Key words Sphaeralcea angustifolia - Malvaceae - anti-inflammatory - arthritis - coumarins -
scopoletin.
Abbreviations
BW: body weight
CFE: λ-carrageenan footpad edema
CFA: complete Freud’s adjuvant
ED50
: median effective dose
IL: interleukin
K/C: kaolin/carrageenan
MS: Murashige and Skoog
PSS: physiological saline solution
SaDM:
Sphaeralcea angustifolia dichloromethane-methanol
TPA: 12-O -tetradecanoyl phorbol-13-acetate
Introduction
Rheumatoid arthritis is a progressive and chronic autoimmune disease that causes systemic
damage and that may occur in all ages, although it entertains a high prevalence in
middle age. The joints (synovial) are the most affected system, but the joints are
also capable of disturbing other organs, such as the kidneys and lungs, among others.
The disease develops as a painful condition, with loss of function and mobility. Later,
the damage dimension is considerable since the quality of life of patients is diminished
and enormous disability is caused [1 ].
Treatment of patients with this illness is based on the use of disease-modifying antirheumatic
drugs and entertains the goal of diminishing the symptoms and preventing structural
damage and disability; examples of these groups include etanercept, adalimumad, and
methotrexate. Also, nonsteroidal anti-inflammatory drugs and glucocorticoids are employed.
However, all of these drugs present several toxicity effects such as hepatotoxicity,
myelosuppression, and an elevated risk of infections (bacterial and viral) [2 ]. There is a need for new treatments with the possibility of decreasing the collateral
toxic effects of chronic therapy for rheumatoid arthritis, thereby improving the life
quality of patients.
Fresh aerial tissues of Sphaeralcea angustifolia (Cav.) G. Don (Malvaceae) are employed to treat inflammatory processes [3 ]
[4 ]
[5 ]. The dichloromethane extract from the aerial tissues of this species was active
in TPA-induced mouse ear edema, CFE in mice, and CFA-induced arthritis in rats. Scopoletin
([Fig. 1 ]) was the active compound detected in the extract [6 ]
[7 ]
[8 ]. Since the collection of S. angustifolia plants from their natural habitat is restricted by the Mexican ministry of the environment
and natural resources (SEMARNAT, NOM-059-ECOL-2001), the cell suspension culture of
this species was established to produce the bioactive compound. Cell suspension developed
in MS medium, with the total nitrate concentration reduced to 2.74 mM, produces the
anti-inflammatory compounds ([Fig. 1 ]) scopoletin, tomentin, and sphaeralcic acid [9 ]. Intraperitoneal administration (100 mg/kg) of the dichloromethane extract from
the medium (42±3%) and the dichloromethane-methanol extract from the biomass (39±9%)
of the S. angustifolia cell suspension showed a similar anti-inflammatory effect on CFE. The dichloromethane-methanol
(9:1, v/v) extract (SaDM) from the biomass exerted a dose-dependent inhibitory effect
with an ED50 of 137.63 mg per kg on CFE [10 ]. The SaDM extract from the biomass (2.0 mg per ear) was also inhibited by TPA-induced
mouse ear edema by 78% [9 ]. Tomentin and sphaeralcic acid were active in both acute inflammatory models: the
anti-inflammatory effects on CFE at a dose of 45.0 mg/kg via the intraperitoneal route
was 67% for sphaeralcic acid and 62% for tomentin. TPA-induced mouse ear edema was
inhibited by sphaeralcic acid (87±3%) and tomentin (48±7%) to 1.0 mg per ear. The
effect of sphaeralcic acid was dose-dependent with an ED50 of 93 mM [9 ]. The anti-inflammatory (52%, 0.50 mg/ear) and antiarthritic effects of scopoletin
isolated from S. angustifolia and Erycibe obtusifolia Benth. (Convolvulaceae) wild
plants have already been reported [8 ]
[11 ]. The cell suspension comprises a controlled culture with a high-quality production
of scopoletin, tomentin, and sphaeralcic acid and is useful as an alternative for
obtaining standardized extracts and for isolating compounds, with the purpose to carry
out their evaluation in a K/C-induced arthritis animal model and to advance in the
knowledge of its beneficial effects.
Fig. 1 Chemical structures scopoletin, 7-hydroxy, 6-methoxycoumarin; tomentin, 5-hydroxy-6,7-dimethoxycoumarin;
and sphaeralcic acid, 2-(1,8-dyhidroxy-4-isopropyl-6-methyl-7-methoxy) naphthoic acid.
