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Effects of Koumine on Adjuvant- and Collagen-Induced Arthritis in Rats Jian Yang,†,‡ Hong-Da Cai,† Yu-Lan Zeng,† Ze-Hong Chen,† Meng-Han Fang,† Yan-Ping Su,†,‡ Hui-Hui Huang,†,‡ Ying Xu,†,‡ and Chang-Xi Yu*,†,‡ †
Department of Pharmacology and ‡Fujian Key Laboratory of Natural Medicine Pharmacology, College of Pharmacy, Fujian Medical University, Fuzhou, Fujian 350108, China
ABSTRACT: To examine the effect of koumine, a Gelsemium alkaloid, on two experimental models of rheumatoid arthritis (RA), rats with adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) were administered koumine (0.6, 3, or 15 mg/kg/day) or vehicle through gastric gavage (i.g.). Clinical evaluation was performed via measurements of hind paw volume, arthritis index (AI) score, mechanical withdrawal threshold, organ weight, and by radiographic and histological examinations. Levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and antitype II collagen (CII) antibody were also examined. In rats with AIA, koumine reduced the AI score and mechanical allodynia of the injected hind paw in a dose-dependent manner and significantly inhibited increase in thymus and liver weights. In rats with CIA, koumine inhibited increase in hind paw volume, AI score, and mechanical allodynia in a dose-dependent manner and reduced joint space narrowing. Furthermore, koumine also attenuated the increase in the expression of IL-1β and TNF-α, as well as the robust increase of serum anti-CII antibodies in response to immunization. These results suggested that koumine effectively attenuated arthritis progression in two rat models of RA and that this therapeutic effect may be associated with its immunoregulatory action.
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and pain.13 In recent years, the pharmacology of G. elegans extract has been widely researched.14,15 In vivo studies using rodents have demonstrated that G. elegans extracts exhibit analgesic and anti-inflammatory properties.16,17 Even though the clinical use of crude G. elegans extract is limited by its narrow therapeutic window,14 literature concerning the isolation of phytochemical constituents from G. elegans is extensive.18,19 More than 90 alkaloids have been isolated from G. elegans, among which koumine (molecular formula C20H22N2O; molecular weight 306.40 g·mol−1; CAS registry number 135876-5; Figure 1) is the most abundant, and its chemical structure is distinct from NSAIDs and other commonly used antiinflammatory agents as we described previously.20,21 Interestingly, the toxicity of koumine is lower than most other active alkaloids.22 Therefore, there have been an increasing number of reports on the pharmaceutical potential of koumine.23,24 We previously established a method to isolate koumine with high purity and concentration from G. elegans25 and have over the
heumatoid arthritis (RA) is a chronic autoimmune disease that affects multiple organ systems, with a prevalence of about 1% in the general population.1,2 Although corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, and disease-modifying antirheumatic drugs (DMARDs) such as methotrexate suppress the symptoms of RA, their clinical use is limited due to the continuous progression of the disease despite ongoing therapy or the emergence of side effects, which discourages long-term compliance or use by patients.3,4 A better understanding of the important role of pro-inflammatory cytokines in the pathophysiology of RA in recent times has vastly improved treatment options, as evidenced by the development of proinflammatory cytokine blockers.5,6 However, these drugs achieve disease remission in only a minority of patients due to infectious adverse events, rebound of symptoms, short halflives, or high treatment costs.7−9 Medicinal plants are important sources of novel therapeutic drugs for the treatment of RA; thus, scientists have focused their attention on discovering novel therapeutic agents from botanicals.10,11 Gelsemium elegans Benth. (G. elegans), a plant native to China and Southeast Asia,12 has been widely used in Chinese folk medicine as a remedy for inflammatory diseases © 2016 American Chemical Society and American Society of Pharmacognosy
