International Immunopharmacology 20 (2014) 110–116
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Norisoboldine induces apoptosis of fibroblast-like synoviocytes from adjuvant-induced arthritis rats Yubin Luo a,1, Zhifeng Wei a,1, Guixin Chou b, Zhengtao Wang b, Yufeng Xia a,⁎, Yue Dai a,⁎ a b
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 2 January 2014 Received in revised form 17 February 2014 Accepted 21 February 2014 Available online 6 March 2014 Keywords: Norisoboldine Rheumatoid arthritis Fibroblast-like synoviocytes Apoptosis
a b s t r a c t Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by pronounced synovial inflammation and hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Norisoboldine (NOR), the main active constituent in the alkaloid fraction isolated from Radix Linderae, was previously demonstrated to alleviate arthritis severity in experimental RA. This study aimed to evaluate the effects of NOR on proliferation and apoptosis of FLS from adjuvant-induced arthritis (AIA) rats to elucidate the mechanism of its inhibitory effect on inflammatory synovial hyperplasia in RA. Our results indicated that NOR exhibited a pro-apoptotic effect on AIA FLS but only slightly affected cell proliferation and the cell cycle. Following treatment with NOR for 24 h, the activation of caspase 3 and caspase 9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in AIA FLS were observed; however, caspase 8 remained unaffected. Meanwhile, a flow cytometric assay revealed that NOR significantly increased the percentage of apoptotic cells, causing the loss of the depolarized mitochondrial membrane potential and the release of cytochrome C. The expression of Bax and Bcl-2 was also regulated by NOR treatment. Additionally, the expression of p53 protein was up-regulated by NOR, and pretreatment with PFT-α, a p53 specific inhibitor, reversed the increase in FLS apoptosis caused by NOR. These findings indicated that NOR-induced apoptosis in AIA FLS is achieved via a mitochondrial-dependent pathway, which may be mediated by promoting the release of cytochrome C and by regulating the expression of Bax and Bcl-2 proteins, and p53 might also be required for NOR-induced apoptosis in AIA FLS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of unknown etiology that leads to fundamental changes in cellular composition and function of various joints [1]. One important characteristic of RA is chronic synovitis and progressive bone and cartilage damage. Fibroblast-like synoviocytes (FLS), which normally populate the thin synovial lining layer, appear to be the key players in changes of the joints based on their hyperplastic growth and their ability to become locally invasive in the adjacent cartilage and bone. Although in situ measurements of the lining layer proliferation rates suggest that indices of proliferation in RA synovium are insufficient to explain the degree of observed synovial hyperplasia [2,3], FLS derived from these tissues show in vitro anchorage-independent growth, loss of contact inhibition and the expression of various growth factors, inflammatory cytokines, oncogenes and cell cycle proteins, which resembles that
Abbreviations: NOR, norisoboldine; RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; AIA, adjuvant-induced arthritis; PARP, poly (ADP-ribose) polymerase. ⁎ Corresponding authors. Tel.: +86 25 83271400; fax: +86 25 85301528. E-mail addresses: [email protected] (Y. Xia), [email protected] (Y. Dai). 1 These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.intimp.2014.02.023 1567-5769/© 2014 Elsevier B.V. All rights reserved.
seen in tumor cells [4]. Recently, insufficient apoptosis of FLS has been recognized as a potential cause for the hyperplastic growth of FLS. RA FLS in vivo are reported to acquire mutations in the tumor suppressor gene p53 [5]. In addition, both nuclear factor-κB (NF-κB) activation and TNF-α stimulation can decrease FLS apoptosis through mechanisms such as resistance to Fas ligand-induced signaling and upregulation of anti-apoptotic molecules [6]. Cells found in the rheumatoid joints, including macrophages and FLS, appear to be resistant to apoptosis, even though Fas and Fas ligand are strongly expressed [7,8]. Also, accumulating evidence has suggested that a reduced level of apoptosis in vivo, especially at sites of invasion into cartilage and bone, is associated with the activation of FLS in RA [6]. For these reasons, the induction of apoptosis has been proposed as a potential therapeutic approach for RA. Radix Linderae, the dry roots of Lindera aggregata, is commonly used in traditional Chinese medicine. These extracts have been reported to possess anti-inflammatory and analgesic properties as well as potential anti-rheumatism activities [9]. Previous studies in our laboratory showed that norisoboldine (NOR), the main active constituent of Radix Linderae, effectively ameliorated synovial inflammation and hyperplasia, and protected joints from destruction in collagen II-induced arthritis (CIA) of mice [10]. One study has shown that boldine, an analog of NOR, induced cell death in a cell-type specific and dose-dependent
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
manner [11]. Therefore, in this study, we investigated whether and how NOR inhibited proliferation and induced apoptosis in FLS to gain insight into its underlying mechanisms for the prevention of synovial hyperplasia 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (6–7 weeks old) were obtained from the Experimental Animal Center of China Pharmaceutical University (Nanjing, China). All rats were acclimatized in the husbandry room, which was maintained at 22 ± 2 °C with a 12-h light:12-h dark cycle, and were allowed free access to food and water. The experimental animals were treated in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals. 2.2. Chemicals and reagents NOR (purity N 98%) was isolated and purified from Radix Linderae by authors, and the structure was established by comparison of its spectral data (UV, IR, MS, 1H- and 13 C NMR) with the literature data [12]. Dulbecco's modified Eagle medium (DMEM), penicillin and streptomycin were purchased from Gibco BRL (Grand Island, NY, USA); Newborn calf serum (NBCS) was purchased from PAA Laboratories GmbH (Austria); Mycobacterium butyricum was purchased from Difco Laboratories (Detroit, MI, USA); 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT), Propidium iodide (PI), Tween 20, bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and phenylmethyl sulfonylfluoride (PMSF) were purchased from Sigma Chemical (St. Louis, MO, USA); The Hoechst 33258 staining kit, AnnexinV-FITC apoptosis detection kit and mitochondrial membrane potential assay kit were purchased from Keygen Bio-tech (Nanjing, China); GAPDH monoclonal antibodies and the peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from KangChen Bio-tech (Shanghai, China); The caspase 3, caspase 9 Activity Assay Kit was purchased from the Beyotime Institute of Biotechnology (Nanjing, China); Anti-caspase-3, anticaspase-8, anti-caspase-9, anti-Bcl-2, anti-Bax, and anti-cyochrome C polyclonal antibodies were purchased from Bioworld Technology (St. Louis Park, MN, USA); all other chemicals and reagents used were of analytical grade. 2.3. Preparation and culture of FLS AIA was initiated by an intradermal injection of 100 μl of a 10 mg/ml Mycobacterium butyricum mineral oil suspension into the base of the right hind paw of the rats. Clinical signs, including swelling of the feet, inability to bend the ankle and the presence of nodules at the base of the tail and ears, were observed by day 13 [13]. The fibroblast-like synoviocytes (FLS) were isolated and cultured as described previously [14]. Briefly, the synovial tissues were obtained from AIA rats at day 21 after arthritis induction and were minced into small pieces and incubated with 0.4% type II collagenase in DMEM medium supplemented with 5% NBCS for 2 h at 37 °C. Non-adherent tissues were digested in serum-starved DMEM containing 0.25% trypsin for 30 min. The tissue suspension was transferred through a sterile 200-mm2 nylon mesh filter and centrifuged at 1500 rpm for 10 min. The collected cells were washed with 5% NBCS-DMEM and cultured in DMEM medium supplemented with 10% NBCS, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. After an overnight culture, the nonadherent cells were removed, and the adherent cells were cultured continuously under the same conditions. The cells were trypsinized and passaged at a 1:3 ratio when the cells were 80–90% confluent. FLS were used from passages 3 to 7 in these experiments when cells were a homogeneous population.