Results
The antiarthritic activity of SaDM extract and tomentin were evaluated in an arthritis
model in mice induced with K/C for a period of 10 days. In this arthritis model, oral
administration of vehicle 1% Tween 20 (negative control) presented an increasing development
of joint edema from the application of immunogens. Maximal inflammation was obtained
on day 3 and this edema remained until day 9 ([Fig. 2 ]). The animals were lethargic and had difficulty walking; the legs of the majority
of these were fissured and secreted a thick yellowish liquid. Tissue inflammation
was evaluated as a percentage with respect to baseline size. On day 9 after the induced
damage, the joint increased its size by 2.25 mm; this parameter was considered 100%
of inflammation ([Table 1 ]). In the PSS group, mice not receiving K/C, develop edema until day 1, which was
lower (0.55±0.08) than that developed by the negative control. Additionally, inflammation
decreased with time and the animals recovered the initial size of their joints ([Fig. 2 ]).
Fig. 2 Effect of oral administration of methotrexate (5.0 mg/kg), SaDM extract, and tomentin
(5.0, 10.0, 15.0, and 20.0 mg/kg) on left joint size in mouse arthritis model induced
with K/C.
Table 1 Effect of SaDM extract from the biomass of S. angustifolia cell suspension and tomentin administered via the oral route on the left joint after
10 days of K/C-induced edema.
Treatment
Dose (mg/kg)
Edema (mm)
Edema inhibition (%)
Negative control (vehicle)
–
2.25±0.13
–
Physiological saline solution (PSS)
–
0
–
Methotrexate
5.0
1.01±0.08**
55
SaDM extract
100.0
0.63±0.10**
72
Tomentin
5.0
1.55±0.03**
31
10.0
1.36±0.06**
40
15.0
0.81±0.09**
64
20.0
0.61±0.08**
73
Mean±SEM (n=12). ** Edema volumes significantly different (p <0.0001) according to the Dunnett test
In the mouse group treated with 5.0 mg/kg per day of methotrexate, maximal inflammation
developed at day 2 and continued to be evident at day 4. After this time, edema was
continuously reduced until day 9 of treatment ([Fig. 2 ] and [Table 1 ]). The mice demonstrated no difficulty in supporting their legs and walking. In the
group treated with 100.0 mg/kg of SaDM extract daily, inflammation remained constant
for 3 days, and from day 4, the inflammation continuously decreased until day 9 ([Table 1 ]). Redness of the leg was minimal, and its appearance was similar to that prior to
the damage being caused.
In mice treated with pure tomentin compound, at 5.0 mg/kg, joint edema increased during
the first 6 days (1.79±0.03 mm), without reaching the edema volume of the negative
control group. In mice treated with 10.0 and 15.0 mg/kg, the initial edema that formed
remained until day 5 (1.68±0.06 and 1.47±0.10 mm, respectively), after which it declined
([Table 1 ]). Only at the 20.0 mg/kg dose did the joint edema in the mice decrease from day
1 of treatment until day 9 ([Fig. 2 ] and [Table 1 ]). Administration of methotrexate, SaDM extract, and tomentin caused a significant
decrease of articular edema when treatments were compared with the negative control
group (p <0.0001). Tomentin exhibited dose-dependent inhibition of joint edema induced with
K/C ([Table 1 ]), with a median ED50 of 10.32 mg/kg (Fig. 1S , Suppporting Information). All groups of mice treated with tomentin showed no cracks
in the legs, and no yellowish fluid secretion was observed.
The mean BW of mice in the PSS group increased during the study period, with a weight
gain at day 9 ([Table 2 ]). Articular administration of K/C caused a decrease in the BW of mice gradually,
until day 9. Methotrexate-administered animals also tended to lose weight in a similar
manner to that of the negative control group. According to the Student’s t-test, the
average weight loss at day 9 was superior (p <0.0001) to that of the negative control. In contrast, in mice treated with the SaDM
extract at 100.0 mg/kg, a lost weight was observed during the first 2 days (−1.92±0.17 g),
with a tendency to recover this from day 3. After 9 days, the weight loss was not
statistically different from the negative control group. Mice treated with 5.0, 10.0,
15.0, and 20.0 mg/kg of tomentin showed a weight reduction only on day 1 of the induced
damage, with a tendency to recover it. Weight loss in the group treated with 5.0 mg/kg
was statistically lower than that registered in the negative control group (Student’s
t-test, p <0.0001). In the groups treated with 10.0, 15.0 and 20.0 mg/kg, there was a slight
increase in the BW. Nevertheless, the weight gain was lower than that of the PSS group
([Table 2 ]).