Received: June 16, 2016 Published: September 22, 2016 2635
DOI: 10.1021/acs.jnatprod.6b00554 J. Nat. Prod. 2016, 79, 2635−2643
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polyarthritis; concurrently, inflammatory edema and mechanical allodynia in the contralateral hind paws had also become significant (two-way ANOVA, P < 0.01 vs the blank control on days 18 and 24, Figure 2C,E). The mean AI score increased slightly during the first 12 d and subsequently rose further, up to 24 d, after immunization (Figure 2A). Administration of koumine did not lead to significant inhibition of the development of joint swelling induced by the adjuvant in the acute phase; the ipsilateral and contralateral hind paw volumes were not significantly different relative to those of rats treated with the vehicle. However, 18 d after immunization (9 d after the termination of the treatment), the mean AI scores for all koumine-treated groups (0.6, 3, and 15 mg·kg−1) were significantly reduced (Kruskal−Wallis test, P < 0.05) relative to the vehicle group. A two-way ANOVA of MWT data of the ipsilateral hind paw indicated a significant difference in results that was dependent on treatment (F = 3193, P < 0.0001) and time (F = 3053, P < 0.0001), as well as significant interaction between treatment and time (F = 265.8, P < 0.0001). Koumine inhibited mechanical allodynia in a dosedependent manner in the ipsilateral hind paw (P < 0.01 for 0.6 mg·kg−1 vs vehicle control at 9, 12, and 18 d; P < 0.01 for 3 mg· kg−1 vs vehicle control at 6, 9, 12, and 18 d; P < 0.01 for 15 mg· kg−1 vs vehicle control at 3, 6, 9, 12, and 18 d), but not in the contralateral hind paw. On the other hand, indometacin significantly restricted the development of AIA, as indicated by hind paw volume, AI score, and MWT (Figure 2). Additionally, the mean weights of the spleen, liver, and thymus of the rats with AIA were increased, suggesting the onset of splenomegaly, hepatomegaly, and thymic enlargement. The increase in liver and thymus weights was significantly (oneway ANOVA, P < 0.01, for both) inhibited by koumine treatment compared to the vehicle, whereas there was no significant change in spleen weight between koumine-treated rats and vehicle control rats (Figure 3). The results show that the progression of AIA followed a biphasic time course, consisting of an acute local inflammatory reaction and a subsequent chronic systemic reaction characterized by polyarthritis of the contralateral paw. The administration of koumine to rats with AIA was designed as an acute treatment and was successful in reducing arthritis score and pain parameters, demonstrating its anti-inflammatory and analgesic effects on AIA. Inflammation and pain characterize the initiation of arthritis;31 thus, although the acute treatment of koumine did not significantly inhibit the development of joint swelling or splenomegaly, we believe the treatment elicited, to a certain extent, a therapeutic effect against arthritis in the AIA model. Effects of Koumine on Rats with CIA. As one animal model by itself cannot reflect the complexity of human RA, another frequently used animal model, i.e., the rat CIA model, was employed to confirm the antiarthritic activity of koumine. Its effects on hind paw volume, AI score, and MWT were also investigated in the rat CIA model. As shown in Figure 4, no significant differences were found in these parameters in all groups before immunization. Hind paw volume increased after immunization (Figure 4B) immediately prior to the treatment. On days 5 and 10 after the initiation of the treatment, the mean AI score was increased (Figure 4A), while the mean MWT was decreased (Figure 4C) in the vehicle control group. A two-way ANOVA of the hind paw volume and MWT indicated the significant effects of treatment (F = 74.30, P < 0.001 for hind paw volume and F = 151.63, P < 0.001 for
Figure 1. Chemical structure of koumine. Molecular formula: C20H22N2O. Molecular weight: 306.40 g·mol−1. CAS registry number: 1358-76-5.
