The Effect of Serum Carrying Total Flavonoids of Rhizoma Drynariae on the Proliferation and Apoptosis of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Objective: To investigate the mechanism of the effects of total flavonoids of Rhizoma Drynariae in different concentrations on synovial inflammation in rheumatoid arthritis, which is expected to provide experimental evidence for clinical application. Methods: Serum carrying total flavonoids of Rhizoma Drynariae was used to treat the in vitro culture of synovial cells isolated from patients with rheumatoid arthritis. Cells were identified and passaged before the cell treatment. The experimental groups included the 20% blank serum group, the 20% high-dose total flavonoids of Rhizoma Drynariae medicated serum group, 20% low-dose total flavonoids of Rhizoma Drynariae medicated serum group, and the 20% tripterygium glucosides medicated serum group. With the serum carrying tripterygium glycosides as a positive control, the drug-carrying serum at a concentration of 20% was added to the third-generation synovial cells, and the cells were continuously cultured. Results: Compared with the blank control serum, the sera carrying total flavonoids of Rhizoma Drynariae could significantly inhibit the expression of proliferating cell nuclear antigen (PCNA) mRNA and Bcl-2 mRNA in fibroblast-like synoviocytes (FLSs) (P<0.0001), and the effect of the treatment at a high dose was more significant. Conclusion: Total flavonoids of Rhizoma Drynariae may play a role in inhibiting the proliferation of FLSs by regulating the expression of PCNA mRNA and Bcl-2 mRNA in FLSs.


Introduction
Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease. Currently, it is generally believed that the dysfunction caused by RA, cartilage damage, and bone invasion are closely related to the lack of apoptosis of fibroblast-like synoviocytes (FLSs), and insufficient apoptosis is an important cause of most tumors and autoimmune diseases [1]. Therefore, inhibition of the apoptosis of FLSs can effectively treat RA [2]. Drynaria is the dry rhizome, which is belonged to epiphytic fern, keel subject.
Drynaria is also named as monkey ginger or turmeric. The efficacy is for reinforcing kidney, strengthening bone and promoting blood circulation and removing blood stasis. Previous studies have shown that total flavonoids of Rhizoma Drynariae have anti inflammatory and osteogenesis effects [3]. However, the effect of total flavonoids of Rhizoma Drynariae on the treatment of RA is rarely reported. In this study, the effect of total flavonoids of Rhizoma Drynariae on RA was observed.

Methods
Preparation of Drug-Carrying Serum: Twenty Wistar rats were randomly divided into the blank control group (gavage with sterilized water), the high-dose total flavonoids of Rhizoma Drynariae group, the low-dose total flavonoids of Rhizoma Drynariae group, and the tripterygium glycosides group. The rats in the blank control group were gavaged with sterilized water (2mL/rat/day).
For the rats in the total flavonoids of Rhizoma Drynariae groups, according to the clinical equivalent dose conversion equation proposed by the US FDA, Qianggu Capsule was administered at 300mg/kg/day (containing approximately 240mg/kg/day of total flavonoids of Rhizoma Drynariae). The total flavonoids of Rhizoma Drynariae were dissolved in sterile water to prepare a 120mg/ mL solution. The low-dose group was given 0.5mL per rat per day, diluted to 2mL for gavage, and the high-dose group was given 2mL per day per rat. The tripterygium glycoside tablets (1.2mg per rat per day) were dissolved in water for gavage. All animals were administered the tablets for three consecutive days, and blood was collected 1 hour after the last administration. The blood was placed in a sterile test tube without an anticoagulant to allow natural coagulation. All tubes were placed in a test tube rack, incubated in a 37 0 C water bath for 1h, and centrifuged at 3000 r/min for 15min to separate the serum, which was placed in another sterile test tube and stored in a -20 0 C refrigerator for later use. Just before use, the serum was inactivated at 56 0 C for 30min, diluted with the serumfree medium and sterilized by filtration.

Isolation, Culture and Passage of Primary Synovial Cells:
Under sterile conditions, the synovial tissue was collected, placed in a 15-mL centrifuge tube containing 5% FBS DMEM, and placed in a sterile Petri dish containing DMEM within 2 hours. Fat and blood clots on the surface were removed under sterile conditions, and the tissue was then washed 3 times. The synovial tissue was cut into small, 1mm 3 pieces and placed in a 6cm 2 culture dish. After adding 4mL of DMEM medium and 500μl of collagenase type I (final concentration of 1mg/mL), the tissue was digested for 4 h in an incubator at 37 0 C and 5% CO 2 . The tissue block was flocculent. The sample was filtered through a 100-mesh stainless steel filter and rinsed with PBS. The filtrate was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 15% FBS DMEM and placed in a 10-cm 2 culture dish, which was incubated at 37 0 C and 5% CO 2 . After the confluence of the cells reached >80%, digestion and passage were performed. Thirdgeneration cells were digested with trypsin for the experiment.