Table 2 Effect of SaDM extract from the biomass of S. angustifolia cell suspension and tomentin administered via the oral route on BW after 10 days
of K/C-induced edema.
Treatment
Dose (mg/kg)
Body weight (day 0)
Body weight (day 9)
Weight differences (g)
Negative control (vehicle)
–
30.52±2.58
29.02±1.78
−2.40±0.29
Physiological saline solution (PSS)
–
28.06±2.87
30.34±2.50
2.28±0.16
Methotrexate
5.0
30.18±2.85
25.83±2.81
−4.35±0.47**
SaDM extract
100.0
31.29±1.39
30.10±1.24
−1.19±0.17
Tomentin
5.0
28.90±1.57
27.59±1.83
−1.31±0.19**
10.0
27.60±2.16
27.78±2.2.23
0.18±0.03**
15.0
27.28±1.74
27.70±1.22
0.42±0.08**
20.0
29.74±2.26
30.65±1.88
0.91±0.06**
Mean±SE (n=12). ** BW values significantly different (p<0.0001) according to Student’s
t-test
To understand how the SaDM extract and tomentin exert their effects on arthritis,
immunoanalyses were performed to examine the participation of inflammatory (TNF-α
and IL-1β) and anti-inflammatory (IL-4 and IL-10) cytokines in joint tissue derived
from K/C-induced arthritic mice with or without treatment. Carrageenan promoted a
significant increase of TNF-α and IL-1β concentration in comparison to the healthy
(SSE) mice. On the other hand, the content of anti-inflammatory cytokines IL-4 and
IL-10 ([Fig. 3 ]) decreased significantly. In animals treated with 5 mg/kg methotrexate daily, levels
of pro-inflammatory cytokines TNF-α and IL-1β decreased in joints when compared with
the negative control group. The content of anti-inflammatory cytokines IL-4 and IL-10
([Fig. 3 ]) did not improve.
Fig. 3 Effect of SaDM and tomentin from S. angustifolia cell suspensions on tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β),
IL-4, and IL-10 in joint edema in mice induced with K/C. Each bar represents the mean±SEM,
n=4. ** Means represent differences of the treatment groups compared with the negative
control group according to Student’s t-test (p <0.0001) and differences of the negative control group compared with the PSS group
according to the Student’s t-test (p <0.0001).
In the group treated with the SaDM extract at a dose of 100 mg/kg, TNF-α and IL-1β
levels were statistically lower than that presented in the negative control. Furthermore,
TNF-α levels were similar to those detected in the healthy mice. The SaDM extract
did not give rise to significant changes to the K/C group in IL-4 joint levels. However,
the SaDM extract was able to increase IL-10 levels relative to the negative control
([Fig. 3 ]).
In mice treated with tomentin, pro-inflammatory TNF-α levels were lower than those
of the negative control group. This effect depended on the dose administered. At the
20-mg/kg dose, the content of this cytokine was statistically similar than that of
mice treated with methotrexate ([Fig. 3 ]). In all groups receiving tomentin, the IL-1β levels were statistically lower than
that of the negative control, and no dose-dependent effect was observed. In addition,
the cytokine levels were similar to those of mice treated with methotrexate. The results
showed that cytokine IL-4 and IL-10 levels increase with increasing doses of tomentin
([Fig. 3 ]).
Discussion
Cell suspension culture is an excellent technique to produce scopoletin, the active
compound identified in the wild plant of S. angustifolia as well as tomentin and sphaeralcic acid anti-inflammatory compounds. The SaDM extract
from S. angustifolia cell suspension is capable of diminishing the edema development induced by diverse
irritating agents, such as TPA or λ-carrageenan [9 ]
[10 ]. However, the SaDM extract and tomentin in the rheumatoid-related diseases treatment
remains unknown. With this purpose in mind, the present study was designed based on
the disease course of K/C-induced arthritis.
