Article

Elucidating the role of protandim and 6-gingerol in protection against osteoarthritis†

Jamilah Abusarah1, Houda Abir Benabdoun1, Qin Shi1, Bertrand Lussier2, Johanne Martel-Pelletier2,
Michel Malo1, Julio C. Fernandes1, Fátima Pereira de Souza3, Hassan Fahmi2, and Mohamed
Benderdour1*

Short title: Role of Nrf2 in osteoarthritis

Key words: Osteoarthritis, Cartilage, Nrf2, GSTA4-4, HNE, Protandim.

1
Orthopedic Research Laboratory, Hôpital du Sacré-Coeur de Montréal and Department of Surgery,
Université de Montréal, 5400 Gouin Blvd. West, Montreal, QC, Canada H4J 1C5
2
Osteoarthritis Research Unit and Research Centre, Centre hospitalier de l’Université de Montréal
(CRCHUM) – Hôpital Notre-Dame, Montreal, QC, Canada H2L 4M1
3
Universidade Estadual Paulista "Júlio de Mesquita Filho", (UNESP), Departamento de Física,
Laboratório de Biologia Molecular, Centro Multiusuário de Inovação Biomolecular (CMIB), 15054-000,
São José Do Rio Preto, SP, Brazil

*Address reprint requests and other correspondence to: Dr. Mohamed Benderdour, Orthopedic
Research Laboratory, Hôpital du Sacré-Cœur de Montréal, Room K-3045, 5400 Gouin Blvd. West,
Montreal (QC) Canada H4J 1C5; Telephone: (514) 338-2222 (Extension 3279); Fax: (514) 338-2694.
Email: mohamed.benderdour@umontreal.ca

†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/jcb.25659]

Received 2 February 2016; Revised 26 July 2016; Accepted 26 July 2016

Journal of Cellular Biochemistry
This article is protected by copyright. All rights reserved
DOI 10.1002/jcb.25659

This article is protected by copyright. All rights reserved

ABSTRACT

Protandim and 6-gingerol, two potent nutraceuticals, have been shown to decrease free radicals

production through enhancing endogenous antioxidant enzymes. In this study, we evaluated the

effects of these products on the expression of different factors involved in osteoarthritis (OA) process.

Human OA chondrocytes were treated with 1 ng/ml IL-1 in the presence or absence of protandim (0-

10 g/ml) or 6-gingerol (0-10 M). OA was induced surgically in mice by destabilization of the medial

meniscus (DMM). The animals were treated weekly with an intraarticular injection of 10 l of vehicle or

protandim (10 g/ml) for 8 weeks. Sham-operated mice served as controls. In vitro, we demonstrated

that protandim and 6-gingerol preserve cell viability and mitochondrial metabolism and prevented 4-

hydroxynonenal (HNE)-induced cell mortality. They activated Nrf2 transcription factor, abolished IL-1-

induced NO, PGE2, MMP-13, and HNE production as well as IL-induced GSTA4-4 down-regulation.

Nrf2 overexpression reduced IL-1-induced HNE and MMP-13 as well as IL-1-induced GSTA4-4

down-regulation. Nrf2 knockdown following siRNA transfection abolished protandim protection against

oxidative stress and catabolism. The activation of MAPK and NF-B by IL-1 was not affected by 6-

gingerol. In vivo, we observed that Nrf2 and GSTA4-4 expression was significantly lower in OA

cartilage from humans and mice compared to normal controls. Interestingly, protandim administration

reduced OA score in DMM mice. Altogether, our data indicate that protandim and 6-gingerol are

essential in preserving cartilage and abolishing a number of factors known to be involved in OA

pathogenesis. This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved 2

INTRODUCTION

Osteoarthritis (OA), a subtype of arthritis, is the most common age-related chronic degenerative

disease of the joints that is considered to be the leading cause of physical disability. OA development

and progression are characterized by cartilage destruction, synovial inflammation, and subchondral

bone remodelling with heightened inflammatory and catabolic responses (Madry et al., 2012; Pereira

et al., 2015).

Among factors involved in OA process are cartilage-degrading metalloproteinase-13 (MMP-13),

inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2), and 4-hydroxynonenal (HNE)

(Pereira et al., 2015; Shi et al., 2014). Our research on OA has shown increased HNE production in

articular tissues, including cartilage and synovial membranes from humans and animals (Morquette et

al., 2006; Shi et al., 2014), compared to control tissues. Interestingly, we showed that the injection of

HNE into dog knee joints induce cartilage lesions, osteophyte formation and cartilage-degrading MMP-

13. In contrast, the administration of HNE-trapping carnosine attenuated OA process. In vitro,

exposure of human chondrocytes to non-toxic doses of HNE elicits a series of catabolic and

inflammatory genes involved in cartilage degradation and bone damage (Morquette et al., 2006;

Vaillancourt et al., 2007). In binding to proteins, HNE activates MMP-13 and increases the vulnerability

of type II collagen (Col II) to degradation (Golizeh et al., 2014; Morquette et al., 2006). We have

clarified the significance of increased HNE-Col II adducts in OA cartilage. We showed that interactions

between chondrocytes and HNE-Col II adducts, via adhesion molecules, strongly evokes signalling

pathways that trigger cell adhesion and viability as well as catabolic and inflammatory responses (El-

Bikai et al., 2010).

There is currently no effective treatment to prevent or stop cartilage destruction during OA and the

primary goal is to alleviate pain and other associated symptoms. Therefore, identification of agents

that down-regulate the expression of matrix degrading enzymes may prove useful for the prevention or

treatment of OA. The present study examines the biological effects of two natural products, namely

protandim and 6-gingerol, on the expression of a number of factors involved in cartilage damage in

vitro and in vivo OA models. Several lines of evidence indicated that both protandim and 6-gingerol

This article is protected by copyright. All rights reserved 3

strongly induce the levels of endogenous antioxidant enzymes through activating the transcription

factor Nrf2 (Joddar et al., 2011; Lisk et al., 2013). Interestingly, the protection of protandim against

oxidative stress was abolished when bovine brain microvascular endothelial cells were transfected

with siRNA Nrf2 (Lisk et al., 2013). Moreover, protandim and 6-gingerol are able to block the

generation of lipid peroxidation, the depletion of antioxidant status, and NF-B-mediated inflammatory

responses (Joddar et al., 2011).

