FLS from CIA rats and healthy rats were purchased from Shanghai Institute of Health Industry Co., Ltd. (Shanghai, China). Daphnetin, extracted from Daphne odora var. marginata by high-speed counter-current chromatography, was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Other reagents were purchased as follows: Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); siRNAs (Ribobio Co., Ltd., Guangzhou, China); Opti-MEM I (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); reverse transcription-quantitative polymerase chain reaction (RT-qPCR) primers (Sangon Biotech Co., Ltd., Shanghai, China); ReverTra Ace qPCR RT kit (Toyobo Life Science, Osaka, Japan); SYBR Green Real Time PCR Master mix (Toyobo Life Science); rabbit anti-Bcl2 antibody conjugated to fluorescein isothiocyanate (FITC; bs0032r-F; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China); annexin V/propidium iodide (PI) kit (KeyGen Biotechology Co. Ltd., Nanjing, China); diethyl pyrocarbonate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); and TransZol (Beijing TransGen Biotech Co., Ltd., Beijing, China).

FLS were cultured and passaged in Dulbecco's Modified Eagle's medium (DMEM) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% foetal bovine serum (FBS, Tianjin Haoyang Biological Manufacture Co., Ltd., Tianjin, China). All cultures were incubated at 37°C in 5% humidified CO₂. Following 2 days incubation, FLS were digested using 0.25% trypsin prior to centrifugation at low speed (112 × g for 10 min at 25°C). Nonadherent granulocytes were removed, and FLS were cultured in 24-or 6-well plates (5×10⁴ or 1×10⁵ cells/ml) in 500 µl or 2 ml DMEM, respectively, containing 10% FBS without penicillin and streptomycin for 24 h following treatment with siRNA (various concentrations) or daphnetin (40 µg/ml, 37°C).

All siRNA sequences were purchased from Ribobio Co., Ltd. (Guangzhou, China). A total of three siRNA sequences specific to Bcl2 were selected: i) si-Bcl2.1, GCC UCC GAC CCU ACG GAA A; ii) siBcl2.2, CCU ACU GAC UCA UGG ACU U; and iii) siBcl2.3, CCU CUU GUC CCA UAC UAU U. siRNAs were chemically synthesised and annealed by Nanjing Anbo Ruila Biotechnology Co., Ltd. Three targeting siRNA was respectively used to silence the Bcl2 gene in FLS of CIA rats. siRNAs labelled with fluorescent cyanine 3 (Cy3; siN05815122149; Ribobio Co., Ltd.) were used to test whether the transfection was successful. An siRNA specific to an invalid gene (siN05815122147; Ribobio Co., Ltd.) was used as a negative silencing control. Experimental groups were used to determine the optimal siRNA concentration, including siRNA transfection negative control group (NC group), si-Bcl2.1-transfection group, si-Bcl2.2-transfected group and si-Bcl2.3-transfection group.

Transfection was conducted according to the manufacturer's protocol. Briefly, 50 µl Opti-Mem I (serum-free, penicillin-free and streptomycin-free) was used in 24-well plates (or 250 µl in 6-well plates) to dilute 5, 2.5, 1.25, 0.75, 0.5 and 0.25 µl annealed siRNA (or 20, 10, 5, 3, 2 and 1 µl siRNA in 6-well plates). Another 50 µl Opti-Mem I was used in 24-well plates (or 250 µl in 6-well plates) to dilute 1 µl Lipofectamine 2000 (or 5 µl in 6-well plates). Subsequently, the diluted siRNA-Lipofectamine 2000 complex was incubated at room temperature for 20 min. This mixture was then added to 400 µl FLS cell culture in 24-well plates (5×10⁴ cells/well), or 1,500 µl in 6-well plates (1×10⁵ cells/well). A concentration gradient of the siRNA was generated as follows: 200, 100, 50, 30, 20 and 10 nM. Following 6 h incubation at 37°C, an equal volume of DMEM supplemented with 10% FBS was added to the cells. When combined with siRNA, daphnetin (final concentration 40 µg/ml) was added to the FLS cell culture as appropriate for each of the treatment groups. Following 24 or 48 h transfection at 37°C, Cy3 fluorescence was observed under a fluorescent microscope to confirm that the transfection was successful. The transfected RAFLS were washed with DMEM and then immediately used for subsequent experiments.

Experimental groups were: i) FLS of untreated healthy Wistar rats (healthy group); ii) untreated FLS of CIA rats (CIA group); iii) daphnetin (40 µg/ml)-treated group (E003 group); iv) si-Bcl2-treated group; v) daphnetin (40 µg/ml) plus si-Bcl2 combined treatment group (E003-B group). Each group of cells was treated by the method of siRNA transfection as described above. Following treatment (5×10⁴/1×10⁵ cells/well at 37°C in 5% humidified CO₂), FLS were cultured for 24, 48 and 72 h, and total RNA was extracted to measure the relative mRNA expression levels of Bcl2 and STAT3. In addition, resuspended FLS were used to measure Bcl2 protein expression levels and to assess apoptosis by flow cytometry.

Total RNA was isolated from FLS (5×10⁴ cells) following gene silencing using TRIzol (All-in gold Biological Co., Ltd.), according to the manufacturer's protocol. cDNA was synthesized using the ReverTra Ace qPCR RT kit (Toyobo Life Science, Osaka, Japan) was used. Briefly, extracted RNA was heat denatured at 65°C for 5 min and immediately cooled on ice. Subsequently, 1 µl RT Enzyme mix, 1 µl Primer mix and 4 µl 5X RT buffer were incubated with 1 µg DNA-free total RNA for 15 min at 37°C. The mixture was incubated for 5 min at 98°C to inactivate the RT enzyme, and the first-strand cDNA was stored at 4°C until use in qPCR.

