Cinnamomi ramulus exhibits anti-proliferative and anti-migration effects on MH7A rheumatoid arthritis-derived fibroblast-like synoviocytes through induction of apoptosis & cell arrest and suppression of matrix metalloproteinase

Background: Rheumatoid arthritis (RA) is a complex chronic inflammatory disease that is associated with the aberrant activation of fibroblast-like synoviocytes (FLS). The extract of Cinnamomi ramulus has been reported to exert alleviates pain, anti-tumor and anti-inflammatory effects. The present study was designed to investigate the effects of Cinnamomi ramulus on RA and explore the underlying mechanisms. Material/methods: TNF-α induced human synoviocyte MH7A cells was performed to evaluate the anti-proliferative and anti-migration effect of Cinnamomi ramulus . The anti-proliferative effect of Cinnamomi ramulus was determined by CCK-8 assay and colony formation assay. Apoptosis was measured by AnnexinV FITC/PI staining and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis and cell cycle-related molecules, including Bcl-2, Bax, C-Caspase-3, CDC2 and Cycylin B1 were determined by Western blotting. Furthermore, the migration and invasion abilities of MH7A cells were determined using scratch wound healing assay and transwell assay. mRNA expressions of (MMP)-1, -2, & -3, P53, P21 and Cyclin D were determined using qRT-PCR analysis. For qualitative analysis on its chemical components, an ultra-high performance liquid chromatography (UPLC) coupled with Q-Exactive MS (QE-MS) was established for rapid separation and structural identification of the constituents in Cinnamomi ramulus . The further computationally study on the relationships between the 9 compounds and the potential target proteins of RA were carried out with molecular docking strategy. Results: Our data demonstrate that Cinnamomi ramulus inhibited proliferation of MH7A cells, induced cell apoptosis, blocked the cell cycle in the G2/M phase and regulated the protein expression of Bcl-2, Bax, C-Caspase-3, CDCD2 and Cyclin B1. Moreover, Cinnamomi ramulus was proven to significantly inhabited migration and invasion of MH7A cells ramulus reduced mRNA levels of CDK4 whereas increased the expression of P53, P21 and CyclinD, implying its regulation effects on apoptosis and cell cycle distribution in MH7A cells. The chromatographic profiling of the extract by UPLC-QE-MS/MS analysis showed 9 compounds are the main components. And the molecular docking strategy results showed that the compounds in Cinnamomi ramulus have good affinity with protein crystal, and benzyl cinnamate may be the main active component of Cinnamomi ramulus to induce cell apoptosis and cycle resistance. Conclusions: exhibits and on