Here, we demonstrate that chondrocyte treatment with protandim and 6-gingerol activates Nrf2 and

reduces HNE toxicity and interleukin-1β (IL-1β)-induced HNE, nitric oxide (NO), MMP-13,

prostaglandin E2 (PGE2) production as well as IL-1β-induced glutathione-s-tranferase A4-4 (GSTA4-4)

down-regulation. These biological effects required Nrf2 expression. Moreover, experiments on a

mouse model of OA disclosed that protandim attenuate OA development, indicating its potential as a

therapeutic agent in OA.

This article is protected by copyright. All rights reserved 4

MATERIALS AND METHODS

Specimen selection

All patients were evaluated by rheumatologists according to American College of Rheumatology

criteria (Altman et al., 1986). Normal articular cartilage was obtained from patients aged 58 ± 6 years

(mean ± SD) requiring hip joint arthroplasty after traumatic injury with no history of arthritis. OA

articular cartilage was sampled from OA patients (64 ± 7 years (mean ± SD) undergoing total knee

arthroplasty. All specimens were collected within the first 24 h post-surgery. Informed consent was

obtained from OA patients to use their tissues for research purposes. The Research Ethics Board of

Hôpital du Sacré-Coeur de Montréal approved the experimental protocol and use of human tissues.

Sample preparation and isolation of chondrocytes

OA knee cartilage samples were collected aseptically from each specimen, spliced and rinsed for

chondrocyte isolation, as described previously (El-Bikai et al., 2010). They were digested by pronase

(1 mg/ml, Sigma-Aldrich, Oakville, ON, Canada) for 1 h at 37ºC and then by type IV collagenase (0.5

mg/ml, Sigma-Aldrich) for 6 h in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies,

Burlington, ON, Canada) completed with 10% heat-inactivated fetal bovine serum (FBS; Life

Technology, Inc.) and a antibiotic mixture containing 100 units/ml of penicillin and 100 μg/ml of

streptomycin (Life Technology, Inc.). Cells were seeded in culture flasks (105/cm2) and cultured at

37ºC in a humidified atmosphere of 5% CO2/95% air.

Cell culture

When the cells were confluent they were seeded in tissue culture plates (105 cells/cm2) and incubated

for 48 h before the experimental procedures were performed. To preserve cell phenotype, only first-

passage chondrocytes were studied. The experiments were undertaken in DMEM containing 1% FBS

and antibiotics with the factors under study. Chondrocytes were pre-treated with protandim (0-10

g/ml, generously provided by Dr. J.M. McCord, University of Colorado, Denville, CO, USA) or 6-

This article is protected by copyright. All rights reserved 5

gingerol (0-10 M, Sigma-Aldrich) for 1 h. They were then treated with either IL-1β (1 ng/ml) or HNE

(20 M) for 24 h.

Cartilage homogenization

Normal and OA cartilage explants (~50 mg) from humans were homogenized on ice in 1 ml of

extraction buffer containing 40 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 M

guanidine hydrochloride, and protease inhibitor cocktail (Sigma-Aldrich), as described by Morquette et

al. (Morquette et al., 2006). Cartilage extracts were dialyzed 24 h against extraction buffer at 4°C and

then conserved at -80°C until use

Protein detection by W estern blotting

20 µg of total proteins from cartilage or chondrocytes extracts were applied to 4-12% sodium dodecyl

sulfate (SDS)-polyacrylamide gel for electrophoresis. They were then electro-blotted onto

nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). After protein transfert,

membranes were blocked using commercial blocking buffer (Pierce) and then incubated with the

following primary antibodies: mouse anti-human -actin (Sigma-Aldrich), mouse anti-human total Nrf2

(R&D Systems, Minneapolis, MN, USA), mouse anti-human GSTA4-4 (Abcam, Inc., Cambridge, MA,

USA), and rabbit anti-human pp38 MAPK, ppJNK1/2, pERK1/2, and NF-B/p65 (Cell Signaling). After

serial washes, the membranes were incubated with anti-mouse IgG conjugated to horseradish

peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Immunoreactive

bands were visualised with SuperSignal blotting substrate (Pierce Biotechnology, Inc., Rockford, IL,

USA) using half-blue x-ray film (Clonex Corporation, Markham, ON, Canada). Data are presented as

fold change over control values

The phosphorylation status of Nrf2

This article is protected by copyright. All rights reserved 6

Experiments were conducted in OA chondrocytes treated with increasing doses of prodandim and 6-

gingerol for 60 min, as described in cell culture section. Cells were then washed with PBS and then

nuclear proteins were extracted, as indicated in our previous report (Vaillancourt et al., 2007). The

levels of phosphorylated Nrf2 was determined in nuclear proteins by Western blot using mouse anti-

phosphorylated Nrf2 (R&D Systems).

Determination of MMP-13, NO, and PGE2 in conditioned

medium

MMP-13 and PGE2 were quantified by human pro-MMP-13 enzyme-linked immunosorbent assay

(ELISA) kit (R&D Systems) and by enzyme immunoassay (Cayman Chemical Company, Ann Arbor,

Ml, USA), respectively, according to the manufacturer’s instructions. Sensitivity of the kits was 8 and 9

pg/ml, respectively. Nitrite, a stable end-product of NO, was quantified by spectrophotometry based on

Griess reaction (Green et al., 1982). All assays were performed in duplicate. Absorbance was

measured by micro-ELISA Vmax photometer (Bio-Tek Instruments, Winooski, VT, USA).

Viability test

Chondrocyte viability was evaluated in 96-well plates by CellTiter 96® Non-Radioactive Cell

Proliferation Assay (Promega) according to the manufacturer's recommendation (Mosmann, 1983).

After 24 and 48 h of incubation at 37ºC, cells were incubated for 4 hours with 20% of Dye Solution. A

volume of 100 μl of solubilization solution was then added to dissolve the formazan salt formed.

Absorbance was measured at 570 nm by micro-ELISA Vmax photometer (Bio-Tek Instruments).

