Ononin induces cell apoptosis and reduces inflammation in rheumatoid arthritis fibroblast-like synoviocytes by alleviating MAPK and NF-κB signaling pathways

1Department of Joint Surgery, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong Province, 510900, China; 2Department of Breast Surgery, The Fifth Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong Province, 510900, China; 3Department of Spine Orthopedics, Shenzhen People’s Hospital, Shenzhen, Guangdong Province, 518055, China; 4Department of Spine Orthopedics, NanFang Hospital, Guangzhou, Guangdong Province, 510515, China; 5Department of Spine Orthopedics, Hangzhou Xiaoshan Hospital of TCM, Hangzhou, Zhejiang Province, 311200, China


INTRODUCTION
Rheumatoid arthritis (RA), one kind of serious chronic autoimmune and inflammatory diseases, is characterized by inflammation of the joints and parenchyma (Guo et al., 2018). Previous study indicates that synovium is the main lesion site of RA, abnormal synovial hyperplasia and inflammatory cells infiltrating the synovium will eventually lead to inflammatory destruction of the joint (Buchbender et al., 2013). RA has a low cure rate and is prone to relapse, which is caused by the abnormal immune responses (Heidari, 2011). Notably, inhibiting inflammation-related pathways can delay the progression of RA (Chimenti et al., 2015). Non-steroidal anti-inflammatory drugs plus anti-rheumatic medicines and corticosteroids are often used in the first-line treatment of RA, but the therapeutic effects still remains to be improved (Kumar & Banik, 2013). Previous study indicates rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells play a key role in the initiation and progression insynovitis induced RA (Bartok & Firestein, 2010). As the inflammation occurs, RA-FLS cells produce apoptosis resistance, excessive proliferation, migration and invasion, resulting in the destruction of the joint tissue (Shi et al., 2016). Thus, exploring the apoptosis of RA-FLS caused by inflammation is of great significance for further investigating the pathogenesis of RA and improving the treatment strategy.
MAPK and NF-κB signaling pathways are considered to be important signaling pathways involved in the development of RA inflammation (Yang et al., 2017). The MAPK family contains three major classes, such as JNK1/JNK2/JNK3, ERK1/ERK2and p38 (Johnson & Lapadat, 2002). Activated MAPKs induces a series of downstream signaling cascade including ERK, JNK and p38 cascades, which implicated inregulating various cellular processes including cell survival/apoptosis, proliferation and inflammatory responses (Kim & Choi, 2010). NF-κB, a collective name for dimeric transcription factors including cRel, RelA (p65), RelB, NF-κB1 and NF-κB2, is a key factor in the transcription of many inflammatory genes (Shih et al., 2015). NF-κB can be activated by a variety of pathogenic stimulation, including bacterial or viral proteins, cytokines, radiation and oxidative stress (Karin et al., 2004). Activated NF-κB leads to the release of diverse pro-inflammatory factors, including MCP-1, cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), IL-1β and IL-6, which are essential for the inflammatory response. (Makarov, 2001) It has also demonstrated that RA is associated with the abnormal activation of the MAPK and NF-κB pathways (Liu et al., 2017).
Ononin (Formononetin 7-O-glucoside) is a natural isoflavone that widely distributed in a variety of foodborne plants such as Astragalus membranaceus, soybean, kudzu, lupine and broccoli (Dong et al., 2017) been reported that Ononin possesses anti-inflammatory effect on zebrafish model induced by lipopolysaccharide, which can reduce the production of proinflammatory cytokines like TNF-α, IL-1β, IL-6 and IFN-γ, increase the expression of anti-inflammatory factor IL-10, and regulate the activity of MAPK pathway (Luo et al., 2019).
In addition, Ononin can inhibit the expression of NO, PGE2, TNF-α, IL-1β and IL-6 by inhibiting the activation of IκBα and MAPKs, and then plays an anti-inflammatory role in LPS stimulated macrophage RAW264.7 (Dong et al., 2017). Besides, Ononin is also reported to exert inhibitory and pro-apoptotic effects on cell proliferation in breast cancer cell by down-regulating PI3K/ AKT/mTOR signaling pathway . However, whether Ononin has anti-inflammatory and pro-apoptotic effects on RA remains unknown. At present, we established the TNF-α-induced RA-FLS and MH7A cell model and found that Ononin alleviated the cell viability, increased cell apoptosis, decreased theproduction of pro-inflammatory cytokines like IL-1β and IL-6, and further inhibited the abnormal activation of NF-κB and MAPK pathways in TNF-α-induced RA-FLS cells. Our results indicated that Ononin was suggested to be a promising agent for the treatment of RA.

