Canine chondrocytes were exposed to the proteasome inhibitor N-acetyl-Leu-Leu-Norleu-al and trea-ted with recombinant TRAIL protein under normoxic and hypoxic condi-tions, measuring chon
Trang 1apoptosis – a possible mechanism of maintaining
homeostasis of normal articular cartilage
J.-W Seol1, H.-B Lee1, Y.-J Lee1, Y.-H Lee2, H.-s Kang1, I.-s Kim1, N.-S Kim1and S.-Y Park1
1 Center for Healthcare Technology Development, Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk, South Korea
2 Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju, Jeonbuk, South Korea
Keywords
chondrocytes; hypoxia; proteasome; reactive
oxygen species; tumour necrosis
factor-related apoptosis-inducing ligand
(TRAIL)
Correspondence
S.-Y Park, College of Veterinary Medicine,
Chonbuk National University, Jeonju,
Jeonbuk 561-756, South Korea
Fax: +82 63 270 3780
Tel: +82 63 270 3886
E-mail: sypark@chonbuk.ac.kr
(Received 21 August 2009, revised 10
October 2009, accepted 20 October 2009)
doi:10.1111/j.1742-4658.2009.07451.x
Hypoxia and hypoxia-related genes are important factors in articular chon-drocytes during cartilage homeostasis and osteoarthritis We have investi-gated the various apoptotic factors that show significance in synovial fluid obtained from normal and experimental osteoarthritic animal models and have evaluated the effect of hypoxia on articular chondrocyte apoptosis induced by these apoptotic factors Mature beagle dogs underwent surgical transections of ligaments and medial meniscectomies to explore the under-lying mechanisms of osteoarthritis Cartilage and synovial fluid obtained from normal animals and those with osteoarthritis were evaluated via pro-teasome inhibition, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) protein expression, mitochondrial transmembrane poten-tial and levels of reactive oxygen species Canine chondrocytes were exposed to the proteasome inhibitor N-acetyl-Leu-Leu-Norleu-al and trea-ted with recombinant TRAIL protein under normoxic and hypoxic condi-tions, measuring chondrocyte cell viability, proteasome activity and levels
of apoptotic factors TRAIL protein expression and ubiquitinated proteins were increased significantly, but the proteasome activity in the synovial fluid of osteoarthritic joints relative to that in normal joints was not Pri-mary cultured articular chondrocytes cotreated with the proteasome inhibi-tor and TRAIL progressed to severe apoptosis under normoxic conditions, but the sensitization caused by the combined treatment was suppressed by exposure to hypoxia Caspase-8 activation, c-Jun N-terminal kinase phos-phorylation, the mitochondrial transmembrane potential and the genera-tion of reactive oxygen species involved in cell death regulagenera-tion were significantly inhibited under hypoxic conditions These findings suggest that proteasome inhibition and TRAIL may be possible mechanisms in cartilage degradation and joint-related diseases Furthermore, the maintenance
of hypoxic conditions or therapy with hypoxia-related genes in the joint may be successful for the treatment of joint-related diseases, including osteoarthritis
Abbreviations
ALLN, N-acetyl-Leu-Leu-Norleu-al; DCFH2-DA, 2¢,7¢-dichlorodihydrofluorescein diacetate; DR-5, death receptor-5; JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢3,3¢-tetraethylbenzimidazol-carbocyanine iodide; JNK, c-Jun N-terminal kinase; JNK-SAPK, c-Jun N-terminal kinase-stress-activated protein kinase; MTP, mitochondrial transmembrane potential; OA, osteoarthritis; ROS, reactive oxygen species; Suc-LLVY-AMC, Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.
