EXPERIMENTAL AND THERAPEUTIC MEDICINE 7: 1408-1414, 2014
1408
Effect of γ‑tocotrienol in counteracting oxidative stress
and joint damage in collagen‑induced arthritis in rats
AMMU RADHAKRISHNAN1, DULANTHI TUDAWE1, SRIKUMAR CHAKRAVARTHI1,
GAN SENG CHIEW2 and NAGARAJA HALEAGRAHARA3
1
Faculty of Medicine and Health Sciences, International Medical University; 2Faculty of Medicine and Health Sciences,
Universiti Tunku Abdul Rahman, Kuala Lumpur 57000, Malaysia; 3Discipline of Physiology and Pharmacology,
School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811, Australia
Received October 4, 2013; Accepted February 13, 2014
DOI: 10.3892/etm.2014.1592
Abstract. Tocotrienols exhibit a significant anti‑inflammatory
and antioxidant effect in numerous human diseases. However,
the anti‑inflammatory and antioxidant effects of tocotrienols
in arthritic conditions are not well documented. Therefore, the
effect of γ‑tocotrienol supplementation against oxidative stress
and joint pathology in collagen‑induced arthritis in rats was
investigated in the present study. Adult female Dark Agouti
rats were randomly divided into groups: Control, γ‑tocotrienol
alone, arthritis alone and arthritis with γ‑tocotrienol. Arthritis
was induced using 4 mg/kg body weight collagen in complete
Freund's adjuvant. The rats were treated orally with 5 mg/kg
body weight of γ‑tocotrienol between day 21 and day 45. After
45 days, serum C‑reactive protein (CRP), tumor necrosis factor
(TNF)‑α, superoxide dismutase (SOD) and total glutathione
(GSH) assays were conducted. γ‑tocotrienol significantly
reduced the arthritis‑induced changes in body weight, CRP,
TNF-α, SOD and the total GSH levels. There was a significant
reduction in the arthritis-induced histopathological changes
in the γ‑tocotrienol treatment group. The data indicated that
administration of γ‑tocotrienol resulted in a significant antioxidant and anti‑inflammatory effect on collagen‑induced
arthritis; therefore, γ‑tocotrienol may have therapeutic potential as a long‑term anti‑arthritic agent in rheumatoid arthritis
therapy.
Correspondence to: Dr Nagaraja Haleagrahara, Discipline of
Physiology and Pharmacology, School of Veterinary and Biomedical
Sciences, Faculty of Medicine, Health and Molecular Sciences,
James Cook University, Building 87, Solander Drive, Douglas,
Queensland 4811, Australia
E‑mail: haleagrahara.nagaraja@jcu.edu.au
Key words: rheumatoid arthritis, oxidative stress, tocotrienols,
tumor necrosis factor
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that is associated with progressive disability
and systemic complications (1,2). RA is able to initiate in any
joint, however, it commonly begins in the smaller joints of
the fingers, hands and wrists. Other joints that are commonly
affected include the hips, knees, ankles, feet, neck, shoulders
and elbows. In addition to joint pain and inflammation, individuals suffering from RA may experience fatigue, occasional
fevers and a general sense of ill health (2‑4). The cellular
and molecular pathology of RA involves chronic inflammation of the synovium as well as synovial proliferation and
infiltration by macrophages, memory T cells and plasma
cells. Furthermore, marked hyperplasia of the synovium and
progressive cartilage destruction occurs, which are mediated
by cytokine‑induced degradative enzymes (4,5). Regardless
of recent improvements in the treatment options for RA, the
pathology underlying inflammatory arthritis and its causative
factors have not been well described.