111
2.4. Cell viability assay Cell viability was determined using the MTT assay. Briefly, FLS were plated at a density of 0.5 × 104 cells/well in 96-well plates and treated with various concentrations of NOR (0, 3, 10, 30, 100 and 300 μM) in complete culture medium. After incubation at 37 °C for the indicated times (20 h, 44 h and 68 h), 20 μl of MTT (5 mg/ml) was added into each well, followed by incubation at 37 °C for an additional 4 h. The supernatant was then removed, and the formazone crystals were dissolved with 150 μl of DMSO. The optical absorbance at 540 nm was read with a Model 1500 Multiskan spectrum microplate reader (Thermo, Waltham, MA, USA). 2.5. Cell proliferation assay The proliferation assay on FLS was performed using a Cell-Light TM EdU DNA Cell Proliferation Kit (Ruibo Biotech, Guangzhou, China) [15]. In brief, FLS (0.5 × 104 cells/well) were cultured in 96-well plates with different concentrations of NOR (10, 30 and 100 μM) for 24 h. The cells were then incubated with EdU for another 8 h. Five random 100 × fields were photographed for each well, and EdU-positive cells were counted, averaged and compared. 2.6. Hoechst 33258 staining FLS (0.3 × 105 cells/well) were seeded in 24-well plates and exposed to various concentrations of NOR (10, 30 and 100 μM) for 48 h. Prior to fluorescent analysis, the cells were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed with PBS again and stained with Hoechst 33258 for 30 min in the dark. After washing with PBS, the cells were observed under a fluorescence microscope for nuclear morphological changes [16]. 2.7. Flow-cytometric analysis of Annexin-V-PI binding Apoptosis was evaluated by using a fluorescein isothiocyanatelabeled Annexin V/Propidium iodide (PI) Apoptosis Detection Kit according to the manufacturer's protocol. Briefly, FLS (1 × 105 cells) in 100-mm culture dishes were grown to 70% confluence and then treated with NOR (10, 30 and 100 μM) for 48 h. The cells were collected by trypsinization and washed with PBS, and then resuspended in 500 μl of binding buffer and 5 μl of Annexin V-FITC solution. Propidium iodide was added into each sample and incubated for 30 min at room temperature. The apoptotic cell population was immediately analyzed by flow cytometry after cells were washed with PBS for 3 times [17]. Data acquisition and analysis were performed in Becton-Dickinson FACSClibur flow cytometer using CellQuest 3.3 software. 2.8. Flow-cytometric analysis of the cell cycle FLS were treated with NOR (10, 30 and 100 μM) for 24 h and then were trypsinized, washed with PBS and centrifuged. The cell pellet was resuspended in 50 μl of ice-cold PBS and fixed in 1 ml of 70% ethanol for 20 min at 4 °C. After centrifugation at 1000 rpm for 5 min, FLS were washed twice with ice-cold PBS and treated with 0.1% Triton X-100 for 10 min. Subsequently, the cells were centrifuged and resuspended in 1 ml of PBS containing PI (50 μg/ml), then incubated for 30 min at 4 °C. The samples were acquired and analyzed in a Becton-Dickinson FACSCalibur flow cytometer using CellQuest 3.3 software. 2.9. Measurement of mitochondrial membrane potential (Δψm) Mitochondrial membrane potential was assessed using JC-1, according to the manufacturer's instructions (Keygen Bio-tech, Nanjing, China). After treatment with different concentrations of NOR (10, 30
112
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
and 100 μM) for 48 h, FLS were washed twice with cold PBS and then collected by centrifugation. After incubation with 500 μl of fluorescence-activated cell-sorting analysis (FACS) buffer containing JC-1 (1 mg/ml) at 37 °C for 30 min, the cells were washed with PBS 3 times, and the mitochondrial membrane potential was determined by flow cytometry [18]. 2.10. Caspase activity assay The caspase colorimetric assay kits specific for caspase-3 or caspase9 were used to detect caspase activation by measuring the cleavage of a synthetic colorimetric substrate. In brief, AIA FLS (1 × 105 cells) were cultured in 100-mm culture dishes and grown to 80% confluence and were then treated with NOR (10, 30 and 100 μM) for 24 h. Cell lysates were prepared in the lysis buffer from the assay kits; the lysates were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected. The synthetic colorimetric substrates were added to equal amounts of proteins from each sample at 37 °C for 2 h, followed by reading the samples at 405 nm on a microplate reader. The fold-increase in caspase-3 or -9 activities was determined by comparing the data of treated samples with those of the control samples. 2.11. Western blot analysis AIA FLS were treated with NOR for various time intervals and washed twice with ice-cold PBS. The cells were lysed with lysis buffer (50 mM Tris–Cl [pH 8.0], 150 mM NaCl, 0.02% NaN3, and 1% NP40). The cytosolic fraction was obtained from the supernatant after centrifugation at 12,000 rpm for 15 min at 4 °C. The cell lysates (20 μg) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% nonfat milk in PBS Tween-20 (PBST) for 1 h, the PVDF membranes were probed with relevant monoclonal antibodies overnight at 4 °C. After washing three times with PBST, the membranes were incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature. The bands were then visualized using enhanced chemiluminescence detection reagents and exposure to X-ray film. 2.12. Statistical analysis Statistical differences were assessed by a one-way analysis of variance (ANOVA) and the Student's t-test. All of the results were expressed as the means ± S.D. P values less than 0.05 were considered statistically significant. 3. Results 3.1. NOR inhibits the viability of AIA FLS After exposing AIA FLS to various concentrations of NOR for different time intervals, the cell viability was evaluated using the MTT assay. As shown in Fig. 1, there was a concentration- and time-dependent inhibition of the viability of AIA FLS after treatment with NOR. Cell viability was only slightly affected by lower concentrations of NOR (3 μM) for 24 and 48 h; however, viability was significantly reduced by the addition of 10 μM NOR for 48 h, and decreased to 41.8% ± 4.4 and 23.4% ± 5.0 in the presence of NOR (100 and 300 μM). Meanwhile, treatment with NOR (100 μM) for different time intervals caused a time-dependent reduction in cell viability to 58.1% ± 3.7 (24 h), 41.8% ± 4.4 (48 h) and 21.2% ± 2.2 (72 h), respectively.
