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Archives of Physiology and Biochemistry

ISSN: 1381-3455 (Print) 1744-4160 (Online) Journal homepage: http://www.tandfonline.com/loi/iarp20

Insulin and vanadium protect againstosteoarthritis development secondary to diabetesmellitus in rats

Abbas O. El Karib, Bahjat Al-Ani, Fahaid Al-Hashem, Mohammad Dallak,Ismaeel Bin-Jaliah, Basiouny El-Gamal, Salah O. Bashir, Refaat A. Eid &Mohamed A. Haidara

To cite this article: Abbas O. El Karib, Bahjat Al-Ani, Fahaid Al-Hashem, Mohammad Dallak,Ismaeel Bin-Jaliah, Basiouny El-Gamal, Salah O. Bashir, Refaat A. Eid & Mohamed A. Haidara(2016) Insulin and vanadium protect against osteoarthritis development secondary todiabetes mellitus in rats, Archives of Physiology and Biochemistry, 122:3, 148-154, DOI:10.3109/13813455.2016.1159698

To link to this article: http://dx.doi.org/10.3109/13813455.2016.1159698

Accepted author version posted online: 04Mar 2016.Published online: 29 Mar 2016.

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Arch Physiol Biochem, 2016; 122(3): 148–154! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/13813455.2016.1159698

ORIGINAL ARTICLE

Insulin and vanadium protect against osteoarthritis developmentsecondary to diabetes mellitus in rats

Abbas O. El Karib1*, Bahjat Al-Ani1*, Fahaid Al-Hashem1, Mohammad Dallak1, Ismaeel Bin-Jaliah1,Basiouny El-Gamal2, Salah O. Bashir1, Refaat A. Eid3, and Mohamed A. Haidara1,4

1Department of Physiology, 2Department of Clinical Biochemistry, and 3Department of Pathology, College of Medicine, King Khalid University, Abha,

Saudi Arabia, and 4Department of Physiology, Kasr al-Aini Faculty of Medicine, Cairo University, Cairo, Egypt

Abstract

Objective: Diabetic complications such as cardiovascular disease and osteoarthritis (OA) areamong the common public health problems. The effect of insulin on OA secondary to diabeteshas not been investigated before in animal models. Therefore, we sought to determine whetherinsulin and the insulin-mimicking agent, vanadium can protect from developing OA in diabeticrats. Methods: Type 1 diabetes mellitus (T1DM) was induced in Sprague–Dawley rats and treatedwith insulin and/or vanadium. Tissues harvested from the articular cartilage of the knee jointwere examined by scanning electron microscopy, and blood samples were assayed foroxidative stress and inflammatory biomarkers. Results: Eight weeks following the induction ofdiabetes, a profound damage to the knee joint compared to the control non-diabetic groupwas observed. Treatment of diabetic rats with insulin and/or vanadium differentially protectedfrom diabetes-induced cartilage damage and deteriorated fibrils of collagen fibers. The relativebiological potencies were insulin + vanadium44insulin4vanadium. Furthermore, there wasabout 2- to 5-fold increase in TNF-a (from 31.02 ± 1.92 to 60.5 ± 1.18 pg/ml, p50.0001) and IL-6(from 64.67 ± 8.16 to 338.0 ± 38.9 pg/ml, p50.0001) cytokines and free radicals measured asTBARS (from 3.21 ± 0.37 to 11.48 ± 1.5mM, p50.0001) in the diabetic group, which wassignificantly reduced with insulin and or vanadium. Meanwhile, SOD decreased (from17.79 ± 8.9 to 8.250.29, p50.0001) and was increased with insulin and vanadium. The relativepotencies of the treating agents on inflammatory and oxidative stress biomarkers wereinsulin + vanadium44insulin4vanadium. Conclusion: The present study demonstrates that co-administration of insulin and vanadium to T1DM rats protect against diabetes-induced OApossibly by lowering biomarkers of inflammation and oxidative stress.

