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. 2022 Feb;13(2):4028-4038.
doi: 10.1080/21655979.2022.2031769.

Methylprednisolone up-regulates annexin A1 (ANXA1) to inhibit the inflammation, apoptosis and oxidative stress of cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model, through the formyl peptide receptor 2 (FPR2) receptors and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway

Chan Yu  1 , Linghui Zhang  2
Affiliations

Methylprednisolone up-regulates annexin A1 (ANXA1) to inhibit the inflammation, apoptosis and oxidative stress of cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model, through the formyl peptide receptor 2 (FPR2) receptors and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway

Chan Yu et al. Bioengineered. 2022 Feb.

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive degenerative disease, of which smoking is the main causer. We carried out this study with the aim of exploring the underlying mechanism of methylprednisolone (MP) treating the COPD. To stimulate COPD in vitro, cigarette smoke extract (CSE)was employed to induce human bronchial epithelial cells BEAS-2B. With the help of MTT and Tunel assays, the viability and apoptosis of BEAS-2B cells after indicated treatment were assessed. The levels of inflammatory response and oxidative stress were determined by the changes of markers basing on their commercial kits. Additionally, annexin A1 (ANXA1) expressions at both protein and mRNA levels were assessed with Western blot and Reverse transcription‑quantitative PCR (RT-qPCR). Moreover, the expressions of apoptosis- and formyl peptide receptor 2 (FPR2) receptors and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway-related proteins were determined with Western blot., related proteins and proteins. As a result, MP up-regulated the ANXA1 expression in CSE-induced BEAS-2B cells. MP enhanced the viability but suppressed the apoptosis, inflammatory response and oxidative stress of CSE-induced BEAS-2B cells via regulating FPR2/AMPK pathway, while ANXA1 knockdown exhibited oppositive effects on them. In conclusion, MP up-regulated ANXA1 to inhibit the inflammation, apoptosis and oxidative stress of BEAS-2B cells induced by CSE, alleviating COPD through suppressing the FPR2/AMPK pathway.

Keywords: ANXA1; FPR2/AMPK pathway; Methylprednisolone; bronchial epithelial cells; chronic obstructive pulmonary disease.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
MP increases the viability of CSE-induced BEAS-2B cells. (a) The viability of BEAS-2B cells treated with 1, 2, 5 and 10% CSE for 24 h was detected by CCK-8 assay. *P < 0.05 and ***P < 0.001 vs. Control group. $$P < 0.01 vs. 2% CSE group. @@P < 0.01 vs. 5% CSE group. (b) The viability of BEAS-2B cells treated with 5% CSE for 12, 24 and 48 h was detected by CCK-8 assay. **P < 0.01 and ***P < 0.001 vs. Control group. ###P < 0.001 vs. 12 h group. $P < 0.05 vs. 24 h group. (c) The viability of BEAS-2B cells treated with 2, 5 and 10 μM MP was detected by CCK-8 assay. (d) The viability of BEAS-2B cells treated MP and CSE was detected by CCK-8 assay. *P < 0.05 and ***P < 0.001 vs. Control group.
Figure 2.
Figure 2.
MP inhibits the apoptosis of CSE-induced BEAS-2B cells. (a and b) The apoptosis of BEAS-2B cells treated MP and CSE was detected by Tunel assay. (c) The expression of apoptosis-related proteins in CSE-induced BEAS-2B cells pre-treated with MP was determined by Western blot. ***P < 0.001 vs. Control group. ###P < 0.001 vs. CSE group. $P < 0.05 and $$$P < 0.001 vs. 2 μM + CSE group. @@P < 0.01 and @@@P < 0.001 vs. 5 μM + CSE group.
Figure 3.
Figure 3.
MP attenuates the inflammatory response and oxidative stress of CSE-induced BEAS-2B cells. The levels of inflammatory factors TNF-α (a), IL-6 (b) and IL-1β (c) in CSE-induced BEAS-2B cells pre-treated with MP were determined by ELISA assay kits. The levels of oxidative stress factors SOD (d), GSH-Px (e) and MDA (f) in CSE-induced BEAS-2B cells pre-treated with MP were determined by assay kits. ***P < 0.001 vs. Control group. #P < 0.05, #P < 0.01 and ###P < 0.001 vs. CSE group. $$P < 0.01 and $$$P < 0.001 vs. 2 μM + CSE group. @@@P < 0.001 vs. 5 μM + CSE group.
Figure 4.
Figure 4.
MP upregulates the ANXA1 expression in CSE-induced BEAS-2B cells and knockdown of ANXA1 promotes the apoptosis of CSE-induced BEAS-2B cells treated with MP. mRNA (a) and protein (b) expression of ANXA1 in CSE-induced BEAS-2B cells treated with MP were respectively detected by RT-qPCR and Western blot. ***P < 0.001 vs. Control group. #P < 0.05, #P < 0.01 and ###P < 0.001 vs. CSE group. $$P < 0.01 vs. 2 μM + CSE group. @@@P < 0.001 vs. 5 μM + CSE group. mRNA (c) and protein (d) expression of ANXA1 in BEAS-2B cells transfected with si-ANXA1#1/2 were respectively detected by RT-qPCR and Western blot. ***P < 0.001 vs. Control group. ###P < 0.001 vs. si-NC group. $$$P < 0.001 vs. si-ANXA1#1 group. (e) The viability of MP+CSE-induced BEAS-2B cells transfected si-ANXA1 was detected by CCK-8 assay. (f and g) The apoptosis of MP+CSE-induced BEAS-2B cells transfected si-ANXA1 was detected by Tunel assay. (h) The expression of apoptosis-related proteins in MP+CSE-induced BEAS-2B cells transfected si-ANXA1 was determined by Western blot. ***P < 0.001 vs. Control group. ###P < 0.001 vs. CSE group. $$P < 0.01 and $$$P < 0.001 vs. MP + CSE group. @@P < 0.01 and @@@P < 0.001 vs. MP + CSE + si-NC group.
Figure 5.
Figure 5.
Knockdown of ANXA1 enhances the inflammatory response and oxidative stress, and suppresses the FPR2/AMPK pathway of CSE-induced BEAS-2B cells treated with MP. (a) The levels of inflammatory factors TNF-α, IL-6 and IL-1β in MP+CSE-induced BEAS-2B cells transfected si-ANXA1 were determined by ELISA assay kits. (b) The levels of oxidative stress factors SOD, GSH-Px and MDA in MP+CSE-induced BEAS-2B cells transfected si-ANXA1 were determined by assay kits. (c) The expression of FPR2/AMPK pathway in MP+CSE-induced BEAS-2B cells transfected si-ANXA1 was analyzed by Western blot. ***P < 0.001 vs. Control group. ###P < 0.001 vs. CSE group. $P < 0.05 and $$$P < 0.001 vs. MP + CSE group. @P < 0.05 and @@@P < 0.001 vs. MP + CSE + si-NC group.
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