Determination of mitochondrial NADP -isocitrate dehydrogenase (mNADP -

ICDH)
The activity of mNADP-ICDH was quantified in isolated human chondrocytes according to our

published method (Shi et al., 2012). Briefly, cells (106/well) were treated for 24 h with 6-gingerol (0-10

M) or protandim (0-10 g) and then mitochondria were isolated with commercial kit according to the

manufacturer's instructions (abcam, Cambridge, MA, USA). They were resuspended in phosphate-

This article is protected by copyright. All rights reserved 7

buffered saline containing 0.1% Triton X-100, sonicated on ice for 5 sec at maximal intensity (40 W),

and centrifuged at 12 000 g for 10 min. The activity of mNADP-ICDH was measured in the

supernatants and expressed as units/mg proteins, where 1 unit is as the amount of enzyme catalyzing

the conversion of 1 μmol substrate/min at 37°C.

RNA extraction and quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from the chondrocytes with TRIzol reagent according to the manufacturer's

instructions (Life Technologies). RiboGreen RNA quantitation kits (Molecular Probes, Eugene, OR,

USA) was used to assess RNA concentration. mRNA was reverse transcribed and amplified using

commercial kits from Qiagen (Mississauga, ON, Canada) and monitored in real time with a Mx3000

PCR system (Stratagen, La Jolla, CA, USA). The qPCR was performed as described previously using

the following specific sense and antisense primers (Bio-Corp Inc., Montreal, QC, Canada),: human

GSTA4-4 5’-CCG GAT GGA GTC CGT GAG ATG G-3’ (forward) and 5'-CCA TGG GCA CTT GTT

GGA ACA GC-3’ (reverse); mouse GSTA4-4, 5 -GGA AGA ACT CAG TGC CCC TGT AC-3’ (forward)

and 5 -TGT AGG AAT GTT GCT GAT TCT TGT C-3’ (reverse); human Nrf2 5'-CCT CAA CTA TAG

CGA TGC TGA AT-3’ (forward) and 5'-AGG AGT TGG GCA TGA GTG AG-3’ (reverse); mouse Nrf2,

5′-AGC AGG ACA TGG AGC AAG TT-3’ (forward) and 5′-TTC TTT TTC CAG CGA GG AGA-3’

(reverse); human GAPDH 5'-CAT CAA GAA GGT GGT GAA GCA G-3’ (forward) and 5'-CGT CAA

AGG TGG AGG AGT GG-3 (reverse); mouse GAPDH, 5′-TGG AAT CCT GTG GCA TCC ATG-3′

(forward) and 5′-TAA AAC GCA GCT CAG TAA CAG TC-3′ (reverse).

Relative mRNA expression in chondrocytes was quantified by the ΔΔCt method, as detailed in our

previous report (El-Bikai et al., 2010). Each PCR was run in triplicate on 2 separate occasions for each

independent experiment (n=3-4).

Mouse model of OA

The study was approved by the local animal care committee of the Hôpital du Sacré-Cœur de

Montréal and in accordance with guidelines of the Canadian Council on Animal Care. 10-week-old

This article is protected by copyright. All rights reserved 8

male mice were recruited to the present protocol. Briefly, anesthesia was induced and maintained by

isoflurane and O2 and OA was induced surgically in 10-week-old male C57BL/6 mice by

destabilization of the medial meniscus (DMM) in the right knee (Amiable et al., 2011). Animal were

divided into three groups (n=6/group) as follow: Sham, DMM, and DMM+protandim. The mice were

anesthetized with isoflurane and O2, and the right knee joint was destabilized by transection of the

anterior medial meniscus attachment to the tibial plateau. Sham operation consists an incision on the

right to the knee without compromising the joint capsule, was also performed on the right knee of 10-

week-old C57BL/6 mice. Each week, mice with DMM received intraarticular injection (10 l) of saline

solution (0.9 g/l NaCl) or protandim at 10 g/ml from day 1 during 8-weeks. They were observed daily

to verify healing and to ensure that they were using their right limb.

Cartilage histology

Operated (sham and OA) mice were euthanized at 8 weeks after surgery. OA severity was assessed

by evaluating isolated knee joints histomorphometrically. The right knee joints were dissected free of

skin tissue, fixed in TissuFix (Chaptec, Inc., Montreal, QC, Canada), decalcified in Rapid Bone

Decalcifier (Apex Engineering, Aurora, IL, USA), and embedded in paraffin (Amiable et al., 2011).

Briefly, frontal histological sections (5 µM) were obtained throughout the knee joint at 80-µM intervals

and deparaffinized in xylene, followed by a graded series of alcohol washes, stained with Safranin O-

fast green (Sigma-Aldrich), and scored by 2 blinded observers according to the OsteoArthritis

Research Society International (OARSI) scoring system (Pritzker et al., 2006), which grades cartilage

structure and PG content on a scale of 0-6, where 0 = normal cartilage, 0.5 = loss of PGs without

structural changes, 1 = superficial fibrillation without structural changes, 2 = vertical clefts and loss of

surface lamina, 3 = vertical clefts/erosion of the calcified layer lesion for 1-25% of quadrant width, 4 =

lesions reach calcified cartilage for 25-50% of quadrant width, 5 = lesions reach calcified cartilage for

50-75% of articular surface, and 6 = lesions reach calcified cartilage for >75% of quadrant width). All

This article is protected by copyright. All rights reserved 9

joint quadrants (medial tibial plateau, medial femoral condyle, lateral tibial plateau, and lateral femoral

condyle) were scored separately and added to obtain summed OA scores for the whole joint.

Plasmids and transient Nrf2 transfection

Empty and expression vectors of Nrf2 were gifted by Dr. Y. Xiong (University of North Carolina at

Chapel Hill, Chapel Hill, NC, USA). Nrf2 small interfering RNA (siRNA) and randomly sequenced

siRNA as negative controls were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Subconfluent chondrocytes were transiently transfected by Lipofectamine 2000TM reagent (Life

Technologies) according to the manufacturer’s protocol. Briefly, transfections were conducted for 6 h

with DNA lipofectamine complexes containing either 100 nM Nrf2 siRNA, randomly sequenced siRNA,

2 µg empty DNA plasmid, or 2 g Nrf2 expression plasmid. After washing, experiments were

performed in 2% FBS fresh medium supplemented or not with 1 ng/ml IL-1β or 10 g protandim. Then,

the following parameters were documented as described above: Nrf2 expression, MMP-13 release,

HNE generation, GSTA4-4 activity.

Cellular level of HNE -protein adducts

Total cellular levels of HNE-protein adducts were assessed by ELISA, as described previously

(Benderdour et al., 2003). HNE-modified bovine serum albumin (Sigma-Aldrich) served as standard.