Trang 2A hallmark of osteoarthritis (OA) is a decrease in the
number of chondrocytes, as they are the only resident
cells in articular cartilage Chondrocytes regulate the
enzymatic breakdown of the extracellular matrix,
thereby maintaining the equilibrium between synthetic
and degradative processes in the cartilage [1]
There-fore, the metabolic and structural changes of
chondro-cytes in the articular cartilage play a significant role in
the initiation and progression of the disease Several
studies have examined cell death in human articular
cartilage affected by OA or in experimental models of
OA [2,3]
Tumour necrosis factor-related apoptosis-inducing
ligands (TRAILs) are type II transmembrane
mole-cules that trigger the apoptotic signal cascade by
bind-ing to cognate receptors expressed on the cell surface
[4,5] TRAIL is highly expressed on the surfaces of
natural killer (NK) cells, as well as on CD4+ and
CD8+ T cells It promotes apoptosis, which may aid
in the resolution of infection and the attenuation of
the development of streptoxotocin-induced diabetes
and collagen-induced arthritis [6–9] Some studies have
reported a role for TRAIL protein in articular joint
disease [10,11] TRAIL alone can induce apoptosis in
primary cultured chondrocytes from different animal
species, such as humans and rats [11,12], but the exact
role of TRAIL in chondrocytes has not been clearly
defined to date
Ubiquitin-proteasome-mediated protein degradation
pathways have been shown to play an important role
in regulating both cell proliferation and cell death [13]
Most recent studies have suggested that
ubiquitin-pro-teasome-dependent proteolysis is also involved in
apoptosis, although its exact role remains controversial
[14] Proteasome inhibitors block the process of
pro-grammed cell death in thymocytes and neurons, but
induce apoptosis in various human cancer cell lines
Proteasome inhibition suppresses growth plate
prolifer-ation and induces chondrocyte apoptosis [15] Human
chondrocytes are also sensitive to proteasome-induced
apoptosis [16] Although the treatment of cells with
this compound causes marked increases in a large
number of cellular proteins, including cyclin-dependent
protein kinase inhibitors, it is not clear how this agent
actually induces apoptosis [17,18] More research is
required to fully characterize the types of cell death in
aging and arthritic cartilage, together with their
respec-tive frequencies
Articular cartilage is an avascular tissue that
func-tions at an oxygen tension lower than that of most
other tissues, and derives both its nutrition and oxygen
supply by diffusion from the synovial fluid and subchondral bone [19,20] It has been estimated that articular chondrocytes in the deepest layers may have access to no more than 1–6% O2 [21,22] Oxygen can
be processed to generate reactive oxygen species (ROS), which play an important role in intracellular signalling and thus in cell physiology and cellular destruction ROS are known to induce a wide range of responses, depending on cell type and levels of ROS within the cell [23,24]
The aims of this study were to investigate the major signalling pathways and effects of hypoxic conditions
in experimental osteoarthritic cartilage degeneration and cell death of primary cultured chondrocytes In particular, we focused on the role of proteasome inhibition and TRAIL in osteoarthritic disease and chondrocyte apoptosis, and the hypoxic inhibition of cartilage and chondrocyte degeneration
Results
Macroscopic and radiographic examination of the articular cartilage after experimentally induced OA
Articular cartilage of the femoral condyles from the experimental joints was examined to assess any macro-scopic damage caused by experimentally induced OA Cartilage damage was visualized on the tibial plateau
of the experimental joints when compared with normal control joints The medial tibia plateau cartilage in the experimental joints was fibrillated with erosive lesions (Fig 1A), and radiographic findings revealed joint distension There was no evidence of sclerosis, erosions
or osteophyte and enthesophyte formation in the experimental joints (Fig 1B)
TRAIL and ubiquitinated protein expression were significantly increased, but proteasome activity was not, in the synovial fluid of osteoarthritic joints
Proteasome activity was assayed in the synovial fluid from experimental osteoarthritic joints via Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) hydrolysis, and was significantly lower than the activity in control joints The ubiquitinated protein lev-els in synovial fluid from osteoarthritic joints were higher than in the control joint group (Fig 2A) We also examined TRAIL protein expression in experi-mental osteoarthritic synovial fluid The protein
Trang 3expression of TRAIL was increased in osteoarthritic
joints compared with the control group (Fig 2B) In
this experiment, the elevated TRAIL protein of
osteo-arthritic synovial fluid may have originated from
vari-ous inflammatory cells, such as lymphocytes, and other
studies support this [6,8]
Combined treatment with proteasome inhibitor
and TRAIL markedly enhanced apoptosis in
cultured canine chondrocytes
To investigate the proteasome inhibition effect on
TRAIL-induced apoptosis in canine chondrocytes,
N-acetyl-Leu-Leu-Norleu-al (ALLN) was used as a
proteasome inhibitor Canine chondrocytes were
exposed to ALLN (10 lm) for 12 h and then treated
with recombinant TRAIL protein for an additional
12 h TRAIL and ALLN alone did not induce
sis, but combined treatment markedly induced
apopto-sis to 60% in canine chondrocytes (Fig 3A) The
