Formononetin Suppresses Interleukin-6 Production in Lipopolysaccharide-Stimulated Macrophages Via Activation of the AMP-Activated Protein Kinase

Background: Interleukin-6 (IL-6), an inammatory cytokine, plays a majoy role in the pathogenesis of inammation and serves as a key marker in the diagnosis and treatment of inammation and related diseases. Formononetin (FMN), an isoavone ingredient in various Chinese herbal medicines, inhibits the production of IL-6 in some cells. However, its mechanism of action has not been clearly established. In this study, we aimed to identify whether FMN inhibits IL-6 production in Lipopolysaccharide (LPS)-stimulated macrophages via AMPK activation. Methods: In the current study, the anti-IL-6 mechanism of FMN was evaluated using a macrophage model with LPS stimulation. An inverted microscope was utilized to obtain images of cells. Nuclear staining assay and CCK-8 assay were used to identify the viability of ANA-1 cells. The expression of IL-6 in cells was investigated by Enzyme-linked immunosorbent (ELISA) and Western blotting. The expression of AMP-activated protein kinase (AMPK) was determined by Western blotting and phosphorylation of AMPK was determined by Western blotting and Immunouorescence assay. Bioinformatic analysis was used to predict potential targets of FMN. Results: we found that FMN reduced the expression of IL-6 in ANA-1 cells and increased the phosphorylation of AMPK. The effect of FMN was similar to that of acadesine, an AMPK activator, which also reduced IL-6 expression in LPS-induced ANA-1 cells and increased AMPK phosphorylation. Its combination with dorsomorphin (an AMPK inhibitor), however, reversed the effects of FMN on AMPK phosphorylation and IL-6 expression. The target of FMN was identied as the cAMP-dependent protein kinase inhibitor alpha, as searched in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and further veried with the STRING database. Conclusions: In conclusion, our ndings suggest that FMN effectively inhibits IL-6 production by activating AMPK in LPS-stimulated macrophages. inhibit


Introduction
Interleukin-6 (IL-6), an important in ammatory cytokine, plays various roles in the human body and is involved in different in ammatory diseases. Hence, it is considered a therapeutic target for such diseases [1][2][3]. As a response to infection or tissue damage, macrophages immediately produce IL-6 via the activation of pattern recognition receptors. Lipopolysaccharide (LPS), which is a bacterial endotoxin present in the cell wall of gram-negative bacteria, can induce an in ammatory response in the body, causing injury, disease, and even death [4][5][6]. The uncontrolled excess or sustained production of IL-6 through LPS induction can lead to the development of various in ammatory diseases and cancers [7]. IL-6 expression is mainly associated with the activation of nuclear factor-κB (NF-κB), nuclear factor-IL-6, tumor-necrosis factor, and other factors [8,9]. The conventional anti-in ammatory drugs currently used have various adverse effects; therefore, it is imperative to nd new anti-IL-6 drugs.
Formononetin (FMN) is a typical iso avone compound. It is extracted from herbs used in traditional Chinese medicines, such as Astragalus membranaceus [10] and Trifolium pratense L. [11]. The molecular formula of FMN is C 16 H 12 O 4 . Its structural formula is shown in Fig. 1. Some studies have shown that FMN has various pharmacological effects such as anti-cancer, anti-oxidation, and anti-in ammatory [12,13]. Moreover, FMN can inhibit LPS-induced IL-6 expression in cells including macrophages [14], microglia [15], and bone marrow-derived dendritic cells [16]. However, the mechanism of its action is not completely clear; the upstream signaling pathways and targets thus require further research.
Studies have reported that FMN can activate 5' AMP-activated protein kinase (AMPK) [17,18], which is a key regulator protease commonly expressed in cells to provide energy. Cells activate AMPK via adenine nucleotide-dependent and other non-classical pathways to meet their energy requirements [19]. Studies have demonstrated that AMPK, which is located upstream of multiple signaling pathways, can inhibit IL-6 expression [20,21]. However, whether FMN exerts an anti-IL-6 production effect via AMPK remains unclear. In this study, we aimed to identify whether FMN inhibits IL-6 production in LPS-stimulated macrophages via AMPK activation.
CCK-8 assay ANA-1 cells in the logarithmic growth phase were inoculated in 96-well plates. The cells were treated with equal volumes of DMSO solvent, the LPS solution, FMN solution, and the LPS + FMN solution for 24 h. Clear medium without cells was used as a blank and the untreated cells were the control. Then, 10 µL of CCK-8 reagent was added into each well and incubated at 37 °C for 1.5 h. A microplate reader was used to measure the absorbance of the sample in each well at 450 nm, and the proliferating percentage of cells was calculated as follows: Cell proliferation percentage (%) = [(experimental group absorbance − blank group absorbance) / (control group absorbance − blank group absorbance)] × 100% Enzyme-linked immunosorbent assay (ELISA) ANA-1 cells were exposed to LPS and FMN at different concentrations for 24 h. The levels of IL-6 in ANA-1 cells supernatants were quanti ed with commercially available ELISA kits (Beyotime Biotechnology, China), following the company's directions.

