Zhike Pingchuan Granule suppresses interleukin (IL)-6 or the medium of M2 macrophages induced apoptosis in human bronchial epithelial cells

Abstract

The aim of this study was to explore the effects and action mechanism of Zhike Pingchuan Granule in human bronchial epithelial cells induced by IL-6 or the supernatant of M2. Upon IL-6 stimulation at different doses, Cell Counting Kit-8 (CCK8) assay and flow cytometry were, respectively, utilized to detect the cell viability and apoptosis levels of 16-HBE cells. ELISA and Western blot were, respectively, used to analyze the inflammatory markers and JAK2/STAT3 signals. Immunofluorescence assay was performed to identify M0 and M2 cells. As shown in results, ZKPC perturbed the expression of IL-6 inducible genes important for apoptosis, oxidative and inflammatory response, which was enhanced by JAK2 inhibitor. Besides the inhibitory effects on the phosphorylation levels of JAK2/STAT3, ZKPC markedly increased cell viability and reduced apoptosis in human bronchial epithelial cells (16-HBE) cultured in the supernatant of M2 cells. Collectively, ZKPC could inhibit the IL-6-induced JAK/STAT3 signaling cascade, increase cell viability and decrease apoptosis induced by the supernatant of M2. A more comprehensive understanding of the action mechanism of ZKPC on JAK2/STAT3 signaling pathway in human bronchial epithelial cells induced by IL-6 or M2 supernatant will enable ZKPC development in the control of asthma.

Keywords: IL-6; JAK2/STAT3; Zhike Pingchuan Granule; asthma.

Figure 1.

(a) IL-6 at different concentration was utilized to stimulate 16-HBE cells for 6 h. (b) ZKPC increased cell viability in IL-6-stimulated 16-HBE cells. *P < 0.05, **P < 0.01, ***P < 0.001. Model: 16-HBE cells received 50 ng/mL IL-6 treatment for 6 h. Fedr: 16-HBE cells were co-treated with IL-6 and Fedratinib. ZKPC: HBE cells were stimulated with IL-6, Fedratinib, and Zhike Pingchuan granule. Each experiment was repeated at least three times. Data was presented as mean±SD

Figure 2.

(a)-(b) The apoptosis levels were analyzed through Flow cytometry in IL-6 induction. (b) Inflammatory markers were assessed by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001. Model: 16-HBE cells received 50 ng/mL IL-6 treatment for 6 h. Fedr: 16-HBE cells were co-treated with IL-6 and Fedratinib. ZKPC: HBE cells were stimulated with IL-6, Fedratinib and Zhike Pingchuan granule. Each experiment was repeated at least three times. Data was presented as mean±SD

Figure 3.

(a)-(c) We detected ROS, SOD, and MDA levels to evaluate oxidative stress through ROS, SOD, or MDA kit, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. Model: 16-HBE cells received 50 ng/mL IL-6 treatment for 6 h. Fedr: 16-HBE cells were co-treated with IL-6 and Fedratinib. ZKPC: HBE cells were stimulated with IL-6, Fedratinib, and Zhike Pingchuan granule. Each experiment was repeated at least three times. Data was presented as mean±SD

Figure 4.

(a) ZKPC and Fedratinib regulated Bax, Bcl-2, and cleaved-caspase 3. (b) ZKPC reduced the phosphorylation levels of JAK/STAT3 signals. *P < 0.05, **P < 0.01, ***P < 0.001. Model: 16-HBE cells received 50 ng/mL IL-6 treatment for 6 h. Fedr: 16-HBE cells were co-treated with IL-6 and Fedratinib. ZKPC: HBE cells were stimulated with IL-6, Fedratinib, and Zhike Pingchuan granule. Each experiment was repeated at least three times. Data was presented as mean±SD

Figure 5.

(a) PMA (10 ng/ml) was added to induce 16-HBE cells. (b) IL-4 and IL-13 were added to induce the polarization of M0 cells into M2 cells

Figure 6.

(a) ZKPC increased 16-HBE cell viability cultured in the supernatant of M2 cells. (b) ZKPC decreased 16-HBE cell apoptosis cultured in the supernatant of M2 cells. (c) ZKPC altered the expression levels of CCR3 and CCR4 and the ratio of p-STAT3/STAT3 in 16-HBE cells cultured in the supernatant of M2 cells. *P < 0.05, **P < 0.01, ***P < 0.001. Model: 16-HBE cells were treated with the supernatant of M2. Fedr: 16-HBE cells were co-treated with the supernatant of M2 and Fedratinib. ZKPC: HBE cells were stimulated with the supernatant of M2, Fedratinib, and Zhike Pingchuan granule. Each experiment was repeated at least three times. Data was presented as mean±SD