examination of cell morphology also supported the
enhancing effect of combined ALLN and TRAIL
treatment in canine chondrocytes (Fig 3B)
To determine whether treatment with the
protea-some inhibitor ALLN affected proteaprotea-some-mediated
degradation in canine chondrocytes, proteasome activ-ity was assayed in cell lysates as a measure of the hydrolysis of the fluorogenic substrate Suc-LLVY-AMC Proteasome activity was significantly inhibited
by treatment with ALLN only and cotreatment with ALLN and TRAIL (Fig 3C) Western blot analysis was also used to investigate whether ALLN induced proteasome inhibition in canine chondrocytes In the absence of ALLN, smears of ubiquitinated proteins were not observed in control and TRAIL-treated canine chondrocytes In the presence of ALLN, marked accumulation of polyubiquitinated proteins was observed in canine chondrocytes (Fig 3D)
Proteasome inhibition increased significantly TRAIL-mediated caspase-8 activation and JNK phosphorylation
To determine the mechanism by which proteasome inhibition enhanced TRAIL-induced apoptosis in canine chondrocytes, we examined caspase-8 activation and death receptor-5 (DR-5) and TRAIL protein
A
B
Fig 1 Evaluation of articular cartilage after experimentally induced
osteoarthritis (A) Photomicrographs of articular cartilage (B) The
evaluation of osteoarthritis in the right and left joints of dogs was
graded 12 weeks after surgery by the evaluation of radiographs
using established parameters OA, osteoarthritis sample.
A
B
Fig 2 TRAIL protein and ubiquitinated protein levels were signifi-cantly higher and proteasome activity was lower in osteoarthritic joints (A) Proteasome activity was measured using the synthetic fluorogenic substrate Suc-LLVY-AMC Fluorescence was measured
at 380 nm excitation and 440 nm emission The fluorescence value for control cells was set at 100%, and the fluorescence values rela-tive to the control are presented The experiments were performed
in triplicate at least twice independently (A, B) Proteins were sepa-rated on an 8–15% SDS gel, and apoptotic proteins were detected
by western blot analysis b-Actin was used to normalize equal pro-tein loading Blot images represent one of three independent exper-iments *P < 0.05 versus normal sample was calculated using Student’s t-test OA, osteoarthritis sample; Ubi, ubiquitin.
Trang 4expression in cells treated with ALLN and⁄ or TRAIL
protein Western blot analysis showed that caspase-8
was slightly activated in control, ALLN-treated and
TRAIL-treated chondrocytes However, under
protea-some inhibition induced by pretreatment with ALLN,
TRAIL treatment unexpectedly increased the
activa-tion of caspase-8 In addiactiva-tion, the phosphorylaactiva-tion of
JNK protein was markedly increased by combined
treatment with ALLN and TRAIL when compared
with other groups The expression of DR-5 and
TRAIL protein was also investigated, but these were
not altered in cells with proteasome inhibition (Fig 4)
Proteasome inhibition and the expression of
TRAIL protein induced the dissipation of the
mitochondrial transmembrane potential (MTP)
and ROS generation
MTP was investigated in order to address the possible
mechanism by which proteasome inhibition enhances
TRAIL-induced apoptosis in canine chondrocytes
MTP evaluation is based on the ability of a fluorescent probe to enter the mitochondria selectively and reversibly change its colour from green to red as the mitochondrial potential increases 5,5¢,6,6¢-Tetrachloro-1,1¢3,3¢-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Molecular Probes, Eugene, OR, USA) exists as
a monomer at low MTP values and shows green fluo-rescence, whereas it forms an aggregate at high MTP and shows red fluorescence The fluoroscopic results presented in Fig 5A show a red and slightly green flu-orescence in cells treated with ALLN and TRAIL alone, but a highly green fluorescence after combined treatment Photomicrographs indicated that ALLN and TRAIL alone induced a small change in MTP, whereas combined treatment with ALLN and TRAIL caused a significant dissipation of MTP relative to neg-ative controls When ROS generation was examined, the results showed that pretreatment of the cells with the proteasome inhibitor increased ROS levels, and that significant ROS generation was induced with TRAIL cotreatment in canine chondrocytes (Fig 5B)
B D
Fig 3 Proteasome inhibition markedly enhanced TRAIL-induced apoptosis and significantly inhibited proteasome activity in primary cultured canine chondrocytes (A) Cell viability was determined by the crystal violet staining method The viability of control cells was set at 100%, and the viability relative to the control is presented The experiments were performed in triplicate at least twice independently The bars describe the standard deviation (B) Cell morphology was photographed (·200) under the various conditions (C) Proteasome activity was measured using the synthetic fluorogenic substrate Suc-LLVY-AMC Fluorescence was measured at 380 nm excitation and 440 nm emis-sion The fluorescence value for control cells was set at 100%, and the fluorescence values relative to the control are presented The experi-ments were performed in triplicate at least twice independently The bars describe the standard deviation (D) Whole-cell lysates were prepared and total protein (40 lgÆmL)1) was electrophoretically resolved on SDS gel Ubiquitin protein levels were detected by western blot-ting analysis b-Actin was used to normalize equal protein loading Blot images represent one of three independent experiments.
