Based on Proteomics Reveals the Anti-inammatory Mechanism of Phillygenin by Inhibition NF-κB Pathway

Background: Inammation is a common pathological phenomenon when homeostasis is seriously disturbed. Phillygenin (PHI) is a lignin component isolated from Forsythiae Fructus, which showed a good anti-inammation effect. However, the mechanisms of PHI on anti-inammation have not yet been systematically elucidated. Methods: In the study, the lipopolysaccharide (LPS)-induced RAW264.7 cell inammation model was established to investigate mechanisms of PHI on inammation. The effect of PHI on LPS-induced IL-1β and PGE2 inammatory factors was detected by ELISA, and the mRNA expression of IL-1β, IL-6 and TNF-α was detected by RT-qPCR. Proteomics studies the signaling pathways that may be affected by PHI. Molecular docking technology was used to study the possible targets of PHI on NF-kB pathway. Western blot was performed to detect progressive changes in protein expression. Results: The research showed that PHI signicantly inhibited the robust increase of IL-1β and PGE2, and lowered the transcriptional level of inammatory genes including IL-6, IL-1β and PGE2 in LPS-stimulated RAW264.7 cells. Proteomics results indicated that PHI was involved in the regulation of multiple signaling pathways. Molecular docking results indicated that PHI has an anity for most proteins in NF-kB pathway. Western blot analysis showed that PHI inhibited LPS-induced NF-κB pathway activation. Conclusion: PHI inhibits the activation of NF-κB pathway, thereby inhibiting the expression of related inammatory genes and the release of cytokines, thus showing remarkable anti-inammatory effect.


Background
In ammation is one of the most common clinical symptoms. It is a defense response of body to ensure removal of harmful stimuli and repair of damaged tissues [1]. When human immune cells are affected by external stimuli, some small molecule soluble proteins or peptides that can transmit information between cells and have speci c immunoregulatory functions are secreted by body itself and participate in or cause in ammatory reactions. These substances are called in ammatory factors, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-a, nitric oxide (NO) and prostaglandin E2 (PGE2) [2]. Nowadays, many studies have reported that in ammatory response can also cause some autoimmune diseases or cancer, and is closely related to many chronic diseases such as arthritis, osteoporosis [3], asthma [4], Alzheimer's disease [5], cardiovascular disease[6], cancer [7] and obesity [8] and so on . Forsythia Fructus ("Lianqiao" in Chinese) is a kind of heat clearing and detoxifying traditional Chinese medicine and is the fruit of Forsythia suspensa (Thunb.) Vahl. [9]. It is often used in modern medicine to treat acute respiratory infections, skin purulent infections, acute nephritis, hepatitis, meningitis and other diseases [10]. Forsythia has more than 150 main chemical components, including lignans, phenylethanoid glycosides, pentacyclic triterpenoids and avonoids and so on. In recent years, the antiin ammatory activity and mechanism of many active ingredients of Forsythia Fructus, such as phillyrin, have been reported [11][12][13][14]. However, another active ingredient in Forsythia Fructus is phillygenin (PHI) (4-[(3S,3aR,6R,6aR)-6-(3,4-dimethoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro [3,4-c]furan-3-yl]-2methoxyphenol) that is often overlooked (Fig. 1). PHI is a glycoside of phillyrin, which is one of the important lignans in Forsythia Fructus and is regarded as the ngerprint component of Forsythia Fructus [15]. Studies have shown that phillyrin was poorly water-soluble and the oral absorption effect is not satisfactory [16]. In addition, it was found that phillyrin was rapidly metabolized to PHI by human intestinal bacteria [17]. Another study suspected that PHI might have the involvement of phillyrin in its anti-in ammatory effects [18]. Pre-study in our group, PHI dose-dependently inhibited the production expression of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages, and PHI also inhibited inductible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression and gene transcription [19]. Although some reports have shown that PHI has made some progress in its antiin ammatory activity in in ammatory diseases, its exact anti-in ammatory mechanism has not been clearly characterized. Proteomics as one of the most promising methods and technologies to predict disease biomarkers can re ect the signal transduction pathway and re ect the mechanism of action of drugs [20]. The combination of proteomics and pharmacology has been proved to be a means to study the mechanism of drug action.
LPS-induced macrophage in ammatory response is a common in ammation model. The NF-kB signaling pathway is the most important downstream pathway in the signaling pathway mediated by LPS [21]. NF-kB consists of two subunits belonging to the Rel family composed of homologous or heterodimers. In resting cells, the NF-kB dimer binds to IkB protein to cover the nuclear localization signal of NF-kB, so that the complex formed by NF-kB and IkB stays in the cytoplasm. When the cell receives external stimulation, IkBa/β is phosphorylated and ubiquitinated and then dissociate with NF-dimer which make the nuclear localization signal of NF-kB exposed. So NF-kB enters cell nucleus and exerts transcriptional regulatory effects which result in the gene transcription of iNOS, COX-2, IL-6, IL-1β and TNF-α and so on [22][23][24].
In this study, we conducted a comprehensive study based on quantitative proteomics and pharmacological experiments to clarify the anti-in ammatory mechanism of PHI. Our results showed that PHI inhibited the production of various in ammatory factors by inhibiting the activation of NF-KB signaling pathway. These results provided new ideas for the signal pathway of PHI to inhibit the in ammatory response, and also supplied the basis for the subsequent development and research of PHI. Elisa kits and PGE2 Elisa kits were obtained from Elabscience Biotechnology Co., Ltd (Wuhan, Hubei, China). Trizol reagent was obtained from Ambion Life Technologies (CA, USA). 5X All-In-One MasterMix and EvaGreen 2XqPCR MasterMix-No Dye were obtained from Applied Biological Materials (abm) Inc (Canada). RIPA lysis buffer, PMSF and BCA Protein Assay Kit were obtained from MultiSciences(Lianke)Biotech Co., Ltd (Hangzhou, Zhejiang, China). iTRAQ kit was obtained from AB Sciex. Rabbit anti-p-p65, mouse anti-p65, rabbit anti-IKKβ, rabbit anti-IκBα, rabbit anti-IκBα, Goat anti-Mouse IgG-HRP and Goat anti-Rabbit IgG-HRP were obtained from Multi Sciences (LIANKE) Biotech Co., Ltd. (Hangzhou, Zhejiang, China).