Rheumatoid arthritis is a symmetric polyarticular arthritis that primarily affects
small diarthrodial joints of the hands and feet [11 ]. K/C-induced arthritis is used as an animal model of human rheumatoid arthritis,
which is difficult to assess in human systems [12 ]. The arthritis-like signs of K/C-induced arthritis in mice resemble several histopathological
features of human rheumatoid arthritis, including mononuclear cell infiltration and
synoviocyte hyperplasia, resulting in pannus formation followed by bone and cartilage
destruction [13 ]
[14 ]. Joint inflammation in experimental arthritis induced with K/C in mice exhibited
maximal joint edema in the first 24 h accompanied by an exudate [15 ]. Methotrexate has long been used in the treatment of rheumatoid arthritis and it
has significantly inhibited joint edema, but the mean BW of these mice dropped, which
may be one of the side effects of methotrexate. The SaDM extract also inhibited joint
edema in K/C-induced mouse arthritis, demonstrating only redness in the left joint.
This effect was higher than that reported for the dichloromethane extract of the wild
plant administered via the intraperitoneal route at the same dose in the model of
chronic inflammation with CFA [8 ]. The BW lost in mice treated with the SaDM extract was similar than that of the
K/C group.
The SaDM extract to 100 mg/kg and tomentin to 20 mg/kg had the highest anti-inflammatory
activity. During the whole period of tomentin administration, the joint edema size
decreased progressively. This phenomenon was only observed during the first 5 days
of SaDM extract administration, while at this time, the extract effect was lower than
that observed by tomentin. Subsequently, there was a rapid decrease in the joint edema
(biphasic effect) and at the last day of treatment with the SaDM extract had a similar
effect to that of tomentin. Each 100 mg of SaDM extract contains 0.10 mg of scopoletin,
0.10 mg of tomentin, and 0.19 mg sphaeralcic acid (Fig. 2S , Supporting Information). The amount of tomentin administered in the SaDM extract
(100 mg/kg) was much lower than when given only tomentin (20 mg/kg). This phenomenon
can be seen as a dose-dependent effect. In addition, the chemical content of the SaDM
extract is complex and it could exert anti-inflammatory activity by different mechanisms
of action associated with the compounds already identified and the presence of others.
Scopoletin, for example, possesses a wide range of pharmacological activities [16 ]
[17 ]
[18 ]
[19 ]. Scopoletin was isolated from S. angustifolia and E. obtusifolia , and the anti-inflammatory effects have already been evaluated in TPA-induced mouse
ear edema [8 ]
[19 ]. In rat CFA-induced arthritis, intraperitoneal administration of scopoletin dose-dependently
reduced both inoculated and non-inoculated joint edema, and elevated the mean BW [20 ]. Scopoletin was capable of ameliorating synovial hyperplasia, reduced the presence
of inflammatory cells in the synovium, and diminished erosive changes in cartilage
and bone in addition to exhibiting angiogenesis inhibition. Also, scopoletin selectively
inhibited the overexpression of IL-6 rather than TNF-α or IL-β in synovial tissues
of rats with CFA-induced arthritis [20 ].
Tomentin also inhibited both inoculated and non-inoculated joint edema in K/C-induced
mouse arthritis and the BW loss was similar to that of the K/C-induced arthritis group.
The anti-inflammatory effect of tomentin is dependent on its coumaric origin. It has
been reported that various compounds of this group, natural or synthetic, act as inhibitors
of the cyclooxygenase and lipoxygenase enzymes, which modulate the biosynthesis of
prostaglandins and leukotrienes, respectively, with the latter participating in the
process of chronic inflammation [21 ].
Injection of 2% carrageenan into the knee joint in rats promotes the activation of
macrophages, dendritic cells, and lymphocytes. These cells in turn produce TNF-α,
IL-1β and IL-6, and other cytokines that are responsible for the inflammatory response.
Once the focus of damage is localized, the production of pro-inflammatory cytokines
ceases and the production and release of anti-inflammatory cytokines such as IL-4
and IL-10 begins, with the latter in turn disabling the effector cells [14 ]
[15 ]. In the results presented here, it was observed that intra-articular administration
of K/C caused widespread damage in mice due to the rise in the pro-inflammatory cytokine
(TNF-α and IL-1β) levels and a decrease in the levels of anti-inflammatory cytokines
(IL-4 and IL-10).