Keywords

Diabetes, T1DM, osteoarthritis, insulin,vanadium, rat model

History

Received 28 November 2015Revised 28 January 2016Accepted 24 February 2016Published online 23 March 2016

Introduction

An estimated 347 million people around the world suffer from

diabetes, which is one of the leading global health care

problems that claims the life of about 3.4 million every year

(Danaei et al., 2011). Type 1 diabetes mellitus (T1DM) is

believed to be caused by autoimmune antibodies targeting the

b cells-producing insulin in the pancreas that leads to the loss

of insulin production and hence the appearance of diabetes

symptoms in children, commonly between the ages of 7–19

years (Atkinson et al., 2014; Narendran et al., 2005; Pugliese,

2004).

Osteoarthritis (OA) is a degenerative joint disease that

involves degradation and destruction of the articular cartilage

structure of the joint leading to pain, swelling and reduced

joint movement; and there is increasing evidence to link

metabolic disturbances such as diabetes to OA (Pottie et al.,

2006, Van Manen et al., 2012). OA of the knee and/or hip is

regarded as one of the most prevalent conditions leading to

disability particularly in the elderly population (Grazio &

Balen, 2009). Knee OA is more important, not only for its

high prevalence rate compared with other types of OA, but

also for its presentation at earlier age groups particularly in

younger age groups of obese women (Bliddal & Christensen,

2009; Hayami, 2008).

Diabetic limited joint mobility syndrome is seen in 8–50%

of T1DM patients. The lack of the bone anabolic actions of

insulin and other pancreatic hormones could be the reason

why T1DM affects the skeleton more severely than type 2

diabetes mellitus (T2DM) (Hamann et al., 2012; Janghorbani

et al., 2007). In a study of patients with T2DM followed over

a course of 20 years it was concluded that longstanding

diabetes per se is detrimental for knee and hip joints, leading

to progressive destruction and joint failure (Schett et al.,

2013).

* These authors contributed equally to this work.

Correspondence: Professor Mohamed Haidara, Department ofPhysiology, College of Medicine, King Khalid University, Abha61421, Saudi Arabia. Tel: 00966540733723. E-mail: haidaram@hotmail.com

Proinflammatory biomarkers IL-6 and TNF-a are signifi-

cant predictors of knee OA (Kou & Wu, 2014; Livshits et al.,

2009) and OA cartilages from DM patients showed increased

responsiveness to IL-1b-induced inflammation via oxidative

stress and polyol pathway thus participate in the increased

inflammation in OA (Laiguillon et al., 2015).

Vanadium is a trace mineral that is widely distributed in

nature and has been reported to ameliorate DM in humans

when administered orally (Gaede et al., 2008). The glucose-

lowering effect of vanadium is attributed to the insulin-like

function of the compound and its ability to enhance insulin

activity and cellular bioavailability (Haidara et al., 2015;

Karmaker et al., 2007). Vanadium induces its effect through

insulin receptor-independent pathways since it has no effect in

activating the insulin receptor tyrosine kinase, a key enzyme

in insulin cell signalling (Shechter et al., 1995). However,

vanadium exerts its effects through inhibition of protein

tyrosine phosphatases, mainly PTEN and activation of

cytosolic tyrosine kinases that augment and activate down-

stream insulin receptor cell signalling (Srivastava & Mehdi,

2005). Studies have shown that vanadyl sulphate can lower

elevated blood glucose, cholesterol and triglycerides in a

variety of diabetic models including the streptozotocin (STZ)

diabetic rat, the Zucker fatty rat and the Zucker diabetic fatty

rat (Gruzewska et al., 2014).