years made several investigations on the pharmacology and toxicity of koumine.24,21,26,27 Our previous studies reported analgesic action of koumine in rodent models of inflammatory pain and diabetic neuropathy.21,26 Since inflammation and pain are two core features of RA,28 we speculate that koumine may also be effective against RA. Different animal models of RA have been used to evaluate and determine the effectiveness of novel therapeutic agents, with adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) being the most common.29 In general, the AIA model is often used to investigate an experimental autoimmune disease with several features of human RA, while CIA is the most widely used model for studying disease pathogenesis and validation of potential therapeutic targets.30 In the present study, we examined the effects of koumine on joint inflammation and pain on rats with AIA and CIA. We further analyzed radiologic and histological evidence for joint damage and inflammation and determined the effects of koumine on antitype II collagen (CII) antibody and proinflammatory cytokines.
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RESULTS AND DISCUSSION Acute Toxicity. In the current study, we evaluated the acute toxicity of koumine in Wistar rats according to an up-and-down procedure (UDP) and found the median lethal dose (LD50) of koumine upon gastric gavage (i.g.) was 300.0 mg·kg−1 (95% confidence interval: 230.8−313.0 mg·kg−1). Death generally occurred within 30 min of administration. Brief generalized colonic convulsions occurred immediately before death in most subjects. Its toxicity signature, i.e., the symptom of poisoning, was similar to that of the crude alkaloidal extract of G. elegans.14 However, the estimated LD50 level of koumine by gastric gavage was much higher than that of crude alkaloidal extract in mice using the same route (15 mg·kg−1),14,16 as well as the range of koumine dosage administered to rats in this study (0.6−15 mg·kg−1), demonstrating that the toxicity of koumine is lower than that of the crude G. elegans extract. Thus, it is exciting to note the promising potential of koumine over crude G. elegans extract in clinical applications. Effects of Koumine on Rats with AIA. To investigate the pharmacological effects of koumine on RA development, the AIA model in Lewis rats was employed. No significant differences were found in hind paw volume, AI score, or mechanical withdrawal threshold (MWT) in all groups prior to the immunization. The injected hind paws showed acute inflammatory swelling, erythema, and mechanical allodynia over a 24 h period in the foot injected with adjuvant. The ipsilateral hind paw volume increased over the next 12 d accompanied by decrease in MWT (two-way ANOVA, P < 0.01 vs the blank control for hind paw volume, Figure 2B; P < 0.01 vs the blank control for MWT, Figure 2D), followed by subsequent chronic 2636
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Figure 2. Effects of repeated intragastric administrations of koumine on disease progression in rats with AIA. Eight-week-old Lewis rats were immunized by a single intradermal injection of 0.1 mL of Freund’s complete adjuvant (CFA) into the right hind paw. Immunized rats received koumine (0.6, 3, or 15 mg·kg−1 per day) or vehicle by gastric gavage 1 h before the induction and daily for the next 9 d. A group of immunized rats receiving indometacin (2.5 mg·kg−1 per day) during the same period served as a positive control. Clinical evaluation was performed prior to the immunization (baseline) and on alternate days after the initiation of koumine/indometacin treatment (postdosing) up to 24 d through standardized scoring of arthritis (A), measurement of edema (B, C), and evaluation of articular nociception (D, E). The results are shown as mean ± SD; n = 10 per group. *P < 0.05, **P < 0.01 vs the vehicle control; ##P < 0.01 vs the blank control. Data were analyzed using two-way ANOVA, followed by Dunnett’s T3 test, or the Kruskal−Wallis with Dunn’s multiple comparisons test.
