NF-kB (panels C, G, K, O and S – ×400), and PGF2α (panels D, H, L, P and T - ×400). The control group showed low amounts of iNOS positive cells, NF-kB, arginase and PGF2α in alveolar tissue sections. In contrast, the distal lung parenchyma of OVA-exposed and vehicle-treated animals showed an increase in the amount of arginase, iNOS-positive cells, NF-kB and PGF2α. The 1400 W and nor-NOR treatments in ovalbumin-exposed animals reduced all these parameters compared to the OVA group. Both treatments in conjunction contributed to a greater PGF2α reduction.