**P < 0.01, *P < 0.05 versus control were calculated using Student’s t-test.
Trang 5To investigate the effects of MTP in cartilage by the induction of OA, chondrocytes were isolated from experimentally induced osteoarthritic cartilage The photomicrographs and fluorescence values indicated that these chondrocytes showed a decrease in MTP compared with normal cartilage (Fig 5C) In addition, the chondrocytes isolated from osteoarthritic joints demonstrated significantly greater ROS generation than did the controls (Fig 5D)
Hypoxia inhibited the apoptosis of primary cultured canine chondrocytes induced by proteasome inhibition and TRAIL treatment
In order to examine the functional role of ALLN and TRAIL in apoptotic cell death under hypoxic condi-tions, canine chondrocytes were exposed to hypoxia and ALLN (10 lm) for 12 h, and were then treated with recombinant TRAIL protein for an additional
12 h under normoxic and hypoxic conditions Hypoxic conditions inhibited significantly the apoptosis of chondrocytes induced by cotreatment of the cells with the proteasome inhibitor and TRAIL (Fig 6A) Chon-drocyte survival under hypoxic conditions was enhanced by 25% compared with the survival of cells
Fig 4 Proteasome inhibition and TRAIL treatment significantly
increased caspase-8 activation and JNK phosphorylation Whole-cell
lysates were prepared and total protein (40 lgÆmL)1) was
electro-phoretically resolved on SDS gel Apoptotic proteins were detected
by western blotting analysis b-Actin was used to normalize equal
protein loading Blot images represent one of three independent
experiments.
A C
B D
Fig 5 The decrease in MTP and ROS generation induced by proteasome inhibition and TRAIL (A, C) MTP was determined using a JC-1 probe The cells were photographed using a fluoroscope The green fluorescence intensity was measured under the conditions described in Materials and methods The experiments were performed in triplicate at least twice independently (B, D) The ROS level was measured using DCFH-DA DCFH fluorescence was determined with a fluorescence plate reader with 490 and 525 nm as excitation and emission wavelengths, respectively The fluorescence value for control cells was set at 100%; fluorescence values relative to the control are pre-sented The experiments were performed in triplicate at least twice independently **P < 0.01, *P < 0.05 versus control were calculated using Student’s t-test MFI, mean fluorescence intensity; OA, osteoarthritis sample.
