Cytokines are vital cellular signaling molecules and mediators of the immune system that monitor and regulate immune and inflammatory responses through complex networks. Unusual cytokine levels can contribute to cytokine storms and disease. The level of cytokines is therefore of great value in the diagnosis and therapy of diseases (Liu et al., 2021). A wealth of studies have shown remarkable anti-inflammatory activity of FMNT on luxuriant inflammatory models induced by manifold inflammagens in vivo and in vitro (Table 1). The primary macrophage-secreted pro-inflammatory cytokine, such as TNF-α and multiple ILs, chemokines, adhesion molecules and NOsynthesized by iNOS via the L-arginine pathway, are widely accepted to be compactly implicated in the development and amplification of inflammation.

It has been reported that FMNT had a neuroprotective effect on brain injury by alleviating neuroinflammation. For example, the researchers found that levels of inflammatory cytokines were not only reduced by FMNT in the injured brain tissues. In addition, the team enriched the intervention effect of FMNT on inflammation in serum and brain tissue of the cortical neurons in follow-up studies. This also reflects the critical role of FMNT in brain disease inflammation. (Li et al., 2014; Li et al., 2018). In addition, FMNT was also able to alleviate side effects of drugs with multiple organ toxicity, and organ damage due to serious diseases. FMNT improved liver dysfunction in rat models of Ritonavir induced hepatotoxicity via inhibiting irregular IL-1β, IL-6, and TNF-α secretion (Alauddin et al., 2018). Besides, researchers demonstrated pro-inflammatory cytokine inhibitory activity of FMNT against acute lung injury induced by LPS. The authors observed a downregulation of increased IL-6 and TNF-α levels in dose-dependent manners after FMNT administration (Ma et al., 2013). Besides that, pre-treatment with FMNT distinctly attenuated the hyperoxia-induced increase of pro-inflammatory cytokine secretion (Chen et al., 2021). Similarly, other researchers found that FMNT administration mitigated the overexpression of TNF-α and IL-1β in cisplatin-induced acute kidney injury models on male Wistar rats (Hao et al., 2021).

In addition to anti-inflammatory cytokine release in vivo, FMNT also has the potential to diminish the overexpression of pro-inflammatory cytokines in inflammatory models in vitro. Recently, researchers evaluated the effect of FMNT in treating osteoarthritis. According to the authors, 1.25 μg/mL FMNT treatment visibly lowered IL-1β secretion in IL-1β stimulated chondrocytes (Xiong et al., 2021). Notably, the FMNT used in the experiments in both in vivo and in vitro in this article is the same concentration, which is inconsistent with the doses of the ordinary article and deserves more rigorous consideration. The potential of FMNT in remedying inflammation has been evaluated using the RAW264.7 macrophage model triggered by LPS. According to the results, FMNT showed reduction of the NOx level in the inflammatory macrophages, exhibiting favorable antioxidant activity (Bottamedi et al., 2021). Other authors stated that 32 μg/mL FMNT significantly reversed the strong inflammatory response caused by streptococcus suis serotype 2 infection in macrophage-like J774 cells (Wang et al., 2020).

The chemokines (or chemotactic cytokines) are a large family of small, secreted proteins that signal via G protein-coupled heptahelical chemokine receptors on the cell surface (Hughes and Nibbs, 2018). They act primarily by stimulating cell migration and are the “primary regulators” of the movement and positioning of many inflammation-associated cells such as macrophages, lymphocytes, neutrophils and dendritic cells in the body. Chemokines therefore play a remarkable role in the development of inflammatory responses in vivo in terms of leukocyte recruitment and are involved in all destructive or protective inflammatory responses (Zlotnik and Yoshie, 2012). The studies reporting the inhibitory actions of FMNT on chemokines are discussed (Table 1).

Some researchers have reported the chemokines inhibitory effect of FMNT. In this study, the authors noted that FMNT treatment reduced eotaxin and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion in JME/CF15 cells after IL-13 stimulation in a dose-dependent manner (Huang et al., 2021). In another study, the chemokine inhibitory features of FMNT also have been displayed in rat chondrocytes induced by IL-1β. According to the authors, FMNT lowered the level of several chemokines, including fractalkine, macrophage inflammatory protein-3α, and monocyte chemoattractant protein-1, and showed brilliant osteoarthritis relief (Cho et al., 2019).

PGE2 produced by mPGES-1 plays a crucial role in inflammation (Chang and Meuillet, 2011; Finetti et al., 2012). Isoform two of cyclooxygenase (COX2) converts arachidonic acid (AA) to PGH2, an unstable metabolite, which is then isomerized by mPGES-1 to inflammatory PGE2. Thus, COX2 inhibition has a specific value in the onset and duration of inflammatory diseases. Several studies reporting COX2 inhibitory properties of FMNT are summarized in this section (Table 2).