Background RA is a chronic autoimmune joint disease characterized by synovial tissue inflammation, destruction of articular cartilage, and deformities of the joints whose exact cause is still not completely known [1,2]. Synovial fibroblasts (SFs) play an important role in cartilage destruction by mediating most relevant pathways. Increasing evidences have suggested that activated synovial fibroblasts can manifest similar properties of tumor-like cells, such as hyperproliferation and insufficient apoptosis [3]. The uncontrolled proliferation of synovial fibroblasts has been thought to contribute to the formation of rheumatoid arthritis (RA) [4]. Meanwhile, Synovial fibroblasts also can spontaneously secrete numerous pro-inflammatory cytokines and matrix metalloproteinases (MMPs), which plays an important role in progressive destruction of articular cartilage [5,6]. Thus, that promoting apoptosis and inhibiting proliferation of synovial fibroblasts is believed to exert potential therapeutic effects on RA.
Cinnamomi ramulus, known as "Guizhi" in China and "Geiji" in Korea, has been traditionally used to reduce chronic joint pain in patients with arthritis [7,8]. it's reported that the extract of Cinnamomi ramulus has a variety of biological activities, including analgesic, anti-tumor and anti-inflammatory activities [9,10]. These bioactivities of Cinnamomi ramulus might be depend on the presence of certain classes of compounds in this plant, such as phenylpropanoids, monoterpenoids, sesquiterpenoids, sterols, etc [ 11].
Although therapeutic potentials of Cinnamomi ramulus for inflammatory reactions have been investigated, its effect in RA is currently poorly understood. In previous studies, we found that the traditional Chinese recipe guizhi-shaoyao-zhimu decoction had a significant anti-arthritic effect [8], and Cinnamomi ramulus one of the most important herbal medicines in this traditional Chinese Medicine (TCM) formula. In addition, there are also many other TCM formulas with good curative effect for RA with Cinnamomi ramulus as the main component, such as huangqi guizhi wuwu decoction, guizhi fuzi decoction, and chai hu guizhi ganjiang decoction, etc [12][13][14]. In addition, some modern studies have confirmed the anti-RA potential of Cinnamomi ramulus, but the detail molecular mechanisms remains unclear.
In this paper, we assessed the anti-arthritic effects of the aqueous extract of Cinnamomi ramulus on a human synovial fibroblast cell line (MH7A), focusing on possible mechanisms associated with anti-inflammatory, suppressing invasion & migration of synovial fibroblasts, and inducing cell cycle arrest and apoptosis. In addition, we used the molecular docking to study the interactions between the main compounds of Cinnamomi ramulus and the rheumatoid arthritis-associated protein targets. Therefore, our present study provides a direction for the mechanism and drug research of anti-rheumatoid arthritis of Cinnamomi ramulus, as well as a reference for the new treatment strategy of rheumatoid arthritis and the clinical application and development of Cinnamomi ramulus. was air-dried and cut into small pieces. The prepared sample was soaked by 300 mL of distilled water for 2 h and then left to boil for 1 h in a closed flask. Afterwards, the filtrates were cooled to room temperature and subsequently dried by frozen (-20 °C) and lyophilized to afford the dry extract (0.6678 g). Then, the Cinnamomi ramulus extract (CRE) was dissolved in DMEM at the appropriate concentrations for the further experiment.

Cell viability assay
Cell viability was determined using the CCK-8 kit according to the instruction of manufacturer. Briefly, MH7A cells were seeded at 5 × 10 3 cells/well in 96-well culture plates in DMEM containing 10% FBS, incubated overnight. Then cells were stimulated with TNF-α (20 ng/mL) and exposed at various concentrations of CRE for 24 h. After that, CCK-8 was added to each well of the plate and incubated at 37 °C for 1. were then kept at -20 °C for 24 h before analysis. Cells were stained with propidium iodide (PI; Sigmar), and the DNA content was determined by FACS Calibur flow cytometry (Becton Scratch wound healing experiment Cell migration assay was performed by wound healing assay. MH7A cells (5 × 10 5 /well) were seeded into a 6-well plate, and when the cells covered the whole bottom surface of the well, the serum-free medium was used to continue the culture for 12 h, so as to eliminate the influence of normal cell growth on the scratch experimental results. After that, a p200 pipet tip was used to make a scratch at the bottom of the well, and the cells were treated with TNF-α (20 ng/mL) and CRE (0.2, 0.4 and 0.6 mg/mL). After 24 hours of scratch, cells were stained with crystal violet, and an inverted microscope (Nikon TS2, Tokyo, Japan) was used to observe and photograph the scratch area, and then the distance change of the scratch area was measured and analyzed.

Transwell experiment
Transwell chamber was used to assess the migration and invasion capacity of cells. The cells (1 × 10 5 /well) were seeded in a serum-free medium in the upper chamber of transwell (Corning Inc, Corning, NY, USA), and a DMEM medium containing 20% serum in the lower chamber,and cells treated with TNF-α (20 ng/mL) or CRE (0.2, 0.4 and 0.6 mg/mL) for 24 h. After that, the transwell chambers were removed, and the cells inside the membrane were gently scraped off with a cotton swab, while the cells outside the membrane were thought to be migratory or invasive. The migratory or invasive cells were fixed with 4% paraformaldehyde at room temperature for 20 min. Wash with PBS three times and stain with crystal violet for 10 min. Finally, an inverted microscope (Nikon TS2, Tokyo, Japan) was used to observe and photograph. In addition, Image J software (version 1.51, nih, MD) was used to count invasive cells. In contrast to the migration experiment, 0.1% matrigel (BD) was spread at the bottom of the transwell chamber in the invasion experiment.
Colony formation assay MH7A cells (1 × 10 3 /well) were seeded in 6-well plates and treated with TNF-α (20 ng/mL) and CRE (0.2, 0.4 and 0.6 mg/mL) for 24 h. The cells were cultured in a new medium for a week, the cells were immobilized and stained with crystal violet, the number of colony formation was counted in a random microscopic field and the photos were taken.

