Polyphyllin I Inhibits Propionibacterium acnes-Induced IL-8 Secretion in HaCaT Cells by Downregulating the CD36/NOX1/ROS/NLRP3/IL-1 β Pathway

Abstract

Acne vulgaris (AV) is a chronic skin disease involving inflammation of the pilosebaceous units. Propionibacterium acnes (P. acnes) hypercolonization is one pathogenic factor for AV. P. acnes that triggers interleukin-1β (IL-1β) by activating the pyrin domain-containing 3 protein (NLRP3) inflammasome of the NOD-like receptor family in human monocytes. Reactive oxygen species (ROS) acts as a trigger for the production of IL-8 and activates theNLRP3 inflammasome. IL-8 promotes the metastasis and multiplication of different cancerous cells, whereas keratinocyte proliferation and migration contribute to the progression of AV. A steroidal saponin called polyphyllin I (PPI) that is extracted from Paris polyphylla's rhizomes has anti-inflammatory properties. This study investigates the regulatory role of P. acnes in the secretion of IL-8 mediated by the CD36/NADPH oxidase 1 (NOX1)/ROS/NLRP3/IL-1β pathway and the effects of PPI on the CD36/NOX1/ROS/NLRP3/IL-1β/IL-8 pathway and human keratinocyte proliferation and migration. HaCaT cells were cultured and stimulated with 10⁸ CFU/ml of P. acnes for 0, 6, 12, 18, 24, 30, and 36 hours. P. acnes induced IL-8 secretion from HaCaT cells via the CD36/NOX1/ROS/NLRP3/IL-1β pathway. PPI inhibited the CD36/NLRP3/NOX1/ROS/IL-8/IL-1β pathway and HaCaT cell proliferation and migration. PPI alleviates P. acnes-induced inflammatory responses and human keratinocyte proliferation and migration, implying a novel potential therapy for AV.

Figure 1

P. acnes inducing IL-8 release and the activation of NLRP3 inflammasome in HaCaT cells. (a) The CD36, NOX1, NLRP1, NLRP3, NLRC4, AIM2, ASC, active caspase-1, and cleaved IL-1β protein levels in HaCaT cells following 10⁸ CFU/ml P. acnes treatment detection done by Western blot. GAPDH was used as the loading control. CD36, NOX1 (b), NLRP1, NLRP3 (c), NLRC4, AIM2 (d), and ASC (e) relative protein levels compared to the GAPDH level, active caspase-1/pro-caspase-1, and cleaved IL-1β/pro-IL-1β are calculated by ImageJ. (h) ELISA was performed for the analysis of IL-8 protein levels in the culture supernatant of HaCaT cells. n = 5/each group. ^∗P < 0.05 and ^∗∗p < 0.01 vs. 0 h group.

Figure 2

P. acnes inducing the CD36/NOX1/ROS/NLRP3/IL-1β/IL-8 pathway in HaCaT cells. (a) The CD36, NOX1, NLRP3, ASC, active caspase-1, and cleaved IL-1β protein levels in HaCaT cells following 10⁸ CFU/ml P. acnes treatment with CD36 siRNA, NOX1 siRNA, DPI, NAC, NC siRNA, or 01% DMSO exposure detected by Western blot. GAPDH is used as the loading control. CD36, NOX1 (b), NLRP3, and ASC (c) relative protein levels compared to GAPDH expression, active caspase-1/procaspase-1 (d), and cleaved IL-1β/pro-IL-1β (e) calculated by ImageJ. (f) The IL-8 protein levels in the culture supernatant of HaCaT cells determined by ELISA. (g) Cytofluorometric profiles representing the distribution of HaCaT cells after staining with DCFH-DA. (h) ROS positive ratio calculated by cytometry flow. n = 5/each group. ^∗∗P < 0.01 vs. control group. ^#P < 0.05 vs. P. acnes treatment group.

Figure 3

PPI inhibiting the P. acnes-induced CD36/NOX1/ROS/NLRP3/IL-8 pathway in HaCaT cells. The CD36, NOX1, NLRP3, ASC, active caspase-1, and cleaved IL-1β protein levels in HaCaT cells following 10⁸ CFU/ml P. acnes treatment with PPI at different concentrations (0.3, 0.6, and 0.9 μg/ml) or 0.1% DMSO exposure detected by Western blot. GAPDH is used as the loading control. CD36, NOX1 (b), NLRP3, and ASC (c) relative protein levels compared to GAPDH expression, active caspase-1/procaspase-1 (d), and cleaved IL-1β/pro-IL-1β (e) calculated by ImageJ. (f) The IL-8 protein levels in the culture supernatant of HaCaT cells determined by ELISA. (g) Cytofluorometric profiles representing the distribution of HaCaT cells after staining with DCFH-DA. (h) ROS positive ratio calculated by cytometry flow. n = 5/each group. ^∗∗P < 0.01 vs. control group. ^#P < 0.05 vs. P. acnes treatment group.

Figure 4

PPI inhibiting proliferation and migration of HaCaT cells. (a) The proliferation of HaCaT cells following P. acnes treatment with PPI (0.9 μg/ml) or 01% DMSO exposure detected by the EdU incorporation assay. The red color represents EdU positive, and the blue color represents DAPI (nucleus). The average ratio of EdU positive HaCaT cells is calculated by ImageJ. (b) The migration of HaCaT cells following P. acnes treatment with PPI (0.9 μg/ml) or 0.1% DMSO exposure detected by the transwell assay. (d) The average number of migrated HaCaT cells calculated by ImageJ. n = 5/each group. ^∗∗P < 0.01 vs. control group. ^##P < 0.01 vs. P. acnes treatment group.

Figure 5

The schematic diagram showing PPI inhibited the P. acnes-induced CD36/NOX1/NLRP3/ROS/IL-1β/IL-8 pathway and HaCaT cell proliferation and migration.