Studies have proven that chondrocyte dysfunction, such as type II collagen degradation and inflammation, is associated with joint disease like rheumatoid arthritis and osteoarthritis (Menu and Vince, 2011). Researchers reported anti-inflammatory role of FMNT via primary Rat Chondrocytes. According to the authors, 25 and 50 μM FMNT markedly downregulated the expression of PGE2 as well as COX2 in primary rat chondrocytes treated with 10 ng/mL IL-1β, in which the specific production of PGE2 was inhibited by 98% ± 3% and 77% ± 2.2%, respectively compared to control group. Results above suggested the counteracting role of FMNT on IL-1β-induced catabolic pro-inflammatory effects in primary rat chondrocytes and may counteract IL-1β-induced articular cartilage degeneration (Cho et al., 2019).

Scholars investigated the protective effect of FMNT on ox-LDL-induced endothelial dysfunction via network pharmacology and experimental validation. According to the authors, FMNT had an effect on anesthetizing ox-LDL-induced inflammation and apoptosis in HUVECs through downregulation of COX2, eNOS, and cleaved caspase-3 expression (Zhang et al., 2021). FMNT treatment also suppressed the level of PGE2 in LPS-treated RAW264.7 macrophage cells in a dose-dependent manner (Kim et al., 2019). What’s more valuable is that the authors of this study synthesized a novel bioinnovative product (formononetin 7-O-phosphate), which has lower cytotoxicity and stronger anti-inflammatory activity compared to FMNT. Moreover, other researchers reported the influence of FMNT on neuroinflammation in LPS-treated BV2 microglia. According to the authors, FMNT dramatically decreased the level of PGE2, iNOS and COX2, providing strong support for the development of new compounds with selective neuroprotective effects in the brain (El-Bakoush and Olajide, 2018). In addition, the COX2 inhibitory properties of FMNT against methotrexate-induced acute kidney injury in Wistar rats were evaluated. The authors verified that rats treated with FMNT had enhanced anti-inflammatory features, compared with positive controls due to its capacity to significantly downregulate COX2 production (Aladaileh et al., 2019). All in all, these results provide evidence that FMNT is capable of blocking the conversion of arachidonic acid to PGE2 by inhibiting COX2, thereby ameliorating the inflammatory response. Additionally, the inhibition of COX2 may be the result of restrain of one or more inflammatory intracellular and intercellular signaling cascades.

NF-κB is a key transcriptional regulator that controls the expression of many inflammatory and immune genes. It is inactivated in the cytoplasm by binding to I-κB. Once activated by stimuli such as viruses, oxidants and inflammatory cytokines, NF-κB dissociates from I-κB and binds to a specific promoter in the nucleus, thereby regulating gene expression and activate the transcription of TNF-α, IL-6, IL-1β and COX2. Excessive activation of NF-κB is associated with cancers, immune cell dysplasia, delayed cell growth as well as many chronic inflammatory diseases, including rheumatoid arthritis, asthma, AIDS, atherosclerosis and neurodegenerative diseases. Thus, in this portion, we review several studies that have reported the inhibitory properties of FMNT on NF-κB (Table 3).

Allergic diseases, which include food allergies, asthma, anaphylaxis, sinusitis and atopic dermatitis, are triggered by allergens and feature adaptive allergic inflammation. In a study, the NF-κB inhibitory role of FMNT was assessed in a mast cell-mediated allergic inflammation model. Results indicated that the properties of FMNT were correlated with a restriction of NF-κB activation and upstream IκKα phosphorylation in a dose-dependent manner (Xu and An, 2017). However, this study was lack of the exploration of the dosage of FMNT, which was addressed in other articles. The NF-κB inhibitory effect of FMNT in an ovalbumin-induced asthmatic female BALB/c mice model was also been evaluated. According to the authors, FMNT treatments appeared to observably inhibit the activation of NF-κB, certifying distinct therapeutic effects of FMNT in attenuating airway inflammation (Yi et al., 2020). A20 is a negative regulator of NF-κB, which plays an integral role in the regulation of immune and inflammatory responses (Priem et al., 2020). In another study, researchers supplied evidence of the anti-inflammatory ability of FMNT against FITC-treated atopic dermatitis mice model as well as siA20 and siGPER-transfected HaCaT cells model. As reported, FMNT (10 mg/kg, i.p.) was capable of attenuating atopic dermatitis by enhancing A20 expression via GPER activation (Yuan et al., 2021). All of the above studies indicated that FMNT had a clear therapeutic effect on allergic inflammation.

A study investigated the anxiolytic role of FMNT against chronic inflammatory pain responses in C57BL/6 male mice. The FMNT administration has shown to inhibit NF-κB signaling and microglial activation in the mice with chronic inflammatory pain, thereby relieving the anxiety (Wang et al., 2019). Meanwhile, FMNT also has a non-negligible role in the regulation of autoimmune diseases. For example, studies showed that FMNT decreased the level of p-NF-κB p65 and increased the level of IκBα, the inhibitor of NF-κB, in the liver from concanavalin-A-induced autoimmune hepatitis mice, showing a powerful immunomodulatory property (Liu et al., 2021). FMNT also possess activity of restraining monocrotaline-treated pulmonary arterial hypertension, whose pharmacological mechanism was related to the downregulation of phosphorylated ERK and NF-κB (Wu et al., 2020). In addition to the inflammation model mentioned above, NF-κB pathway plays a critical role in mastitis. Scholars determined the protective effect of FMNT in LPS-induced mastitis in female lactating C57BL/6 mice and mouse mammary epithelial cells EpH4-Ev, and they observed an obvious inhibition of NF-κB signaling pathway activation in both in vitro and in vivo experiments (Xiang et al., 2022).