Effect of Drug-Carrying Serum on the Proliferation of FLSs In Vitro
The third-generation osteoblasts were adjusted to a concentration of 2×10 3 cells/mL and seeded in a 96-well culture plate at 100μl/well. After culture with medium containing 2% fetal bovine serum for 24 hours, the cells were divided into 4 groups: a) The 20% blank serum group; b) The 20% high-dose total flavonoids of Rhizoma Drynariae medicated serum group; c) The 20% low-dose total flavonoids of Rhizoma Drynariae medicated serum group and d) The 20% tripterygium glucosides medicated serum group, with 10 wells for each group.
After culture in an incubator at 37 0 C and 5% CO 2 for 24h, 100μl of the culture solution was discarded and 20μl of thiazolyl tetrazolium (MTT) solution (5mg/mL) was added to each well, followed by incubation at 37 0 C for 4h. The supernatant was discarded, and the crystals were dissolved in 150μl of dimethyl sulfoxide (DMSO) per well. The absorbance (wavelength at 490nm) was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer. The calculation performed was as follows: cell viability % = (OD of the tested well / OD of the control well) × 100%.

Effect of Drug-Carrying Serum on the Expression of Proliferating Cell Nuclear Antigen (PCNA) mRNA and Bcl-2 mRNA in FLSs in vitro
a) The third-generation osteoblasts were digested with 0.25% trypsin to prepare a single cell suspension by repeated pipetting; the cell concentration was adjusted to 2×10 4/mL, and the cells were seeded onto a 12-well cell culture plate in 1mL per well. The cells were cultured in an incubator at 37 0 C and 5% CO 2 for 24 h. The culture medium in the well was removed completely and replaced with culture medium containing 1% fetal bovine serum for 12h; then, the culture medium in the well plate was aspirated and replaced with 20% blank control serum, 20% high-dose total flavonoids of Rhizoma Drynariae drug-carrying serum, 20% lowdose total flavonoids of Rhizoma Drynariae drug-carrying serum or 20% of tripterygium glycosides drug-carrying serum, followed by culture for another 24h.
b) The total RNA of the FLSs was extracted following the instructions of the Trizol one-step reagent, and 1μg of total RNA was used as the template to synthesize cDNA by reverse transcription.
c) The primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.; the primer sequences are provided in Table 1. After sealing, the reaction mixture was put into a real-time PCR analyzer (ABI 7900) for PCR to determine the CT values for PCNA and Bcl-2 mRNA expression. The expression level was calculated based on the 2 -ΔCT equation (Table 1).

Statistical Analysis:
Statistical analysis was performed on the experimental data using SPSS 18.0 software. The normally distributed data are presented as means ± standard deviations. If the variance of each treatment group was homogeneous, the LSD-t test was used for the comparison between the two groups. When the mean values of the two groups of samples were compared, if the variance was homogeneous, the t-test was used; if the variance was heterogeneous, the t' test was used. Differences with P<0.05 were considered statistically significant.  Note: Compared with the blank control group, **P<0.0001; a t=11.325; b t = 6.125; c t = 6.335. Table 2, compared with the blank control group, treatment with sera containing 20% high-dose and low-dose total flavonoids of Rhizoma Drynariae and tripterygium glycosides could significantly inhibit the proliferation of FLSs (P<0.0001), with no significant differences between the three groups ( Table 2).

Effect of Drug-Carrying Serum on the Expression of PCNA mRNA in the in vitro Cultured FLSs
As shown in Table 3

Effect of Drug-Carrying Serum on the Expression of Bcl-2 mRNA in the in vitro Cultured FLS
As shown in Table 3, compared with the blank control group, treatment with sera containing 20% high-dose and low-dose total flavonoids of Rhizoma Drynariae and tripterygium glycosides could significantly inhibit the expression of Bcl-2 mRNA in FLS (P<0.0001), with no statistically significant differences between the three groups (Table 3).

Conclusion
Total flavonoids of Rhizoma Drynariae may inhibit the proliferation of FLSs by regulating the expression of the PCNA and Bcl-2 genes, thereby inhibiting inflammation and reducing bone loss.

Discussion
FLSs in patients with RA can play an important role in inflammation and joint destruction by secreting various proteases, arachidonic acid metabolites and their cytokines [4][5][6]. They are the ultimate target cells for the pathological changes of RA, and their excessive proliferation may result in synovial hyperplasia, which is one of the main pathological features of RA [7,8]. Currently, the hypothesis of insufficient apoptosis of FLS received the most attention. When the number of cells in inflammatory synovial tissue is multiplied, under normal conditions, the body can eliminate these excess cells through apoptosis to maintain a balance, but insufficient cell apoptosis may lead to hyperplasia of the synovial tissue and even a series of pathological changes in arthritis.
Therefore, the excess hyperplasia and insufficient apoptosis of FLSs are the main mechanisms of RA and inhibiting the proliferation of FLSs and inducing their apoptosis are considered to be effective methods for treating RA [2,9]. Traditional plant extracts, such as celastrol [10] and curcumin [11], can inhibit the proliferation of

Consent for Publication
Not applicable.

Availability of Data and Material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing Interests
All the authors declare that they have no conflict of interest.

Author's Contributions
LS and XL contributed to the study design. All authors collected the data and performed the data analysis. All authors prepared the manuscript.