Selection of the appropriate medication for RA is
difficult due to the range of factors that contribute to the
development of the disease. The drugs used for RA treatment
include disease‑modifying anti‑rheumatic drugs, such as
methotrexate, sulfasalazine, hydroxychloroquine sulfate and
azathioprine (5‑7). These may also be described as slow‑acting
anti‑rheumatic drugs; they suppress inflammation and may
impede the development of joint erosion. The mechanism by
which the drugs act in patients with arthritis is not currently
well understood; the majority of these drugs do not effect
the progression of the disease; however, they may relieve
the arthritic symptoms. The administration of these drugs is
often limited due to an increased risk of cardiovascular events
and upper gastrointestinal complications, such as gastric
ulcers (4‑6). The long‑term effects of the anti‑inflammatory
therapeutic agents on the joint require further investigation;
there is significant interest in supplements, nutraceuticals and
novel therapeutic agents, which have the potential to reduce
arthritic symptoms and impede the progression of the disease.
Vitamin E includes a family of lipophilic micronutrients
consisting of four forms of tocopherols and tocotrienols (α, β,
γ and δ), which consist of a chromanol ring and a side chain.
RADHAKRISHNAN et al: ANTI‑ARTHRITIS EFFECTS OF TOCOTRIENOLS
Tocopherols and tocotrienols are found in various components
of the human diet (8‑9); tocopherols are primarily present in
nuts and vegetable oils, while tocotrienols are minor plant
constituents particularly abundant in rice bran, cereal grains
and palm oil. Tocopherols have a saturated phytyl tail, whereas
tocotrienols have an unsaturated phytyl tail. The individual
isoforms of tocopherols and tocotrienols differ in the number
and position of the methyl groups attached to the aromatic
ring (10). The specific forms of vitamin E exhibit different
biopotency; in the vitamin E group, α‑tocopherol demonstrates the highest biological activity (11‑12). Tocopherols
have previously been investigated for their antioxidative, anti‑
inflammatory, anticancer and antineurodegenerative effects;
however, investigations concerning the antioxidant and anti‑
inflammatory effects of tocotrienols are limited. γ-tocotrienols
have recently become a point of interest due to improved
therapeutic potential when compared with tocopherols. This
specific isomer has been identified as exhibiting significant
physiological activity within cell line and animal studies
and γ‑tocotrienol possesses antioxidant, anti‑inflammatory,
cardioprotective and neuroprotective properties (13,14).
Previous studies have identified that the γ‑tocotrienols and
tocotrienol-rich fractions exhibit significant anti‑inflammatory
properties (13,15,16); however, to the best of our knowledge,
no studies exist concerning the antioxidant and anti‑inflammatory effects of γ‑tocotrienols in arthritis. The present study
investigated the anti‑inflammatory and antioxidant properties
of γ‑tocotrienols against collagen‑induced arthritis in Dark
Agouti rats.
Materials and methods
Chemicals. Complete Freund's adjuvant (CFA), type II collagen
and γ‑ tocotrienol were purchased from Sigma‑Aldrich
(St. Louis, MO, USA). ELISA kits, obtained for the determination of superoxide dismutase (SOD) and total glutathione
(GSH), were purchased from the Cayman Chemical Company
(Ann Arbor, MI, USA). A C‑reactive protein (CRP) assay kit
was purchased from AssayPro (St. Charles, MI, USA) and a
tumor necrosis factor (TNF)‑α assay kit was obtained from
eBioscience (San Diego, CA, USA). The remaining chemicals
and reagents were obtained from Sigma‑Aldrich (St. Louis,
MO, USA).
Animals. Female dark Agouti rats (age,10 weeks; weight,
120‑140 g) were obtained from the Institute of Medical
Research (Kuala Lumpur, Malaysia). The rats were housed
in individual ventilation cages with food and water provided
ad libitum. The experimental procedures were conducted
according to internationally approved ethical guidelines for
the care of laboratory animals and study gained approval from
the International Medical University, Kuala Lumpur research
and ethics committee.
The animals were randomly assigned into the following
groups: i) Control; ii) arthritis; iii) γ‑tocotrienol alone; and
iv) arthritis with γ‑tocotrienol.