It has been mentioned that carrageenan-induced arthritis models possess many features
of human rheumatoid arthritis. The administration of this substance resulted in localized
inflammation associated with the production of a number of mediators such as TNF-α
and IL-1β [22 ]. In a study in our laboratory, it has been shown that the intra-articular administration
of carrageenan, as an experimental procedure, induced an important local increment
of TNF-α and IL-1β [23 ]. It has been demonstrated that TNF-α induces cartilage and bone degradation, and
the penetration of lymphocytes, macrophages, and neutrophils to the joint cavity.
IL-1β in vitro induces cytokine production by synovial mononuclear cells, and when
it is blocking IL-1β and components of the receptor for IL-1β, it has been shown to
be effective in reducing inflammation and, particularly, articular damage in several
rodent models of rheumatoid arthritis. These pro-inflammatory cytokines and interleukins
are involved in osteoclast differentiation, inflammation, and bone erosion [24 ], enhance the synthesis of metalloproteinases and proliferation of synovial cells
resulting in cartilage degradation [25 ], and are capable of stimulating the production and secretion of other cytokines
such as IL-6, which also promotes cartilage degradation. In the K/C-induced arthritis
animal model, this cytokine was also elevated in the articulation [22 ]. While IL-4 and IL-10 are regulators that control the progression of rheumatoid
arthritis, the level of these interleukins may lead to protection in order to prevent
cartilage destruction and bone erosion [26 ].
The SaDM extract and tomentin were also capable of modulating cytokine concentrations.
IL-1β and TNF-α levels decrease, accompanied by an increase of interleukins IL-4 and
IL-10, activity that can prevent the damage induced by K/C controlling the inflammation
and progression of rheumatoid arthritis. The reduction of TNF-α and increase of IL-10
was tomentin doses-dependent. In rats with CFA-induced arthritis and treated with
the dichloromethane extract of the aerial parts from the S. angustifolia wild plant, the levels of pro-inflammatory interleukins IL-1β and IL-6 were also
reduced. Furthermore, the anti-inflammatory cytokine IL-10 level was increased [7 ]. It is proposed that IL-10 has a downregulation activity on the levels of the pro-inflammatory
cytokines like TNF-α.
The histopathology analyses for the dichloromethane:methanol extract and tomentin
are a perspective of this experimental project. It will also be important to isolate
and identify other components with anti-inflammatory activity in the SaDM extract,
as well as to evaluate the effect of sphaeralcic acid and the interaction of the active
compounds identified in the extract to determine if there is synergy in their effects
in the K/C-induced arthritis animal model. These advances are important for the development
of a phytomedication with the standardized SaDM extract in the active components for
a clinical trial.
Materials and Methods
Plant material
Plants and fruits of Vara de San Jose plants were collected in Hidalgo State, Mexico.
Plant samples were authenticated by Abigail Aguilar, M.Sc., Head of the Herbarium
at the Instituto Mexicano del Seguro Social in Mexico City [IMSSM], as S. angustifolia , and vouchers were stored for reference under #14294.
Plant cell culture
Suspension-cultured cells in batches of S. angustifolia were grown in 250 mL flasks with 80 mL of whole MS liquid medium [27 ], with 27.4 mmol of total nitrates (NH4 NO3 15.9 mmol, and KNO3 11.5 mmol) supplied with 1 mg/L of α-naphthalene acetic acid in combination with
kinetin (0.1 mg/L) and supplemented with 30 g/L of sucrose. The flasks were placed
in an orbital shaker at 110 rpm (New Brunswick Scientific Co., Inc.) and incubated
at 26±2°C during a light:dark (16 h:8 h) photoperiod under 50 μM/m2 /sec warm white fluorescent light intensity (9–10). The biomasses were changed to
fresh medium under sterile conditions every 16 days, utilizing an inoculum of 4% (w/v)
of fresh biomass.
Stimulation of scopoletin, tomentin, and sphaeralcic acid production
Utilizing the same inoculum, cell suspensions cultivated in whole MS medium were transferred
into 1 L flasks with 400 mL of MS medium, with the total nitrate concentration reduced
to 2.74 mmol (NH4 NO3 1.59 mmol, and KNO3 1.15 mmol) and were incubated under previously described conditions. Cultures were
arrested on day 16 of the culture to obtain the biomass [9 ]
[10 ].
Extraction and tomentin isolation
Suspension-cultured cells from flasks were filtered and the biomasses were pooled
and dried at room temperature. Then, the dry biomass was extracted 3 times by maceration
at room temperature with a mixture of grade-reactive solvent (CH2 Cl2 :CH3 OH 9: 1; Merck) at a ratio of 1:20 (w/v) at 24 h for each. The dichloromethane-methanol
extracts were filtered, pooled, and concentrated to dryness under reduced pressure.