Trang 6that were cotreated with ALLN and TRAIL under
normoxic conditions Moreover, photomicrographs
revealed that cells showed a decreased death rate under
hypoxic conditions when they were cotreated with
ALLN and TRAIL (Fig 6A)
Hypoxia inhibited chondrocyte apoptosis through
the inhibition of caspase activation, JNK
phosphorylation, restoration of MTP loss and
ROS generation
To examine why hypoxia inhibited the combined
effects of ALLN and TRAIL in canine chondrocytes,
western blot analysis was performed It was shown
that cotreatment of cells with ALLN and TRAIL
increased caspase-8 activation and JNK
phosphoryla-tion However, both caspase-8 activation and JNK
phosphorylation were inhibited under hypoxic condi-tions (Fig 6B) MTP and ROS were investigated in order to address the inhibitory mechanism exerted by hypoxic conditions Canine chondrocytes were exposed to ALLN (10 lm) for 12 h and were then treated with recombinant TRAIL protein for an addi-tional 12 h under normoxic and hypoxic conditions The fluoroscopic results presented in Fig 6C show that the cells fluoresce green after cotreatment with ALLN and TRAIL, indicating lower MTP under normoxic conditions However, the green fluorescence indicating lower MTP declined under hypoxic condi-tions (Fig 6C) Cotreatment of cells with ALLN and TRAIL induced ROS generation significantly under normoxic conditions, but hypoxia prevented ROS generation after cotreatment with ALLN and TRAIL (Fig 6D)
B D
Fig 6 Hypoxia inhibited chondrocyte death and apoptosis-related signals induced by proteasome inhibition and TRAIL (A) Cell viability was determined using the crystal violet staining method The control cell viability was set at 100%; viability relative to the control is presented The experiments were performed in triplicate at least twice independently The cell morphology was photographed (·200) (B) Whole-cell lysates were prepared and total protein (40 lgÆmL)1) was electrophoretically resolved on SDS gel and then tested for apoptotic proteins by western blotting analysis b-Actin was used to normalize equal protein loading; Blot images represent one of three independent experiments (C) MTP was determined using a JC-1 probe The cells were photographed using a fluoroscope The green fluorescence intensity was mea-sured under the conditions described in Materials and methods (D) The ROS level was meamea-sured using DCFH-DA The fluorescence value for control cells was set at 100%; fluorescence values relative to the control are presented The experiments were performed in triplicate at least twice independently **P < 0.01, *P < 0.05 versus control were calculated using Student’s n-test Nor, normoxia; Hypo, hypoxia; A, ALLN; T, TRAIL; MFI, mean fluorescence intensity.
Trang 7TRAIL is a good candidate for cancer therapy as it
selectively induces apoptosis in tumour cells, with little
or no effect on normal cells [25] It has recently been
reported that rheumatoid arthritis synovial tissue and
fibroblasts both express high levels of DR5
(TRAIL-R2), are highly susceptible to DR5-mediated apoptosis,
and DR5 may be a selective marker for rheumatoid
arthritis [10] In addition, TRAIL protein is produced
in rat arthritic cartilage and plays an important role in
the pathogenesis of OA [11] Our study showed that
TRAIL and ubiquitin protein expression in the
syno-vial fluid from osteoarthritic joints was increased
com-pared with that in control joints Changes in TRAIL
and ubiquitin levels may be linked to the progression
of inflammation and may be detected in the synovial
fluid These changes could be associated with a natural
history of OA and may be beneficial in the detection
of patients at risk of rapidly progressing disease
Articular cartilage is an avascular tissue that
func-tions at an oxygen tension lower than that of most
other tissues Articular cartilage derives both its
nutri-tion and oxygen supply through diffusion from the
synovial fluid and the subchondral bone [19,20,26] It
has been reported that the partial pressure of oxygen
in the synovial fluid of joints affected by OA is
between 40 and 85 mmHg, corresponding to an
oxy-gen concentration of approximately 6–11% [27] It has
been estimated that articular chondrocytes in the
deep-est layers have access to no more than 1–6% O2
[20,22] Moreover, mitochondria are sparse in the
articular chondrocytes, occupying only 1–2% of the
intracellular volume [28], compared with 15–20% in
other typical animal cells (for example, the liver)
Marcus [29] and Otte [30] observed that chondrocytes
