Tamaractam, a New Bioactive Lactam from Tamarix ramosissima, Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA) treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam (1), together with the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2) and trans-N-feruloyl-3-O-methyldopamine (3). The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS) and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS) were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G1 fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G1 fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima.


Introduction
Tamarix ramosissima, commonly known as tamarisk or rose willow, is a shrub or dungarunga belonging to the family Tamaricaceae.The Tamarix species have strong adaptability to the arid desert environment and saline and alkaline soil.They are not only excellent sand fixing plants, but also a good species for soil and water conservation and afforestation in saline-alkali soil [1].Various species of Tamarix have been used as herbal medicines in the treatment of inflammation, leucoderma, spleen troubles, and eye diseases [2].Tender branches and leaves of T. ramosissima are one of the herbal medicines with a long history of use for the treatment of rheumatoid arthritis (RA) in northwest China, especially in the Ningxia Hui Autonomous Region.Previous pharmacological study revealed that this plant has antioxidant and antimicrobial activities [1].In a chemical constituents study, this plant has been found to be rich in polyphenolic compounds such as flavonoids, phenolic acids, hydrolyzable tannins, and coumarins [3][4][5].
In our pursuit to find new, potential, natural anti-RA compounds, the chemical investigation of T. ramosissima was carried out.The study resulted in the identification of three structurally related compounds, including the new compound tamaractam (1) and the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2) and trans-N-feruloyl-3-O-methyldopamine (3).The anti-proliferation effects of the three compounds were assessed on RA fibroblast-like synoviocytes (RA-FLS), and the apoptosis-inducing effects of compound 1 were further investigated.
desert environment and saline and alkaline soil.They are not only excellent sand fixing plants, but also a good species for soil and water conservation and afforestation in saline-alkali soil [1].Various species of Tamarix have been used as herbal medicines in the treatment of inflammation, leucoderma, spleen troubles, and eye diseases [2].Tender branches and leaves of T. ramosissima are one of the herbal medicines with a long history of use for the treatment of rheumatoid arthritis (RA) in northwest China, especially in the Ningxia Hui Autonomous Region.Previous pharmacological study revealed that this plant has antioxidant and antimicrobial activities [1].In a chemical constituents study, this plant has been found to be rich in polyphenolic compounds such as flavonoids, phenolic acids, hydrolyzable tannins, and coumarins [3][4][5].
In our pursuit to find new, potential, natural anti-RA compounds, the chemical investigation of T. ramosissima was carried out.The study resulted in the identification of three structurally related compounds, including the new compound tamaractam (1) and the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2) and trans-N-feruloyl-3-O-methyldopamine (3).The anti-proliferation effects of the three compounds were assessed on RA fibroblast-like synoviocytes (RA-FLS), and the apoptosis-inducing effects of compound 1 were further investigated.

In Vitro Anti-RA Activity of Compounds 1-3
Synovial hyperplasia is recognized as one of the key pathological characteristics of RA, which causes marginal bone erosion and joint destruction [9,10].RA-FLS exhibit aggressive features, including hyperproliferation, apoptosis resistance, and high invasiveness [11,12].Evidence suggested that RA-FLS play a pivotal role in the pathological process of synovitis and joint destruction in RA [13][14][15].Therefore, RA-FLS were recruited for the investigation of anti-RA effect of compounds 1-3 in vitro.

In Vitro Anti-RA Activity of Compounds 1-3
Synovial hyperplasia is recognized as one of the key pathological characteristics of RA, which causes marginal bone erosion and joint destruction [9,10].RA-FLS exhibit aggressive features, including hyperproliferation, apoptosis resistance, and high invasiveness [11,12].Evidence suggested that RA-FLS play a pivotal role in the pathological process of synovitis and joint destruction in RA [13][14][15].Therefore, RA-FLS were recruited for the investigation of anti-RA effect of compounds 1-3 in vitro.