Collagen‑induced arthritis. Following an acclimatization
period, the rats were injected with collagen that was emulsified
in CFA, as reported in previous investigations (17,18). Briefly,
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5 mg collagen was dissolved in 2.5 ml cold, 0.1 M acetic acid.
This mixture was emulsified with 2.5 ml CFA and the solution
was mixed using a glass homogenizer (Fisher Scientific, Kuala
Lumpur, Malaysia) for ~15 min. The procedure for preparing
this solution was conducted on ice to ensure the proteins in
the emulsion were not denatured. Prior to receiving the injection of the collagen‑CFA mixture, the rats were anesthetized
with diethyl ether (Sigma‑Aldrich, St Louis, MO, USA) and
4 mg/kg body weight collagen‑CFA emulsion was injected
intradermally into the four paws of the rat as well as in to the
base of its tail.
γ‑tocotrienol treatment. The rats in the γ‑tocotrienol group
were fed orally with 5 mg/kg of γ‑tocotrienol from day 21 of
the experiment and this treatment continued, by daily gavage,
until day 45. The rats were fed normally and had access to
water ad libitum.
Evaluation of arthritis. The severity of arthritis was assessed
via measurement of paw thickness, the changes were recorded
from eight joints using a digital vernier caliper (TESA,
Ludwigsburg, Germany). The thickness of two joints was
measured in each of the four paws and the changes were
recorded on alternating days.
CRP assay. The CRP level in the plasma of the experimental animals was quantified using a rat CRP ELISA kit,
in accordance with the manufacturer's instructions. Briefly,
the lyophilised biotinylated rat CRP was dissolved in 4 ml of
enzyme immunoassay diluent solution and 25 µl of the diluted
samples were added to the respective wells in duplicates.
Subsequently, 25 µl diluted biotinylated rat CRP was added
to each of the wells. The plate was incubated for 20˚C 2 h;
50 µl diluted streptavidin peroxidase conjugate was added to
each of the wells in addition to 50 µl chromogen substrate,
which was added to each well to enable color development.
The stop solution was added to each well and the absorbance
was read at 450 nm using a microplate reader (TECAN,
Männedorf, Switzerland); the CRP concentration of each
sample was calculated based on the standard curve obtained.
TNF‑ α assay. The concentration of TNF-α in the plasma was
quantified using a rat TNF- α ELISA kit. Briefly, the plate
was coated with the appropriately diluted capture antibody
(pretitrated, purified antibody) one night prior to conducting
the assay. On the following day, 100 µl standard solution and
the sample was added to the respective wells in duplicate.
Following this, 100 µl diluted detection antibody solution was
added to the wells and the plate was incubated for 1 h. Diluted
avidin‑horseradish peroxidase (100 µl) was added to the wells
and incubated for 30 min in the dark followed by the addition
of 100 µl substrate solution. Stop solution, 2N (H2SO4; 50 µl),
was added to the wells and the absorbance was read at 450 nm
using a microplate reader. The standard curve was used to
calculate the TNF-α levels in each sample.
SOD assay. The SOD concentration was quantified using a
SOD assay kit, in accordance with the manufacturer's instructions. Briefly, the assay buffer, sample buffer, radical detector,
xanthine oxidase (XO), plasma samples and standard solu-
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EXPERIMENTAL AND THERAPEUTIC MEDICINE 7: 1408-1414, 2014
tions were prepared in accordance with the manufacturer's
instructions. Subsequently, 200 µl diluted radical detector was
added to the wells and 10 µl standard solution was added to
the relevant wells in duplicate. Diluted plasma samples (10 µl)
were added to the relevant sample wells and the reaction
was initiated by the addition of 20 µl XO into each well. The
absorbance was read at 450 nm using a microplate reader. The
standard curve was used to calculate the SOD level of each
sample.
GSH assay. The total GSH activity in the plasma of the
experimental rats was quantified using a total GSH assay kit.