Cell Viability Assay
To evaluate the effect of PHI on the viability of RAW264.7 cells. RAW264.7 cells were seeded at a density of 1 × 10 5 cells / mL on a 96-well cell culture plate, and DMEM solutions containing different concentrations of PHI or DEX (0, 1, 5, 10, 25, 50, 150 and 200 µg/mL) were added for 24 hours, respectively. MTT solution was added to each well to incubate for additional 4 hours. And then carefully removed the supernatant and added 150 µL of dimethyl sulfoxide (DMSO) solution to each well. After the crystals in the culture plate were completely dissolved, the absorbance was measured at 570 nm through a microplate reader and calculated the cell proliferation rate.

ELISA for IL-1β and PGE2
RAW 264.7 cells were plated at a density of 1 × l0 5 cells/mL in 24-well plates and divided into six groups. The control group was only treated with DMEM. LPS group was treated with 10 ng/mL LPS. In LPS + PHI group, DMEM solution containing LPS (10 ng / mL) and PHI (5, 50, 100 µg / mL) was added; In the positive control group, DMEM containing LPS (10 ng / mL) and DEX (50 µg / mL) was added. Cells were incubated in the cell incubator for 24 h. Cell culture supernatants were used to determinate the concentrations of IL-1β and PGE2 according to the instructions of the ELISA kit and measured each OD value at 450 nm and then calculated the cytokine release level based on the OD value.

RNA Extraction and RT-qPCR Analysis
The mRNA expression of TNF-α, IL-6, IL-1β was measured by RT-qPCR. After treatment, supernatant was discarded and cells were washed twice with PBS. Total RNA was extracted by Trizol Reagent. The experiment was performed according to the procedure of the ampli cation kit, and GAPDH was used as an internal reference. The formula of 2-ΔΔCt was used to calculate the expression changes of target genes. The gene primer sequences were shown in Table 1. Table 1 Primers used for RT-qPCR in this study.