produced ATP mostly through substrate-level
phos-phorylation during glycolysis However, oxygen
tensions below 1% inhibit both glucose uptake and
lactate production, as well as cellular RNA synthesis
[29,30] This indicates that chondrocytes need at least
some oxygen for their basal metabolic activity
There-fore, hypoxia is considered to be a key factor in the
growth and survival of chondrocytes
Hypoxia is known to regulate the expression of
many genes, but little is known about its role in either
apoptosis or anti-apoptosis, especially in canine
chon-drocytes In this article, we investigated the possible
effects of proteasome inhibition on TRAIL-induced
apoptosis under normoxic and hypoxic conditions
We found that TRAIL and ALLN alone did not
induce apoptosis, but combined treatment of the cells
with ALLN and TRAIL increased apoptosis markedly
to 60% in canine chondrocytes However, ALLN⁄ TRAIL cotreatment-induced apoptosis of canine chondrocytes was inhibited significantly under hypoxic conditions This suggests that hypoxia can inhibit apoptotic activity in canine chondrocytes, and may therefore suppress the development and progression of OA
Cell death in osteoarthritic cartilage possesses cer-tain features of apoptosis or programmed cell death [31] Apoptosis is mediated by a cascade of aspartate-specific cysteine proteases or caspases, and increased caspase expression has been correlated with reduced cell density in human osteoarthritic cartilage [32] The present study demonstrated that ALLN pretreatment with TRAIL increased the activation of caspase-8 under normoxic conditions, but that caspase-8 activation was inhibited under hypoxic conditions In addition, the enhancing effect of proteasome inhibition
on TRAIL-induced apoptosis was completely inhibited
by the pan-caspase inhibitor, z-VAD-fmk Taken together, our data indicated that proteasome inhibition enhanced TRAIL-induced cell death via the caspase pathway, the key regulator of the TRAIL-induced cell death pathway in canine chondrocytes Furthermore, cotreatment of cells with both ALLN and TRAIL increased JNK phosphorylation under normoxic condi-tions, but this increase was inhibited under hypoxic conditions This suggests that the protective role of hypoxia involves the inhibition of caspase activation and JNK phosphorylation
Mitochondria are central regulators of apoptosis [33,34] and may also be involved in chondrocyte death during bone development The activities of respiratory chain complexes II and III and the mitochondrial membrane potential are significantly reduced in cul-tured human chondrocytes from osteoarthritic donors when compared with normal donors [35] In this study,
we demonstrated that hypoxic conditions prevented ROS generation and restored the loss of MTP seen after cotreatment with ALLN and TRAIL These find-ings suggest that the mitochondrial respiratory chain complexes are probable sites of ROS production, and that the inhibition of depolarization of MTP during hypoxia probably induces a decrease in ROS levels in canine chondrocytes
In conclusion, the present study has demonstrated that proteasome activity, ubiquitinated protein, TRAIL and ROS are altered significantly in synovial fluid acquired from experimentally induced osteoar-thritic joints At the cellular level, proteasome inhibi-tion markedly enhances TRAIL-induced apoptosis through the activation of caspase-8, the phosphoryla-tion of JNK protein, a decrease in MTP and the
Trang 8gener-ation of ROS in primary cultured canine chondrocytes.
However, the enhanced apoptosis of chondrocytes
induced by this combined treatment is inhibited under
hypoxic conditions All these findings suggest that
pro-teasome inhibition and TRAIL play a pivotal role in
canine chondrocyte death and cartilage degradation
These findings indicate that the maintenance of
hypoxic conditions in cartilage inhibits articular
chon-drocyte apoptosis and may suppress the progression
of arthritis
Materials and methods
Induction of OA
1.4 ± 0.4 years and a mean ± SD weight of 10.2 ± 1.4 kg
were used A right stifle joint medial arthrotomy was
per-formed The cranial cruciate and the medial collateral
liga-ments were transected and a medial meniscectomy was
performed The experimental animals were given
intrave-nous crystalloid fluids (10 mLÆkg)1Æh)1) The surgical area
was shaved and prophylactic antibiotic, cephalexin
(Methi-lexin Inj; Union Korea Pharm Co Ltd., Seoul, South
Korea), 25 mgÆkg)1 intravenously, was administered 1 h
before surgery The experimental animals were