Statistical analyses

Statistical analysis was conducted with 2-tailed paired Student’s t test, and differences less than or

equal to 0.05 were considered significant. All statistics were generated using GraphPad Prism

software (GraphPad Software, San Diego, CA, USA).

This article is protected by copyright. All rights reserved 10

RESULTS

Cytotoxicity studies of protandim and 6-gingerol

To reach our first objective, we conducted initial experiments to ascertain the cytotoxicity of protandim

and 6-gingerol. Chondrocytes were first treated with these substances at increasing concentrations for

24 and 48 h. The results obtained and depicted in Figure 1A show no change in cell viability with

protandim (0-10 g/ml) and 6-gingerol (0-10 M).

To examine the protective effect of protandim and 6-gingerol against oxidative stress-induced cell

death, cells were incubated with HNE at 20 M in the presence or absence of both products. We

report that up to 10 M HNE did not alter cell viability, but 20 M HNE was cytotoxic and significantly

decreased cell viability by approximately 50% (Vaillancourt et al., 2008). Cell viability assay, depicted

in Figure 1B, revealed dose-dependent protection by protandin and 6-gingerol against HNE-induced

cell death. Maximum protection was observed with 10 M 6-gingerol and reached 105% in comparison

to HNE-treated cells. As an indicator of mitochondrial energy, we documented the activity of mNADP-

ICDH in the isolated mitochondria. As illustrated in Figure 1C, the incubation of treated chondrocytes

with increasing doses of protandim and 6-gingerol had no effect on mNADP-ICDH activity, suggesting

that both products preserved mitochondrial function. Taken together, these data indicate that

protandim and 6-gingerol at the tested doses are safe, protect cells from the cytotoxicity of oxidative

stress, and maintain mitochondrial energy metabolism.

Protandim and 6-gingerol induce Nrf2 expression and phosphorylation

We performed additional experiments to demonstrate that protandim and 6-gingerol induce Nrf2

expression and phosphorylation in isolated human OA chondrocytes. Cells were treated for 24 h with

increasing doses of protandim (0-10 g) or 6-gingerol (0-10 M). The chondrocyte treatment with

either protandim or 6-gingerol significantly enhanced Nrf2 expression at protein (Figs. 2A & 2C) and

mRNA (Figs. 2B & 2D) levels in a dose-dependent manner. At 10 g, protandim was the most potent

inducer of Nrf2 expression (380% vs untreated cells, P<0.001). Moreover, Nrf2 requires

This article is protected by copyright. All rights reserved 11

phosphorylation for its transactivation and DNA binding. Thus, we analyzed the phosphorylation status

of Nrf2 in protandim and 6-gingerol-treated chondrocytes. As seen in Figures 2E and 2F, the addition

of these products to cultured cells elicited Nrf2 phosphorylation, in dose-dependent manner.

Collectively, our data strongly show that protandim and 6-gingerol are potent Nrf2 activators in human

OA chondrocytes.

Protandim and 6-gingerol inhibit IL-1-induced catabolic and inflammatory mediators

In this study, we tested the influence of protandim and 6-gingerol on IL-1β-induced release of

inflammatory and catabolic mediators, such as PGE2, NO and MMP-13 in isolated human OA

chondrocytes. Cells were pre-incubated with increasing concentrations of protandim or 6-gingerol for 1

h, followed by stimulation with 1 ng of IL-1β for 24 h. Interestingly, as seen in Figure 3, protandim or 6-

gingerol dose-dependently reduced IL-1β-induced NO (A), PGE2 (B) and MMP-13 (C). At 10 µM, 6-

gingerol was the most potent inhibitor of these mediators compared to protandim. Altogether, our

findings suggest that inhibition of NO, PGE2 and MMP-13 by protandim and 6-gingerol may contribute

to reduction of inflammation and catabolism.

Protandim and 6-gingerol prevent IL-1-induced oxidative stress

To investigate the oxidative stress elicited by IL-1, human OA chondrocytes were pre-treated or not

with 10 g/ml of protandim or 10 M 6-gingerol for 1 h and then exposed to 1 ng/ml IL-1 for 24 h.

Nrf2 and HNE-detoxifying GSTA4-4 mRNA expression was measured by qPCR and HNE level was

ascertained by ELISA. As illustrated in Figure 4, incubation of cells with protandim or 6-gingerol

abolished IL-1-induced Nrf2 and GSTA4-4 down-regulation (Fig. 4A & 4B) and HNE production (Fig.

4C). These findings suggest that protandim and 6-gingerol are essential to restore redox status in

human OA chondrocytes, probably through Nrf2 activation.

Nrf2 overexpression attenuates IL-1β-induced oxidative stress and catabolism

This article is protected by copyright. All rights reserved 12

This experiment was designed to investigate whether Nrf2 overexpression is essential to block IL-1β-

induced GSTA-4-4 down-regulation as well as MMP-13 and HNE production. Briefly, human OA

chondrocytes were transiently transfected with Nrf2 expression vector or empty vectors and then

treated or not with 1 ng/ml IL-1β for 24 h. Nrf2 overexpression (Fig. 5A) significantly abolished IL-1β-

induced GSTA4-4 down-regulation (Fig. 5B), HNE generation (Fig. 5C), and MMP-13 release (Fig.

5D). Taken together, our data confirm that Nrf2 is a key transcription factor that regulates cellular

redox status and cartilage-degrading MMP-13.

Nrf2 silencing abolishes protandim effects

To determine whether protandim and 6-gingerol effects required Nrf2 expression, isolated

chondrocytes are transfected with sc-siRNA (control) or Nrf2 siRNA and then treated with 1 ng/ml IL-

1 for 24 h in the presence or absence of 10 g/ml protandim. Figure 5E showed specific siRNA-

mediated ablation of Nrf2. As illustrated in Figure 5F, the addition of protandim reduced HNE

production and enhanced GSTA4-4 expression in sc-siRNA transfected cells (control). In contrast, the

ablation of Nrf2 by siRNA blocked the protective effect of protandim against IL-1-induced oxidative

stress.