Fig. 1. Effect of norisoboldine (NOR) on the activity of AIA FLS. The MTT assay was used to detect cell viability after treatment with various concentrations of NOR for 24 h, 48 h and 72 h, respectively. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎, ##, $$ p b 0.01 versus control.
concentrations of NOR for 24 h, cell proliferation was not significantly changed, although it was decreased to some extent in the presence of NOR (100 μM). Consistent with this finding, FACS analysis on the cell cycle suggested that there was little change in any phase of the cell cycle after treatment with various concentrations of NOR (data not shown). Based on the above results, we postulated that the inhibition of NOR on cell viability may be due to the induction of apoptosis. As shown in Fig. 2B, the nuclear morphology of untreated AIA FLS was normal, whereas condensation of nuclei and fragmentation of cells were observed in FLS treated by NOR (10, 30 and 100 μM). This finding was mirrored by the apoptosis detection by Annexin V and PI staining. Fig. 2C shows that NOR (10, 30 and 100 μM) increased the percentage of apoptotic FLS from 3.6% to 11.0%, 27.2% and 33.6%, respectively. The percentage of annexin V-positive cells was proportional to that of dead cells detected by MTT assay, indicating that the cell death induced by NOR was apoptotic cell death. 3.3. NOR-induced apoptotic cell death in AIA FLS involves the processing of caspase-9, -3 and cleavage of PARP To clarify the death signaling pathway underlying the NOR-induced apoptosis in AIA FLS, we assessed the activation of the caspase cascade, including caspase-3, -8 and -9. Caspase-3 and -9 were both processed after NOR treatment in a concentration- and time-dependent manner, whereas, the expression of caspase-8 remained unchanged (Fig. 3A & B). It is well recognized that cleavage of PARP protein considered as an important biomarker of apoptosis occurs while active caspase 3 proteolytically cleaves and activates. In this study, the activities of caspase-3 and -9 were detected by measuring the cleavage of a synthetic colorimetric substrate. As shown in Fig. 3C, the activities of caspase-3 and -9 in AIA FLS were significantly decreased by the addition of NOR (100 μM) for 24 h. Moreover, concurring with the process of caspase 3, PARP cleavage was observed in the samples treated with NOR (10, 30 and 100 μM). Additionally, pretreatment with Z-LEHD-FMK, an inhibitor of caspase-9 activity, reversed the pro-apoptotic action of NOR (Fig. 3D). In sum, our results suggested that NOR-induced cell death in AIA FLS takes place in a caspase-dependent manner.
3.2. NOR induces apoptosis of AIA FLS
3.4. NOR induces mitochondrial membrane potential loss and the release of cytochrome C in AIA FLS
To further understand how NOR inhibited AIA FLS viability, we investigated the effect of NOR on FLS proliferation as well as on the cell cycle. As shown in Fig. 2A, after incubation with increasing
Disruption of mitochondrial integrity is one of the early events leading to apoptosis. Mitochondrial membrane potential (Δψm) loss and the release of cytochrome C are biomarkers of mitochondrial function
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
113
Fig. 2. Norisoboldine (NOR) has no effect on FLS proliferation but induces apoptotic cell death. (A) AIA FLS were treated with various concentrations of NOR for 24 h. The cells were then stained with EdU, and the proliferation rates were measured as described in Materials and methods. (B) The effects of NOR on the nuclear morphology of AIA FLS. The cells were treated with various concentrations of NOR for 48 h and then stained with Hoechst 33258. The nuclear morphology was observed under a fluorescent microscope. (C) Induction of apoptosis was measured by an Annexin-V/PI double-staining assay. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
homeostasis. We detected both the mitochondrial membrane potential loss and cytochrome C expression in AIA FLS. As shown in Fig. 4A, NOR (10, 30 and 100 μM) treatment for 24 h caused a concentrationdependent rapid decline of Δψm. The release of cytochrome C from the mitochondria was increased by NOR (10, 30 and 100 μM) in a concentration-dependent manner (Fig. 4B). These results indicated that the NOR-induced apoptosis of AIA FLS is associated with disruption of the mitochondrial membrane and subsequent cytochrome C activation. The release of cytochrome C is tightly regulated by members of the Bcl-2 family that are known to have an important role in the regulation of apoptosis [19]. A decrease in the level of Bcl-2 or an increase in the level of Bax can trigger a signal to initiate apoptosis. As shown in Fig. 4C, the ratio of Bax/Bcl-2, which is crucial for the activation of the
mitochondrial apoptotic pathway, was increased in FLS treated with NOR, which was the consequence of the increased expression of the apoptotic protein Bax and the decreased expression of the anti-apoptotic protein Bcl-2. 3.5. NOR-induced apoptosis is linked to p53 Because the p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair and apoptosis [20,21], we examined whether p53 was required for NOR-induced apoptosis in AIA FLS. Western blot analysis revealed that exposure to different concentrations of NOR (10, 30, 100 μM) significantly increased p53 expression in FLS (Fig. 5A). However, pretreatment with PFT-α, an inhibitor of p53 transcriptional activity, markedly attenuated the apoptosis of FLS induced
114
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
Fig. 3. Norisoboldine (NOR) induces apoptotic cell death by processing caspase-3, -9 and cleaving PARP in AIA FLS. (A) AIA FLS were treated with NOR (100 μM) for various times or (B) were treated with indicated concentrations of NOR for 24 h. The whole cell extracts were analyzed by SDS-PAGE and western blotting with antibodies against caspase-3, -8, -9 and cleavage PARP. GAPDH was used as the internal control. (C) AIA FLS were treated with concentrations of NOR for 24 h. The enzymatic activities of the respective caspases were analyzed by using the caspase activity assay kit, as described in the Materials and methods section. The relative activity per milligram of protein was determined by calculating the fold increase of caspase-3 and -9 activities in untreated cells. (D) After pretreatment with Z-LEHD-FMK for 1 h, AIA FLS were treated with various concentrations of NOR for 48 h and then apoptotic cell death was quantified by flow cytometry, as described in the Materials and methods section. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
by NOR (100 μM) (Fig. 5B), suggesting that p53 activation may contribute to NOR-induced apoptosis in AIA FLS. 4. Discussion Synovial hyperplasia is one of the pathological features of RA and accounts, in part, for the persistence of the disease and local joint destruction. Although the accumulation of cells in the intimal lining may result from the ingress of cells from the blood, local proliferation, or insufficient deletion through apoptosis, a substantial body of evidence has suggested that many pro-apoptotic genes are either defective or minimally expressed in RA, including p53 [21]. Therefore, interventions designed to increase programmed cell death of synoviocytes have been proposed as one of the promising strategies for treating RA. AIA is a model of experimental arthritis commonly used for in vivo studies. It shares several histopathological features with human RA, including mononuclear cell proliferation and synoviocyte hyperplasia as well as pannus formation followed by bone and cartilage destruction [22]. In the present study, FLS from AIA rats were used to assess the effects of NOR on the proliferation and apoptosis of FLS to elucidate the mechanisms by which NOR exerted its anti-hyperplasia activity on inflammatory synovial membranes in arthritis. It was found that