Briefly, sample deproteination was conducted and 50 µl standard solution and 50 µl sample was added to the respective
wells. Following this, 150 µl of the assay cocktail (mixture
of N‑morpholino ethanesulphonic acid buffer, NADP+ and
glucose 6‑phosphate, glutathione reductase and glucose
6‑phosphate dehydrogenase mixture and 5,5'dithio‑bis‑
2‑nitrobenzoic acid) was added to each well. The absorbance
was measured at time intervals of 5 min for a total of 30 min,
using a microplate reader with a 450‑nm filter. The absorbance
value of the standard solution was subtracted from the values
obtained from the standard and the sample solution. A graph
was plotted with the corrected absorbance values of each of the
standard solutions as a function of the concentration of total
GSH, thus the total GSH value for each sample was calculated.
Histopathological analysis. Following the sacrifice of the rats
with overdose of anaesthesia (pentobarbital sodium), their
joints were harvested, the flesh was removed from the bone
and the joint samples were stored in 10% formalin solution
for three weeks in specimen bottles. Blocks were prepared
following the decalcification of the joints for 48 hours, the
blocks were sectioned at 3‑4 µm thickness and slides were
prepared and stained with hematoxylin and eosin (H&E). The
joints were evaluated and analyzed according to the grading
system adopted in a previous study (19).
Statistical analysis. Values were expressed as the mean ± standard error of the mean. The differences were analyzed
for significance using one‑way analysis of variance with
Bonferroni post hoc multiple comparisons, which were used
to assess the differences observed between the independent
groups. P<0.05 was considered to indicate a statistically
significant difference.
Results
Body weight. There was a significant increase in body weight
in the normal control group and in the group with γ‑tocotrienol
alone (P<0.05). The arthritis alone group exhibited a significant decrease in body weight throughout the duration of the
experiment (P<0.05). On day 35 and 45, the arthritis group
exhibited a significant decrease in body weight compared with
the other groups (P<0.05; Fig. 1).
Paw thickness. The arthritis alone group showed significant, macroscopic signs of severe arthritis such as swelling,
redness, deformity and ankylosis in the hind paw and ankle
joints; however, these symptoms were less pronounced in the
Figure 1. Body weight changes in the arthritis group and following
γ‑tocotrienol treatment. Data are presented as the mean ± standard error of
six rats per group. *P<0.05 vs. the control group.
A
B
Figure 2. Effect of γ‑tocotrienol on paw edema in the arthritis group. (A) Left
paw and (B) Right paw. Data are presented as the mean ± standard error of
six rats per group. *P<0.05 vs. the control group and +P<0.05 vs. the arthritis
group.
forelimbs. There was a significant decrease in the hind paw
thickness and edema of the g‑tocotrienol treated arthritis rats
(P<0.05) compared to arthritis alone rats. At the end of the
experimental period, the γ‑tocotrienol treated arthritis rats
exhibited a hind paw thickness that was analogous to that of the
normal control rats. No significant changes in paw thickness
were observed in the γ‑tocotrienol alone group and compared
with the γ‑tocotrienol alone group, the rats with arthritis and
γ‑tocotrienol (treatment from day 1) exhibited a significant
reduction in paw thickness (P<0.05; Figs. 2A,B and 3).
RADHAKRISHNAN et al: ANTI‑ARTHRITIS EFFECTS OF TOCOTRIENOLS
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Figure 3. Effect of γ‑tocotrienol on hind paw edema. (A) Control rats without arthritis without any paw edema; (B) Arthritis rats showing severe paw edema;
(C) Arthritis rats treated with γ‑tocotrienol exhibiting a significant reduction in the paw edema.
Figure 4. Effect of γ‑tocotrienol on C‑reactive protein (CRP) concentration.
Data are presented as the mean ± standard error of six rats per group. *P<0.05
vs. the control group and +P<0.05 vs. the arthritis group.
Figure 6. Effect of γ‑tocotrienol on serum superoxide dismutase (SOD). Data
are presented as the mean ± standard error of six rats per group. *P<0.05 vs.
the control group and +P<0.05 vs. the arthritis group.
