Natural drugs are potential resources for anti-RA treatment [20, 21]. A growing number of studies have demonstrated that natural drugs play an important role in anti-inflammation therapies. For example, TG, represented by triptolide, has been widely used in anti-RA treatment as one of the DMARDs. Our study found that FMN showed similar activities of anti-inflammation effects. The results showed that the therapeutic effects of FMN on the arthritis of CIA mice were achieved by inhibiting the NF-κB signaling pathway, by down-regulating IL-6, and TNF-α expression and release, as well as by upregulating IL-10.
The CIA model is one of the most classical and widely used animal models in anti-arthritis drug studies and is used in the study of disease pathogenesis and validation of potential therapeutic targets [22, 23]. Hence, in this study, we adopted the CIA model by inducing inflammation in C54 background mice with chicken collagen type II. By scoring the paw swelling and the arthritis index during the experiment, we monitored the disease progression  and evaluated the anti-arthritis activity of FMN. The results showed that 100 mg/kg FMN reduced the paw volume of CIA mice and significantly decreased the arthritis index, which was functionally equivalent to treatment with 10 mg/kg TG. On day 49 following disease induction, the mice paw swelling and arthritis index scores in the FMN-M group were significantly lower than those on day 21, indicating that FMN could significantly delay the disease progression of CIA. Additionally, animals in the RA group (without anti-RA drug treatment) suffered from weight reduction due to loss of appetite, disturbance of the immune system and persistent inflammation, which can also be used as an acute clinical signal to reflect the severity of the disease [25, 26]. Thus, increased feeding is usually taken to counteract or prevent weight loss in RA animals . However, the FMN treatment did, at least in part, prevent the weight loss of CIA mice in our experiment, with FMN-M displaying the strongest effect. Furthermore, the disruption of joint and synovial tissue usually reflects RA progression and thus becomes one of the most important targets of RA therapy [27, 28]. Our data showed that FMN alleviated the inflammation and destruction of joint and synovial tissues of CIA mice at an optimal dose of 100 mg/kg body weight. Histopathological analysis demonstrated that no synovial inflammation and hyperplasia, or cartilage destruction in the mice of the healthy control group, while in the RA group mice had obvious inflammation, immune cell infiltration, synovial hyperplasia, and cartilage destruction. In the FMN groups and the TG group, drug treatment significantly alleviated synovial inflammation, synovial hyperplasia, and articular cartilage destruction.
At present, synovial inflammation and joint destruction are the two main problems faced by RA patients. TNF-α overexpression leads to excessive production of many cytokines, such as IL-6 [29, 30]. TNF-α and IL-6 are also the two key inflammatory cytokines media in the process of RA sustainable development . IL-6 promotes the aggregation of osteoclasts and inhibits the synthesis of proteoglycan . Osteoclasts are the key factor causing joint erosion , while proteoglycan is the main component of articular cartilage, and the reduction of proteoglycan leads to the degradation of articular cartilage. Moreover, IL-10 is an important multifunctional cytokine with anti-inflammatory activity, capable of inhibiting the cytokine producing Th1 cells . Therefore, in this study, we analyzed the expression levels of pro-inflammatory cytokines TNF-α and IL-6, as well as of anti-inflammatory factor IL-10 in serum of CIA mice. The content of TNF-α and IL-6 significantly decreased in the FMN-treated group, while the content of IL-10 increased dramatically.
Although our data indicated that FMN had a significant therapeutic effect on the CIA mice, one phenomenon has attracted our attention, that is, mice treated with the high dose (200 mg/kg) of FMN were not better off than the mice treated with a middle or low dose of FMN. Importantly, only the middle dose (100 mg/kg) of FMN optimally increased the level of the anti-inflammatory factor IL-10 in the serum and reduced the swelling degree of paws, while these effect were lost at a high dose of FMN. Treatment with the middle dose of FMN also led to a lower content of pro-inflammatory cytokines of IL-6 and TNF-α in the serum compared with mice treated with the high or low dose of FMN. The results also indicated that with an increase in FMN dose, the content of TNF-α in serum showed a dose-dependent increase, although the reason for this is still unclear.
NF-κB is the most classical pathway involved in the activation and regulation of inflammatory responses . Recent studies have shown that the activated NF-κB pathway promoted the release of many inflammatory cytokines such as TNF-α and IL-6 in stimulated macrophages and synovial cells, thus playing an essential role in the pathogenesis and progression of RA [36, 37]. Research data have shown that TIPE-2 was preferentially expressed by immune cells, and could inhibit the NF-κB pathway activation in T cells and macrophages . The downregulation of TIPE-2 expression activated the NF-κB pathway, a process that might be related to the occurrence and development mechanisms of RA . Therefore, we considered the NF-κB pathway as a potential therapeutic target in RA given the anti-inflammatory effects of inhibiting IκB-α, TIPE-2 degradation, and blocking the activation of the NF-κB pathway. Our study investigated the inhibitory effects on the progression of RA of the members of the NF-ĸB pathway, including p65, p-p65, IκB-α, TIPE-2, and PCNP in both the RA group and the drug-treated groups. The results showed that FMN could down-regulate the expression of p-p65 and PCNP, and inhibit the degradation of IκB-α and TIPE-2. Again, the data revealed that the middle dose of FMN appeared to yield optimal effects and there was no benefit to treating with high dose FMN. In summary, FMN plays an anti-RA role mainly by inhibiting the activation of the NF-κB pathway.