Western blotting
The treated cells were collected at the indicated time points and lysed with cell lysis buffer containing protease inhibitors. The total protein was quanti ed using the BCA method. The total proteins were subjected to 12% SDS-PAGE and transferred onto nitrocellulose (NC) membranes. The NC membranes were incubated with β-actin (1:1500), IL-6 (1:1000), p-AMPK (1:1000), and AMPK (1:1000) antibodies at ANA-1 cells in the logarithmic growth phase were inoculated in 96-well plates. The cells were treated with FMN solution and the LPS + FMN solution for 24 h. The cells were xed with 4% paraformaldehyde for 1 h and then permeabilized with 1% Triton X-100 for 30 min and blocked with 1% BSA in PBS for 60 min.
Cells were then incubated with p-AMPK (1:200) antibodies at 4 °C overnight. Next, cells were incubated with DyLight 488 anti-rabbit IgG (1:500) for 2 h at 20 °C. The cells were washed three times with PBS between each step. Then, the HCS was used to obtain images of cells at 200 × magni cation and determine the cell average FITC intensity in each group.

Bioinformatic analysis
The Computational Systems Biology Laboratory (TCMSP -Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, https://tcmspw.com/tcmsp.php) database was used to search potential targets of FMN, and the STRING website (STRING -functional protein association networks, https://string-db.org/) was used to analyze protein interactions.

Statistical analysis
All experiments were repeated at least three times. The data were analyzed using the one-way ANOVA and Student's t-test with GraphPad Prism 7.04 software. Results with a p value < 0.05 were considered statistically signi cant.

LPS increases IL-6 levels in ANA-1 cells
To determine whether the proliferation of ANA-1 cells is inhibited by LPS, the cells were treated with LPS (0.05, 0.1, 0.5, 1, and 2 µg/mL) for 24 h and evaluated using the HCS. ANA-1 cells were observed under an inverted microscope and appeared to have an irregular shape with enlargement, protrusion, and lm-like pseudopods (Fig. 2a). The total cell count decreased as the concentration of LPS increased (Fig. 2b). In addition, the results revealed that the incubation of ANA-1 cells with varying concentrations of LPS for 24 h increased IL-6 expression in a dose-dependent manner (Fig. 2c-2e). The above results suggest that LPS is toxic to ANA-1 cells and can induce ANA-1 cells to produce excessive amounts of IL-6.