Figure 5. Effect of γ‑tocotrienol on tumor necrosis factor‑α (TNF‑α) level.
Data are presented as the mean ± standard error of six rats per group. *P<0.05
vs. the control group and +P<0.05 vs. the arthritis group.
Figure 7. Effect of γ‑tocotrienol on serum glutathione levels. Data are presented as the mean ± standard error of six rats per group. *P<0.05 vs. the
control group and +P<0.05 vs. the arthritis group.
Plasma levels of CRP, TNF‑ α, SOD and GSH. ELISAs were
performed to quantify the CRP levels in the plasma. There was
a significantly elevated CRP concentration observed in the
untreated arthritis group and the arthritis group treated with
γ‑tocotrienol, when compared with the control rats (P<0.05).
However, the CRP level was significantly decreased in the
γ‑tocotrienol group when compared with the CRP levels of
the untreated arthritis group (P<0.05; Fig. 4). The untreated
arthritis group showed a significantly higher concentration
of TNF‑α compared with the γ‑tocotrienol-treated group
(P<0.05). Treatment with γ‑ tocotrienol to arthritis rats
resulted in a significant reduction in TNF‑α when compared
with the untreated arthritis group (P<0.05; Fig. 5). There was
a significant decrease in the SOD in the arthritis group and
EXPERIMENTAL AND THERAPEUTIC MEDICINE 7: 1408-1414, 2014
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A
B
C
D
Figure 8. Histopathological analysis of joint morphology (magnification, x200). (A) Control group exhibiting a normal joint. (B) γ‑tocotrienol treatment group,
exhibiting normal joint morphology. (C) Arthritis group exhibiting synovial fibrosis, congestion and hyperplasia. (D) Arthritis with γ‑tocotrienol treatment
group exhibiting mild synovial hyperplasia.
the γ‑tocotrienol‑treated group indicated high levels of SOD
concentration when compared with the arthritis only group
(Fig. 6). The γ‑tocotrienol-treated arthritis group exhibited
significantly elevated levels of total GSH when compared with
the arthritis group (P<0.05; Fig. 7).
Histopathological analysis. To evaluate the treatment of
γ‑tocotrienol against collagen-induced arthritis, histopathological analysis was conducted using an adapted method
from a previous study (19). The arthritis only group showed a
severity of grade three, while the γ‑tocotrienol-treated arthritis
group exhibited the characteristics of grade two severity. The
significance between these groups, in terms of pathological
conditions based on their grading, was calculated against the
untreated arthritis group. The γ‑tocotrienol-treated arthritis
group exhibited a significant reversal in the histopathological
changes compared with the untreated group (Fig. 8).
Synovial hyperplasia was observed in the untreated
arthritis group; the rats appeared to develop extensive edema
resulting in a narrowing of the joint space. There was inflammation, to the extent of forming panni, observed in numerous
locations within the rats in the untreated arthritis group. The
panni were composed of a granulomatous accumulation of
chronic inflammatory cells, such as lymphocytes, plasma cells,
macrophages and multinucleated giant cells. In the arthritis
treated with γ‑tocotrienol group, it was observed that synovial
hyperplasia was moderately present in addition to inflammation and vascular dilation. The inflammation was moderate
and was observed as scattered clusters of chronic inflammatory cells, with few focal attempts at granuloma formation. In
the control group the fore‑ and hind limbs were identified as
exhibiting a normal joint orientation. There was no evidence
of edema, cellular infiltration, joint narrowing, synovial hyperplasia, fibrosis or erosion. In the untreated arthritis rats, the
joints exhibited extensive edema with narrowing of the joint
spaces and the surface of the joint margins exhibited degenerative changes. In the arthritis group treated with γ‑tocotrienol
there was narrowing of the joint space, however, it was to a
lesser extent than that in the untreated arthritis group (Fig. 8).