6-Gingerol had no effect on IL-1-induced MAPKS and NF-B activation

To elucidate the signalling pathways that are regulated by IL-1, we analyzed members of the MAPKs

and NF-B pathways by Western blotting with protein samples from human OA chondrocytes that had

been preincubated with 10 M 6-gingerol for 1 h, followed by 5, 15, 30, 60 min of stimulation with 1

ng/ml IL-1 or preincubated with increased dose of 6-gingerol (0-10 M) followed by another

incubation for 30 min in the presence of 1 ng/ml IL-1. As illustrated in Figure 6, and combined with IL-

1, 6-gingerol at 10 M had no effect on IL-1-induced phosphorylation of p38 MAPK, JNK1/2, ERK1/2,

and p65-NF-B in time (Fig. 6A) and dose (Fig. 6B) dependent manner.

This article is protected by copyright. All rights reserved 13

Nrf2 and GSTA4-4 expression decreases in articular OA cartilage

This part of the present study was designed to investigate the expression profile of both Nrf2 and

GSTA4-4 in OA cartilage from humans and mice. Our findings are reported in Figure 7. Nrf2 and

GSTA4-4 protein (Fig. 7A) and mRNA (Fig. 7B and 7C) expression levels were ~50% lower in OA

cartilage than in normal controls. These observations strongly suggest that alteration of Nrf2 and

GSTA4-4 expression would contribute to HNE accumulation in articular tissues and, consequently,

predisposes to cartilage degradation and synovium inflammation.

Intra-articular injection of protandim reduces cartilage degradation in the DMM mouse model of

OA

To further evaluate the overall protective capability of protandim, we tested the influence of intra-

articular injection of this product in the surgical DMM model of OA. Each week, mice were received or

not 10 l of vehicle or of protandim (10 g/ml) for 8 weeks post-surgery. After sacrifice, histological

cartilage samples from the knee were assessed for signs of cartilage degradation, such as fibrillation

with loss of chondrocytes and aggrecan. Each sample was assigned a score according to the OARSI

scoring system. Figure 8B reports that intra-articular injection of protandim is associated with lower

OARSI scores and much lower cartilage degradation than vehicle (Fig. 8A). Figure 8C shows no

proteoglycans loss in mice knee joints (sham control joint).

This article is protected by copyright. All rights reserved 14

DISCUSSION

The present study was designed to investigate the beneficial effects of protandim and 6-gingerol in

OA. In isolated human OA chondrocytes, we showed that both products suppress HNE accumulation

and upregulate the key endogenous phase II antioxidant GSTA4-4. Furthermore, treatment of cultured

isolated human chondrocytes with protandim or 6-gingerol resulted in Nrf2 up-regulation and

activation, cell protection against HNE-induced toxicity and IL-1-induced MMP-13, NO and PGE2.

Then, we conducted additional experiments to investigate whether changes in Nrf2 expression have

an impact on oxidative stress as well as catabolic and inflammatory responses. In OA chondrocytes,

overexpression of this transcription factor offered significant protection against IL-1-induced HNE

accumulation, GSTA4-4 inhibition, and MMP-13 production. Moreover, Nrf2 overexpression confers

chondrocyte resistance to HNE toxicity (data not shown). Augmented cell resistance to HNE in Nrf2-

transfected cells could be attributed to an increased rate of HNE metabolism by GSTA4-4 up-

regulation. Interestingly, the ablation of Nrf2 by siRNA suppresses the effect protandim. Collectively,

these data support the notion that Nrf2 activation by protandim and 6-gingerol in OA chondrocytes

could be essential to restore redox status and to inhibit a number of mediators involved in cartilage

degradation. In vivo, we provided evidence that both products attenuate the initiation of cartilage

lesions in experimental mouse OA induced by DMM. Moreover, the expression profile of Nrf2 and

Nrf2-regulating GSTA4-4 is lower in OA cartilage as comparared to normal. Collectively, our study

provides the strongest evidence to date that protandim and 6-pingerol suppress cartilage damage,

most likely through Nrf2 activation.

Several natural and synthetic compounds are being investigated and/or developed in an attempt to

regulate and enhance Nrf2 transcriptional activity. Though activation occurs mainly by Nrf2 release

from Keap1, several scenarios have been proposed (Huang et al., 2002). It has been shown that PKC

catalyzes Nrf2 phosphorylation and promotes Nrf2 dissociation from Keap1. The anticipated

subsequent increase in expression of cytoprotective molecules endows such activators with great

benefit, especially in inflammatory disorders. Finding a specific natural Nrf2 activator that is

pharmacologically effective for a specific disorder is still a huge struggle (Hybertson et al., 2011).

This article is protected by copyright. All rights reserved 15

Amongst the promising synthetic molecules being investigated as specific activators of Nrf2, the

derivative of oleanolic acid and the triterpenoid derivative of dihydro-CDDO-trifluoroethyl

amide (dh404), for example, hold great therapeutic potential in several pathological conditions

(Ichikawa et al., 2009). The authors reported that dh404 was able to enhance Nrf2 nuclear

translocation, to induce Nrf2-driven transcription, and to abolish angiotensin II-induced oxidative stress

in isolated cardiomyocytes. The ablation of Nrf2 almost blocked the anti-oxidative effect of dh404.

By investigation of different signaling pathways, we obtained data indicating that activation of Nrf2 by

6-gingerol had no effect on IL-1-induced activation of p38 MAPK, JNK1/2, ERK1/2, and NF-B. These

findings suggest that inhibition of downstream target genes expression of Nrf2 is not related to MAPKs

and NF-B signaling pathways. Indeed, Nrf2 activation has been shown to regulate AP-1 and NF-B

expression and directly modify their binding to DNA (Yang et al., 2005).

Among the natural Nrf2 activators studied, both protandim and 6-gingerol hold great potential

(Hybertson et al., 2011; Lee et al., 2011). Protandim is a commercial dietary supplement composed of

five phytochemicals with antioxidant properties. The mixture of these natural Nrf2 activators enhances

the expression of endogenous antioxidant enzymes through multiple kinase pathways (Velmurugan et

al., 2009). Moreover, dietary protandim administration has a chemopreventive effect in mice along with

reduction of oxidative stress and LPO markers (Liu et al., 2009).

6-Gingerol also seems to be a potent Nrf2 activator. Lee et al. (Lee et al., 2011) have reported that 6-

gingerol exhibits preventive and/or therapeutic potential in the management of Alzheimer’s disease via

the augmentation of antioxidant capacity and Nrf2 transactivation. In a recent investigation, Mohamed

et al. (Mohamed et al., 2015) noted that ginger extract containing 7% gingerol has an antioxidant

protective effect against lead hepatotoxicity via the induction of GST expression and the reduction of

malondialdehyde, another LPO product.

