Erythromycin attenuates oxidative stress-induced cellular senescence via the PI3K-mTOR signaling pathway in chronic obstructive pulmonary disease

Abstract

Background and Purpose: Chronic obstructive pulmonary disease (COPD) is proposed to hasten lung aging. Erythromycin protects against oxidative stress and inflammatory responses. However, the potential anti-senescence effect of erythromycin remains disclosed. In the present study, we investigated whether erythromycin influenced oxidative stress-induced cellular senescence and investigated its related mechanisms. Methods: A cigarrete smoke (CS) -induced emphysema mouse model and a H₂O₂-induced premature senescence model in human bronchial epithelial cell line (BEAS-2B) were established. Senescence-related markers (P53, P21 and SA-β-Gal activity), and levels of oxidative stress biomarkers (MDA, SOD and ROS) were measured. Additionally, cells were pretreated with rapamycin (mTOR inhibitor) or erythromycin, and the expression levels of components of the PI3K-mTOR signaling pathway were measured in BEAS-2B cells. Results: Exposed to H₂O₂, increased SA-β-gal activity was observed in BEAS-2B cells suggesting premature senescence. Erythromycin inhibited the expression of P53 and P21 in the CS-induced emphysema mouse model. MDA levels significantly increased and SOD levels decreased in the CS-exposed mice and H₂O₂-induced BEAS-2B cells. Rapamycin and erythromycin significantly suppressed the expression of P53 and P21. Additionally, rapamycin and erythromycin inhibited the PI3K-mTOR signaling pathway. Conclusion: Our findings suggest that erythromycin ameliorates oxidative stress-induced cellular senescence via the PI3K-mTOR signaling pathway. Hence, we establish a theoretical foundation for the clinical application of erythromycin for COPD prevention and treatment.

Keywords: PI3K-mTOR signaling pathway; cellular senescence; chronic obstructive pulmoanry disease; erythromycin; oxidative stress.

FIGURE 1

MLI and IHC staining of P53 and P21 in murine lungs (A–B) Alveolar MLI. Magnification: ×200. (C–D) IHC staining of P53. Magnification: ×400. (E–F) IHC staining of P21. Magnification:×400. (G) Scatter plot of correlation analysis for mean density of P53 and P21 staining with MLI in murine lungs. ***p < 0.0001. **p < 0.001. CS, cigarrete smoke. EM,erythromycin. MLI, mean linear intercept. IHC, immunohistochemistry.

FIGURE 2

Erythromycin inhibited the expression of P53 and P21 in mice with CS-induced emphysema. (A)Protein expression levels of P53 and P21. (B‐C) mRNA expression levels of P53 and P21. ***p < 0.0001, **p < 0.001.

FIGURE 3

Antioxidant effect of erythromycin in vivo and in vitro. (A) Fluorescence photomicrographs of different groups of BEAS-2B cells. BEAS-2B cells were treated with 100  $\mu$ M H₂0₂,10 mg/m1 erythromycin or lOnM rapamycin. (B–C) SOD and MDA levels in mice. (D–E) SOD and MDA levels in BEAS-2B cells. ***p < 0.0001, **p < 0.001, *p < 0.05. EM, erythromycin. Rapa, rapamycin.

FIGURE 4

Effects of H₂0₂ in BEAS-2B cells. (A–B) SA- $\beta$ -gal staining of BEAS-2B cells treated with different concentration of H₂0₂. Magnification: ×200. (C) Cell viability assay of BEAS-2B cells treated with different concentration of H₂0₂. (D–E) Effects of H₂0₂ on P53 and P21 protein and mRNA expression. ***p < 0.0001, **p < 0.001.

FIGURE 5

Effects of erythromycin in H₂0₂-treated BEAS-2B cells. (A–B) SA-13-gal staining of BEAS-2B cells treated with or without erythromycin and H₂0₂. Magnification: ×200. (C) Cell viability assay of BEAS-2B cells treated with different concentrations of erythromycin. (D–E) Expression of senescence markers P53 and P21. ***p < 0.0001, **p < 0.001,*p < 0.005.

FIGURE 6

The PI3K-mTOR signaling pathway contributed to H₂0₂-induced cellular senescence in BEAS-2B cells. (A–B) SA-13-gal staining of BEAS-2B cells treated with or without rapamycin and H202. Magnification: ×200. (C) Cell viability assay of BEAS-2B cells treated with different concentrations of rapamycin. (D–E) Protein and mRNA levels of P53 and P21. (F) The expression level of proteins of the PI3K-mTOR pathway. **p < 0.001, *p < 0.005.

FIGURE 7

Erythromycin inhibited H₂0₂-induced BEAS-2B cellular senescence via the PI3K-mTOR signaling pathway. The expression level of proteins of the PI3K-mTOR pathway was assessed using western blot. ***p < 0.0001, **p < 0.001,*p < 0.005.