NOR treatment for different times (24, 48 and 72 h) caused a concentration- and time-dependent inhibition of FLS viability. Interestingly, this compound had little effect on cell proliferation and the cell cycle, but it substantially induced the apoptosis of FLS as evidenced by morphological changes in the nucleus and Annexin V-FITC/PI staining, suggesting that pro-apoptotic activity rather than anti-proliferation activity contributes to the anti-hyperplasia mechanisms of NOR. It is well accepted that apoptosis requires the activation of a series of caspases, including initiator and effector caspases. There are two central pathways that mediate apoptosis: the mitochondrial/stress pathway, triggered by diverse cytotoxic conditions, which leads to activation of initiator caspase-9, and the death receptor pathway, triggered by aggregation on the plasma membrane of receptors of the TNF family, which leads to activation of initiator caspase-8 [23]. The activation of caspase-3 plays a crucial role in the initiation of apoptosis. Therefore, it is feasible to differentiate them by evaluating the activities of these proteins. In this study, the cleavage of PARP and the activation of caspase-3 and caspase-9 were observed after NOR treatment. NOR did not alter the expression of caspase-8 or the activity of caspase-3, but it markedly decreased the activation of caspase-9. Additionally, pretreatment with Z-LEHD-FMK, an inhibitor of caspase-9 activity, reversed the pro-apoptotic activity of NOR. Mitochondrial dysfunction also
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
115
Fig. 4. Effect of norisoboldine (NOR) on the mitochondrial membrane potential (Δψm) and the subsequent release of cytochrome C in the cytoplasm. (A) AIA FLS were treated with various concentrations of NOR for 24 h. The cells were then stained with JC-1 and subjected to flow cytometric analysis for Δψm. (B) and (C) AIA FLS were treated with NOR (10, 30 and 100 μM) for 24 h, and then, the cell extracts were prepared as described in the Materials and methods section. Lysates of cytosolic fractions were analyzed by SDS-PAGE followed by western blotting with antibodies against cytochrome c, Bax and Bcl-2. GAPDH was used as the internal control. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
promotes the activation of caspases by regulating the Bcl-2 family members in the mitochondrial outer membranes. An increase in the Bax/Bcl2 ratio induces a significant activation of caspases and results in cell death [24,25]. In our study, NOR-induced cell apoptosis in AIA FLS appeared to accompany the decrease in mitochondrial membrane potential loss and the release of cytochrome C. Additionally, NOR decreased the expression of Bcl-2 but increased the expression of Bax in AIA FLS, leading to a marked increase of the Bax/Bcl-2 ratio. These results indicated that NOR triggers apoptosis of AIA FLS in a mitochondriadependent pathway by regulating the expression of known apoptosisrelated proteins. Recent studies have suggested that p53 plays critical role in the cellular response to DNA damage and apoptosis induced by reactive oxygen species. In the intrinsic mitochondria-initiated apoptotic pathway, p53-target gene products are involved in the release of cytochrome C and the resultant activation of caspase-9, which in turn activates execution-type caspase-3. In this study, we found that NOR enhanced p53 expression in AIA FLS in a concentration dependent manner. In
addition, inhibition of p53 activity by PFT-α attenuated apoptosis in AIA FLS induced by NOR, suggesting that p53 participates, at least in part, in the apoptosis induced by NOR. In conclusion, NOR, the main active constituent responsible for the anti-RA properties of Radix Linderae, exerts its pro-apoptotic effects by converging on the mitochondrial signaling pathway in FLS of AIA rats. The current findings provide a plausible explanation for the therapeutic benefit of NOR on inflammatory synovial hyperplasia, pannus formation and growth, and joint destruction that occurs in human RA.
Acknowledgments This work was supported by the Research Fund for the Doctoral Program of Higher Education of China (Grant number: 20090096110007) and the Priority Academic Program Development of Jiangsu Higher Education Institutions, and was partially funded by the Program for
116
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
Changjiang Scholars and Innovative Research Team in University (Grant number: IRT1193). References
Fig. 5. Norisoboldine (NOR)-induced apoptotic death is linked to the p53 signaling pathway. (A) AIA FLS were treated with various concentrations of NOR for 24 h; the cell extracts were then analyzed by SDS-PAGE followed by western blotting with antibodies against p53. GAPDH was used as the internal control. (B) After pretreatment with PFT-α for 1 h, AIA FLS were treated with various concentrations of NOR for 48 h, then apoptotic cell death was quantified by flow cytometry as described in the Materials and methods section. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
[1] Firestein GS. Evolving concepts of rheumatoid arthritis [review]. Nature 2003;423:356–61. [2] Henderson B, Pettipher ER. The synovial lining cell: biology and pathobiology. Semin Arthritis Rheum 1985;15:1–32. [3] Zvaifler NJ. Relevance of the stroma and epithelial-mesenchymal transition (EMT) for the rheumatic diseases. Arthritis Res Ther 2006;8:210. [4] Firestein GS. Invasive fibroblast-like synoviocytes in rheumatoid arthritis: passive responders or transformed aggressors? [review]. Arthritis Rheum 1996;39:1781–90. [5] Tak PP, Zvaifler NJ, Green DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today 2000;21:78–82. [6] Baier A, Meineckel I, Gay S, Pap T. Apoptosis in rheumatoid arthritis. Curr Opin Rheumatol 2003;15:274–9. [7] Schedel J, Gay RE, Kuenzler P, Seemayer C, Simmen B, Michel BA, et al. FLICEinhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis. Arthritis Rheum 2002;46:1512–8. [8] Asahara H, Hasumuna T, Kobata T, Yagita H, Okumura K, Inoue H, et al. Expression of Fas antigen and Fas ligand in the rheumatoid synovial tissue. Clin Immunol Immunopathol 1996;81:27–34. [9] Chou G, Li Q, Wang Z, Xu L. Compositions and anti-rheumatism effect of LEF fraction from the root of Lindera aggregate (Sims) Kosterm. J Plant Resour Environ 1999;8:1–6. [10] Luo Y, Liu M, Xia Y, Dai Y, Chou GX, Wang ZT. Therapeutic effect of norisoboldine, an alkaloid isolated from Radix Linderae, on collagen-induced arthritis in mice. Phytomedicine 2010;17:726–31. [11] Gerhardt D, Horn AP, Gaelzer MM, Frozza RL, Delgado-Cañedo A, Pelegrini AL, et al. Boldine: a potential new antiproliferative drug against glioma cell lines. Invest New Drugs 2009;27:517–25. [12] Chou GX, Norio N, Ma CM, Wang ZT, Masao H. Isoquinoline alkaloids from Lindera aggregata. Chin J Nat Med 2005;5:272–5. [13] Pan R, Dai Y, Gao X, Xia Y. Scopolin isolated from Erycibe obtusifolia Benth stems suppresses adjuvant-induced rat arthritis by inhibiting inflammation and angiogenesis. Int Immunopharmacol 2009;9:859–69. [14] Li Y, Dai Y, Liu M, Pan R, Luo Y, Xia Y, et al. Scopoletin induces apoptosis of fibroblastlike synoviocytes from adjuvant arthritis rats by a mitochondrial-dependent pathway. Drug Dev Res 2009;70:378–85. [15] Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA in vivo. Proc Natl Acad Sci U S A 2008;105:2415–20. [16] Chen XL, Cao LQ, She MR, Wang Q, Huang XH, Fu XH. Gli-1 siRNA induced apoptosis in Huh7 cells. World J Gastroenterol 2008;14:582–9. [17] Meshkini A, Yazdanparast R. Induction of megakaryocytic differentiation in chronic myelogenous leukemia cell K562 by 3-hydrogenkwadaphnin. J Biochem Mol Biol 2007;40:944–51. [18] Liu E, Du X, Ge R, Liang T, Niu Q, Li Q. Comparative toxicity and apoptosis induced by diorganotins in rat pheochromocytoma (PC12) cells. Food Chem Toxicol 2013;60:302–8. [19] Madan E, Prasad S, Roy P, George J, Shukla Y. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells. Biochem Biophys Res Commun 2008;377:1232–7. [20] Ko LJ, Prives C. p53: puzzle and paradigm. Genes 1996;10:1054–72. [21] Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323–31. [22] Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol 1992;27:134–42. [23] Perlman H, Pagliari LJ, Volin MV. Regulation of apoptosis and cell cycle activity in rheumatoid arthritis. Curr Mol Med 2001;1:597–608. [24] Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;75:241–51. [25] Tamm I, Schriever F, Dorken B. Apoptosis: implications of basic research for clinical oncology. Lancet Oncol 2001;2:33–42.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Norisoboldine induces apoptosis of fibroblast-like synoviocytes from adjuvant-induced arthritis rats Yubin Luo a,1, Zhifeng Wei a,1, Guixin Chou b, Zhengtao Wang b, Yufeng Xia a,⁎, Yue Dai a,⁎ a b