In the present study, we demonstrated that Nrf2 activation and overexpression control HNE generation

and GSTA4-4 expression. Our results concur with those in the literature, that GSTs are regulated by

Nrf2 transactivation (Wu et al., 2012). Loss or disturbance of Nrf2 activity greatly affects the inducible

expression of GSTs and leaves cells more vulnerable to oxidative stress (Kensler et al., 2007). These

This article is protected by copyright. All rights reserved 16

enzymes play a key role in cellular metabolism and detoxification. GST facilitates conjugation between

the GSH molecule and an electrophilic center like HNE, preventing their interaction with cellular

components. In fact, GST is over 600 times more active than non-enzymatic Michael addition

reactions (Siems and Grune, 2003). Increased GST activity neutralizing HNE can lead to a

concomitant, fast drop in intracellular glutathione (GSH) concentrations (Siems and Grune, 2003).

GSTs could also be instrumental in reducing hydrogen peroxide formation during the LPO process,

lowering HNE generation (Awasthi et al., 2004). Among different GST isoforms, GSTA4-4 is

instrumental in HNE detoxification in tissues because of its high catalytic efficiency with the toxic

aldehyde (Balogh and Atkins, 2011). On the one hand, studies on OA have determined that loss of

GSTA4-4 enhances the cytotoxic effect of HNE on chondrocytes. On the other hand, the enzyme’s

overexpression protects against HNE-induced cell death (Vaillancourt et al., 2008).

The GSTA4-4 isoform is important in HNE detoxification and in protecting cells against HNE-induced

apoptosis (Balogh and Atkins, 2011; Vaillancourt et al., 2008). Interestingly, HNE can trigger Nrf2

release from Keap1, ultimately inducing GSTA4-4 expression through Nrf2-ARE activity (Raza and

John, 2006; Zheng et al., 2014). This adaptive cellular mechanism is programmed to counteract HNE

accumulation. Upon activation, Nrf2 can indirectly inhibit HNE production and subsequently the effect

of free HNE on signal transduction pathways in OA chondrocytes by stimulating GSTA4-4 gene

transcription.

Data from our current study indicate a profound reduction of both Nrf2 and GSTA4-4 protein and

mRNA levels in OA cartilage from humans and mice compared to the controls, and that IL-1

promotes their inhibition in human OA chondrocytes. From these findings, we speculate that Nrf2 and

GSTA4-4 down-regulation in OA could be attributed to enhanced pro-inflammatory cytokines,

including IL-1.Furthermore, our results prompted us to hypothesize that loss of Nrf2 in articular

cartilage could reduce GSTA4-4 expression and activity, and consequently induce an oxidative stress

phenotype in chondrocytes. Until now, the role of Nrf2 and GSTA4-4 in OA has not been reported.

Disruption of GSTA4-4 genes enhances HNE tissue levels, as verified experimentally in 129/sv

GSTA4-4-/- mice (Engle et al., 2004). GSTA4-4 null mice have a short lifespan, underscoring the

This article is protected by copyright. All rights reserved 17

possible relationship between HNE and aging. According to our unpublished data on the DMM mouse

model of OA, GSTA4-4 null mice are susceptible to rapid OA development compared to wild type

(WT) controls (study in progress). The involvement of the transcription factor Nrf2 in arthritis has been

reported in Nrf2 null mice; an experimental model of rheumatoid arthritis (RA) (Maicas et al., 2011). It

has been shown that Nrf2 deficiency significantly enhances the severity of arthritis. COX-2, iNOS, and

peroxynitrite expression in the joints is higher in Nrf2 deficiency, whereas heme-oxygenase-1 is down-

regulated (Maicas et al., 2011). While OA is a degenerative disease, RA, on the other hand, is a

systemic disorder. Though both conditions have different pathologies, they share the presence of high

level inflammation.

CONCLUSION

Our observations revealed promising outcomes of protandim and 6-gingerol in the prevention of OA

cartilage degradation, mainly via Nrf2 activation. They seem to attenuate oxidative stress and

catabolic as well as inflammatory responses through GSTA4-4 activation and inhibition of HNE

production. As well, our study resulted in greater understanding of the biochemical and molecular

mechanisms involved in HNE accumulation. These mechanisms include declines of GSTA4-4 as well

as Nrf2, an important transcription factor involved in GSTA4-4 regulation and cell function. Future

studies will probably lead to the development of the first molecular diagnostic tools to identify

individuals at risk of developing OA. Other approaches include the development of innovative

therapeutics to maintain or reactivate Nrf2 expression and, consequently, GSTA4-4 in articular

cartilage by pharmacological interventions, as proposed in Figure 10.

ACKNOWLEDGMENTS

This study was supported by Canadian Institutes of Health Research grant (#IMH 131570 for Dr

Mohamed Benderdour) and by the Centre of Excellence for Arthroplasty Research (for Dr Mohamed

Benderdour).