6, 8, and 10 d; P < 0.01 for 3 and 15 mg·kg−1 at 2, 4, 6, 8, and 10 d; methotrexate: P < 0.01 at 6, 8, and 10 d) relative to the vehicle. Both koumine and methotrexate significantly reduced the AI score (Kruskal−Wallis test; koumine: P < 0.01 for 15 mg·kg−1 at 5 d; P < 0.01 for 3 and 15 mg·kg−1 at 10 d; methotrexate: P < 0.01 at 10 d) relative to the vehicle after the initiation of the treatment. The CIA model often results in chronic arthritis and has been used by numerous researchers to investigate RA pathogenesis and in the search for new drugs to treat inflammatory arthritis.32 In the present study, koumine was
MWT) and time (F = 448.66, P < 0.001 for hind paw volume and F = 97.03, P < 0.001 for MWT), as well as significant interaction between treatment and time (F = 58.88, P < 0.001 for hind paw volume and F = 32.67, P < 0.001 for MWT). The effect of koumine was both dose-dependent (two-way ANOVA; F = 6.38, P < 0.01 for hind paw volume; F = 60.73, P < 0.001 for MWT) and time-dependent (two-way ANOVA; F = 344.79, P < 0.001 for hind paw volume; F = 61.75, P < 0.001 for MWT). Both koumine and methotrexate significantly reduced hind paw volume (P < 0.01 for all at 5 and 10 d) and mechanical allodynia (koumine: P < 0.01 for 0.6 mg·kg−1 at 4, 2637
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Figure 3. Effect of repeated intragastric administrations of koumine on organ weight in rats with AIA. The animals were treated as described in Figure 2. The liver, spleen, and thymus were removed and weighed after sacrifice by exsanguination under deep anesthesia 24 d after immunization. Relative weights (g/100g body weight) of the spleen (A), liver (B), and thymus (C) are showed as mean ± SD; n = 10 in each group. ##P < 0.01 compared with the blank control; *P < 0.05, **P < 0.01 compared with the vehicle control. Data were analyzed using one-way ANOVA followed by Student−Newman−Keuls post hoc test.
joints (arrows in Figure 5) compared to blank control rats. In koumine- or methotrexate-treated rats, however, bone erosion and degradation were scarcely detected, and the extent of the narrowing of joint space was observed to be relatively small. Histologic and radiologic examinations showed that administration of 15 mg·kg−1 koumine per day ameliorated the degeneration of bone and cartilage in joints. Here we investigated the effects of intragastric treatment of koumine on two different models, comprising rats with AIA and CIA, which are well-established models of RA strongly driven by inflammation, with each reflecting the diverse pathophysiologic mechanisms involved in the disease.29,32 Our results demonstrated that koumine inhibited inflammation and pain in the treated animals and led to inhibition of thymic enlargement and hepatomegaly in rats with AIA and histological and radiological features of joint degeneration and synovial inflammation in rats with CIA. These results demonstrate the protective effect of koumine against the development of arthritis in RA animal models and serve as the first pieces of evidence that koumine exhibits antirheumatic properties in rats with AIA and CIA. Therefore, these findings strongly suggest the potential of koumine as an alternative source and template for the development of novel antiarthritic drugs for RA; this discovery has been granted a United States invention patent.36
administrated during the chronic stage of arthritis in CIA. It effectively suppressed the development of macroscopic symptoms including swelling, erythema, and ankylosis of the hind paw and ankle joints, which were quantified by hind paw volume and AI score. The effect of koumine was dosedependent; administration of 15 mg·kg−1 koumine reduced inflammatory and pain parameters to a level comparable to that of the blank control. Pain management, a priority in the medical care of RA patients,33 remains a major challenge despite major advances in treatment strategies over the past few decades.34 In the current study, koumine exhibited a dose-dependent analgesic activity on MWT in both AIA and CIA models. This finding is consistent with the results of our previous report that koumine attenuated acetic acid-, formalin-, and Freund’s complete adjuvant (CFA)-induced inflammatory pain in mice.21 Effects of Koumine on Joint Destruction and Inflammation in Rats with CIA. Destruction of the periarticular bone and articular cartilage is a dominant feature of structural damage and is radiographically reflected by bone erosion and joint space narrowing.35 X-ray and histological examinations performed in this study showed that the immunized rats exhibited apparent degeneration of joint structure and narrowed joint space in the hind paw ankle 2638
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Figure 4. Effects of repeated intragastric administrations of koumine on disease progression in rats with CIA. Seven-week-old Wistar rats were immunized with chicken CII in Freund’s complete adjuvant (CFA) and Freund’s incomplete adjuvant (IFA). Immunized rats received koumine (0.6, 3, or 15 mg·kg−1 per day) or vehicle by gastric gavage for 10 consecutive days, starting 20 d from the first CII injection. A group of immunized rats receiving methotrexate (1 mg·kg−1 every 3 d) during the same period was included as a positive control. Clinical evaluation was performed prior to the immunization (baseline), immediately prior to the treatment (predosing), and on alternate days after the initiation of koumine/methotrexate treatment (postdosing) through standardized scoring of arthritis (A), measurement of edema (B), and evaluation of articular nociception (C). The results are shown as mean ± SD; n = 10 per group. **P < 0.01 vs the vehicle control, ##P < 0.01 vs the blank control. Data were analyzed using twoway ANOVA, followed by Dunnett’s T3 test, or the Kruskal−Wallis with Dunn’s multiple comparisons test.
