Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway

Abstract

Plants rich in luteolin have been used as Chinese traditional medicines for inflammatory diseases, hypertension, and cancer. However, little is known about the effect of luteolin on the apoptosis or autophagy of the macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of luteolin. The viability of the cells was determined by an MTT assay, apoptosis was determined by flow cytometric analysis, the level of cell autophagy was observed by confocal microscopy, and the expression levels of apoptotic or autophagic and antiapoptotic or antiautophagic proteins were detected by Western blot analysis. The results showed that luteolin decreased the viability of ANA-1 cells and induced apoptosis and autophagy. Luteolin induced apoptosis accompanied by downregulation of the expression of Bcl-2 and upregulation of the expression of caspase 3 and caspase 8. And luteolin increased FITC-LC3 punctate fluorescence accompanied by the increased expression levels of LC3-I, ATG7, and ATG12, while it suppressed the expression level of Beclin-1. Luteolin treatment resulted in obvious activation of the p38, JNK, and Akt signaling pathways, which is important in modulating apoptosis and autophagy. Thus, we concluded that luteolin induced the apoptosis and autophagy of ANA-1 cells most likely by regulating the p38, JNK, and Akt pathways, inhibiting the activity of Bcl-2 and Beclin-1 and upregulating caspase 3 and caspase 8 expression. These results provide novel insights into a therapeutic strategy to prevent and possibly treat macrophage-related diseases through luteolin-induced apoptosis and autophagy.

Figure 1

The cell viability of ANA-1 cells treated with luteolin. (a) Cell viability was analyzed with MTT assay after exposing to luteolin for 24 h. (b) Cell viability was analyzed with MTT assay after exposing to luteolin for 48 h. All data are expressed as the percentage of change in comparison with that of the control group, which were assigned 100% viability. The values are given as mean ± SD (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01 versus the control.

Figure 2

Luteolin induces the change of DNA content in the ANA-1 cells. (a) The representative image from flow cytometry to analyze cellular DNA content. (b) The statistical analysis of the data from (a) to show the cellular DNA content at 24 hours post treatment. (c) The statistical analysis of the data from (a) to show the cellular DNA content at 48 hours post treatment. The values are shown as mean ± SEM. ^∗p < 0.05 versus the control. ANA-1 macrophages were exposed to different concentrations of luteolin for 24 h or 48 h. The DNA content of ANA-1 cells in sub-G1 was assayed by flow cytometry.

Figure 3

Luteolin induces the ANA-1 macrophages apoptosis using TUNEL assay and Annexin V-FITC/PI staining. (a) The images of TUNEL-positive cells were captured by FCM. (b) The flow cytometry data show one representative Annexin V-FITC/PI staining result. (c) The ANA-1 macrophages were exposed to different concentrations of luteolin for 48 h. The values are given as mean ± SD.^∗p < 0.05 and ^∗∗p < 0.01 versus the control. (b) The flow cytometry data show one representative TUNEL assay result. (d-e) The ANA-1 macrophages were exposed to different concentrations of luteolin for 24 h and 48 h. The rates of early- and late-stage apoptotic cell death were detected by staining cells with Annexin V-FITC and PI and analyzing by flow cytometry. The values are given as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 versus the control.

Figure 4

Luteolin increased the ANA-1 macrophage autophagosome using immune fluorescence combined with CLSM. (a) Immunofluorescence staining of LC3.The ANA-1 cells were seeded on glass cover slips in 24-well plates with luteolin for 48 h. The fluorescence images were captured with a confocal microscopy. Magnification, ×1000. (b) Average intracellular autophagosome in macrophages treated with luteolin for 48 h. The values are given as mean ± SEM. ^∗p < 0.05 and ^∗∗p < 0.01 versus the control.

Figure 5

The effects of luteolin on apoptotic and antiapoptotic proteins in ANA-1 cells. (a) Representative Western blot results showed the rate of Bcl-2/Bax, caspase 3, and caspase 8 expression in the ANA-1 cells after incubation with luteolin for 48 h (b and c). The relative expression of proteins compared with the control. The values are given as mean ± SEM.^∗p < 0.05 versus the control.

Figure 6

The effects of luteolin on autophagic and antiautophagic proteins in ANA-1 cells. (a) Representative Western blot results showed LC3, Beclin-1, ATG7, and ATG12 expression in the ANA-1 cells after incubation with luteolin for 48 h. (b–d) The relative expression of proteins compared with the control. The values are given as mean ± SEM.^∗p < 0.05 and ^∗∗p < 0.01.

Figure 7

The effects of luteolin on ERK/p38/Akt in the ANA-1 cells. (a) Representative Western blot results showing phospho-ERK1/2/ERK1/2, phospho-p38/p38, and phospho-Akt/Akt expression in the ANA-1 cells. (b) The relative expression of proteins compared with the control. ^∗p < 0.05 versus the control.