This article is protected by copyright. All rights reserved 18

REFERENCES

Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R,
Hochberg M, et al. 1986. Development of criteria for the classification and reporting of osteoarthritis.
Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the
American Rheumatism Association. Arthritis Rheum 29:1039-1049.
Amiable N, Martel-Pelletier J, Lussier B, Kwan Tat S, Pelletier JP, Boileau C. 2011. Proteinase-
activated receptor-2 gene disruption limits the effect of osteoarthritis on cartilage in mice: a novel
target in joint degradation. The Journal of rheumatology 38: 911-920.
Awasthi YC, Yang Y, Tiwari NK, Patrick B, Sharma A, Li J, Awasthi S. 2004. Regulation of 4-
hydroxynonenal-mediated signaling by glutathione S-transferases. Free radical biology & medicine
37:607-619.
Balogh LM, Atkins WM. 2011. Interactions of glutathione transferases with 4-hydroxynonenal. Drug
Metab Rev 43:165-178.
Benderdour M, Charron G, DeBlois D, Comte B, Des Rosiers C. 2003. Cardiac mitochondrial NADP+-
isocitrate dehydrogenase is inactivated through 4-hydroxynonenal adduct formation: an event that
precedes hypertrophy development. J Biol Chem 278:45154-45159.
El-Bikai R, Welman M, Margaron Y, Cote JF, Macqueen L, Buschmann MD, Fahmi H, Shi Q, Maghni
K, Fernandes JC, Benderdour M. 2010. Perturbation of adhesion molecule-mediated chondrocyte-
matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis. Arthritis
research & therapy 12:R201.
Engle MR, Singh SP, Czernik PJ, Gaddy D, Montague DC, Ceci JD, Yang Y, Awasthi S, Awasthi YC,
Zimniak P. 2004. Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-
hydroxynonenal: generation and analysis of mGsta4 null mouse. Toxicol Appl Pharmacol 194:296-
308.
Golizeh M, Abusarah J, Benderdour M, Sleno L. 2014. Covalent binding of 4-hydroxynonenal to matrix
metalloproteinase 13 studied by liquid chromatography-mass spectrometry. Chem Res Toxicol
27:1556-1565.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. 1982. Analysis of
nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126:131-138.
Huang HC, Nguyen T, Pickett CB. 2002. Phosphorylation of Nrf2 at Ser-40 by protein kinase C
regulates antioxidant response element-mediated transcription. J Biol Chem 277:42769-42774.
Hybertson BM, Gao B, Bose SK, McCord JM. 2011. Oxidative stress in health and disease: the
therapeutic potential of Nrf2 activation. Mol Aspects Med 32:234-246.
Ichikawa T, Li J, Meyer CJ, Janicki JS, Hannink M, Cui T. 2009. Dihydro-CDDO-trifluoroethyl amide
(dh404), a novel Nrf2 activator, suppresses oxidative stress in cardiomyocytes. PLoS One 4:e8391.
Joddar B, Reen RK, Firstenberg MS, Varadharaj S, McCord JM, Zweier JL, Gooch KJ. 2011.
Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-
dependent pathway. Free radical biology & medicine 50:700-709.
Kensler TW, Wakabayashi N, Biswal S. 2007. Cell survival responses to environmental stresses via
the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 47:89-116.
Lee C, Park GH, Kim CY, Jang JH. 2011. [6]-Gingerol attenuates beta-amyloid-induced oxidative cell
death via fortifying cellular antioxidant defense system. Food Chem Toxicol 49:1261-1269.
Lisk C, McCord J, Bose S, Sullivan T, Loomis Z, Nozik-Grayck E, Schroeder T, Hamilton K, Irwin DC.
2013. Nrf2 activation: a potential strategy for the prevention of acute mountain sickness. Free radical
biology & medicine 63:264-273.
Liu J, Gu X, Robbins D, Li G, Shi R, McCord JM, Zhao Y. 2009. Protandim, a fundamentally new
antioxidant approach in chemoprevention using mouse two-stage skin carcinogenesis as a model.
PLoS One 4:e5284.
Madry H, Luyten FP, Facchini A. 2012. Biological aspects of early osteoarthritis. Knee Surg Sports
Traumatol Arthrosc 20:407-422.

This article is protected by copyright. All rights reserved 19

Maicas N, Ferrandiz ML, Brines R, Ibanez L, Cuadrado A, Koenders MI, van den Berg WB, Alcaraz
MJ. 2011. Deficiency of Nrf2 accelerates the effector phase of arthritis and aggravates joint disease.
Antioxid Redox Signal 15:889-901.
Mohamed OI, El-Nahas AF, El-Sayed YS, Ashry KM. 2016. Ginger extract modulates Pb-induced
hepatic oxidative stress and expression of antioxidant gene transcripts in rat liver. Pharm Biol
54:1164-72 .
Morquette B, Shi Q, Lavigne P, Ranger P, Fernandes JC, Benderdour M. 2006. Production of lipid
peroxidation products in osteoarthritic tissues: new evidence linking 4-hydroxynonenal to cartilage
degradation. Arthritis Rheum 54:271-281.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation
and cytotoxicity assays. J Immunol Methods 65:55-63.
Pereira D, Ramos E, Branco J. 2015. Osteoarthritis. Acta Med Port 28:99-106.
Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, Salter D, van den Berg WB.
2006. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 14:13-29.
Raza H, John A. 2006. 4-hydroxynonenal induces mitochondrial oxidative stress, apoptosis and
expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells. Toxicol Appl
Pharmacol 216:309-318.
Shi Q, Abusarah J, Baroudi G, Fernandes JC, Fahmi H., Benderdour M. 2012. Ramipril attenuates
lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis. Arthritis
research & therapy 14:R223.
Shi Q, Abusarah J, Zaouter C, Moldovan F, Fernandes JC, Fahmi H, Benderdour M. 2014. New
evidence implicating 4-hydroxynonenal in the pathogenesis of osteoarthritis in vivo. Arthritis
Rheumatol 66:2461-2471.
Siems W, Grune T. 2003. Intracellular metabolism of 4-hydroxynonenal. Mol Aspects Med 24:167-175.
Vaillancourt F, Fahmi H, Shi Q, Lavigne P, Ranger P, Fernandes JC, Benderdour M. 2008. 4-
Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of
glutathione-S-transferase. Arthritis research & therapy 10:R107.
Vaillancourt F, Morquette B, Shi Q, Fahmi H, Lavigne P, Di Battista JA, Fernandes JC, Benderdour M.
2007. Differential regulation of cyclooxygenase-2 and inducible nitric oxide synthase by 4-
hydroxynonenal in human osteoarthritic chondrocytes through ATF-2/CREB-1 transactivation and
concomitant inhibition of NF-kappaB signaling cascade. J Cell Biochem 100:1217-1231.
Velmurugan K, Alam J, McCord JM, Pugazhenthi S. 2009. Synergistic induction of heme oxygenase-1
by the components of the antioxidant supplement Protandim. Free radical biology & medicine 46:430-
440.
Wu KC, Cui JY, Klaassen CD. 2012. Effect of Graded Nrf2 Activation on Phase-I and -II Drug
Metabolizing Enzymes and Transporters in Mouse Liver PLoS One. 7: e39006.
Yang H, Magilnick N, Lee C, Kalmaz D, Ou X, Chan JY, Lu, SC. 2005. Nrf1 and Nrf2 regulate rat
glutamate-cysteine ligase catalytic subunit transcription indirectly via NF-kappaB and AP-1. Molecular
and cellular biology 25:5933-5946.
Zheng R, Heck DE, Mishin V, Black AT, Shakarjian MP, Kong AN, Laskin DL, Laskin JD. 2014.
Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end
product. Toxicol Appl Pharmacol 275:113-121.