Gene
Forward ( The experiment was divided into control group, LPS group (10 ng / mL LPS), LPS + PHI group (10 ng / mL LPS + 100ug / mL PHI). After raw264.7 cells were cultured for 24 hours with LPS in the presence or absence of PHI, the total protein was isolated using ice-cold RIPA lysate. The protein concentration was determined by BCA method. The extracted proteins were digested with trypsin and labeled according to the instructions of the iTRAQ kit.

Mass Spectrometry Detection
The extracted samples were detected by Q Exactive™ Plus Combined Quadrupole Orbitrap™ Mass Spectrometer. A 75 µm id × 12 cm C18 column was used for the experiment. The composition of mobile phase A was 0.1% FA + 2% acetonitrile + 98% water; the composition of mobile phase B was 0.1% FA + 98% acetonitrile + 2% water; the liquid ow rate was 300 nL / min. The liquid phase gradient was set as follows: 5-8% B for 8 min, 8-22% B for 50 min, 22-32% B for 12 min, 32-90% B for 1 min, 90% B for 7 min and 90 − 5% B for 2 min.
The ion source voltage was set to 2 kV, the automatic gain control (AGC) was set to 3e6, the scanning range was 300-1400 m / z, and the resolution was 70,000. The strongest ion peaks with 2 charges were selected for further high energy fragmentation, collision capacity is set to 27%, and dynamic exclusion time is set to 18 s.

Data Analysis
The les were searched using MaxQuant (version 1.5.2.8). The lling of data gaps, normalization, and difference screening (P < 0.05%) were all performed using the standard software set of perseus.

Molecular docking
Docking study was performed by the Discovery Studio 3.5 software. The proteins in NF-κB signaling pathway (P65, IκBα, IKKβ, GSK-3β, CBP, JNK, NIK, P38, TAK1, CBC13, RSK1, NAK) were selected as the research objects. The three-dimensional structure of all protein and the corresponding known ligand structure were obtained from the Protein Data Bank (https://www.rcsb.org). Table 2 showed information on target proteins and corresponding ligands of NF-κB pathway. The downloaded proteins and ligands structures were imported into Discovery Studio 3.5 software and performed molecular docking optimization rstly, and then molecular docking work was implemented to get consistency score. If the consistency score of PHI with the target protein was no less than that of the ligand with the target protein, the protein was considered to have a good a nity with PHI.

Western Blot Analysis
After treatment, cells were washed twice with PBS. 380 µL RIPA lysate buffer supplemented with PMSF, protein phosphatase inhibitor and protease inhibitor was added on cells to lyse for 10 min on ice. The lysates were collected with a cell scraper and the cells were broken by ultrasonic cell crusher. Then the supernatant was obtained through centrifugation at -4 ℃,12000 rpm condition and the protein concentration was measured with BCA protein assay reagent according to the manufacturer's instructions. Samples were separated by 10% SDS-PAGE electrophoresis and then electrotransferred to a polyvinylidene uoride (PVDF) membrane in ice bath. Then the PVDF membrane was blocked in 5% skim milk at room temperature for 2 hours. After blocking for 2 h, the membrane was incubated with primary antibodies diluted 1000 times through 5% BSA solution overnight at 4 ℃ in a shaking incubator. Finally, the primary antibodies were recycled and the membrane was incubated with the horseradish peroxidaselabeled secondary antibody (antibody : 5% skim milk = 1: 5 000) at 37 ℃ for 1 hour. After washing, the protein bands were dectected and visualized with the chemiluminescence kit using a chemiluminescence detection system.

Statistical Analysis
Date were presented as means ± SD and SPSS 25.0 was used to statistical analyses. Comparison between groups was performed by one-way analysis of variance (ANOVA). All the experiments were repeated at least three times. The results were considered statistically signi cant if p value < 0.05.

Effects of PHI and DEX on the proliferation of RAW264.7 cells
MTT method was used to investigate the changes of cell proliferation activity of RAW264.7 cells treated with different concentrations of PHI and DEX. As shown in Fig. 2, compared with normal control, PHI had no signi cant inhibitory effect on cell proliferation at concentrations up to 100 µg/mL, and DEX had no signi cant inhibitory effect on cell proliferation at a concentration of 0 to 50 µg/mL. Therefore, in the subsequent experiments, low, medium and high concentrations (5, 50, 100 µg/mL) of PHI that did not signi cantly inhibit cell proliferation were selected for further research, and DEX (50 µg /mL) was used as a positive control.