Interestingly, neither the effect of insulin nor its mimicking

agent, vanadium on diabetes-induced OA in animal models

has been addressed before. Therefore, we were interested to

study the potential protective effect of insulin and or

vanadium against OA of the knee joint secondary to T1DM,

and to see if vanadium adjuvant treatment with insulin can

augment the proposed protection. To answer these questions,

we developed an animal model of T1DM-induced OA in

Sprague–Dawley rats and used the model to evaluate the

effect of insulin and vanadium on OA.

Methods

Animals

The experiments were performed on 36 male Sprague–

Dawley rats of 10 weeks old and weighting 200–250 g. The

rats were fed with standard laboratory diets, given water ad

libitum and maintained under laboratory conditions of

temperature (22 ± 3 �C), with 12-h light and 12-h dark

cycle. All experimental procedures involving the handling

and treatment of animals were approved by the Research

Ethical Committee of King Khalid University (Abha, KSA)

and were conducted in accordance with the National Institute

of Health’s Guide for the Care and Use of Laboratory

Animals.

Animal protocol

After 1-week adaptations, animals were classified and

randomly allocated into 6 groups (n¼ 6) as follows: control

group (Control): non-diabetic, non-treated rats that were

injected intraperitoneally (i.p.) once with citrate buffer (0.1 M,

pH 4.5); vanadium-treated group (Van.): non-diabetic, van-

adium-treated rats that were i.p. injected with citrate buffer as

control group, and received vanadyl sulphate of 0.64 mmol/kg

body weight, freshly dissolved in 1 ml of distilled water daily

through a nasopharyngeal tube (Haidara et al., 2014);

Diabetic type 1 group (D1): DM was induced in rats by a

single i.p. injection of STZ in a dose of 65 mg/kg. Rats

showed glucose level above 300 mg/dl were considered to be

diabetics and included in the experiments (El Karib, 2014;

Haidara et al., 2004); T1DM and insulin group (D1 + Ins.):

rats were made type 1 diabetic as in T1DM group and

received Mixtard insulin subcutaneously in a dose of 0.75 IU/

100 g weight in 0.75 ml volume once daily (Haidara et al.,

2015), after 48 h of diabetic induction; T1DM and vanadium

group (D1 + Van.): T1DM rats received the same dose of

vanadium as in vanadium group after 48 h of induction of

T1DM; T1DM and vanadium and insulin group

(D1 + Van. + Ins.): T1DM rats received both vanadium and

insulin with the same doses as in previous groups.

At the end of the 8th weeks of the experiment, 5 ml retro-

orbital blood samples was obtained under anaesthesia using

40 mg kg�1 sodium thiopentane, i.p., after fasting. Blood was

collected into plain tubes, then allowed to clot for 20 min then

centrifuged at 14 000 rpm for 10 min for serum separation.

Then sera were stored at �80 �C, for subsequent measure-

ments of biochemical parameters. After withdrawal of the

blood samples, the knee joints were opened, dissected, and

fixed in glutaraldehyde and kept for electron microscopy

examinations.

Measurement of IL-6, TNF-a cytokines

Rat interleukin-6 ELISA kit, IL-6 for serum, plasma and

tissue culture supplements (Ray Biotech Inc., Mfr. No. ELR-

IL6-001) and rat tumour necrosis factor alpha (TNF-a ELISA

kit BIOTANG Inc., Cat. No. R6365 were used as recom-

mended by the manufacturer.

Measurements of SOD and TBARS

Superoxide dismutase (SOD) assay kit, rat, Cayman

Chemical, Cat. No. 706002 and thiobarbituric acid reactive

substances (TBARS) assay kit, Cayman Chemical, Item

Number 10009055 were used as recommended by the

manufacturer.