This article is protected by copyright. All rights reserved 20

FIGURE LEGENDS

Figure 1: Protandim and 6-gingerol are safe, attenuate HNE-induced cell death and preserve

mitochondrial metabolism. Human OA chondrocytes were incubated with protandim or 6-gingerol at

different concentrations for 24 or 48 h (A) or for 1 h, followed by another incubation for 24 h in the

presence or absence of 20 M HNE (B). Cell viability was evaluated by MTT assay. (C) Cells were

treated with increasing dose of protandim or 6-gingerol and then mNADP-ICDH activity was measured

in isolated mitochondria as indicated in Matherial and Methods. Data are the means ± SEM of 4

independent experiments. *P<0.05, **P<0.01, ***P<0.001 (compared to untreated cells); #P<0.05,

&
P<0.01, @P<0.001 (compared to HNE).

Figure 2: Protandim and 6-gingerol induce Nrf2 expression and phosphorylation. Human OA

chondrocytes were pre-incubated with Protandim or 6-gingerol for 24 h. Total Nrf2 protein (A & C) and

phosphorylated Nrf2 (E & F) was ascertained by Western blotting. Nrf2 mRNA (B & D) expression was

determined by qPCR. Data are the means ± SEM of 4 independent experiments. *P<0.05, **P<0.01,

***P<0.001 (compared to untreated cells).

Figure 3: Protandim and 6-gingerol reduce IL-1-induced NO, PGE2 and MMP-13 release.

Isolated human OA chondrocytes were pre-treated with increasing concentrations of Protandim or 6-

gingerol for 1 h. The cells were then treated with 1 ng/ml of IL-1β for 24 h. Cell culture medium was

collected and assayed for (A) NO, (B) PGE2, and (C) MMP-13. Values represent the means ± SEM of

4 separate experiments performed in duplicate. *P<0.05, **P<0.01, ***P<0.001 (compared to

untreated cells). #P<0.05, &P<0.01, @P<0.001 (compared to IL-1β-treated cells).

Figure 4: Protandim and 6-gingerol abolishe IL-1β-induced Nrf2 and GSTA4-4 down-regulation

and HNE production. Isolated chondrocytes were pre-incubated for 1 h with 10 µg/ml of Protandim or

10 M of 6-gingerol, followed by incubation for 24 h in the presence or absence of 1 ng/ml IL-1β. Nrf2

and GSTA4-4 mRNA expression was quantified by qPCR (A & B). HNE (C) levels in cellular extracts

were measured by ELISA. Values represent the means ± SEM of 4 separate experiments performed

This article is protected by copyright. All rights reserved 21

in duplicate. *P<0.05, **P<0.01, ***P<0.001 (compared to untreated cells); #P<0.05 (compared to IL-

1β-treated cells).

Figure 5: Nrf2 regulates IL-1-induced GSTA4-4 down-regulation and MMP-13 and HNE as well

as protandim effects. Human OA chondrocytes were transiently transfected with empty or Nrf2

expression vector (1 ng/106 cells) for 6 h and then treated or not with 1 ng/ml IL-1β for 24 h. Cellular

extracts were subjected to Western blotting of Nrf2 (A), GSTA4-4 mRNA expression measured by

qPCR (B), and HNE level by ELISA (C). MMP-13 was measured in culture media by ELISA (D).

Isolated human chondrocytes were transfected with control (sc-siRNA) or Nrf2 siRNA for 6 h and then

treated with 10 mg of protandim for 24 h. The protein expression of Nrf2 was assessed by Western

blot (E). HNE generation and GSTA4-4 expression (F) were determined by in-house ELISA and

qPCR, respectively. 2-tailed paired Student’s t test (n=3). Values represent the means ± SEM of 4

separate experiments performed in duplicate. *P<0.05, **P<0.01, ***P<0.001 (compared to untreated

cells). NS: not significant.

Figure 6: 6-gingerol had no effect on IL-1-induced MAPKs and NF-B activation. Human OA

chondroyctes were (i) pre-treated with 10 M 6-gingerol for 60 min, followed by another treatment with

1 ng/ml IL-1 for 5, 15, 30, and 60 min or (ii) preincubated with increased dose of 6-gingerol (0-10 M)

followed by another incubation for 30 min in the presence of 1 ng/ml IL-1. Cellular extracts were then

subjected to Western blotting using the specific polyclonal antibodies anti-phospho p38 MAPK, anti-

phospho JNK1/2, anti-ERK1/2, and anti-phospho NF-B/p65 (n=3 separate experiments).

Figure 7: Nrf2 and GSTA4-4 expression profiles in OA cartilage. (A) Cartilage extracts from

healthy subjects and OA patients were subjected to Western blotting using monoclonal anti-human

Nrf2 and GSTA4-4. (B & C) Nrf2 and GSTA4-4 mRNA levels were ascertained by qPCR in cartilage

from humans or sham and DMM WT mice at 8 weeks post-surgery. 2-tailed paired Student’s t test:

Values represent the means ± SEM of 3-6 separate experiments performed in duplicate. **P<0.01 (OA

vs normal; DMM vs sham).

This article is protected by copyright. All rights reserved 22

Figure 8: Protandim reduces cartilage degradation in the DMM mouse model of OA. Histological

section of medial tibial plateaus and femoral condyles in DMM WT mice received an intraarticular

injection per week of (A) vehicle and (B) 10 g/ml protandim for 8 weeks post-surgery. (C) Sham-

operated mice served as controls. Arrows indicate areas of fibrillation with loss of chondrocytes and

Safranin-O staining (loss of aggrecan). F indicates femoral condyle, and T, tibial plateaus (original

magnification X40). P values were assessed by 2-tailed paired Student’s t test comparing DMM mice +

drug to DMM mice + vehicle (n=4). **P<0.01.

This article is protected by copyright. All rights reserved 23

This article is protected by copyright. All rights reserved 24
This article is protected by copyright. All rights reserved 25
This article is protected by copyright. All rights reserved 26
This article is protected by copyright. All rights reserved 27
This article is protected by copyright. All rights reserved 28
This article is protected by copyright. All rights reserved 29
This article is protected by copyright. All rights reserved 30
This article is protected by copyright. All rights reserved 31