Fig 1.
PCA2 suppressed LPS-induced macrophage activation.
(A) The chemical structure of PCA2. (B) Cells were cultured with LPS for 18 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except for the control group. Determination of nitrite levels by the Griess assay (n = 5). (C) Cells were cultured with LPS for 6 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except the control group. After cells were incubated with DAF-FM (1 μM) for 1 h, collected to flow tube and monitored by the flow cytometry (n = 3). (D) Statistical analysis fluorescence intensity of DAF-AM (n = 3). (E) Cells were cultured with LPS for 18 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except for the control group. iNOS and COX-2 protein expression were tested by western blotting (n = 3). (F) Statistical analysis of the expression of the protein (n = 3). (G, H) Cells were cultured with LPS for 2 h after treated with PCA2 (80 μM) for 1 h, except for the control group. The mRNA expression of COX-2 and iNOS tested by qRT-PCR (n = 3). ***p < 0.001 vs. LPS group.
Fig 2.
PCA2 inhibited pro-inflammatory cytokines release and gene expression.
Cells were cultured with LPS for 18 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except for the control group. (A) IL-6, (B) TNF-α, and (C) PGE2 were investigated by ELISA kits (n = 5). Cells were cultured with LPS for 2 h after treated with PCA2 (80 μM) for 1 h, except for the control group. The mRNA expression of (D) IL-6 and (E) TNF-αwere tested by qRT-PCR (n = 3). *p < 0.05, p < 0.01, ***p < 0.001 vs. LPS group.
Fig 3.
PCA2 decreased LPS-induced ROS generation.
(A) LPS was used to induce cells for 0, 2, 4, 8, 10 h, and then cells were incubated with DCFH2-DA (1 μM) for 0.5 h, collected to flow tube and monitored by the flow cytometry (n = 3). (B) Cells were cultured with LPS for 8 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except the control group and only treated with PCA2 group. After cells were incubated with DCFH2-DA (1 μM) for 0.5 h, collected to flow tube and monitored by the flow cytometry (n = 3). (C) Statistical analysis of the fluorescence intensity of DCFH2-DA (n = 3). (D) Cells were cultured with LPS for 8 h after treated with PCA2 (20, 40, 80 μM) for 1 h, except the control group and only treated with PCA2 group. Master Reaction Mix was placed into a cell plate and incubate for 1 h. Fluorescence intensity measured by a fluorescence microplate reader (n = 4). (E) The cell was exposed to PCA2 (80 μM) 1 h before cultured with LPS for 8 h, except for the control group. For the measurement of ROS, DCFH2-DA (10 μM) was loaded for 0.5 h and images were acquired with a fluorescence microscope (n = 3). ***p < 0.001 vs. LPS group.
Fig 4.
PCA2 reversed LPS-induced mitochondrial-membrane-potential (MMP) loss and calcium influx.
(A) The cell was exposed to PCA2 (80 μM) for 1 h and cultured with LPS for 8 h, except for the control group. For the measurement of MMP, JC-1 (10 μg/mL) was loaded for 0.5 h and images were acquired with a fluorescence microscope (n = 3). (B) Cells were co-cultured with LPS for 6 h before treated with PCA2 (20, 40, 80 μM) for 1 h, except the control group and only treated with PCA2 group. After cells were incubated with Fluo-3/AM (1 μM) for 1 h, collected to flow tube and monitored by the flow cytometry (n = 3). (C) Statistical analysis of the fluorescence intensity of Fluo-3/AM (n = 3).**p < 0.01, *** p < 0.001 vs. LPS group.
Fig 5.
PCA2 blocked LPS-induced NF-κB/p65 translocation.
(A) The LPS-induced group cells were exposed to LPS for 2 h, while the experiment group cells were exposed to PCA2 (80 μM) for 1 h and cultured with LPS for another 1 h. Subsequently, cells incubated with NF-κB p65 antibody and secondary antibody for 1 h independently. Later, we used Hoechst 33342 to stain nuclei for a quarter. Images were taken with the confocal laser scanning microscopy. (B) The cell was exposed to PCA2 (80 μM) for 1 h and cultured with LPS for 2 h, except for the control group. The protein expressions of p65 were detected by western blotting (n = 3). (C) Statistical analysis of the p65 protein expression in the cytoplasm and the nucleus (n = 3). *** p < 0.001 vs. LPS group.
Fig 6.
PCA2 ameliorated LPS-induced inflammatory activity through the NF-κB/MAPK pathways.
The cell was exposed to PCA2 (20, 40, 80 μM) for 1 h and cultured with LPS for 8 h, except for the control group. Protein expression of (A) NF-κB and (B) MAPK pathway including (C) p-IKKα/β, (D) p-p65, (E) p-IκBα, (F) p- JNK1/2, (I) p-ERK1/2, and (J) p-p38 was measured by western blotting (n = 3). *p < 0.05, *** p < vs. LPS group 0.01.
Fig 7.
PCA2 attenuated LPS-induced inflammatory activity through Nrf2 pathways.
The cell was exposed to PCA2 (20, 40, 80 μM) for 1 h and cultured with LPS for 8 h, except for the control group. The protein expression of the (A) Nrf2 pathway including (B) Nrf2, (C) Keap-1, (D) HO-1 was measured by western blotting. Cells were co-cultured with LPS for 2 h before treated with PCA2 (80 μM) for 1 h, except for the control group. (E) Nrf2, (F) HO-1 mRNA level was determined by qRT-PCR (n = 3). (G) The LPS-induced group cells were exposed to LPS for 2 h, while the experiment group cells were exposed to PCA2 (80 μM) for 1 h and cultured with LPS for another 1 h. Subsequently, cells incubated with Nrf2 antibody and secondary antibody for 1 h independently. Later, we used Hoechst 33342 to stain nuclei for a quarter. Images were taken with the confocal laser scanning microscopy. (H) The cell was exposed to PCA2 (80 μM) for 1 h and cultured with LPS for 2 h, except for the control group. The protein expressions of Nrf2 were detected by Western blotting (n = 3). (I) Statistical analysis of the Nrf2 protein expression in the cytoplasm and the nucleus (n = 3). *p < 0.05, *** p < 0.001 vs. LPS group.
Fig 8.
The schematic diagram of the mechanism of PCA2’s anti-inflammatory and anti-oxidative effects.
PCA2 has an anti-inflammatory and anti-oxidant effect on LPS-induced RAW264.7 cells. The potential mechanisms involved in suppressing the NF-κB, MAPK, and Nrf2 pathway though decreases ROS generation, NO and Ca2+ exclusion, and the release of pro-infammatory cytokines such as IL-6, TNF-α and PGE2.