Western blot analysis (WB)
MH7A cells (5 × 10 5 /mL) were treated with TNF-α (20 ng/mL) and various concentrations of CRE (0.2, 0.4, 0.6 mg/ml) for 24 h. RIPA lysis buffer (containing protease and phosphatase inhibitors) was used to collect protein from cell samples. The supernatant of lysate was boiled, and total protein was measured using a BCA kit. Proteins were separated by SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% BSA (Bovine SerumAlbumin) in TBST (Tris-Buffered Saline and Tween 20) at room temperature for 1 h, followed by exposure to the corresponding primary antibodies overnight at 4 °C. After washing with TBST for three times, the membranes were incubated with the secondary antibodies. Proteins were scanned using the ECL detection system, the gel images were analyzed using the Image J (v1.51) image processing software.

RNA extraction and quantitative real-time PCR (qPCR)
According to the manufacturer's instructions, total RNA was extracted using Trizol reagent according to the manufacturer's instructions and each sample was reverse transcribed using the cDNA synthesis kit according to the manufacturer's protocol. The mRNA expressions of MMP-1, 2 & 3, P53, P21, CDK4 and Cyclin D were determined by qRT-PCR assays using SYBR Green PCR Premix Ex Taq II reagents on a Light Cycler 480 II real-time system (Roche, Mannheim, Germany). GAPDH, a house-keeping gene, was used for was used as a quantitative control for RNA levels. Relative gene expression was calculated by the ΔΔCt method. The sequences for the relevant primers are listed in Table 1. qPCR was run with an initial denaturation step at 95 °C for 30 s, followed by extension step at 57 °C and 30 s for 44 cycles.

Statistical analysis
All results were presented as mean ± standard deviation (SD). Statistical significance between groups was analyzed by Student-t test or ANOVA of SPSS software (version 19).
Statistical significance was defined as a p < 0.05.

Result
Cinnamomi ramulus extracts inhibits the proliferation of synovial Furthermore, colony formation is an in vitro cell survival assay that is based on the ability of a single cell to grow into a colony. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division, and to measure the proliferation capacity of the cells [15]. interestingly, similar to the results of CCK-8 assay, colony formation assay showed that CRE (0.2, 0.4 and 0.6 mg/mL) significantly and concentration-dependently inhibited the colony formation rate (P<0.001), compared to the control cells (Fig. 2).

Cinnamomi ramulus extracts induces apoptosis in synovial fibroblasts
In confocal microscope to exhibit early and late apoptotic characteristics (Fig. 4).

Cinnamomi ramulus extracts inhibits cell migration and invasion in synovial fibroblasts
The wound healing and transwell assays carried out to determine the cell migration and invasion in MH7A cells, and the results showed that CRE (0.2, 0.4, 0.6 mg/mL) can inhibit cell migration and invasion of MH7A cells with a concentration-dependent manner. In the wound healing assay (Fig. 8A), we found that the scratch prepared by pipette tip in the normal and TNF-α stimulated groups were almost fully filled with MH7A cells, however, the migratory ability of cells in the CRE-treated cells were decreased compared with the control MH7A cells (P<0.001). Similar to the results of wound healing assay, significant decreased cells were detected in CRE (0.2, 0.4, 0.6 mg/mL) treated MH7A cells for the transwell migration experiment (P<0.01), compared to the control MH7A cells (Fig. 8B).  Figure 11 showed the MS total ion chromatograms (TIC) provided by analysis of the aqueous extracts in positive ionization modes. To qualitative investigate the main constituents of water extracts of Cinnamomi ramulus, we compared individual retention times (t R ), the online MS spectra and the reference standards in the literature to confirm the identity of the analyte. Peaks 1-9 were unequivocally identified as anisic acid, coumarin, 2-methoxycinnamic acid, coniferyl aldehyde, azelaic acid, cinnamic acid, cinnamaldehyde, 4-methoxycinnamaldehyde and benzyl cinnamate, respectively. Reference standards were used to confirm the retention times, accurate mass and fragment ions. Tentatively identified compounds in the aqueous extracts from Cinnamomi ramulus (Fig. 11) and the main parameters that support their identification are presented in Table 2.

