An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

Abstract

Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKβ/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products.

Keywords: FoxO1/3 pathway; IKKβ/IκB-α/NF-κB pathway; MAPK pathway; anti-inflammatory; carnosic acid; proteomics.

FIGURE 1

Carnosic acid down-regulated the levels of pro-inflammation mediators in LPS-induced RAW264.7 cells. (A) Chemical structure of carnosic acid (CA). (B,C) RAW264.7 cells were treated with various concentration of CA (2.5, 5, 10, and 20 μM) in the absence or presence of LPS (1 μg/ml) for 24 h. Then the cell viability (B) and nitric oxide production (C) were determined by MTT and Griess methods, respectively. (D) RAW274.7 cells were treated with CA and LPS as in (B,C) for 4 h, then the TNF-α level was detected by ELISA. (E) RAW264.7 cells were treated with 1 μg/ml of LPS containing CA (2.5, 5, 10, and 20 μM) or not for 24 h. The protein expression of COX2 was detected by western blot assay. Data are expressed as mean ± SEM from three individual experiments. ^∗∗P < 0.001 vs. LPS group; ^##P < 0.01 vs. control group; N.S., not significant by ANOVA with Bonferroni’s post hoc test.

FIGURE 2

Carnosic acid down-regulated the levels of pro-inflammation gene expression in LPS-stimulated RAW264.7 cells. (A–D) Cells were treated with LPS (1 μg/ml) for 6 h with or without CA (5, 10, and 20 μM). The relative mRNA expressions of Nos2 (A), Tnfα (B), Cox2 (C), and Mcp1 (D) were detected by real-time PCR analysis, respectively. Data are expressed as mean ± SEM (n = 3). ^∗P < 0.05, ^∗∗P < 0.01 vs. LPS group; ^##P < 0.01 vs. control group by ANOVA with Bonferroni’s post hoc test.

FIGURE 3

Outputs of proteomic analysis for CA-altered proteins. (A) Venn diagram showing numerical distribution of proteins identified in different RAW264.7 cell lysates by the nanoLC-LTQ-Orbitrap MS/MS approach. Cells were treated with vehicle (control group), LPS (1 μg/ml, LPS group), and LPS with 20 μM of CA (LPS+CA group) for 6 h, respectively. (B–D) Proteins significantly up-regulated and down-regulated by CA were classified into different cellular components (B), molecular functions (C), and BPs (D).

FIGURE 4

Bioinformatics analysis of the signaling networks negatively regulated by CA. (A,B) Pathway enrichment analysis for the differentially expressed proteins predicted the significantly canonical pathways targeted by CA in LPS-stimulated RAW264.7 cells. The gray columns plotting on the left Y-axis depict the number of identified proteins found in each pathway. The olive yellow columns plotting on the right Y-axis depict the percentage of identified proteins over the total proteins in that pathway. (C) BP enrichment analysis for differentially expressed proteins in CA-treated RAW264.7 cells.

FIGURE 5

CA restrained the activation of ERK, JNK, and p38 MAPKs in LPS-challenged RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylations of ERK, JNK, and p38 protein were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of ERK (B), JNK (C), and p38 MAPK (D) was performed by normalizing to the control group. Data are expressed as mean ± SEM from three individual experiments. ^∗P < 0.05, ^∗∗P < 0.01 vs. LPS group. ^##P < 0.01 vs. control group by ANOVA with Bonferroni’s post hoc test.

FIGURE 6

CA suppressed LPS-induced IKKβ/IκB-α/NF-κB signaling activation in RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylation and total expressions of IKKβ, IκBα, and NF-κB p65 were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of IKKβ (B), IκB-α (C), and NF-κB p65 (D) was performed by normalizing to the control group. (E) The nuclear translocation of NF-κB p65 was detected by immunofluorescence assay. Representative images were displayed with NF-κB p65 (red) and nucleus (blue). Typical apoptotic neurons were labeled with white arrows. Scale bar = 40 μm. Data are expressed as mean ± SEM from three individual experiments. ^∗P < 0.05, ^∗∗P < 0.01 vs. LPS group. ^##P < 0.01 vs. control group by ANOVA with Bonferroni’s post hoc test.

FIGURE 7

CA prevented FoxO1 and FoxO3 proteins translocating into the nucleus and promoted their degradation under LPS-challenged situation.chi-tang ho (A,B) RAW264.7 cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 24 h. The total expressions of FoxO1 and FoxO3 were determined by western blot assay. (C) Cells treated as in (A) for 6 h were collected and subjected for real-time PCR experiment to detect the relative mRNA expressions of FoxO1 and FoxO3. (D,E) The total expressions of FoxO1 and FoxO3 in RAW264.7 cells treated with LPS (1 μg/ml), CA (20 μM), and MG132 (1 μM) for 24 h were determined by western blot assay. (F–I) RAW264.7 cells were treated as in (A) for 1 h, then the nuclear and cytoplasmic expressions of FoxO1 and FoxO3 were detected by western blot assay. Histone H3 and α-tubulin were utilized as internal controls for nuclear and cytoplasmic proteins, respectively. Data are expressed as mean ± SEM from three individual experiments. ^∗P < 0.05, ^∗∗P < 0.01 vs. LPS group. ^##P < 0.01 vs. control group by ANOVA with Bonferroni’s post hoc test.