Macrophages are immune effector cells and play a very important role in body's immune system. They are also central cells that initiate the production of inflammatory mediators in body [26–28]. LPS is the main product of Gram-negative bacteria. It can stimulate the activation of immune cells such as macrophages and cause systemic inflammation. The LPS model is a classic inflammation model[29, 30]. Therefore, in this study, we selected LPS-stimulated RAW264.7 cells as a model for inflammation research. PGE2 is an important inflammatory mediator involved in the inflammatory process and can mediate arterial dilatation and increase microvascular permeability. IL-1β is a marker of body's early inflammation. It can also induce the production of inflammatory factors such as IL-6 and IL-8 while mediating the inflammatory response. As a multifunctional cytokine, IL-6 has two-way functions of anti-inflammatory and pro-inflammatory. Its effect is related to the content in the tissue, and excessive production of IL-6 will cause a series of inflammatory damage. TNF-α is a classic inflammatory indicator, which has the coordination and regulation effect on IL-1β, IL-6, etc., and is at the center of the inflammatory cascade. In our study, we found that PHI significantly reduced the IL-1β and PGE2 production and inhibited IL-6, IL-1β and TNF-α mRNA expression. At the same time, our study found that the inhibitory effect of medium concentration PHI on the IL-1β and PGE2 production and IL-6, IL-1β and TNF-α mRNA expression was very similar to that of the positive control drug DEX, and the inhibitory effect of high concentration PHI was stronger than DEX, suggesting that PHI has great development value in future study.
Nowadays, the proteomics technology has become an important support for the rapid development of modern biotechnology. Proteomics essentially refers to studying the characteristics of proteins on a large scale and understanding key pathways through information network analysis [31]. In this work, a comprehensive approach combining proteomics and pharmacological experimental studies is used to research the anti-inflammatory effects of PHI and mechanism of action. Through GO analyses, we observed that the biological processes were quite active in the differentially expressed proteins, including cellular response to interferon-beta, innate immune response, response to virus. Functionally, they were mainly involved in nucleotide binding, RNA binding, and protein binding. Key pathways that might be affected by pathway enrichment analysis included RIG-I-like receptor signaling pathway, NF-κB signaling pathway, and Chagas disease (American trypanosomiasis). RIG-I-like receptor was one of the recognition receptors of innate immunity. It activated and regulated the intrinsic immunity of cells by recognizing the characteristics of RNA and DNA that were usually absent in the host transcriptome and was a key antiviral pathway [32]. Chagas disease was an anthropozoonosis which resulted in cardiomyopathy, arrhythmia and so on, and American continent was the epicenter of the disease[33]. NF-kB, a multi-directional nuclear transcription factor, regulated the expression of various cytokines, enzymes and other genes, and adjusted body's immune, inflammatory response and cell proliferation, as well as affected the expression of inflammatory mediators, adhesion molecules, etc., subsequently to regulate protein synthesis [34–36]. In the results of pathway enrichment, NF-κB signaling pathway was closely related to the occurrence of inflammation. Therefore, subsequent experiments were carried out around NF-κB signaling pathway.
Molecular docking study is an effective way to understand the interaction between ligands and proteins [37]. The purpose of ligand-protein docking is to explore the major binding modes when a ligand binds to a protein with a known three-dimensional structure [38]. Based on the fact that PHI has been confirmed to have a good anti-inflammatory effect and the results of proteomic analysis, molecular docking experiments were performed to further study the effect of PHI on the binding of each target protein of NF-κB signaling pathway. The results showed that the consistency scores of PHI docking with P65, IκBα, IKKβ, GSK-3β, CBP, NIK, P38, CBC13, RSK1 protens were not lower than the consistency scores of these proteins with their corresponding ligands. These indicated that PHI had a good affinity for most proteins in NF-κB pathway and anti-inflammatory effects might be derived from NF-kB pathway. Of course, binding energies of proteins and molecules by the molecular docking alone is insufficient to judge PHI molecular mechanism. Therefore, comprehensive evaluation needs to be combined with biological experiments.
According to molecular docking and proteomics analysis, PHI might regulate NF-KB signaling pathway. To further confirm there results, we performed western blot experiments. P65 is a very important protein in NF-κB family and is a key signaling molecule in the inflammatory process. It is also a relatively well-known activated macrophage pathway protein that can secrete a large number of various inflammatory factors after activation [39–41].The results indicated that PHI inhibited the activation of LPS-induced NF-KB signaling pathway by inhibiting the expression of IKKβ and the phosphorylation of P65 and IκBα. The results were consistent with molecular docking and proteomics analysis. At the same time, our study found that the inhibitory effect of medium concentration PHI on LPS-activated IKKβ and the phosphorylation levels of P65, IκBα was very similar to that of the positive control drug DEX and the inhibitory effect of high concentration PHI was stronger than DEX which suggested that PHI had a great inhibition on LPS-activated NF-κB pathway.