Scanning electron microscopy

Different specimens were taken from articular cartilage of

all treated rats and subjected for ultrastructural examin-

ations with scanning electron microscope. Ultra-structural

micrographs are representative of the changes seen in

different treated groups. The specimens were fixed with

2.5% (wt/vol) sodium cacodylate-buffered glutaraldehyde,

pH 7.2 at 4 �C for 2 h. Specimens were also post fixed in

1% sodium cacodylate-buffered osmium tetroxide, pH 7.2

for 1 h. After washing and dehydration in ascending series

of ethanol, critical-point drying was performed using the

EMITECH-K850 critical-point drying unit. The specimens

were mounted on aluminium stubs with double-sided tape

and silver glue and then sputter coated with gold by BOC

EDWARDS SCANCOAT. The specimens were observed

using a Jeol field emission scanning electron microscope

JSM-6390LV.

DOI: 10.3109/13813455.2016.1159698 Insulin and vanadium protect against OA 149

Statistical analysis

Data are presented as mean ± SD. Comparison of data was

analysed using Graph pad Prism software, version 5.

Comparison between the groups was performed by one-way

analysis of variance, followed by Tukey’s post-hoc test.

Probability (p) values of 50.05 were considered to be

significant.

Results

Changes in blood glucose level

Figure 1 shows a significant (p50.0001) increase in blood

glucose levels in diabetic group (371 ± 27 mg/dl) in compari-

son to the control group (92 ± 5 mg/dl). Compared to diabetic

group, administration of vanadium and or insulin caused a

significant (p50.0001) decrease in glucose levels. In add-

ition, insulin alone or with vanadium, but not vanadium alone,

were able to lower blood glucose to levels comparable to the

control group.

Osteoarthritis development in rats by induction ofdiabetes

Eight-week post-induction of diabetes, tissue preparations for

histological examination under scanning electron microscopy

(Figures 2 and 3) from the articular cartilage of the knee joint

of the sacrificed T1DM rats (Figures 2C and 3C) revealed OA

development compared to the non-diabetic healthy control

rats (Figures 2A and 3A). The pathological changes revealed

a massive destruction of the articular cartilage caused by

T1DM such as atrophic chondrocytes inside distorted lacunae

that are surrounded by damaged territorial matrix

(Figure 2C), compared to normal structures in control tissues

(Figure 2A). Furthermore, scanning electron micrographs of

T1DM rats’ collagen fibers showed extensive damage and

deterioration in fibrils forming sheets (Figure 3C), whereas

normal fibrils forming sheets of collagen fibers were depicted

in control rats (Figure 3A).

Insulin and vanadium protect the knee joint againstT1DM-induced osteoarthritis

A substantial protection was obtained when insulin was given

to diabetic rats (Figures 2D and 3D). 48 h after induction of

diabetes, treating diabetic group with insulin for 8 weeks

protected the OA ultrstructure of the articular cartilage to

become much less damaged comparable to the control group.

Scanning electron micrographs of insulin-treated tissues

showed a normal territorial matrix and restoration of the

leaving lacunae most of its normal size and regular shape,

with a clear improvement in the healing of chondrocytes

(Figure 2D). In addition, collagen fibers showed intact fibril

sheets, but still with some focal damage (Figure 3D).

Administration of diabetic group with vanadium for 8

weeks differentially protected the ultrastructure of the articu-

lar cartilage (Figures 2E and 3E). Electron microscopy results

revealed a better protection of leaving lacunae and

territorial matrix than chondrocytes and collage fibers

(Figures 2E and 3E). However, focal damage of leaving

lacunae and atrophic chondrocytes are still shown. Strikingly,

co-administration of insulin and vanadium protects the

articular cartilage of the diabetic rats where it showed

normal chondrocytes, leaving lacunae, territorial matrix and

collagen fibres (Figures 2F and 3F). The above treatments

were compared to control groups, non-diabetic ‘‘vehicle-

treated’’ (Figures 2A and 3A) and non-diabetic ‘‘vanadium-

treated’’ rats (Figures 2B and 3B) that showed normal

structures of the articular cartilage.