Effects of PHI on levels of IL-1β and PGE2
The contents of in ammatory factors IL-1β and PGE2 in the supernatant of cell culture were determined by ELISA. When LPS acted on RAW264.7 cells, the contents of IL-1β and PGE2 in cell supernatant were increased signi cantly compared with the normal control. The contents of IL-1β and PGE2 in cell supernatant of LPS + PHI groups at different concentrations were signi cantly lower than those in LPS groups and were dose-dependent. The contents of IL-1β and PGE2 in the supernatant of the DEX group were signi cantly lower than those in the LPS group. There was no signi cant difference in IL-1β and PGE2 content between 50 µg/mL PHI group and 50 µg/mL DEX group. The results were shown in Fig. 3. 3.3 Effect of PHI on LPS-induced in ammatory factor mRNA expression in RAW264.7 cells In order to further explore the effect of PHI on LPS-induced in ammatory factor gene expression, RT-qPCR was used to detected the levels of IL-6, IL-1β and TNF-α. As shown in Fig. 4, compared with the normal control, LPS induced signi cantly expression of IL-6, IL-1β and TNF-α mRNA. Compared with the LPS group, IL-6, IL-1β and TNF-α mRNA expression was down-regulated in a concentration-dependent manner by treating PHI (5, 50, 100 µg/mL). Compared with dexamethasone, IL-1β was no signi cant difference in mRNA expression between PHI and dexamethasone at the same concentration of 50 µg/mL. These results indicated that PHI suppressed the in ammatory response by down-regulating the expression of IL-6, II-1β and TNF-α mRNA in LPS-induced RAW2644.7 cells in ammation and showed a strong antiin ammatory effect.

Effect of PHI on LPS-induced RAW264.7 cell proteome changes
The proteomic pro les were analyzed by Q ExactiveTM Plus Combined Quadrupole OrbitrapTM Mass Spectrometer to research differences of protein expression in LPS-induced Raw264.7 cells in ammation after PHI treatment. The results showed that a total of 4318 proteins were identi ed and contained quantitative information in this study. By comparing the expression levels of total proteins, the differentially expressed proteins in the three groups were screened. The fold change was more than 1. Gene Ontology (GO) is an important bioinformatics analysis method and tool for expressing various properties of genes and gene products. GO annotations are divided into three rst-level categories including Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), which explain the biological role of proteins from different perspectives. We conducted statistics on the distribution of the selected differential proteins in the GO secondary annotations. As shown in Fig. 6, according to the analysis, the cellular composition of these proteins was mainly cytoplasm (51 proteins), cytosol (16 proteins) and nucleoli (10 proteins). In biological processes, these proteins were mainly involved in the cell's response to interferon-β (8 proteins), innate immune response (14 proteins), response to viruses (8 proteins) and so on. The molecular functions mainly included nucleotide binding (22 proteins), RNA binding (13 proteins) and protein binding (34 proteins).

Protein pathway enrichment analysis
In order to visualize the pathways with signi cant enrichment of differentially expressed proteins by PHI treatment, Cytocape software was used to examine the enrichment of 92 PHI-regulated differential proteins in the KEGG pathway database, REACTOME pathway database and Wiki pathway database. Figure 7 depicted a global pathway network enriched for GO terms at P < 0.05. The results showed that PHI mainly affected three pathways, including RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, and Chagas disease (American trypanosomiasis).

Molecular docking
Molecular docking experiments was performed to further predict if PHI exerted a anti-in ammatory effect through NF-κB signaling pathway. Figure 8 showed the consistency score results of docking of the target protein with PHI or ligand molecules in NF-κB pathway. The results showed that the consistency scores of PHI docking with P65, IκBα, IKKβ, GSK-3β, CBP, NIK, P38, CBC13, RSK1 was higher than the consistency score of these proteins with their corresponding ligands, while consistency scores of PHI docking with JNK, TAK1 and NAK was lower than that of with their corresponding ligands.