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 2 January 2014 Received in revised form 17 February 2014 Accepted 21 February 2014 Available online 6 March 2014 Keywords: Norisoboldine Rheumatoid arthritis Fibroblast-like synoviocytes Apoptosis
a b s t r a c t Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by pronounced synovial inflammation and hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Norisoboldine (NOR), the main active constituent in the alkaloid fraction isolated from Radix Linderae, was previously demonstrated to alleviate arthritis severity in experimental RA. This study aimed to evaluate the effects of NOR on proliferation and apoptosis of FLS from adjuvant-induced arthritis (AIA) rats to elucidate the mechanism of its inhibitory effect on inflammatory synovial hyperplasia in RA. Our results indicated that NOR exhibited a pro-apoptotic effect on AIA FLS but only slightly affected cell proliferation and the cell cycle. Following treatment with NOR for 24 h, the activation of caspase 3 and caspase 9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in AIA FLS were observed; however, caspase 8 remained unaffected. Meanwhile, a flow cytometric assay revealed that NOR significantly increased the percentage of apoptotic cells, causing the loss of the depolarized mitochondrial membrane potential and the release of cytochrome C. The expression of Bax and Bcl-2 was also regulated by NOR treatment. Additionally, the expression of p53 protein was up-regulated by NOR, and pretreatment with PFT-α, a p53 specific inhibitor, reversed the increase in FLS apoptosis caused by NOR. These findings indicated that NOR-induced apoptosis in AIA FLS is achieved via a mitochondrial-dependent pathway, which may be mediated by promoting the release of cytochrome C and by regulating the expression of Bax and Bcl-2 proteins, and p53 might also be required for NOR-induced apoptosis in AIA FLS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of unknown etiology that leads to fundamental changes in cellular composition and function of various joints [1]. One important characteristic of RA is chronic synovitis and progressive bone and cartilage damage. Fibroblast-like synoviocytes (FLS), which normally populate the thin synovial lining layer, appear to be the key players in changes of the joints based on their hyperplastic growth and their ability to become locally invasive in the adjacent cartilage and bone. Although in situ measurements of the lining layer proliferation rates suggest that indices of proliferation in RA synovium are insufficient to explain the degree of observed synovial hyperplasia [2,3], FLS derived from these tissues show in vitro anchorage-independent growth, loss of contact inhibition and the expression of various growth factors, inflammatory cytokines, oncogenes and cell cycle proteins, which resembles that
Abbreviations: NOR, norisoboldine; RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; AIA, adjuvant-induced arthritis; PARP, poly (ADP-ribose) polymerase. ⁎ Corresponding authors. Tel.: +86 25 83271400; fax: +86 25 85301528. E-mail addresses: [email protected] (Y. Xia), [email protected] (Y. Dai). 1 These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.intimp.2014.02.023 1567-5769/© 2014 Elsevier B.V. All rights reserved.
seen in tumor cells [4]. Recently, insufficient apoptosis of FLS has been recognized as a potential cause for the hyperplastic growth of FLS. RA FLS in vivo are reported to acquire mutations in the tumor suppressor gene p53 [5]. In addition, both nuclear factor-κB (NF-κB) activation and TNF-α stimulation can decrease FLS apoptosis through mechanisms such as resistance to Fas ligand-induced signaling and upregulation of anti-apoptotic molecules [6]. Cells found in the rheumatoid joints, including macrophages and FLS, appear to be resistant to apoptosis, even though Fas and Fas ligand are strongly expressed [7,8]. Also, accumulating evidence has suggested that a reduced level of apoptosis in vivo, especially at sites of invasion into cartilage and bone, is associated with the activation of FLS in RA [6]. For these reasons, the induction of apoptosis has been proposed as a potential therapeutic approach for RA. Radix Linderae, the dry roots of Lindera aggregata, is commonly used in traditional Chinese medicine. These extracts have been reported to possess anti-inflammatory and analgesic properties as well as potential anti-rheumatism activities [9]. Previous studies in our laboratory showed that norisoboldine (NOR), the main active constituent of Radix Linderae, effectively ameliorated synovial inflammation and hyperplasia, and protected joints from destruction in collagen II-induced arthritis (CIA) of mice [10]. One study has shown that boldine, an analog of NOR, induced cell death in a cell-type specific and dose-dependent
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
manner [11]. Therefore, in this study, we investigated whether and how NOR inhibited proliferation and induced apoptosis in FLS to gain insight into its underlying mechanisms for the prevention of synovial hyperplasia 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (6–7 weeks old) were obtained from the Experimental Animal Center of China Pharmaceutical University (Nanjing, China). All rats were acclimatized in the husbandry room, which was maintained at 22 ± 2 °C with a 12-h light:12-h dark cycle, and were allowed free access to food and water. The experimental animals were treated in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals. 2.2. Chemicals and reagents NOR (purity N 98%) was isolated and purified from Radix Linderae by authors, and the structure was established by comparison of its spectral data (UV, IR, MS, 1H- and 13 C NMR) with the literature data [12]. Dulbecco's modified Eagle medium (DMEM), penicillin and streptomycin were purchased from Gibco BRL (Grand Island, NY, USA); Newborn calf serum (NBCS) was purchased from PAA Laboratories GmbH (Austria); Mycobacterium butyricum was purchased from Difco Laboratories (Detroit, MI, USA); 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT), Propidium iodide (PI), Tween 20, bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and phenylmethyl sulfonylfluoride (PMSF) were purchased from Sigma Chemical (St. Louis, MO, USA); The Hoechst 33258 staining kit, AnnexinV-FITC apoptosis detection kit and mitochondrial membrane potential assay kit were purchased from Keygen Bio-tech (Nanjing, China); GAPDH monoclonal antibodies and the peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from KangChen Bio-tech (Shanghai, China); The caspase 3, caspase 9 Activity Assay Kit was purchased from the Beyotime Institute of Biotechnology (Nanjing, China); Anti-caspase-3, anticaspase-8, anti-caspase-9, anti-Bcl-2, anti-Bax, and anti-cyochrome C polyclonal antibodies were purchased from Bioworld Technology (St. Louis Park, MN, USA); all other chemicals and reagents used were of analytical grade. 2.3. Preparation and culture of FLS AIA was initiated by an intradermal injection of 100 μl of a 10 mg/ml Mycobacterium butyricum mineral oil suspension into the base of the right hind paw of the rats. Clinical signs, including swelling of the feet, inability to bend the ankle and the presence of nodules at the base of the tail and ears, were observed by day 13 [13]. The fibroblast-like synoviocytes (FLS) were isolated and cultured as described previously [14]. Briefly, the synovial tissues were obtained from AIA rats at day 21 after arthritis induction and were minced into small pieces and incubated with 0.4% type II collagenase in DMEM medium supplemented with 5% NBCS for 2 h at 37 °C. Non-adherent tissues were digested in serum-starved DMEM containing 0.25% trypsin for 30 min. The tissue suspension was transferred through a sterile 200-mm2 nylon mesh filter and centrifuged at 1500 rpm for 10 min. The collected cells were washed with 5% NBCS-DMEM and cultured in DMEM medium supplemented with 10% NBCS, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. After an overnight culture, the nonadherent cells were removed, and the adherent cells were cultured continuously under the same conditions. The cells were trypsinized and passaged at a 1:3 ratio when the cells were 80–90% confluent. FLS were used from passages 3 to 7 in these experiments when cells were a homogeneous population.