Figure 5. Effects of repeated intragastric administrations of koumine on synovial inflammation and bone destruction in rats with CIA. The animals were treated as described in Figure 4. The hind limbs of rats from all groups were examined by X-ray and H&E staining of the ankle joint upon completion of treatment and all behavioral tests. Upper panel: Representative radiographs. Lower panel: Representative H&E histology staining photographs. Original magnification: 100×.
Effects of Koumine on Cytokine Levels. A number of conceivable reasons contribute to the efficacy of koumine as an antiarthritic agent. One possibility is the reduction of proinflammatory cytokine production by koumine.6,37 Interleukin (IL)-1β is often associated with the onset and acceleration of
CIA progression in rats, whereas tumor necrosis factor (TNF)α is thought to be responsible for the severity of arthritis.38−40 Hence, inhibition of these cytokines is expected to elicit antiinflammatory effects.41 In our study, immunization of the rats increased the expression of IL-1β (Figure 6A,C) and TNF-α 2639
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Figure 6. Effects of repeated intragastric administrations of koumine on the level of anti-CII IgG in the serum of rats with CIA. The animals were treated as described in Figure 4. Serum samples collected after the completion of treatment were assayed for anti-CII antibody using a commercially available ELISA kit. Data are presented as mean ± SD; n = 10 per group. *P < 0.05, **P < 0.01 vs the vehicle control; ##P < 0.01 vs the blank control.
which was reduced by administration of 15 mg·kg−1 koumine (P < 0.01, for both). Effects of Koumine on Anti-CII Antibody Levels. Some other mechanisms may also contribute to the antiarthritic effects of koumine. An alternative mechanism by which koumine exerts its antirheumatic effects is via the inhibition of anti-CII antibody production. Autoantibodies such as rheumatoid factor and anti-CII IgG are commonly detected in RA patients, and their concentrations correlate with disease severity and joint damage.39,42 Serological quantification of antiCII IgG by enzyme-linked immunosorbent assay (ELISA) in this study showed that the immunization increased the production of the antibody (one-way ANOVA, P < 0.01 vs blank control). Methotrexate significantly attenuated the
(Figure 6B, C) in both serum and joint tissue (one-way ANOVA, P < 0.01 vs blank control, for both). Methotrexate significantly attenuated the increase in serum IL-1β (P < 0.01) level, but had an insignificant effect on the level of serum TNFα. In contrast, koumine significantly inhibited the increase in the serum levels of both IL-1β at 3 and 15 mg·kg−1 (P < 0.01 vs vehicle control) and TNF-α at 15 mg·kg−1 (P < 0.05). At 15 mg·kg−1, koumine also exhibited a significant inhibitory effect against the increase in the concentration of both cytokines in joint tissue (P < 0.01). As shown in Figure 6D, quantitative real-time polymerase chain reaction (qPCR) results revealed that the immunization induced mRNA expression of both IL1β and TNF-α (one-way ANOVA, P < 0.01 vs blank control), 2640
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Figure 7. Effects of repeated intragastric administrations of koumine on the expression of IL-1β and TNF-α in rats with CIA. The animals were treated as described in Figure 4. Serum and joint tissue samples were collected after the completion of treatment. Expressions of IL-1β and TNF-α were examined by ELISA at the protein level and by qPCR at the mRNA level (presented as fold-change over the blank control). Data are presented as mean ± SD; n = 10 per group. *P < 0.05,**P < 0.01 vs the vehicle control; ##P < 0.01 vs the blank control. standard rodent food and water, except during behavioral experiments. Rats were habituated for 7 d prior to use in experiments. All experimental protocols were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985) and were approved by the Committee of Ethics of Fujian Medical University, China. In Vivo Acute Toxicity Determination. Acute oral toxicity of koumine was estimated according to a UDP described in the Organisation for Economic Cooperation and Development guideline 425 (OECD 2001) in ten 7-week-old male Wistar rats. A sequential