Discussion
The present study demonstrated that γ‑tocotrienols are an
effective inhibitor of arthritis‑induced oxidative stress and
TNF- α secretion. To the best of our knowledge, this is the
first study to identify the anti‑arthritic effect of γ‑tocotrienols,
against collagen‑induced arthritis, in Dark Agouti rats.
Female Dark Agouti rats, aged 6‑10 weeks, were injected
with type II collagen emulsified with CFA, which induced
an immunological hypersensitivity reaction to the collagen
within the rats, leading to the development of chronic inflammatory arthritis. The arthritis developed within 2‑3 weeks of
primary immunization and exhibited characteristic arthritic
pathology comparable to that of human RA (20,21). A significant decrease in the body weight of the rats was observed in
the arthritis group compared with the other groups, which
confirmed the observations in previous studies where collagen‑
induced arthritis significantly decreased body weight, and
where the body weight was reduced following three weeks
of immunization (22,23). Body weight loss is the hallmark
symptom of inflammatory arthritis, where a gradual decrease
in weight gain is observed as the disease progresses (24). The
γ‑tocotrienol‑treated arthritis group experienced a significant
recovery of body weight following the second immunization.
Therefore the γ‑tocotrienol supplement to arthritis rats may
have decreased the production of reactive oxygen species
within the tissues and inhibited the metabolic rate of arthritic
rats; γ‑tocotrienol was able to impede the metabolism of the
body, thus favoring fat accumulation (25).
CRP is an inflammatory marker, which is a member of the
group of acute phase proteins and the level of CRP increases
in response to inflammation (26,27). The CRP assay is used
as an optimal laboratory test for the observation of inflammation resulting from RA and other inflammatory diseases. It is
RADHAKRISHNAN et al: ANTI‑ARTHRITIS EFFECTS OF TOCOTRIENOLS
an effective indicator of tissue damage and the concentration
of CRP in serum is associated with disease activity (27,28).
In the present study, an increased CRP level was observed
in the circulation of rats with arthritis and treatment with
γ‑tocotrienol significantly inhibited the arthritis-induced CRP
changes observed. The higher level of CRP observed in the
arthritis group confirmed the pathology of the joint and the
CRP production may have increased as a result of the activated macrophages and fibroblasts within the synovium of the
inflamed joints. The production of CRP is also controlled by
inflammatory mediators within the joints including IL (interleukin)‑1 and IL‑6, thus the reversal of CRP levels following
supplementation indicates a significant decrease in the activation of synovial macrophages and fibroblasts (29).
Various inflammatory mediators are released, which
are responsible for pain in addition to swelling in the joints
observed in cases of severe arthritis. The most common
inflammatory mediators are IL‑1β and TNF- α (30,31). A
series of inflammatory changes develop following the administration of collagen in arthritic rats; joint swelling, infiltration
of inflammatory cells, bone destruction and cartilage erosion
were the significant arthritic changes that were observed in the
present study. In inflammatory arthritis, CD4+ T helper cells
are activated in the joints that stimulate the production of cytokines and other inflammatory mediators. TNF‑α is produced
by macrophages and the synovial lining, and is present at
higher concentration in individuals suffering from arthritis;
TNF-α modulates the secretion of proinflammatory cytokines
(IL‑1 and IL‑6) within the synovial joints (31‑33). TNF‑α acts
synergistically with IL‑1β in the production of matrix metalloproteinases, the expression of cell adhesion molecules and
the secretion of prostaglandins and these changes result in the
joint destruction that is associated with arthritis. The present
study demonstrated that γ‑tocotrienol supplementation in
arthritic conditions attenuated the arthritis‑induced elevation
of the TNF-α level. Furthermore, activation of transcription
factor, nuclear factor kappa‑light‑chain‑enhancer of activated
B cells (NF‑κ B) is considered to be key in TNF‑α-induced
inflammatory processes, including the upregulation of IL‑6.