111
2.4. Cell viability assay Cell viability was determined using the MTT assay. Briefly, FLS were plated at a density of 0.5 × 104 cells/well in 96-well plates and treated with various concentrations of NOR (0, 3, 10, 30, 100 and 300 μM) in complete culture medium. After incubation at 37 °C for the indicated times (20 h, 44 h and 68 h), 20 μl of MTT (5 mg/ml) was added into each well, followed by incubation at 37 °C for an additional 4 h. The supernatant was then removed, and the formazone crystals were dissolved with 150 μl of DMSO. The optical absorbance at 540 nm was read with a Model 1500 Multiskan spectrum microplate reader (Thermo, Waltham, MA, USA). 2.5. Cell proliferation assay The proliferation assay on FLS was performed using a Cell-Light TM EdU DNA Cell Proliferation Kit (Ruibo Biotech, Guangzhou, China) [15]. In brief, FLS (0.5 × 104 cells/well) were cultured in 96-well plates with different concentrations of NOR (10, 30 and 100 μM) for 24 h. The cells were then incubated with EdU for another 8 h. Five random 100 × fields were photographed for each well, and EdU-positive cells were counted, averaged and compared. 2.6. Hoechst 33258 staining FLS (0.3 × 105 cells/well) were seeded in 24-well plates and exposed to various concentrations of NOR (10, 30 and 100 μM) for 48 h. Prior to fluorescent analysis, the cells were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed with PBS again and stained with Hoechst 33258 for 30 min in the dark. After washing with PBS, the cells were observed under a fluorescence microscope for nuclear morphological changes [16]. 2.7. Flow-cytometric analysis of Annexin-V-PI binding Apoptosis was evaluated by using a fluorescein isothiocyanatelabeled Annexin V/Propidium iodide (PI) Apoptosis Detection Kit according to the manufacturer's protocol. Briefly, FLS (1 × 105 cells) in 100-mm culture dishes were grown to 70% confluence and then treated with NOR (10, 30 and 100 μM) for 48 h. The cells were collected by trypsinization and washed with PBS, and then resuspended in 500 μl of binding buffer and 5 μl of Annexin V-FITC solution. Propidium iodide was added into each sample and incubated for 30 min at room temperature. The apoptotic cell population was immediately analyzed by flow cytometry after cells were washed with PBS for 3 times [17]. Data acquisition and analysis were performed in Becton-Dickinson FACSClibur flow cytometer using CellQuest 3.3 software. 2.8. Flow-cytometric analysis of the cell cycle FLS were treated with NOR (10, 30 and 100 μM) for 24 h and then were trypsinized, washed with PBS and centrifuged. The cell pellet was resuspended in 50 μl of ice-cold PBS and fixed in 1 ml of 70% ethanol for 20 min at 4 °C. After centrifugation at 1000 rpm for 5 min, FLS were washed twice with ice-cold PBS and treated with 0.1% Triton X-100 for 10 min. Subsequently, the cells were centrifuged and resuspended in 1 ml of PBS containing PI (50 μg/ml), then incubated for 30 min at 4 °C. The samples were acquired and analyzed in a Becton-Dickinson FACSCalibur flow cytometer using CellQuest 3.3 software. 2.9. Measurement of mitochondrial membrane potential (Δψm) Mitochondrial membrane potential was assessed using JC-1, according to the manufacturer's instructions (Keygen Bio-tech, Nanjing, China). After treatment with different concentrations of NOR (10, 30
112
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
and 100 μM) for 48 h, FLS were washed twice with cold PBS and then collected by centrifugation. After incubation with 500 μl of fluorescence-activated cell-sorting analysis (FACS) buffer containing JC-1 (1 mg/ml) at 37 °C for 30 min, the cells were washed with PBS 3 times, and the mitochondrial membrane potential was determined by flow cytometry [18]. 2.10. Caspase activity assay The caspase colorimetric assay kits specific for caspase-3 or caspase9 were used to detect caspase activation by measuring the cleavage of a synthetic colorimetric substrate. In brief, AIA FLS (1 × 105 cells) were cultured in 100-mm culture dishes and grown to 80% confluence and were then treated with NOR (10, 30 and 100 μM) for 24 h. Cell lysates were prepared in the lysis buffer from the assay kits; the lysates were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected. The synthetic colorimetric substrates were added to equal amounts of proteins from each sample at 37 °C for 2 h, followed by reading the samples at 405 nm on a microplate reader. The fold-increase in caspase-3 or -9 activities was determined by comparing the data of treated samples with those of the control samples. 2.11. Western blot analysis AIA FLS were treated with NOR for various time intervals and washed twice with ice-cold PBS. The cells were lysed with lysis buffer (50 mM Tris–Cl [pH 8.0], 150 mM NaCl, 0.02% NaN3, and 1% NP40). The cytosolic fraction was obtained from the supernatant after centrifugation at 12,000 rpm for 15 min at 4 °C. The cell lysates (20 μg) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% nonfat milk in PBS Tween-20 (PBST) for 1 h, the PVDF membranes were probed with relevant monoclonal antibodies overnight at 4 °C. After washing three times with PBST, the membranes were incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature. The bands were then visualized using enhanced chemiluminescence detection reagents and exposure to X-ray film. 2.12. Statistical analysis Statistical differences were assessed by a one-way analysis of variance (ANOVA) and the Student's t-test. All of the results were expressed as the means ± S.D. P values less than 0.05 were considered statistically significant. 3. Results 3.1. NOR inhibits the viability of AIA FLS After exposing AIA FLS to various concentrations of NOR for different time intervals, the cell viability was evaluated using the MTT assay. As shown in Fig. 1, there was a concentration- and time-dependent inhibition of the viability of AIA FLS after treatment with NOR. Cell viability was only slightly affected by lower concentrations of NOR (3 μM) for 24 and 48 h; however, viability was significantly reduced by the addition of 10 μM NOR for 48 h, and decreased to 41.8% ± 4.4 and 23.4% ± 5.0 in the presence of NOR (100 and 300 μM). Meanwhile, treatment with NOR (100 μM) for different time intervals caused a time-dependent reduction in cell viability to 58.1% ± 3.7 (24 h), 41.8% ± 4.4 (48 h) and 21.2% ± 2.2 (72 h), respectively.