Flow cytometry analysis
Cell apoptosis was measured by FACS Calibur instrument (BD Biosciences, USA), equipped with CellQuest Pro 5 MacOS 9 software. Briefly, RA-FLS and MH7A (1x10 5 cells) were stimulated by TNF-α (10 ng/ml, Pep-roTech) and Ononin with various concentrations of 5, 10 and 50 µM for 48 h. Then the collected cells were washed by cold PBS buffer, subsequently fixed with 70% alcohol under 4°C for 12 h and stained with propidium iodide under 37°C for 30 min prior to flow cytometry (BD Biosciences, USA) analysis.

Ononin alleviated the viability in TNF-α-induced RA-FLS and MH7A cells
To evaluate the therapeutic effect of Ononin, TNF-αinduced RA-FLS and MH7A cells model was constructed. The molecular formula of Ononin was displayed in Fig. 1A. As shown in Fig. 1B, MTT assay showed that TNF-α could significantly induce the cell proliferation in RA-FLS and MH7A cells (p<0.01). However, compared to TNF-α stimulation group, Ononin treatment could significantly alleviate the cell viability both in RA-FLS and MH7A cells (p 0.05 and p<0.01). Additionally, Ononin treatment alleviated cell viability in a concentrationdependent manner. Thus we assumed Ononin could significantly relieve TNF-α-induced RA-FLS and MH7A cells.

Ononin promoted the apoptosis inTNF-α-induced RA-FLS and MH7A cells
The cell apoptosis was assessed by flow cytometry analysis. Compared with control group, TNF-α stimulation would not affect the cell apoptosis both in TNF-αinduced RA-FLS and MH7A cells, which was consistent with previous study (Yamaguchi et al., 2004). However, after Ononin treatment, the cell apoptosis ratios were significantly increased compared to control and Ononin untreated cells (Fig. 2). These results verified Ononin may play a protective effect by attenuating the cell apoptosis in TNF-α-induced RA-FLS and MH7A cells.

Ononin regulated apoptosis protein expression in TNFα-induced RA-FLS and MH7A cells
To further evaluate the protective effect of Ononin on TNF-α-induced cell proliferation in TNF-α-induced RA-FLS and MH7A cells, the expression of cell apoptosis-related proteins was detected by using western blot assay. Previous study indicated that apoptosis-related proteins like B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cleaved caspase-3 and cleaved caspase-9, are modulated during the apoptosis process (Russo et al., 2018). The results showed that compared to control and TNF-α-stimulated RA-FLS and MH7A cells, the apoptosis-related protein expression of Bcl-2 was reduced by Ononin treatment, and a down-regulated tendency was found with the increase of Ononin concentration. However, the expression of Bax, Cleaved caspase-3 and Cleaved caspase-9 proteins was increased by Ononin treatment, and Ononin treatment caused this phenomenon in a dose dependent manner (Fig. 3). Taken togeth-

Ononin reduced inflammatory cytokine production in TNF-α-induced RA-FLS and MH7A cells
Both IL-6 and IL-1β are important pro-inflammatory cytokines responsible for the inflammation of rheumatoid arthritis (Alunno et al., 2017). As shown in Fig. 4, TNF-α could significantly induce the inflammatory cytokines release of IL-1β and IL-6 both in RA-FLS and MH7A cells. In addition, Ononin treatment significantly down-regulated the production of IL-1β in TNF-αinduced RA-FLS and MH7A cells when compared to control and TNF-α stimulation group. And this downregulation manner showed a downward trend with the increased of Ononin concentration (Fig. 4A). Similarly, the expression of IL-6 both in RA-FLS and MH7A cells were also reduced following Ononin challenge and displayed a dose dependent decrease (Fig. 4B). The results suggested Ononin reduced the release of inflammatory cytokines of IL-1β and IL-6 both in TNF-α-induced RA-FLS and MH7A cells.