In previous studies, γ‑tocotrienol was shown to exhibit an
inhibitory effect on the NF‑κ B activation pathway (33‑35). In
addition, Wu et al (2008) identified that the tocotrienol‑rich
fraction was capable of inhibiting proinflammatory cytokines
in human monocyte cells (36). Non‑steroidal anti‑inflammatory drugs, glucocorticoids and other immunosuppressants,
that are commonly used in the treatment of RA, inhibit the
NF-κ B pathway and the expression of different inflammatory‑associated genes. At present, inhibitors of NF‑κ B are
considered to be the optimum anti‑inflammatory drug in the
therapeutic treatment of arthritis (33,37). In the present study,
γ‑tocotrienol significantly inhibited the TNF‑α level observed
in the circulation of the rats, which may be a result of its
suppressive effect on the activation of the NF‑κ B pathway
within the joints. The findings provide support for the use of
γ‑tocotrienol as an anti‑inflammatory candidate for the treatment of arthritis; moreover, to the best of our knowledge, there
are no known side effects as a result of prolonged treatment.
Free radicals are significant in the induction of RA (38);
activation of mono‑ and polymorphonuclear cells in the
articular joints result in oxidative damage within the joints.
1413
Increased oxidative stress is indicated by decreased concentrations of SOD and total GSH; two significant antioxidant
enzymes within the circulation. A case of chronic inflammatory arthritis reduces the antioxidant capacity of the
body and leads to an imbalance in the oxidant‑antioxidant
system (39,40). The significant decline in the level of SOD and
GSH in the present study indicated an increase in the accumulation of the reactive oxygen species within the synovium
and that these antioxidant enzymes were depleted due to
quenching of the free radicals (41). Tocotrienols possess a
potent antioxidant property, thus treatment with γ‑tocotrienol
enabled an increase in SOD and total GSH levels in the
blood, which aided with reducing oxidant‑induced joint tissue
damage. Furthermore, tocotrienols exhibit superior antioxidant and anti-lipid peroxidation effects when compared
with tocopherols, therefore tocotrienols have gained interest.
Previous studies identified that low doses of tocotrienols were
exhibiting an improved antioxidant and free radical scavenging effect, when compared with α‑tocopherols (42,43).
γ‑tocotrienol exhibits significant antioxidant activity due
to an ability for greater distribution within the membrane
bilayer (15,41,43). It exhibits an improved ability to trap free
radicals as a result of the unsaturated double bonds within
the chemical structure. The restoration of the two antioxidant
enzyme levels with γ‑tocotrienol supplementation may be
attributed to the ability of γ‑tocotrienol to elevate the mRNA
expression of these enzymes.
The histopathology of collagen‑induced arthritis in Dark
Agouti rats indicated cartilage destruction and extensive pannus
formation, bone resorption and synovitis. Histopathological
and biomarker changes correlated with the changes observed
in paw edema. The suppression of vascularity, congestion,
pannus formation and joint space narrowing, as a result of
treatment, indicated the anti‑arthritis effect of γ‑tocotrienol.
The γ‑tocotrienols may have suppressed the progression of
arthritis by inhibiting the chronic inflammatory phase and
decreasing the free radical accumulation within the joints,
thus reducing the incidence of cartilage destruction (6).
In conclusion, the results of the present study indicated
that γ‑tocotrienol was capable of reducing the oxidative stress
and inflammation that was observed in the collagen-induced
arthritic rats. The γ‑tocotrienol treatment increased the
antioxidant enzyme levels and decreased the TNF‑ α levels
observed in arthritic rats, which provided protection against
arthritis‑induced joint damage. Histopathology indicated
that the administration of γ‑tocotrienol protected the joints
and prevented the destruction of cartilage, thus significantly
improving the arthritic symptoms. Therefore, γ‑tocotrienol
may be an effective, long‑term anti‑arthritic agent for reducing
the serious side effects of synthetic, anti‑arthritis drugs.
Acknowledgements
The present study was supported by grants from the
International Medical University (Kuala Lumpur, Malaysia).
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