premedicat-ed with atropine sulfate (Atropin Sulfate Inj; Dai Han
Pharm Co Ltd., Seoul, South Korea), 0.05 mgÆkg)1,
sub-cutaneously Anaesthesia was induced with propofol
(Ane-pol Inj; Hana Pharm Co Ltd., Seoul, South Korea),
6 mgÆkg)1 intravenously, and maintained with enflurane
and oxygen During surgery, the jaw reflex, ocular reflex,
heart rate (using electrocardiogram) and respiratory rate
(using capnography) were monitored Based on these data,
we changed the vaporizer settings if the experimental
ani-mals were in deep or light anaesthesia After surgery,
post-operative treatment was given with butophanol (Butopan
Inj; Hana Pharm Co Ltd.), 10 mgÆkg)1intramuscularly,
every 12 h for 7 days for pain relief After 7 days, no
anal-gesic drug was given as the progress of OA was graded
using a clinical scoring system, such as lameness, joint
mobility and weight bearing All procedures employed in
the animal experiments were approved by the Standard
Operation Procedure of the Institutional Animal Care and
Use Committee, Jeonju, South Korea
Evaluation of OA
Experimental animals were sacrificed at 12 weeks to
evalu-ate the severity of OA after surgery Levels of macroscopic
synovial inflammation and cartilage damage were evaluated
with digital high-resolution photographs The severity of
synovial inflammation was graded on the basis of colour,
angiogenesis and fibrillation: grade 0, no inflammation;
grade 1, slight inflammation; grade 2, strong inflammation The cartilage damage severity of the femoral condyles and tibial plateau was graded from 0 to 4: grade 0, smooth surface; grade 1, slight fibrillation; grade 2, fibrilla-tion with shallow grooves; grade 3, deep and sharp grooves; grade 4, deep and sharp grooves with surrounding damage
Radiographic examinations were also performed The severity of osteophyte formation in the femoropatellar, lat-eral femorotibial, medial femorotibial and central femoroti-bial joints was graded from 0 to 3: grade 0, absent; grade
1, mild; grade 2, moderate; grade 3, severe The degree of synovial effusion was graded from 0 to 3: grade 0, absent; grade 1, mild; grade 2, moderate; grade 3, severe Two independent observers assigned individual scores, and all values were averaged and used in the statistical analyses
Synovial fluid preparation
Synovial fluid was collected 12 weeks after the induction of
OA Briefly, experimental animals were sedated with ace-promazine (Sedazect Inj; Samwoo Pharm Co Ltd., Seoul, South Korea), 0.2 mgÆkg)1 intravenously, and placed in ventrodorsal recumbency with the right stifle joints flexed Digital pressure was applied to the medial side of the straight patellar ligament A 21-gauge spinal needle was inserted through the fat pad into the intercondylar space lateral to the straight patellar ligament
Chondrocyte isolation
Normal canine knee cartilage was obtained from the knee joints of beagles (2-year-old females) The cartilage surfaces were first rinsed with sterile NaCl⁄ Pi The cartilage slices were chopped and incubated with 0.25% trypsin for
30 min, followed by 0.1% collagenase (Sigma-Aldrich, St Louis, MO, USA; #C6885) treatment for 6 h in Dulbecco’s modified Eagle’s medium (Invitrogen-Gibco, Grand Island,
NY, USA) supplemented with 10% (v⁄ v) fetal bovine serum (Invitrogen-Gibco) and antibiotics (100 lgÆmL)1 gentamycin and 100 lgÆmL)1penicillin–streptomycin) Cells were filtered through a 70 lm cell strainer (Falcon, Frank-lin Lakes, NJ, USA), washed twice with NaCl⁄ Piand then seeded into tissue culture flasks The total cell number was calculated using a haemocytometer
Cell viability test
Canine chondrocytes were adjusted to 1.0· 106cells per well in 12-well plates, pretreated with ALLN (Sigma,
St Louis, MO, USA) for 12 h, and then further incubated with recombinant TRAIL protein for 12 h under normoxic (21% O2) and hypoxic (1% O2) conditions at the indicated doses Cellular morphology was photographed under light
Trang 9microscopy (Nikon, Tokyo, Japan), and cell viability was
determined using the crystal violet staining method, as
described previously [36] Briefly, the cells were stained for
10 min at room temperature with staining solution (0.5%
crystal violet in 30% ethanol and 3% formaldehyde),
washed four times with water and then dried The cells
were then lysed with 1% SDS solution and the absorbance
was measured at 550 nm The cell viability was calculated
based on the relative dye intensity compared with
controls
Western blot assay
To prepare whole-cell lysates, cells were harvested and
resuspended in lysis buffer (25 mm Hepes, pH 7.4, 100 mm
