Silymarin attenuates cigarette smoke extract-induced inflammation via simultaneous inhibition of autophagy and ERK/p38 MAPK pathway in human bronchial epithelial cells

Diandian Li, Jun Hu, Tao Wang, Xue Zhang, Lian Liu, Hao Wang, Yanqiu Wu, Dan Xu, Fuqiang Wen, Diandian Li, Jun Hu, Tao Wang, Xue Zhang, Lian Liu, Hao Wang, Yanqiu Wu, Dan Xu, Fuqiang Wen

Abstract

Cigarette smoke (CS) is a major risk of chronic obstructive pulmonary disease (COPD), contributing to airway inflammation. Our previous study revealed that silymarin had an anti-inflammatory effect in CS-exposed mice. In this study, we attempt to further elucidate the molecular mechanisms of silymarin in CS extract (CSE)-induced inflammation using human bronchial epithelial cells. Silymarin significantly suppressed autophagy activation and the activity of ERK/p38 mitogen-activated protein kinase (MAPK) pathway in Beas-2B cells. We also observed that inhibiting the activity of ERK with specific inhibitor U0126 led to reduced autophagic level, while knockdown of autophagic gene Beclin-1 and Atg5 decreased the levels of ERK and p38 phosphorylation. Moreover, silymarin attenuated CSE-induced upregulation of inflammatory cytokines TNF-α, IL-6 and IL-8 which could also be dampened by ERK/p38 MAPK inhibitors and siRNAs for Beclin-1 and Atg5. Finally, we validated decreased levels of both autophagy and inflammatory cytokines (TNF-α and KC) in CS-exposed mice after silymarin treatment. The present research has demonstrated that CSE-induced autophagy in bronchial epithelia, in synergism with ERK MAPK pathway, may initiate and exaggerate airway inflammation. Silymarin could attenuate inflammatory responses through intervening in the crosstalk between autophagy and ERK MAPK pathway, and might be an ideal agent treating inflammatory pulmonary diseases.

Figures

Figure 1. Effect of silymarin on cell…
Figure 1. Effect of silymarin on cell viability in CSE-exposed Beas-2B, as determined by CCK8 assay.
Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group. S, silymarin.
Figure 2. Autophagy activation in response to…
Figure 2. Autophagy activation in response to CSE and silymarin treatment.
Beas-2B cells were treated with 6%CSE for different time period, indicated concentrations of CSE for 24 h or both 6%CSE and silymarin (10 μM, 20 μM) for 24 h. Formation of pEGFP-LC3 puncta in Beas-2B cells was analyzed by immunofluorescence under fluorescence microscopy (×400) (A–C). Expressions of LC3I and LC3II were measured by Western Blot (D,F,H). Densitometry was performed and the ratio of LC3II/I were calculated (E,G,I). Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group. S, silymarin.
Figure 3. Effect of silymarin on ERK/p38…
Figure 3. Effect of silymarin on ERK/p38 MAPK pathway in CSE-exposed Beas-2B cells.
Cells were pretreated with silymarin (20 μM), U0126 (10 μM) or SB203580 (10 μM) before exposed to 6%CSE for 24 h. (A) Phosphorylated and total levels of ERK and p38 were measured by Western Blot. (B) Densitometry was performed and the ratio of p-p38/t-p38 and p-ERK/t-ERK were calculated. Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group.
Figure 4. ERK MAPK pathway regulated autophagy…
Figure 4. ERK MAPK pathway regulated autophagy activation in CSE-treated Beas-2B cells.
Cells were pretreated with U0126 (10 μM) or SB203580 (10 μM) before exposed to 6%CSE for 24 h. (A) Expressions of LC3I and LC3II were measured by Western Blot. (B) Densitometry was performed and the ratio of LC3II/I were calculated. Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group.
Figure 5. Blocking autophagy suppressed CSE-induced ERK…
Figure 5. Blocking autophagy suppressed CSE-induced ERK and p38 phosphorylation in Beas-2B cells.
Cells were transfected with Atg5 siRNA (100 nM), Beclin-1 siRNA (100 nM) or negative control siRNA before exposed to 6%CSE for 24 h. (A) Expressions of LC3I and LC3II, phosphorylated and total levels of ERK and p38 were measured by Western Blot. Densitometry was performed and (B) the ratio of LC3II/I, (C) p-p38/t-p38 and p-ERK/t-ERK were calculated. Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group.
Figure 6. Silymarin attenuated CSE-stimulated inflammatory cytokine…
Figure 6. Silymarin attenuated CSE-stimulated inflammatory cytokine release in an autophagy- and ERK/p38 MAPK-dependent manner.
(A) Beas-2B cells pretreated with silymarin (20 μM), (B) ERK/p38 MAPK inhibitors U0126(10 μM) and SB203580(10 μM), or (C) negative control/Atg5/Beclin-1 siRNA (100 nM) were exposed to 6%CSE for 24 h. The secretion of TNF-α, IL-6 and IL-8 in supernatants were detected by ELISA. Data were averaged from a duplicate of each sample and from three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group.
Figure 7. Effect of silymarin on autophagy…
Figure 7. Effect of silymarin on autophagy activation and inflammatory cytokine release in mice lungs.
(A) Expressions of LC3I and LC3II were measured by Western Blot. (B) Densitometry was performed and the ratio of LC3II/I were calculated. (C) The secretion of TNF-α, IL-6 and KC in BALF were detected by ELISA. Results are representative of three independent experiments. Values are expressed as mean ± SEM (n = 3). *P < 0.05 with respect to the control group; #P < 0.05 with respect to the CSE-exposed group.

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