Fig. 1. Effect of norisoboldine (NOR) on the activity of AIA FLS. The MTT assay was used to detect cell viability after treatment with various concentrations of NOR for 24 h, 48 h and 72 h, respectively. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎, ##, $$ p b 0.01 versus control.
concentrations of NOR for 24 h, cell proliferation was not significantly changed, although it was decreased to some extent in the presence of NOR (100 μM). Consistent with this finding, FACS analysis on the cell cycle suggested that there was little change in any phase of the cell cycle after treatment with various concentrations of NOR (data not shown). Based on the above results, we postulated that the inhibition of NOR on cell viability may be due to the induction of apoptosis. As shown in Fig. 2B, the nuclear morphology of untreated AIA FLS was normal, whereas condensation of nuclei and fragmentation of cells were observed in FLS treated by NOR (10, 30 and 100 μM). This finding was mirrored by the apoptosis detection by Annexin V and PI staining. Fig. 2C shows that NOR (10, 30 and 100 μM) increased the percentage of apoptotic FLS from 3.6% to 11.0%, 27.2% and 33.6%, respectively. The percentage of annexin V-positive cells was proportional to that of dead cells detected by MTT assay, indicating that the cell death induced by NOR was apoptotic cell death. 3.3. NOR-induced apoptotic cell death in AIA FLS involves the processing of caspase-9, -3 and cleavage of PARP To clarify the death signaling pathway underlying the NOR-induced apoptosis in AIA FLS, we assessed the activation of the caspase cascade, including caspase-3, -8 and -9. Caspase-3 and -9 were both processed after NOR treatment in a concentration- and time-dependent manner, whereas, the expression of caspase-8 remained unchanged (Fig. 3A & B). It is well recognized that cleavage of PARP protein considered as an important biomarker of apoptosis occurs while active caspase 3 proteolytically cleaves and activates. In this study, the activities of caspase-3 and -9 were detected by measuring the cleavage of a synthetic colorimetric substrate. As shown in Fig. 3C, the activities of caspase-3 and -9 in AIA FLS were significantly decreased by the addition of NOR (100 μM) for 24 h. Moreover, concurring with the process of caspase 3, PARP cleavage was observed in the samples treated with NOR (10, 30 and 100 μM). Additionally, pretreatment with Z-LEHD-FMK, an inhibitor of caspase-9 activity, reversed the pro-apoptotic action of NOR (Fig. 3D). In sum, our results suggested that NOR-induced cell death in AIA FLS takes place in a caspase-dependent manner.
3.2. NOR induces apoptosis of AIA FLS
3.4. NOR induces mitochondrial membrane potential loss and the release of cytochrome C in AIA FLS
To further understand how NOR inhibited AIA FLS viability, we investigated the effect of NOR on FLS proliferation as well as on the cell cycle. As shown in Fig. 2A, after incubation with increasing
Disruption of mitochondrial integrity is one of the early events leading to apoptosis. Mitochondrial membrane potential (Δψm) loss and the release of cytochrome C are biomarkers of mitochondrial function
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
113
Fig. 2. Norisoboldine (NOR) has no effect on FLS proliferation but induces apoptotic cell death. (A) AIA FLS were treated with various concentrations of NOR for 24 h. The cells were then stained with EdU, and the proliferation rates were measured as described in Materials and methods. (B) The effects of NOR on the nuclear morphology of AIA FLS. The cells were treated with various concentrations of NOR for 48 h and then stained with Hoechst 33258. The nuclear morphology was observed under a fluorescent microscope. (C) Induction of apoptosis was measured by an Annexin-V/PI double-staining assay. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
homeostasis. We detected both the mitochondrial membrane potential loss and cytochrome C expression in AIA FLS. As shown in Fig. 4A, NOR (10, 30 and 100 μM) treatment for 24 h caused a concentrationdependent rapid decline of Δψm. The release of cytochrome C from the mitochondria was increased by NOR (10, 30 and 100 μM) in a concentration-dependent manner (Fig. 4B). These results indicated that the NOR-induced apoptosis of AIA FLS is associated with disruption of the mitochondrial membrane and subsequent cytochrome C activation. The release of cytochrome C is tightly regulated by members of the Bcl-2 family that are known to have an important role in the regulation of apoptosis [19]. A decrease in the level of Bcl-2 or an increase in the level of Bax can trigger a signal to initiate apoptosis. As shown in Fig. 4C, the ratio of Bax/Bcl-2, which is crucial for the activation of the
mitochondrial apoptotic pathway, was increased in FLS treated with NOR, which was the consequence of the increased expression of the apoptotic protein Bax and the decreased expression of the anti-apoptotic protein Bcl-2. 3.5. NOR-induced apoptosis is linked to p53 Because the p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair and apoptosis [20,21], we examined whether p53 was required for NOR-induced apoptosis in AIA FLS. Western blot analysis revealed that exposure to different concentrations of NOR (10, 30, 100 μM) significantly increased p53 expression in FLS (Fig. 5A). However, pretreatment with PFT-α, an inhibitor of p53 transcriptional activity, markedly attenuated the apoptosis of FLS induced
114
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
Fig. 3. Norisoboldine (NOR) induces apoptotic cell death by processing caspase-3, -9 and cleaving PARP in AIA FLS. (A) AIA FLS were treated with NOR (100 μM) for various times or (B) were treated with indicated concentrations of NOR for 24 h. The whole cell extracts were analyzed by SDS-PAGE and western blotting with antibodies against caspase-3, -8, -9 and cleavage PARP. GAPDH was used as the internal control. (C) AIA FLS were treated with concentrations of NOR for 24 h. The enzymatic activities of the respective caspases were analyzed by using the caspase activity assay kit, as described in the Materials and methods section. The relative activity per milligram of protein was determined by calculating the fold increase of caspase-3 and -9 activities in untreated cells. (D) After pretreatment with Z-LEHD-FMK for 1 h, AIA FLS were treated with various concentrations of NOR for 48 h and then apoptotic cell death was quantified by flow cytometry, as described in the Materials and methods section. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
by NOR (100 μM) (Fig. 5B), suggesting that p53 activation may contribute to NOR-induced apoptosis in AIA FLS. 4. Discussion Synovial hyperplasia is one of the pathological features of RA and accounts, in part, for the persistence of the disease and local joint destruction. Although the accumulation of cells in the intimal lining may result from the ingress of cells from the blood, local proliferation, or insufficient deletion through apoptosis, a substantial body of evidence has suggested that many pro-apoptotic genes are either defective or minimally expressed in RA, including p53 [21]. Therefore, interventions designed to increase programmed cell death of synoviocytes have been proposed as one of the promising strategies for treating RA. AIA is a model of experimental arthritis commonly used for in vivo studies. It shares several histopathological features with human RA, including mononuclear cell proliferation and synoviocyte hyperplasia as well as pannus formation followed by bone and cartilage destruction [22]. In the present study, FLS from AIA rats were used to assess the effects of NOR on the proliferation and apoptosis of FLS to elucidate the mechanisms by which NOR exerted its anti-hyperplasia activity on inflammatory synovial membranes in arthritis. It was found that