dosing protocol with a dose progression factor of 1/0.75 and a starting dose of 300 mg·kg−1 based on the preliminary limit test was used. Rats were rank-ordered based on random numbers assigned by weight. Food was withdrawn overnight before intragastric administration of koumine (10 mL·kg−1) through gastric gavage. Survival was monitored 30 min after the administration, at 4 h intervals during the first 24 h and twice a day thereafter over the next 13 d. Dosage for the next rat was adjusted to 400 or 225 mg·kg−1 depending upon whether the rat survived or died within 30 min of gastric gavage. The experiment was ended using the rules specified by the AOT425StatPgm program, version 1.0 (USEPA, 2001). The values of LD50 and the 95% confidence interval were calculated using the same program. Complete Freund’s Adjuvant-Induced Arthritis in Lewis Rats. AIA was induced in Lewis rats by a single intradermal injection of 0.1 mL of CFA containing 10 mg of heat-killed Mycobacterium tuberculosis in 1 mL of paraffin oil into the right hind paw according to the method of Prakashet et al.46 Rats receiving a single dose of 0.1 mL of paraffin oil−water emulsion without M. tuberculosis in the same location were included as blank control. Immunized rats received koumine (0.6, 3, or 15 mg·kg−1 per day through gastric gavage) or vehicle 1 h before the induction on day zero (D0) and daily for the next 9 d. A group of immunized rats receiving indomethacin (2.5 mg· kg−1 per day) during the same period was included as a positive control. Clinical evaluation was performed prior to the immunization (baseline) and on alternate days after the initiation of koumine/ indomethacin treatment (postdosing) up to 24 d, through standardized scoring of arthritis, measurement of edema, and evaluation of articular nociception. Collagen-Induced Arthritis in Wistar Rats. Chicken CII (2 mg· mL−1 in 0.1 M acetic acid) was vortexed overnight at 4 °C and emulsified in an equal volume of CFA preprepared by dissolving heatinactivated Bacillus Calmette-Guerin in IFA to a final concentration of 0.5 mg·mL−1. Each rat received two intradermal injections of 0.3 mL of CII emulsion at three sites, separated by 7 d.47 Rats receiving 0.05 M acetic acid were included as blank control. Immunized rats received koumine (0.6, 3, or 15 mg·kg−1 per day through gastric gavage) or vehicle, starting from 20 d after the first CII injection, for 10 consecutive days. A group of immunized rats receiving methotrexate (1 mg·kg−1 every 3 d) during the same period was included as a positive control. Clinical evaluation was performed prior to the immunization (baseline), immediately prior to the treatment (predosing), and on alternate days after the initiation of koumine/
increase in serum anti-CII IgG level (P < 0.01 vs vehicle), as did koumine at 3 mg·kg−1 (P < 0.05 vs vehicle) but not at 0.6 mg· kg−1. At a dose of 15 mg·kg−1, the magnitude of the inhibitory action of koumine on serum anti-CII IgG production (P < 0.01 vs vehicle) was similar to that of methotrexate (Figure 7). Collagen-specific antibodies produced by B lymphocytes could form immune complexes with antigens and interact with complement components to initiate inflammation in the joint.43 Previous studies had indicated a possible link between autoantibody responses and curative effects, in which alleviation of clinical symptoms was associated with the suppression of autoantibody levels.44,45 We showed herein that koumine inhibited the elevation of serum anti-CII antibody level induced by immunization. Taken together, these observations suggest the potential immunoregulatory property of koumine, which may be the primary mechanism underlying its therapeutic effect against CIA in rats. In conclusion, the current study demonstrated that koumine, the most abundant alkaloid of G. elegans, significantly inhibited the progression of arthritis in two different rat models of RA by modulation of various parameters. Furthermore, koumine also exhibited lower toxicity than the crude extract, indicating that it could serve as a suitable template in future research endeavors to develop novel drugs against RA. Further investigations into the underlying mechanisms of its activity are needed to establish a more convincing rationale for clinical trials.