Effect of PHI on LPS-induced NF-κB pathway in RAW264.7 cells
Proteomics results and molecular docking indicated that PHI might be able to regulate NF-KB signaling pathway. To con rm this result, we investigated the effect of PHI on the expression of key proteins in NF-κB signaling pathway induced by LPS through western blot analysis. The results showed that the expression of IKKβ protein increased signi cantly after LPS stimulation, and PHI could signi cantly reverse the increase. After LPS stimulation, the levels of P65 and IκBα phosphorylation were up-regulated signi cantly, while the levels of P65 and IκBα phosphorylation were down-regulated signi cantly by PHT treatment. Between 50 µg / mL PHI and 50 µg / mL DEX, there was no signi cant difference in the inhibitory effects on the expression of IKKβ, P-IkBa and P-P65. These results showed that PHI inhibited the activation of LPS-induced NF-κB signaling pathway by inhibiting the expression of IKKβ and the phosphorylation of P65 and IκBα. The results were shown in Fig. 9.

Discussion
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 in ammatory mediators in body [26][27][28]. LPS is the main product of Gram-negative bacteria. It can stimulate the activation of immune cells such as macrophages and cause systemic in ammation. The LPS model is a classic in ammation model [29,30]. Therefore, in this study, we selected LPS-stimulated RAW264.7 cells as a model for in ammation research. PGE2 is an important in ammatory mediator involved in the in ammatory process and can mediate arterial dilatation and increase microvascular permeability. IL-1β is a marker of body's early in ammation. It can also induce the production of in ammatory factors such as IL-6 and IL-8 while mediating the in ammatory response. As a multifunctional cytokine, IL-6 has two-way functions of antiin ammatory and pro-in ammatory. Its effect is related to the content in the tissue, and excessive production of IL-6 will cause a series of in ammatory damage. TNF-α is a classic in ammatory indicator, which has the coordination and regulation effect on IL-1β, IL-6, etc., and is at the center of the in ammatory cascade. In our study, we found that PHI signi cantly 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-in ammatory 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 multidirectional nuclear transcription factor, regulated the expression of various cytokines, enzymes and other genes, and adjusted body's immune, in ammatory response and cell proliferation, as well as affected the expression of in ammatory mediators, adhesion molecules, etc., subsequently to regulate protein synthesis [34][35][36]. In the results of pathway enrichment, NF-κB signaling pathway was closely related to the occurrence of in ammation. 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 con rmed to have a good anti-in ammatory 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 a nity for most proteins in NF-κB pathway and anti-in ammatory effects might be derived from NF-kB pathway. Of course, binding energies of proteins and molecules by the molecular docking alone is insu cient 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 con rm 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 in ammatory process. It is also a relatively wellknown activated macrophage pathway protein that can secrete a large number of various in ammatory factors after activation [39][40][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.

Conclusion
In summary, this study demonstrated the anti-in ammatory effect and mechanism of PHI on LPSinduced RAW264.7 cell in ammation. Firstly, we demonstrated that PHI inhibited the release of LPSinduced in ammatory factors and the transcription of related in ammatory genes. The signaling pathways that PHI might affect have been studied through proteomics and bioinformatics analysis. And then through molecular docking technology to study the a nity between PHI and NF-κB in ammatory pathway target protein, it was found that PHI had a good a nity for NF-κB pathway. Finally, western blot was used to further verify that PHI inhibited the activation of LPS-induced NF-KB signaling pathway by inhibiting the expression of IKKβ and phosphorylation of P65 and IκBα. On the whole, PHI had shown its anti-in ammatory effect by inhibiting the activation of the NF-κB pathway, thereby suppressing the expression of related in ammatory genes and the release of cytokines. The research deeply explored the effects, targets, and mechanism of PHI on in ammation to provide scienti c references for the study of PHI in the future and improve the overall understanding of PHI. Availability of data and materials

List Of Abbreviations
The datasets in this study are available to provide by corresponding authors on reasonable request. Effect of Phillygenin and dexamethasone on proliferation of RAW264. 7 cells. Cell viability was determined by MTT assay. RAW264.7 Cells were treated with PHI or DEX. Compare with control, * P 0.

Figure 3
Effect of PHI on LPS-induced release of in ammatory factors in raw264.7 cells. RAW264.7 cells were treated with various concentrations of PHI (5,50,100 ug/mL) or 50 ug/mL DEX in the presence of 10ng/mL LPS for 24 hours. Then the IL-1β and PGE2 level were determined by ELISA, respectively.