FMN attenuates LPS-induced toxicity in ANA-1 cells
To determine whether FMN affects cell proliferation, the cells were treated with FMN at different concentrations (0.016, 0.08, 0.4, 2, 10, and 50 µmol/mL) for 24 h, and the cell proliferation rate was determined using the CCK-8 kit. Compared to that in the control group, an increase in the FMN concentration did not affect the cell proliferation rate. Furthermore, there was no signi cant difference between the DMSO solvent and control groups (Fig. 3a). On the other hand, the proliferation rate of LPSstimulated cells was increased with FMN treatment at varying concentrations (0.08, 0.4, 2, 10, and 50 µmol/mL) for 24 h, and the result was statistically signi cant at the concentrations of 10 and 50 µmol/mL FMN (Fig. 3b).
FMN reduces IL-6 levels in LPS-stimulated ANA-1 cells We next investigated the effect of FMN on IL-6 expression induced by LPS. LPS-stimulated ANA-1 cells were exposed to FMN at 0.4, 2, and 10 µmol/mL concentrations for 24 h. We found that FMN decreased the IL-6 levels in LPS-stimulated cells in a dose-dependent manner (Fig. 4), con rming that FMN can inhibit IL-6 production.
FMN increases the phosphorylation of AMPK in ANA-1 cells We then explored how FMN inhibits IL-6 expression. AMPK, a potent inhibitor of IL-6 expression, is present upstream of multiple signaling pathways. We investigated the regulatory effect on AMPK by exposing ANA-1 cells to FMN at concentrations of 0.08, 0.4, 2, and 10 µmol/mL for 24 h. The western blotting results revealed that the p-AMPK level in ANA-1 cells was increased with FMN treatment at concentrations of 0.4, 2, and 10 µmol/mL. However, FMN has no effect on the expression levels of the total AMPK protein (Fig. 5a, 5b, and 5e). Furthermore, FMN dose-dependently increased the level of p-AMPK (Fig. 5c,5d, 5f, and 5 g) in ANA-1 cells when they were co-treated with varying FMN concentrations (0.4, 2, and 10 µmol/mL) and 1 µg/mL LPS for 24 h. These results suggest that FMN might inhibit IL-6 production by activating AMPK.

Acadesine increases the phosphorylation of AMPK in ANA-1 cells and reduces the levels of IL-6
Considering the close association between IL-6 and AMPK, we explored the effect of acadesine (AICAR) on these two proteins. We found that AICAR at varying concentrations (20,40, and 80 µmol/mL) increased the levels of p-AMPK in ANA-1 cells (Fig. 6a). Furthermore, treatment with 20, 40, and 80 µmol/mL AICAR for 24 h increased the levels of p-AMPK compared to those with LPS treatment in ANA-1 cells and reduced the levels of IL-6 in a dose-dependent manner (Fig. 6c). Overall, the activation of AMPK can inhibit the production of IL-6 in LPS-stimulated ANA-1 cells.

Dorsomorphin reverses the effect of FMN on AMPK phosphorylation and IL-6 protein expression in LPSstimulated ANA-1 cells
We con rmed the relationship between IL-6 and p-AMPK using an AMPK inhibitor, dorsomorphin (compound C, CC). ANA-1 cells were treated with LPS, LPS + CC, LPS + FMN, and LPS + CC + FMN. Western blotting was performed to determine the levels of p-AMPK and IL-6. Compared to that with LPS treatment, LPS + CC treatment decreased the p-AMPK levels and increased the IL-6 levels in ANA-1 cells, whereas the LPS + FMN treatment increased the levels of p-AMPK and reduced the levels of IL-6 in ANA-1 cells. Compared to that with LPS + FMN treatment, LPS + FMN + CC treatment suppressed p-AMPK expression and increased IL-6 expression simultaneously (Fig. 7a, 7b). These results suggest that the inhibitory effect of FMN on the production of IL-6 in ANA-1 cells was blocked by the inactivation of AMPK, indicating that AMPK signaling is involved in the inhibition of IL-6 production by FMN.