In Vitro Anti-RA Activity of Compounds 1-3
Synovial hyperplasia is recognized as one of the key pathological characteristics of RA, which causes marginal bone erosion and joint destruction [9,10].RA-FLS exhibit aggressive features, including hyperproliferation, apoptosis resistance, and high invasiveness [11,12].Evidence suggested that RA-FLS play a pivotal role in the pathological process of synovitis and joint destruction in RA [13][14][15].Therefore, RA-FLS were recruited for the investigation of anti-RA effect of compounds 1-3 in vitro.
To evaluate the effects of the isolated compounds on cell viability, RA-FLS were incubated with various concentrations of compounds 1-3 (0.01, 0.1, 1, 5, and 10 μM) for 24 or 48 h, and then cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.It is found that, compared with vehicle-treated control cells, 0.1~10 μM compound 1 treatment could significantly suppress the cell viability of RA-FLS in both time-and dose-dependent manners (p < 0.05); treatment with 0.01, 0.1, 1, 5 and 10 μM of compound 1 decreased cell survival by 91%, 80%, 65%, 54%, and 49%, respectively, after 24 h and by 89%, 74%, 56%, 42%, and 39% of the control viability level after 48 h, respectively (Figure 3).However, only 5 and 10 μM of compounds 2-3 treatment could induce significant suppression of cell viability (Figure 3).Based on these results, 0.1 and 1 μM of compound 1 were employed for subsequent apoptosis inducing experiments.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to evaluate whether compound 1 treatment can influence apoptotic cell death of RA-FLS.It was found that treatment with 0.1 and 1 µM of compound 1 for 48 h significantly increased the number of TUNEL-positive cells compared to vehicle control cells (Figure 4A,B; 14.83% ± 3.76% and 23.52% ± 5.94%, respectively).Then the levels of activated caspase-3/7 were examined with a Caspase-Glo kit.It was found that, after 24 h incubation, compound 1 could significantly increase the levels of activated caspase-3/7 (Figure 4C; 0.1 and 1 µM are about 2.6-and 3.9-fold higher than untreated vehicle, respectively), indicating its potent pro-apoptotic effect [16].Furthermore, the induction of apoptosis was confirmed by determination of sub-G 1 fractions with a flow cytometric approach.After treatment with 0.1 or 1 µM of compound 1 for 48 h, cells were harvested and assessed by flow cytometry.The remarkably increased sub-G 1 peak of apoptotic cells in the cell cycle indicated that compound 1 could potently induce apoptosis in RA-FLS (Figure 4D; for 0.1 and 1 µM, sub-G 1 fractions were about 23.12% and 49.60%, respectively, while untreated vehicles represented 2.57%) [17].
Molecules 2017, 22, 96 5 of 9 Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to evaluate whether compound 1 treatment can influence apoptotic cell death of RA-FLS.It was found that treatment with 0.1 and 1 μM of compound 1 for 48 h significantly increased the number of TUNEL-positive cells compared to vehicle control cells (Figure 4A,B; 14.83% ± 3.76% and 23.52% ± 5.94%, respectively).Then the levels of activated caspase-3/7 were examined with a Caspase-Glo kit.It was found that, after 24 h incubation, compound 1 could significantly increase the levels of activated caspase-3/7 (Figure 4C; 0.1 and 1 μM are about 2.6-and 3.9-fold higher than untreated vehicle, respectively), indicating its potent pro-apoptotic effect [16].Furthermore, the induction of apoptosis was confirmed by determination of sub-G1 fractions with a flow cytometric approach.After treatment with 0.1 or 1 μM of compound 1 for 48 h, cells were harvested and assessed by flow cytometry.The remarkably increased sub-G1 peak of apoptotic cells in the cell cycle indicated that compound 1 could potently induce apoptosis in RA-FLS (Figure 4D; for 0.1 and 1 μM, sub-G1 fractions were about 23.12% and 49.60%, respectively, while untreated vehicles represented 2.57%) [17].Synovial hyperplasia is recognized as one of the major pathological characteristics of RA, which leads to marginal bony erosions and resultant joint destruction.RA-FLS play pivotal roles both in the initiation and the perpetuation of RA.These cells have been linked most prominently to the progressive destruction of articular cartilage [18].There are several lines of evidence suggesting Synovial hyperplasia is recognized as one of the major pathological characteristics of RA, which leads to marginal bony erosions and resultant joint destruction.RA-FLS play pivotal roles both in the initiation and the perpetuation of RA.These cells have been linked most prominently to the progressive destruction of articular cartilage [18].There are several lines of evidence suggesting that rheumatoid synovia shows tumor-like expansion attributed to the resistance of RA-FLS to the apoptotic process [19,20].Thus compounds with pro-apoptotic activity may provide a potent therapeutic approach for the treatment of RA.Our findings revealed that compound 1 had a remarkable apoptosis-inducing effect on RA-FLS in vitro.The in vivo anti-RA effect and the underlining mechanisms need further investigation.

Biological Materials
Tender branches and leaves of T. ramosissima were collected from sandy land near Yellow River, Yinchuan, China.The samples were identified by Dr. Yunsheng Zhao, School of Pharmacy of Ningxia Medical University.A voucher sample was kept at Department of Medical Chemistry, School of Basic Medical Science, Ningxia Medical University under the registration code No. 2014050201.