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EXPERIMENTAL SECTION
General Experimental Procedures. Koumine was isolated from G. elegans by one of the authors (Y.-P.S.) using our previously established method of pH-zone-refining counter-current chromatography25 and provided with a purity of 99.0% by high-pressure liquid chromatography. For the current study, it was dissolved in sterile saline before use. Freund’s complete adjuvant (CFA), Freund’s incomplete adjuvant (IFA), and Chicken CII were purchased from Sigma-Aldrich (St. Louis, MO, USA). Heat-killed Mycobacterium tuberculosis was acquired from Shanghai Biochemical Factory (Shanghai, China). Rat serum IL-1β ELISA kit and rat serum TNF-α ELISA kit were purchased from R&D Systems China Co. Ltd. (Shanghai, China). Rat serum anti-CII antibody ELISA kit was purchased from Chondrex (Redmond, WA, USA). RNeasy micro spin columns and QuantiTect reverse transcription kit were purchased from Qiagen GmbH (Hilden, Germany). All-in-One qPCR Mix was purchased from GeneCopoeia (Rockville, MD, USA). Animals. Female Lewis rats (8 weeks old, 120−140 g) and male Wistar rats (7 weeks old, 130−150 g) were obtained from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and housed in a clean small animal facility at 25 ± 2 °C, with a 12 h light/12 h dark cycle (lights on from 08:00 to 20:00 h). Rats had unlimited access to 2641
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Statistical Analysis. Data are presented as mean ± standard deviation (SD). For hind paw volume and MWT, data were analyzed using two-way analysis of variance (ANOVA) (treatment × time), followed by Dunnett’s T3 test for post hoc comparisons. For analysis of organ weights, serum anti-CII antibody levels, serum or joint tissue IL1β and TNF-α levels, and IL-1β and TNF-α mRNA expression, oneway ANOVA and Student−Newman−Keuls test for post hoc comparisons were used. For comparison of AI score, the Kruskal− Wallis with Dunn’s multiple comparisons test was performed. Statistical significance was set at P < 0.05. All analyses were performed with Statistic Package for Social Science version 19.0 software (SPSS Inc., Chicago, IL, USA).