Ononin regulated MAPK and NF-κB signaling pathways
Abnormal activation of MAPK and NF-κB signaling pathways are involved in the initiation and progression of RA (Yang et al., 2017). Herein, we tried to evaluate the potential therapeutic effects of Ononin on MAPK and NF-κB signaling pathways. Previous study reported that TNF-α effectively activated MAPK and NF-κB signaling pathways as illustrated by increasing the expression of p-p38, p-JNK, p-ERK1/ ERK2 and p-NF-κB (p-p65) (Shi & Sun, 2018). Similarly, Ononin challenge also significantly reduced the phosphorylated level of p38 (p-p38), p-JNK, p-ERK and p-NF-κB in TNF-αinduced RA-FLS and MH7A cells in a dose-dependent manner (Fig. 5).

DISCUSSION
As one kind of serious chronic autoimmune and inflammatory diseases, RA causes many chronic diseases including nephropathy, vasculopathy, pulmonary and cutaneous disorders as a result of the destruction of the cartilage and bone (Gupta et al., 2014). At present, about 0.5-1% of the population suffer from RA, it thus has become a serious threat leading to death (Ahmed, 2010). Even though the exact pathogenesis of RA is still unknown, the possible pathogenic factors of RA contain inflammation, genetic and environmental factors and so on (Mavrogeni et al., 2014). The combination of non-steroidal drugs coids, immunosuppressants and biological therapies have been widely used to treat RA in clinic (Quan et al., 2008). But RA is prone to relapse, above drugs for RA are difficult to meet clinical needs. Therefore, it is urgent to develop more effective therapeutic agents for RA. In this study, we developed a TNF-α-induced RA-FLS and MH7A cell model, and found Ononin could obviously attenuated inflammation and promoted apoptosis in TNF-α-induced RA-FLS and MH7A cells via MAPK and NF-κB pathways. We therefore thought Ononin might be a promising agent for RA treatment.
Ononin, one kind of isoflavone, is a natural plant compound that is similar to 17-β-estradiol in chemical structure and is widely found in Fabaceae/Leguminosae family like soy beans, soy foods, and legumes (Ko, 2014). Ononin has multiple physiological functions, such as antioxidant, antimicrobial and anti-inflammatory activities (Yu et al., 2016). Additionally, people also found Ononin could promote skin growth, scaveng oxygen free radicals, inhibit lipid peroxidation, maintain the concentration of nitric oxide in blood, protect ischemia-reperfusion injury and enhance immunity (Wang et al., 2019). In view of its so many important physiological functions, we speculated that it had a special effect in fighting against RA. Our experimental results confirmed the significant effects of Ononin on the viability and apoptosis of TNF-α-induced RA-FLS and MH7A cells.
In this study, we noticed that Ononin induced apoptosis and attenuated inflammation in TNF-α-induced RA-FLS and MH7A cellsvia inhibiting MAPK and NF-κB signaling pathways. Previous study suggessted above signaling pathways play key roles in inflammatory diseases. For instance, Xia and others (Xia et al., 2018) found inhibiting NF-κB pathway could significantly promote cell apoptosis and inhibit cell proliferation of human FLS cells in RA. Gao et al., found tetrandrine suppressed the developmet of RA inflammation by decreasing the release of inflammatory cytokines via blocking NF-κB (Gao et al., 2016). Liu et al., reported that anhuienoside C could ameliorate collagen-induced RA inflammation by suppressing MAPK and NF-κB Pathways. (Liu et al., 2017) Similarly, we found Ononin reduced cell proliferation and improved apoptosis by blocking these pathways, further confirmed MAPK and NF-κB signaling pathways were important in the progression and pathogenesis of inflammation related diseases like RA. However, the precise effects of Ononin on RA and the potential regulatory mechanisms still need further study.
In summary, we established a TNF-α-induced RA-FLS cell model and found the effects of Ononin on apoptosis in these cells. Our data confirmed that Ononin alleviated the cell viability, increased the apoptosis and decreased the cytokines release like IL-1β and IL-6, and further inhibited the abnormal NF-κB and MAPK pathways activation in TNF-α-induced RA-FLS and MH7A cells. We therefore thought Ononin could be used as a promising agent for RA treatment in the future.