NaCl, 1 mm EDTA, 5 mm MgCl2, 0.1 mm dithiothreitol
and protease inhibitor mixture) Synovial fluid was diluted
10 times with NaCl⁄ Pi Proteins were electrophoretically
resolved on an 8–15% SDS gel, and western blots were
per-formed as described previously [37] Equal amounts of the
lysate protein were also resolved on an 8–15% SDS-PAGE
gel and then electrophoretically transferred to a
nitrocellu-lose membrane The immunoreactivity was detected
through sequential incubation with horseradish
peroxidase-conjugated secondary antibodies and ECL reagents
(Amer-sham corp., Burlington, MA, USA) The antibodies used
for western blotting analyses were caspase-8 (AAP-118)
(Stressgen, Victoria, Canada), Ubiquitin (Cell Signaling
Technology, Danvers, MA, USA), TRAIL (Santa Cruz
Biotechnology, Santa Cruz, CA, USA; sc-8440), c-Jun
N-terminal kinase–stress-activated protein kinase (JNK)
and the phosphorylated form (p-JNK) (Upstate
Biotechnol-ogy, Lake Placid, NY, USA)
Proteasome activity test
Proteasome activity was measured as described previously
[38] The cells were collected by centrifugation and the
synovial fluid was diluted 10 times with NaCl⁄ Pi Protein
concentrations of synovial fluid and cytoplasm were
deter-mined using the Bradford protein assay kit (Bio-Rad,
Her-cules, CA, USA) Two hundred micrograms of synovial
fluid protein and cytoplasm protein were added to the assay
buffer (20 mm Tris⁄ HCl, pH 8.0, 1 mm ATP, 2 mm MgCl2)
in the presence of the synthetic fluorogenic substrate
Suc-LLVY-AMC to a final concentration of 60 lm
(Sigma-Aldrich) suspended in a final volume of 1 mL The tubes
were incubated at 30C for 30 min, after which the
reac-tion was terminated through the addireac-tion of 1 mL of cold
ethanol The lysate was spun at 12 000 g for 10 min at
4C Fluorescence was measured at 380 nm excitation and
440 nm emission using a fluorescence plate reader
(Spectra-Max fluorometer with the softmax program; Molecular
Probes, Eugene, OR, USA)
Evaluation of MTP
The level of MTP was determined using a lipophilic cation, JC-1 (Molecular Probes) Briefly, chondrocytes were iso-lated from cartilage obtained from osteoarthritic joints The cells were collected by centrifugation, washed twice with NaCl⁄ Piand resuspended in 500 lL of NaCl⁄ Picontaining JC-1 at a concentration of 10 lm After 30 min of incuba-tion at 37C, the cells were photographed using a micro-scope (ECLIPSE 80 i, Nikon), and red fluorescence was monitored with a fluorescence plate reader (SpectraMax fluorometer with the softmax program; Molecular Probes), with 490 and 590 nm as excitation and emission wave-lengths, respectively
Determination of ROS
ROS levels, particularly the levels of intracellular hydroper-oxides, were assessed using the oxidant-sensitive dye 2¢,7¢-dichlorodihydrofluorescein diacetate (DCFH2-DA) The cells treated with ALLN and TRAIL for 12–24 h were washed twice with NaCl⁄ Pi and incubated with 10 lm DCFH2-DA in sodium pyruvate containing Dulbecco’s modified Eagle’s medium for 1 h at 37C After DCFH2
-DA incubation, the cells were washed and further incubated
in sodium-containing medium for 10 min to allow de-esteri-fication to occur The cells were then collected, and the flu-orescence signals corresponding to intracellular ROS were monitored at 490 nm excitation and 525 nm emission using
a fluorescence plate reader (SpectraMax fluorometer with the softmax program, Molecular Probes)
Hypoxic conditions
A sealed chamber was used to culture the chondrocytes at low oxygen tension (1%) A gas mixture of 1% O2, 5%
CO2 and 94% N2 was added to the sealed chamber, and ambient air was evacuated through an outlet tube The oxygen flow was allowed to stream through the chamber for 2–3 min to maintain the desired oxygen tension inside the chamber Culture plates were incubated in sealed cham-bers containing 1% O2 at 37C For the normoxic condi-tion (21% O2tension), the chondrocytes were incubated at
37C in a 95% humidified atmosphere with 5% CO2 There were two controls (normoxia and hypoxia) in this experiment The hypoxia control was handled in the same type of sealed unit as used for 1% O2
Statistical evaluation
All data are expressed as the mean ± SD, and were com-pared using Student’s t-test and the ANOVA Duncan test with the sas statistical package The results were considered
to be significant at P < 0.05 and P < 0.01
Trang 10This work was supported by a Korea Research
Foun-dation Grant from the Regional Research Universities
Program⁄ Center for Healthcare Technology
Develop-ment
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