For qPCR, reactions were performed in a volume of 20 µl SYBR⁻Green Real Time PCR Master mix (Toyobo Life Science). The primers used in this study were as follows: Bcl2, forward 5′-GGGATGCCTTTGTGGAACTAT-3, reverse 5′-AGGTATGCACCCAGAGTGATG-3′ (124 bp); STAT3, forward 5′-GGGCACAAACACAAAAGTGAT-3′, reverse 5′-CAGTCACAATCAGGGAAGCAT-3′ (140 bp); β-actin, forward 5′-TGACAGGATGCAGAAGGAGA-3′, reverse 5′-TAGAGCCACCAATCCACACA-3′ (106 bp). Thermocycling conditions included initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec and extension at 72°C for 45 sec. qPCR was performed on an ABI 7500 PCR instrument. A relative quantitative assay was adopted (19), and the melting curve increased following amplification. The reference gene β-actin, and Bcl2 and STAT3 were simultaneously amplified in the samples of each experimental group. Each sample was tested in triplicate. Samples were obtained from ≥3 independent experiments to calculate the mean and standard deviation. The formula 2^−ΔΔCq was used to calculate the relative expression of Bcl-2, STAT3mRNA (19).

Bcl2 protein expression analysis and the apoptotic rate of FLS from CIA rats were performed on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For the Bcl2 protein, a FITC-conjugated rabbit anti-rat Bcl2 antibody (Biosynthesis Corporation, Beijing, China) was used. FLS cells (1×10⁵ cells/well) were treated as aforementioned, collected by trypsinization following 72 h incubation, fixed by 1% paraformaldehyde at room temperature for 15 min, and resuspended with 100 µl or 500 µl PBS. Following collection, cells were stained with (FITC)-labeled rabbit anti-rat Bcl2 antibody (1:100) and 70% ethanol added in the dark for 15 min at room temperature for permeabilisation. Bcl2 protein expression was assessed by determining the average fluorescence intensity. Apoptosis of FLS was examined using a FITC-labeled Annexin V/propidium iodide (PI) Apoptosis Detection kit (Nanjing KeyGen Biotechology Co. Ltd., Nanjing, China) according to the manufacture's protocol. Cells (1×10⁵ cells/well) treated as aforementioned for 72 h were collected by trypsinization, fixed by 70% ethanol in the dark for 15 min at 4°C and washed twice with PBS. The cells were resuspended in 500 µl binding buffer. Then 5 µl Annexin-V-FITC and 5 µl PI-FITC were added and incubated in the dark for 30 min at room temperature. Data acquisition and analysis were performed in a Becton Dickinson FACSCalibur flow cytometer using Cell Quest software version 6.1.2 (BD Biosciences, Franklin Lakes, NJ, USA).

All data are expressed as the mean ± standard deviation, and were analysed by one-way analysis of variance followed by LSD post hoc multiple comparison test using SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). A Student's t-test was used to determine the significances of differences in multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Cy3-labeled si-Bcl2 was transfected into FLS. To determine whether the transfection was successful, the fluorescence was observed under a microscope at 24 and 48 h post-transfection (Fig. 1A and B, respectively).

Identification of the optimal siRNA concentration and incubation time was examined, as well as determining the optimal treatment time for daphnetin. All experimental conditions were designed to assess the mRNA expression levels of the antiapoptotic gene Bcl2, which was determined using RT-qPCR. The results revealed that Bcl2 mRNA expression in the si-Bcl2.1 group was significantly lower compared with the NC, si-Bcl2.2, and si-Bcl2.3 groups (Fig. 2A). As demonstrated in Fig. 2B, 100 nM si-Bcl2.1 inhibited Bcl2 mRNA expression most following transfection for 48 h compared with the NC group (Fig. 2B). In a previous study, it was determined that 40 µg/ml daphnetin significantly promoted FLS apoptosis (13). Therefore, the present study focused on determining the most suitable length of time required to downregulate Bcl2 mRNA expression. The results demonstrated that Bcl2 mRNA expression in FLS cells following daphnetin treatment for 48 or 72 h was significantly lower compared with cells treated for 24 h and untreated CIA FLS cells (Fig. 2C). However, daphnetin treatment for 48 h was not statistically significant (P>0.05) compared with for 72 h, so cells were treated for 48 and 72 h to observe Bcl2 mRNA expression in the later combined experiment.

In the optimal conditions, si-Bcl2.1 was transfected into FLS and daphnetin was added to FLS cultures 6 h following gene silencing. Bcl2 and STAT3 mRNA expression levels were determined by RT-qPCR (Fig. 3A and B, respectively). Bcl2 protein expression was assessed by determining the average fluorescence intensity using flow cytometry (Fig. 3C). The results demonstrated that Bcl2 mRNA expression in the E003-B group was significantly lower than that in the other treatment groups. In addition, Bcl2 mRNA expression in the E003-B group treated for 48 h was significantly lower compared with that in the same group treated for 72 h (P<0.05), and no significant difference was observed in expression between the E003-B group treated for 48 h and the healthy group treated for 48 h (P>0.05; Fig. 3A). It suggested that daphnetin combined with si-Bcl2 for the treatment of RA was effective, particularly as in the treatment for 48 h, Bcl2 mRNA was downregulated most. Consistent with Bcl2 mRNA expression, STAT3 mRNA expression in the E003-B group was also decreased compared with the other treatment groups (P<0.05; Fig. 3B). Bcl2 protein expression in the E003-B group was significantly lower compared with expression in the other treatment groups and the CIA group (P<0.05; Fig. 3C).

The apoptotic rate of RAFLS derived from CIA rats was also determined by flow cytometry. The results demonstrated that the apoptotic rate of FLS in the E003-B group was significantly higher than that of other groups (P<0.05; Fig. 4).