The scopoletin, tomentin, and sphaeralcic acid content in the SaDM extract was determined
by HPLC [9 ]
[10 ].
To purify the tomentin compound, the SaDM extract was fractionated by silica gel column
chromatography (9×28 cm, 70–230 mesh; Merck) using an n-hexane-ethyl acetate-methanol
gradient system (grade-reactive solvents; Merck) with 5% polarity increments. Aliquots
of 500 mL were collected, and samples with a similar TLC profile were integrated into
10 fractions (1–10). The fraction with tomentin was through an open silica gel RP-18
column (1.5×28 cm, 40–63 mesh; Merck) with an H2 O:CH3 CN elution system (grade-reactive solvents; Merck) with an increasing polarity of
10%, and the compound was isolated from pooled subfractions (70:30, H2 O:CH3 CN) [9 ]. The structure of tomentin was confirmed by comparison of spectroscopic data (Table 1S , Supporting Information) 1 H NMR [9 ].
HPLC conditions
HPLC analyses were carried out in a Waters system (2 695 Separation Module) coupled
to a diode array detector (2996) with a 190–600 nm detection range and operated by
the Manager Millennium software system (Empower 1; Waters Corp.). Separations were
performed in a Spherisorb® RP-18 column (250×4.6 mm, 5 µm; Waters) employing a constant temperature of 25°C
during analyses. Samples (20 μL) were eluted at a 1.2 mL/min flow rate with (A) high-purity
H2 O (TFA-1.0%) and (B) high-purity CH3 CN gradient mobile phases (Merck), and were detected by monitoring absorbance at 340 nm.
The mobile phase was started with water (100%) and was maintained for 1 min. Then,
the concentration of solvent B was gradually increased to 15% (at 1 min), 37% (at
10 min), and 85% (at 2 min). During the next 2 min, solvent B was increased to 100%
and this proportion was maintained for 3 min. Finally, the next 3 min were utilized
to return the mobile phase to the initial condition. The chromatographic method had
a 22-min run time. Calibration curves were constructed with a standard solution of
2, 4, 8, 12 and 16 µg/mL. Scopoletin presented a regression equation of Y=21 985(X)–117 889
and R2 =0.9997, while for tomentin theses were Y=46 018(X)–394 683 and R2 =0.9992 and for sphaeralcic acid, these were Y=2 576(X)-23 696 and R2 =0.9996. The retention times (rt) of compounds tomentin at λ=343 nm (≥93%), scopoletin
at λ=343 nm (99%; Sigma-Aldrich Química México), and sphaeralcic acid (≥98%) at λ=357 nm
were 13.37 min, 13.65, min and 18.00 min, respectively. Sphaeralcic acid was isolated
and purified at our Laboratory from S. angustifolia cell suspensions according to the procedure already reported [9 ]. Compound identifications were performed by comparing their rt and absorbance spectra
[9 ]
[10 ].
Animals
We used ICR outbred-strain female mice weighting 25.0–30.0 g each. The experiments
were performed according to Mexican Official Norm 062-ZOO-1999 (Technical Specifications
for the Production, Care and Use of Laboratory Animals) and international ethical
guidelines for the care and use of laboratory animals. The animals were maintained
at a temperature of 22±3°C, 70±5% humidity, and 12-h/12-h light/dark cycles with water
and food ad libitum.
The experimental protocol was evaluated by the committee CLIEIS-1701 (Comité Local
de Investigación y Ética en Investigación y Salud) from the Instituto Mexicano del
Seguro Social (IMSS, Mexico), and approved January 1, 2011, with the register number
2011-1701-3.
Arthritis model induced by kaolin/carrageenan
This experimental design lasted 10 days. There were 8 groups of 12 mice each as follows:
initially, measurements were taken from the baseline size of the left joint with a
Mitutoyo-brand digital micrometer (Micrometric Digimatic Calibration MDC-1”SB; Mitutoyo
Products). Then, the animals were anesthetized with pentobarbital sodium administered
i.p. at a dose of 55 mg/kg. Later, these mice were slowly injected with a kaolin (4%,
40 μL) solution in the joint cavity of the left knee. Consecutively, flexions and
extensions were performed during 15 min in the joint administered with the drug. Immediately,
the mice were injected in the same left knee joint cavity with 40 μL of carrageenan
at 2%. Similarly, flexions were carried out for an additional 5 min [28 ].