The MAPK signaling pathway, a highly conserved serine/threonine protein kinase, is one of the most fundamental regulatory mechanisms in eukaryotic cells and part of a vital signaling system (Krishna and Narang, 2008). There are three well-characterized subfamilies of MAPKs in mammals: the ERKs, the JNKs and the p38 kinases (Cargnello and Roux, 2011). Of the three types of MAPKs, ERKs and JNKs are closely associated with some physiological processes, such as immunity, inflammation and tissue growth.

Several studies have shown the inhibition of FMNT to the activation of MAPK signaling pathways (Table 4). For instance, researchers revealed that FMNT administration was capable of down-regulating the expression of JNK1, ERK1 and p38α, which are compactly associated with MAPK pathway in LPS-induced inflammatory zebrafish models, thus showing a dramatic anti-inflammatory activity (Luo et al., 2019). Besides, in another experiment, the potential of FMNT for ameliorating cholestasis has been assessed. This study found that FMNT attenuated alpha-naphthyl isothiocyanate-induced inflammatory response through inactivation of JNK inflammatory pathway in a PPARα-dependent manner (Yang et al., 2019).

JAK/STAT signaling pathway, one of the key central communication nodes for many cellular functions and processes, is a means of intracellular signaling in response to growth hormones and cytokines (Darnell, 1997). Its aberrant phosphorylation triggers the overexpression of key mediators associated with various autoimmune and inflammatory diseases (Liongue and Ward, 2013; Schwartz et al., 2017). Several of these studies presenting the JAK-STAT inhibitory effects of FMNT are reviewed in this section (Table 4).

In a study, researchers tested anti-inflammatory properties of FMNT on cerebral ischemia-reperfusion injury model in SD rats. Using Western blotting they found that FMNT inhibited the protein expression of p-JAK2 and p-STAT3 at a dose of 30 mg/kg body weight, indicating prominent JAK2/STAT3 signaling pathway inhabitation (Yu et al., 2022). In another study, FMNT was shown to lessen constitutive STAT3 and STAT5 activation in U266 and RPMI 8226 cells belonging to myeloma cell line after FMNT therapy. More interestingly, the raised production of reactive oxygen species (ROS) due to a GSH/GSSG imbalance was involved in above effect (Kim et al., 2018).

Inflammasome is a group of intracellular polymeric protein complexes that activate inflammatory caspase-1. Its activation is a major inflammatory pathway and a key participant in innate immunity discovered in recent years (Franchi et al., 2009). Several inflammasomes have been identified, including NLRP2, NLRP7 and NLRP12, of which NLRP3 is the best characterized (Khare et al., 2012; Vladimer et al., 2012; Minkiewicz et al., 2013). NLRP3 is essential for host immune defense and is closely related to a variety of autoimmune and autoinflammatory diseases when it is disrupted (Menu and Vince, 2011). Several of these studies presenting the NLRP3 inflammasome inhibitory effects of FMNT are reviewed in this section (Table 4).

In a recent study, scholars explored therapies for lung injury and fibrosis and found that FMNT nanomedicines could block macrophage pyrodeath involved in NLRP3, thereby improving lung function and longevity in mice (Ouyang et al., 2023). FMNT was also found to suppress streptozotocin-induced NLRP3 protein overexpression in diabetic male C57BL/6 J mice (Wang et al., 2018). Other scholars assessed the NLRP3 inflammasome signaling pathway inhibitory activity of FMNT in vitro. They discovered that FMNT (25 μM, 50 μM) treatment to acute injury model of human colon carcinoma cell line (HCT-116) induced by TNF-α for 12 h led to the downregulation of NLRP3 inflammasome expression levels. Furthermore, they also performed simultaneous in vivo experiments and found that NLRP3 expression was diminished in the colon tissue of dextran sulfate sodium-induced acute colitis mice after FMNT treatment. This finding is consistent with the in vitro experiments (Wu et al., 2018).

The above evidence suggests that FMNT is a very promising anti-inflammatory agent that can inhibit the release of cytokines and chemokines by down-regulating the over-activated COX2/PGE2 pathway, NF-κB pathway, MAPK pathway, JAK-STAT pathway and reducing the production of NLRP3 inflammasome. It is well known that FMNT can be used in the treatment of multiple diseases such as liver injury, kidney injury, central nervous system and airway inflammation. Based on in vitro and in vivo studies of the mechanisms involved, we have summarized schematically the interaction pathways for the anti-inflammatory activity of FMNT as shown in Figure 2.