Discussion
In this study, we found that FMN, an iso avone compound found in many Chinese herbal medicines, reduced the cytotoxicity and IL-6 production in the LPS-stimulated murine macrophage ANA-1 cells. The result was consistent with previous research on other types of cells [14][15][16]. Previous studies have shown that FMN can exert anti-in ammatory effects via the NF-κB signaling pathway [23], the cytokine-activated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway [24], and the nuclear factor erythroid 2-related factor/heme oxygenase-1 signaling pathway [25], among others. In this study, we sought to explore the underlying molecular mechanism of the cytoprotective effect of FMN in the presence of LPS and anticipated more novel and promising discoveries using this experimental model. The inhibition of IL-6 production was reported to occur via AMPK activation [20,21]. We found that FMN increased AMPK phosphorylation in a dose-dependent manner in the model. To validate our results, the inhibitory role of AMPK in IL-6 production was con rmed with an activator of AMPK, acadesine. Furthermore, we performed an inhibition experiment with a speci c AMPK inhibitor, dorsomorphin. As expected, the inhibitory effect of FMN on IL-6 production was attenuated by the addition of the AMPK inhibitor.
Overall, we have demonstrated that FMN inhibits IL-6 production by activating AMPK. To determine whether FMN directly activates AMPK, we used the TCMSP database to search potential targets of FMN and found no evidence of a direct effect of FMN on AMPK. We found that Pkia might serve as a potential target of FMN and then con rmed the association between Pkia and AMPK. Furthermore, Pkia was reported as a speci c inhibitor of the cyclic-AMP-dependent protein kinase A (PKA). Several studies have con rmed that PKA can activate AMPK [26,27]. Therefore, we speculated that FMN reduced the inhibitory effect on PKA by binding to Pkia (a way to consume Pkia) and promoted AMPK activation via the PKA pathway.
IL-6 is a type of cytokine mainly secreted by macrophages and is known to play an important role in immune regulation and in ammation. In ammation plays a key role in the pathogenesis of various human chronic diseases [28] such as cancer [29], diabetes [30], cardiovascular diseases [31], and metabolic diseases [17,32]. Upon in ammatory stimulus, a large amount of IL-6 is released. IL-6 is a proin ammatory cytokine that can cause immune disorders and amplify in ammation. Thus, blocking IL-6 could be exploited as a new treatment strategy for in ammatory diseases [33]. FMN certainly deserves attention and has great therapeutic potential because of its effective inhibitory effect on IL-6 generation in this study.
Furthermore, many studies suggest that AMPK is a potential therapeutic target for in ammation, cancer, diabetes, and other diseases [34][35][36][37]. We proposed that FMN, an activator of AMPK, could not only serve as a candidate drug for the treatment of IL-6-mediated diseases but might also be a novel therapeutic agent against other diseases in which AMPK activity represents a potential therapeutic strategy. Therefore, in future studies, we aim to identify the targets of FMN that are involved in the activation of AMPK and verify the anti-in ammatory effect of FMN using animal models.

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Competing interests Figure 1 Chemical structure of FMN. Effects of LPS on cell count, morphology, and IL-6 expression in ANA-1 cells. The morphology of ANA-1 cells was observed under an inverted microscope (a). ANA-1 cells were counted using an HCS (b). IL-6 protein expression level in supernatants from cells was assayed via ELISA (c). Western blotting was performed to determine the levels of IL-6 in cells, with β-actin used as the control (d, e). The numerical data were analyzed using the one-way ANOVA and the protein level data were analyzed using the t-test. Data are represented as the mean ± SD (n = 3). *p < 0.05 and **p < 0.01, compared with the control group.  Effect of FMN on the expression of IL-6 in LPS-stimulated ANA-1 cells. Western blotting was performed to determine the levels of IL-6 in cells, with β-actin used as the control (a, b). IL-6 protein expression level in supernatants from cells was assayed via ELISA (c). Data were analyzed using the t-test. Data are presented as the mean ± standard deviation (n = 3). **p < 0.01, compared with the control group. #p < 0.05 and ##p < 0.01, compared with the LPS group.  Effect of compound C on the phosphorylation of AMPK and IL-6 in ANA-1 cells. Western blotting was performed to determine the effect of compound C on the levels of p-AMPK and IL-6 in ANA-1 cells with βactin used as the control (a, b). Data were analyzed using the t-test. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01, compared with the LPS group. #p < 0.05 and ##p < 0.01, compared with different groups.