methotrexate treatment (postdosing) through standardized scoring of arthritis, measurement of edema, and evaluation of articular nociception. Arthritis Scoring. A standardized method of arthritis scoring defined as AI score was used to evaluate the degree of swelling and erythema of all four paws,48 in which 0 = normal, 1 = erythema and mild swelling confined to the ankle joint and toes, 2 = erythema and mild swelling extending from the ankle to the midfoot, 3 = erythema and severe swelling extending from the ankle to the metatarsal joints, and 4 = ankylosing deformity with joint swelling, totaling a maximum score of 16. Edema Measurement. Arthritic edema was assessed by measuring the hind paw volume with a plethysmometer (Yi Yan Technology Development Co., Ltd., Jinan, China). For rats with AIA, the volumes of the injected (ipsilateral) primary-response and uninjected (contralateral) secondary-response hind paws were measured and recorded separately; for rats with CIA, the measured volume was the average of both hind paws. Antinociceptive Activity. Pain was assessed through an MWT test 1 h after administration of drug, as described previously.21 Rats were habituated to the test apparatus for 30 min immediately prior to the session and were placed in individual plastic cubicles. Mechanical stimulation was applied to the center of the hind paw in an upward motion using a von Frey filament until foot withdrawal. The maximum strength of the stimulus was 55 g. The procedure was repeated twice at 10 min intervals for each hind paw. Measurements of MWT of the hind paws for rats with either AIA or CIA were performed as described in Edema Measurement. Measurement of Organ Weight of Rats with AIA. Rats with AIA were sacrificed by exsanguination under deep anesthesia 24 d after the adjuvant injection. The spleen, liver, and thymus were removed, and the weight of the organs was recorded and corrected for body weight. Radiographic and Histological Examination of Rats with CIA. At the end of the treatment period and after the completion of all behavioral tests on rats with CIA, two or three rats were selected randomly from each group and anesthetized with sodium pentobarbital (50 mg·kg−1, i.p.). X-ray examination of the left hind paw was carried out with a Multi Soft X-ray film shooting apparatus (50 kV and 0.08 s exposure; Philips Eleva, DA Best, The Netherlands). After radiographic examination, all rats were sacrificed by exsanguination under deep anesthesia. The left hind paw joints were fixed in 10% formalin solution and decalcified in 10% ethylenediaminetetraacetic acid for 28 d at 4 °C. Specimens were then paraffin embedded, sectioned (5 μm) for hematoxylin and eosin (H&E) staining, and observed under a light microscope. Cytokine Measurement in Rats with CIA by ELISA. Trunk blood was collected after animal sacrifice. Serum samples were stored at −70 °C. The right hind paw joint tissues were pooled, cut into small pieces, suspended (200 mg of tissue per mL of phosphate-buffered saline), and homogenized in 50 U·mL−1 aprotinin. The homogenate was centrifuged at 3000 rpm for 20 min at 4 °C. IL-1β and TNF-α levels in serum sample and joint tissue supernatant were determined using specific commercially available ELISA kits according to the manufacturer’s instructions and analyzed on a ELISA-plate reader (RT-6000; BioTek, Winooski, VT, USA). Serum anti-CII IgG antibody was also measured by ELISA using a rat serum anti-CII antibody ELISA kit. Measurement of IL-1β and TNF-α mRNA Expression in Rats with CIA. Total RNA was extracted using RNeasy micro spin columns from ankle synovial tissue homogenate and reverse transcribed using a QuantiTect reverse transcription kit. Equivalent volumes of complementary DNA were analyzed by qPCR using All-in-One qPCR mix. The primers (Sangon Biological Co., Shanghai, China) used were 5′TCGTGCTGTCTGACCCATGT-3′ (sense) and 5′-CAGGGATTTTGTCGTTGCTTGT-3′ (antisense) for IL-1β and 5′-GGGTGATCGGTCCCAACA-3′ (sense) and 5′-TGGGCTACGGGCTTGTCA-3′ (antisense) for TNF-α. The assay was conducted in triplicate; the results are shown as fold difference relative to the blank control using the 2−ΔΔCt method.49
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the First Affiliated Hospital of Fujian Medical University for supporting radiographic examination and our colleagues from the Department of Pharmacology and Fujian Key Laboratory of Natural Medicine Pharmacology for koumine preparations and in vivo studies. This work was supported by the National Natural Science Foundation of China [No. 81273493], the Natural Science Foundation of Fujian Province of China [No. 2016J01367], and the Key Program of Scientific Research of Fujian Medical University [No. 09ZD009].
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