NOR treatment for different times (24, 48 and 72 h) caused a concentration- and time-dependent inhibition of FLS viability. Interestingly, this compound had little effect on cell proliferation and the cell cycle, but it substantially induced the apoptosis of FLS as evidenced by morphological changes in the nucleus and Annexin V-FITC/PI staining, suggesting that pro-apoptotic activity rather than anti-proliferation activity contributes to the anti-hyperplasia mechanisms of NOR. It is well accepted that apoptosis requires the activation of a series of caspases, including initiator and effector caspases. There are two central pathways that mediate apoptosis: the mitochondrial/stress pathway, triggered by diverse cytotoxic conditions, which leads to activation of initiator caspase-9, and the death receptor pathway, triggered by aggregation on the plasma membrane of receptors of the TNF family, which leads to activation of initiator caspase-8 [23]. The activation of caspase-3 plays a crucial role in the initiation of apoptosis. Therefore, it is feasible to differentiate them by evaluating the activities of these proteins. In this study, the cleavage of PARP and the activation of caspase-3 and caspase-9 were observed after NOR treatment. NOR did not alter the expression of caspase-8 or the activity of caspase-3, but it markedly decreased the activation of caspase-9. Additionally, pretreatment with Z-LEHD-FMK, an inhibitor of caspase-9 activity, reversed the pro-apoptotic activity of NOR. Mitochondrial dysfunction also
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
115
Fig. 4. Effect of norisoboldine (NOR) on the mitochondrial membrane potential (Δψm) and the subsequent release of cytochrome C in the cytoplasm. (A) AIA FLS were treated with various concentrations of NOR for 24 h. The cells were then stained with JC-1 and subjected to flow cytometric analysis for Δψm. (B) and (C) AIA FLS were treated with NOR (10, 30 and 100 μM) for 24 h, and then, the cell extracts were prepared as described in the Materials and methods section. Lysates of cytosolic fractions were analyzed by SDS-PAGE followed by western blotting with antibodies against cytochrome c, Bax and Bcl-2. GAPDH was used as the internal control. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
promotes the activation of caspases by regulating the Bcl-2 family members in the mitochondrial outer membranes. An increase in the Bax/Bcl2 ratio induces a significant activation of caspases and results in cell death [24,25]. In our study, NOR-induced cell apoptosis in AIA FLS appeared to accompany the decrease in mitochondrial membrane potential loss and the release of cytochrome C. Additionally, NOR decreased the expression of Bcl-2 but increased the expression of Bax in AIA FLS, leading to a marked increase of the Bax/Bcl-2 ratio. These results indicated that NOR triggers apoptosis of AIA FLS in a mitochondriadependent pathway by regulating the expression of known apoptosisrelated proteins. Recent studies have suggested that p53 plays critical role in the cellular response to DNA damage and apoptosis induced by reactive oxygen species. In the intrinsic mitochondria-initiated apoptotic pathway, p53-target gene products are involved in the release of cytochrome C and the resultant activation of caspase-9, which in turn activates execution-type caspase-3. In this study, we found that NOR enhanced p53 expression in AIA FLS in a concentration dependent manner. In
addition, inhibition of p53 activity by PFT-α attenuated apoptosis in AIA FLS induced by NOR, suggesting that p53 participates, at least in part, in the apoptosis induced by NOR. In conclusion, NOR, the main active constituent responsible for the anti-RA properties of Radix Linderae, exerts its pro-apoptotic effects by converging on the mitochondrial signaling pathway in FLS of AIA rats. The current findings provide a plausible explanation for the therapeutic benefit of NOR on inflammatory synovial hyperplasia, pannus formation and growth, and joint destruction that occurs in human RA.
Acknowledgments This work was supported by the Research Fund for the Doctoral Program of Higher Education of China (Grant number: 20090096110007) and the Priority Academic Program Development of Jiangsu Higher Education Institutions, and was partially funded by the Program for
116
Y. Luo et al. / International Immunopharmacology 20 (2014) 110–116
Changjiang Scholars and Innovative Research Team in University (Grant number: IRT1193). References
Fig. 5. Norisoboldine (NOR)-induced apoptotic death is linked to the p53 signaling pathway. (A) AIA FLS were treated with various concentrations of NOR for 24 h; the cell extracts were then analyzed by SDS-PAGE followed by western blotting with antibodies against p53. GAPDH was used as the internal control. (B) After pretreatment with PFT-α for 1 h, AIA FLS were treated with various concentrations of NOR for 48 h, then apoptotic cell death was quantified by flow cytometry as described in the Materials and methods section. Each data point shows the mean ± S.D. of three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.01, versus control.
[1] Firestein GS. Evolving concepts of rheumatoid arthritis [review]. Nature 2003;423:356–61. [2] Henderson B, Pettipher ER. The synovial lining cell: biology and pathobiology. Semin Arthritis Rheum 1985;15:1–32. [3] Zvaifler NJ. Relevance of the stroma and epithelial-mesenchymal transition (EMT) for the rheumatic diseases. Arthritis Res Ther 2006;8:210. [4] Firestein GS. Invasive fibroblast-like synoviocytes in rheumatoid arthritis: passive responders or transformed aggressors? [review]. Arthritis Rheum 1996;39:1781–90. [5] Tak PP, Zvaifler NJ, Green DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today 2000;21:78–82. [6] Baier A, Meineckel I, Gay S, Pap T. Apoptosis in rheumatoid arthritis. Curr Opin Rheumatol 2003;15:274–9. [7] Schedel J, Gay RE, Kuenzler P, Seemayer C, Simmen B, Michel BA, et al. FLICEinhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis. Arthritis Rheum 2002;46:1512–8. [8] Asahara H, Hasumuna T, Kobata T, Yagita H, Okumura K, Inoue H, et al. Expression of Fas antigen and Fas ligand in the rheumatoid synovial tissue. Clin Immunol Immunopathol 1996;81:27–34. [9] Chou G, Li Q, Wang Z, Xu L. Compositions and anti-rheumatism effect of LEF fraction from the root of Lindera aggregate (Sims) Kosterm. J Plant Resour Environ 1999;8:1–6. [10] Luo Y, Liu M, Xia Y, Dai Y, Chou GX, Wang ZT. Therapeutic effect of norisoboldine, an alkaloid isolated from Radix Linderae, on collagen-induced arthritis in mice. Phytomedicine 2010;17:726–31. [11] Gerhardt D, Horn AP, Gaelzer MM, Frozza RL, Delgado-Cañedo A, Pelegrini AL, et al. Boldine: a potential new antiproliferative drug against glioma cell lines. Invest New Drugs 2009;27:517–25. [12] Chou GX, Norio N, Ma CM, Wang ZT, Masao H. Isoquinoline alkaloids from Lindera aggregata. Chin J Nat Med 2005;5:272–5. [13] Pan R, Dai Y, Gao X, Xia Y. Scopolin isolated from Erycibe obtusifolia Benth stems suppresses adjuvant-induced rat arthritis by inhibiting inflammation and angiogenesis. Int Immunopharmacol 2009;9:859–69. [14] Li Y, Dai Y, Liu M, Pan R, Luo Y, Xia Y, et al. Scopoletin induces apoptosis of fibroblastlike synoviocytes from adjuvant arthritis rats by a mitochondrial-dependent pathway. Drug Dev Res 2009;70:378–85. [15] Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA in vivo. Proc Natl Acad Sci U S A 2008;105:2415–20. [16] Chen XL, Cao LQ, She MR, Wang Q, Huang XH, Fu XH. Gli-1 siRNA induced apoptosis in Huh7 cells. World J Gastroenterol 2008;14:582–9. [17] Meshkini A, Yazdanparast R. Induction of megakaryocytic differentiation in chronic myelogenous leukemia cell K562 by 3-hydrogenkwadaphnin. J Biochem Mol Biol 2007;40:944–51. [18] Liu E, Du X, Ge R, Liang T, Niu Q, Li Q. Comparative toxicity and apoptosis induced by diorganotins in rat pheochromocytoma (PC12) cells. Food Chem Toxicol 2013;60:302–8. [19] Madan E, Prasad S, Roy P, George J, Shukla Y. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells. Biochem Biophys Res Commun 2008;377:1232–7. [20] Ko LJ, Prives C. p53: puzzle and paradigm. Genes 1996;10:1054–72. [21] Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323–31. [22] Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol 1992;27:134–42. [23] Perlman H, Pagliari LJ, Volin MV. Regulation of apoptosis and cell cycle activity in rheumatoid arthritis. Curr Mol Med 2001;1:597–608. [24] Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;75:241–51. [25] Tamm I, Schriever F, Dorken B. Apoptosis: implications of basic research for clinical oncology. Lancet Oncol 2001;2:33–42.
