Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema

Abstract

Background: Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD). Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner.

Methods: We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20) and 40 mg/mL (E40) papain.

Results: The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p < 0.05) and E40 (52.27 ± 4.7; crossed the alveolar intercepts) groups. As compared to the control group, there was a reduction on body (171.6 ± 15.9 g) and muscle weight (251.87 ± 24.87 mg) in the E20 group (157.5 ± 10.3 mg and 230.12 ± 23.52 mg, for body and muscle weight, respectively), which was accentuated in the E40 group (137.4 ± 7.2 g and 197.87 ± 10.49 mg, for body and muscle weight, respectively). Additionally, the thiobarbituric acid reactive substances (TBARS), tert-butyl hydroperoxide-initiated chemiluminescence (CL), carbonylated proteins, and chymotrypsin-like proteolytic activity were elevated in the E40 group as compared to the E20 group (p < 0.05 for all comparisons). The severity of emphysema significantly correlated with the progressive increase in CL (r = -0.95), TBARS (r = -0.98), carbonyl proteins (r = -0.99), and chymotrypsin-like proteolytic activity (r = -0.90). Furthermore, augmentation of proteolytic activity correlated significantly with CL (r = 0.97), TBARS (r = 0.96), and carbonyl proteins (r = 0.91).

Conclusions: Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of oxidative stress in a severity-dependent manner.

Figure 1

Histological images from lungs of papain- and saline-treated hamsters. (A) control + saline (CS): animals treated with approximately 0.3 mL of saline only; (B) emphysema 20 mg/mL (E20): animals treated with approximately 0.3 mL of 20 mg/mL papain in saline; and (C) emphysema 40 mg/mL (E40): animals treated with approximately 0.3 mL of 40 mg/mL papain in saline. Animals were euthanized after 60 days. Hematoxylin and eosin (H & E) images are shown at 50× magnification. Alveolar destruction was determined by the number of times that a predetermined group of coherent lines (1.25 mm² of total area and 1.50 mm of total length) crossed the parenchymal structures. The lesser these structures are crossed, the more extensive is the lesion.

Figure 2

Effect of muscle loss on gastrocnemius muscle lipid hydroperoxide and antioxidant levels in emphysemic hamsters and controls. (A) Tert-butyl hydroperoxide-initiated chemiluminescence was monitored continuously for 40 min. The area under the curve for each animal was determined for comparisons between groups. (B) Total Reactive Antioxidant Potential in gastrocnemius muscles of emphysema and control hamsters. The bars represent the means of 8 animals. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, with p < 0.05 considered significant. *p < 0.001 to CS; ^#p < 0.001 relative to E20, as detected by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 3

Effect of emphysema on skeletal muscle in terms of thiobarbituric acid reactive substances (TBARS) and carbonyl proteins in hamsters. (A) TBARS levels in muscle homogenates of hamsters treated with 2 different doses of papain. (B) Levels of protein carbonylation in muscle homogenates from control and emphysema hamsters. Results are presented as mean ± standard error of the mean (SEM; n = 8). Statistical differences were detected by one-way ANOVA followed by Bonferroni’s multiple comparison test. Control + saline (CS): animals treated with approximately 0.3 mL of saline only; emphysema 20 mg/mL (E20): animals treated with approximately 0.3 mL of 20 mg/mL papain in saline; and emphysema 40 mg/mL (E40): animals treated with approximately 0.3 mL of 40 mg/mL papain in saline. *p < 0.001 relative to CS, as detected by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 4

Chymotrypsin-like proteolytic activity was evaluated in the gastrocnemius muscle of hamsters subjected to intratracheal instillation of papain or saline. *p < 0.001 to CS; ^#p < 0.001 relative to E20, as detected by one-way ANOVA followed by Bonferroni’s multiple comparison test.