Administration of the treatments was conducted each day by oral route, and was initiated
1 day after damage induction until day 10 (9 days of treatment). The negative control
group (group 1) received oral administration of the vehicle without any drugs (negative
control); the positive control group (group 2) was administered with 5.0 mg/kg of
methotrexate (≥98% purity; Sigma-Aldrich Química México). In group 3, mice were treated
with 100 mg/kg of SaDM extract standardized in its content of scopoletin, tomentin,
and sphaeralcic acid, according to previous reports of dichloromethane-methanol extract
from cell suspension biomasses evaluated in acute inflammation models and DE50 determined in a subplantar edema model in mice. In groups 4, 5, 6, and 7, mice were
treated with 5.0, 10.0, 15.0, and 20.0 mg/kg, respectively, of tomentin (93% purity).
A baseline group of healthy animals was also employed. These animals were injected
with PSS into the left joint (group 8) as a substitute of K/C.
Each day, BW and left joint size (mm) in the controls and treated groups were determined
with respect to the baseline size of the left knees, and the percentage of inhibition
of edema development was calculated utilizing the following formula:
Homogenization of joint tissue
The left joint was liquefied to a temperature of 4°C and, under this condition, the
samples were manipulated for analysis. The bone tissue was placed into a mortar and
covered with dry ice. Then, the tissue was pulverized and completely disintegrated
until the dry ice was sublimated. The disintegrated tissue was placed in a vial with
2 mL of PBS (pH 7.4) with phenylmethylsulfonyl fluoride at 0.01% dissolved in isopropyl
alcohol (Merck, México). The joint was completely homogenized with a T-10 Ultra Turrax
Homogenizer during 15 s for its disintegration. It was allowed to rest for 30 s, and
the procedure was repeated 5 additional times [29 ]. Afterward, the samples were placed into a centrifuge at 14 000 rpm for 5 min. We
obtained 300 μL aliquots in centrifuged microtubes, which were immediately stored
at −70°C for cytokine analyses.
Cytokine analyses
Quantification of cytokines IL-1β, TNF-α, IL-10, and IL-4 was carried out by the ELISA
method employing a kit (OptEIA™ ELISA sets; BD Biosciences) and following the manufacturer’s
instructions. On 96-well plates, 100 μL/well of the antibody were added and incubated
for 12 h at 4°C. Once this time had elapsed, the plate was washed 3 times with 300 μL/well
of PBS buffer. Subsequently, 100 μL of PBS with FBS at 10% and pH 7.0 were added and
preserved during 1 h at room temperature. The contents were discarded and the plate
was washed 3 times with 300 μL/well of PBS buffer. Finally, 100 μL of the standard,
target (PBS with FBS), and test samples were added to the corresponding wells. The
plate was incubated for 2 h at room temperature. The contents were discarded and the
plate was washed 3 times with 300 μL/well of PBS buffer. For TNF-α, IL-4, and IL-10,
100 μL/well of antibody detection were added plus a streptavidin-horseradish peroxidase
enzyme, which was incubated for 1 h. After that, the plate was washed 7 times with
300 μL/well of PBS buffer.
For IL-1β, 100 µL/well of antibody detection were added, which was incubated for 1 h
and washed with PBS 0.05% of Tween 20, 300 µL/well 5 times, followed by the streptavidin-horseradish
peroxidase enzyme (100 µL/well), which was incubated at room temperature and washed
with PBS 0.05% of Tween 20, 300 µL/well 7 times.
For all cytokines, 100 µL with O -phenylendiamine substrate (1 OPD tablet with 1 of urea dissolved in 20 mL of distilled
water) were added to the wells. These ELISA plaques were incubated for 30 min at room
temperature, under total darkness, and then a stop solution (2 N H2 SO4 ) was added. Each plaque was read at a 450 nm wavelength in a Stat Fact Awareness
Technologies 2100 spectrophotometer.
Statistical analysis
Data obtained from joint edema (inhibition %) in the left joints and BW were analyzed
by one-way analysis of variance (ANOVA), and p values of ≤0.05 were considered significant. For joint edema and BW, significant
differences among treatment means were calculated by the Dunnett0.05 test. For cytokines (pro- and anti-inflammatory) levels, Student’s t-test was applied
to compare each treatment vs. the negative control.