Molecular docking study
In order to study the active substance basis of Cinnamomi ramulus for its anti-RA effects, the molecular docking strategy was carried out to screen the identified 9 compounds. The active targets were proteins or genes reported in previous paper, and ultimately nine potential protein targets and their target protein-ligand crystal complexes were downloaded from the PDB ( Table 3). The experimental results showed that compound 9 (benzyl cinnamate) had a good affinity with these selected protein targets, and its docking score was much higher than that of the prototype ligand of the target proteins, suggesting that compound 9 might be an important active component of Cinnamomi ramulus for its anti-RA effects. The results are shown in Fig. 12. Table 3 Information of the 9 potential target proteins investigated in the present study

Discussion
At present, the pathogenesis of RA is considered to be a multifactorial interaction process, and its occurrence and expression are influenced by many risk factors such as heredity and environment [22]. The inflammatory reactions of RA are related to numerous cell types, and FLS have been identified as the cells responsible for and destruction of cartilage and bone. RA is characterized by the proliferation of synoviocytes in inflamed synovia. Moreover, synovial fibroblasts show evidence of transformation indicated by excessive proliferation, loss of contact inhibition and increased migration [23].
Unfortunately, although there are many RA treatment options available, including traditional disease-modifying antirheumatic drugs (DMARDs) and currently available biologicals agents, the general effectiveness of the drugs has been far from satisfaction [24]. Therefore, we should pay more attention to the development of novel drugs for treating RA. Numerous previous reports indicated that the Cinnamomi ramulus extracts (CRE) has a variety of biological actions, including anti-microbial, anti-inflammation, and anti-RA activities. On the basis of the known anti-inflammatory and anti-RA effects of Cinnamomi ramulus, we investigated the effects of Cinnamomi ramulus on RA using human synovial cell line MH7A cells and its underlying mechanisms.
In the present study, CCK-8 assays results showed that Cinnamomi ramulus can significantly inhibit the cell viability and proliferation in a dose-dependent manner, indicating that Cinnamomi ramulus exerts potent anti-proliferative effect on MH7A cells.
To investigate the mechanism by which Cinnamomi ramulus inhibits synovial cell proliferation, we evaluated the ability of Cinnamomi ramulus to induce MH7A cells apoptosis and cycle arrest. Promoting programmed cell death (apoptosis) is an important strategy for RA therapy [25,26]. It's known that there are two main ways for apoptosis: The death receptor way and the intrinsic mitochondrial way. The intrinsic mitochondrial mediated apoptosis is considered to be the more critical way of the two [27,28].
Mitochondrial mediated apoptosis is largely regulated by the Bcl-2 protein family. It is well known that the Bcl-2 family proteins, anti-apoptotic proteins such as Bcl-2, inhibit apoptosis, while pro-apoptotic proteins, such as Bax, activate apoptosis in RA [29]. In this study, our results showed that Cinnamomi ramulus increased the expression of Bax in MH7A, as well as decreased the expression of Bcl-2 protein. Moreover, the expressions of cleaved caspases 3 was significantly up-regulated in MH7A cells by Cinnamomi ramulus.
These results collectively indicated that Cinnamomi ramulus has obvious pro-apoptotic effect on MH7A cells.
Cell cycle control is the major regulatory mechanism of cell growth, and central to this process are the cyclin-dependent kinases (Cdks), which complex with the cyclin proteins [30]. In G2-M transition, the cyclin-dependent protein kinase complex, CDC2-Cyclin B1 complex could be used as a marker of G2/M phase arrest [31]. P53 protein was a key tumor suppressor in cells. Through downregulating the expression of gene products which are essential for progression through the cell cycle, activation of the P53 tumor suppressor can lead to cell cycle arrest [32]. Furthermore, P21 also act cyclindependent kinase inhibitors able to arrest cells in the G2/M phase [33]. The Cyclin D-CDK4 complex plays a role in G1 phase, and the cell cycle enters G1 phase when CDK4 and CDK6 form active complexes with D-type cyclins. However, a recent study found that CDK4 plays an unexpected role in the G2/M checkpoint [34,35]. In this study, we detected the