Characterization of Compounds 1-3
Tamaractam (1): White crystal; m.p. 249.4~250.Human fibroblast-like synoviocytes from RA patients (RA-FLS) were purchased from Cell Applications, Inc., (San Diego, CA, USA) and cultured with a synoviocyte growth medium (Cell Applications).RA-FLS obtained from the 3rd to 5th passages were used for experiments.The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Ningxia Medical University (Project identification code: 2014-036).

Assessment of Cell Viability Using MTT Assay
Compounds 1-3 was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St. Louis, MO, USA).RA-FLS were seeded in 48-well plates at a density of 3 × 10 4 cells/well, and were treated with 0.01, 0.1, 1, 5, and 10 µM of compounds 1-3 or DMSO vehicle only in a serum free medium.After 24 and 48 h of incubation, 2.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution (Sigma-Aldrich) was added to the wells, and the cells were then incubated for 2 h.The absorbance of each well was measured by a Bio-Rad 680 microplate reader (Bio-Rad laboratories, Hercules, CA, USA) at 570 nm.Based on MTT assay results, compound 1 was employed in the subsequent apoptosis-inducing experiments.

TUNEL Assay
Apoptosis in RA-FLS cells was measured by TUNEL assay (Roche Diagnostics, Mannheim, Germany), according to the manufacturer s protocol.Briefly, RA-FLS were incubated with 0.1 and 1 µM of compound 1 for 48 h and then fixed with 4% paraformaldehyde for 30 min at room temperature.After washing with phosphate buffer saline (PBS), permeabilization solution (0.1% sodium citrate, 0.1% Triton X-100) was added for a 2 min reaction and cells were incubated with terminal deoxynucleotidyl transferase (TdT) and biotin-11-dUTP for 1 h at 37 • C. The nuclear morphology was observed by fluorescence microscopy IX71 (Olympus, Tokyo, Japan).The apoptosis index was calculated based on the percentage of TUNEL-positive cells in 1000 RA-FLS.

Measurement of Caspase-3/7 Activity
The levels of activated caspase-3/7 in RA-FLS were further assessed with a Caspase-Glo kit (Promega, Madison, WI, USA) [21], according to the manufacturer's protocol.Briefly, cells were plated at 1 × 10 4 cells/well in 96-well plates and treated with 0.1 and 1 µM of compound 1 for 48 h.After incubation, cells were treated for 2 h with reconstituted Caspase 3/7-Glo reagent.Then the

Figure 4 .
Figure 4. Effects of compound 1 on RA-FLS apoptosis.(A) After treatment with compound 1 (0.1 or 1 μM) or dimethyl sulphoxide (DMSO) vehicle for 48 h, apoptotic cell death measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (×100).Bright spots in the lower panels are TUNEL-positive apoptotic cells.Corresponding phase contrast microscopy images are shown in the upper panels; (B) The apoptosis index of RA-FLS treated with 0.1 or 1 μM of compound 1 or DMSO vehicle.Data were shown as means ± S.E.M. of three independent experiments (* p < 0.05); (C) Effects of compound 1 on the levels of active caspase-3/7.RA-FLS cells were treated with 0.1 or 1 μM of compound 1 for 48 h and then processed to measure using the Caspase-Glo kit.Data were shown as means ± S.E.M. of three independent experiments (* p < 0.05); (D) Effects of compound 1 on sub-G1 fractions of RA-FLS.After treatment with compound 1 for 48 h at the concentration of 0.1 or 1 μM, cells were harvested by trypsinization, and analyzed using flow cytometry.

Figure 4 .
Figure 4. Effects of compound 1 on RA-FLS apoptosis.(A) After treatment with compound 1 (0.1 or 1 µM) or dimethyl sulphoxide (DMSO) vehicle for 48 h, apoptotic cell death measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (×100).Bright spots in the lower panels are TUNEL-positive apoptotic cells.Corresponding phase contrast microscopy images are shown in the upper panels; (B) The apoptosis index of RA-FLS treated with 0.1 or 1 µM of compound 1 or DMSO vehicle.Data were shown as means ± S.E.M. of three independent experiments (* p < 0.05); (C) Effects of compound 1 on the levels of active caspase-3/7.RA-FLS cells were treated with 0.1 or 1 µM of compound 1 for 48 h and then processed to measure using the Caspase-Glo kit.Data were shown as means ± S.E.M. of three independent experiments (* p < 0.05); (D) Effects of compound 1 on sub-G 1 fractions of RA-FLS.After treatment with compound 1 for 48 h at the concentration of 0.1 or 1 µM, cells were harvested by trypsinization, and analyzed using flow cytometry.