Insulin and vanadium inhibit T1DM-induced cytokinesand oxidative stress

Proinflammatory cytokines are known to be involved in the

pathology of diabetes (Senn et al., 2002; Shoelson et al.,

2006; Southern et al., 1990) and OA (Doss et al., 2007,

Stannus et al., 2010). To investigate whether vanadium and/or

insulin can suppress the release of the proinflammatory

cytokines in T1DM, we measured the blood level of TNF-aand IL-6, 8 weeks after T1DM induction. T1DM caused a 2-

fold increase in TNF-a and 4-fold increase in IL-6 that were

significantly inhibited by insulin or vanadium or vanadium

plus insulin (Figure 4A and B).

Administration of insulin and vanadium significantly

decreased IL-6 in comparison either to diabetic rats admin-

ister either insulin (p50.0001) or vanadium (p50.001)

alone. Using post-hoc testing, least significant difference

(LSD) for IL-6, it was found that there was a significance

difference between diabetic group that received insulin and

vanadium in comparison to either diabetes and insulin

(p50.0001) or diabetes and vanadium (p50.0001) alone.

Insulin and vanadium administration to diabetic group

significantly decreased TNF-a in comparison either to

diabetic rats administer either insulin (p50.0001) or van-

adium (p50.0001) alone. Using post-hoc testing (LSD) for

Figure 1. Changes in blood glucose level. Control, control vehicleinjected rats; Van., vanadium-treated non-diabetic rats; D1, streptozoto-cin-injected rats (type 1 diabetic group); D1 + Ins., diabetic group treatedwith insulin; D1 + Van., diabetic group treated with vanadium;D1 + Van. + Ins., diabetic group treated with vanadium and insulin. Allresults are the mean (±SD) of three experiments. Significance indicated:p50.05, P1: significant in comparison to control group, P2: significantin comparison to vanadium group, P3: significant in comparison todiabetic group.

150 A. O. El Karib et al. Arch Physiol Biochem, 2016; 122(3): 148–154

TNF-a, it was found that there was a significance difference

between diabetic group that received insulin and vanadium in

comparison to either diabetes and insulin (p50.0001) or

diabetes and vanadium (p50.0001) alone.

To determine whether insulin and/or vanadium can

modulate the oxidant and anti-oxidant biomarkers known to

be affected by diabetes (Molinar-Toribio et al., 2014) and OA

(Gutteridge, 1995), we measured the free radicals as TBARS,

and SOD as biomarkers of oxidative stress by ELISA. Our

results showed that diabetic induction augmented TBARS and

suppressed SOD (Figure 5A and B). Insulin significantly

augmented SOD and inhibited TBARS, whereas vanadium on

its own was a weak stimulant to SOD and exerted no

significant inhibitory effect on TBARS levels in T1DM group.

Furthermore, co-administration of insulin with vanadium

completely restored the levels of SOD and TBARS to the

control levels (Figure 5A and B).

Administration of insulin and vanadium significantly

decreased TBARS in comparison either to diabetic rats

administer either insulin (p50.0001) or vanadium

(p50.0001) alone. Using post-hoc testing (LSD) for

TBARS, it was found that there was a significance difference

between diabetic group that received insulin and vanadium in

comparison to either diabetes and insulin (p50.0001) or

diabetes and vanadium (p50.0001) alone.

Insulin and vanadium administration to diabetic group

significantly increased SOD in comparison either to diabetic

rats administer either insulin (p50.0001) or vanadium

(p50.0001) alone. Using post-hoc testing (LSD) for SOD,

it was found that there was a significance difference between

diabetic group that received insulin and vanadium in

comparison to either diabetes and insulin (p50.0001) or

diabetes and vanadium (p50.0001) alone.

Discussion

The principal finding of our study was that insulin in

combination with the insulinomimetic agent, vanadium

inhibited OA induced by T1DM and suppressed the bio-

markers of inflammation and oxidative stress known to be

induced in diabetes and OA. Furthermore, insulin on its own

was more potent than vanadium to protect against OA. To the

best of our knowledge, these studies are the first to report a

clear protective role for insulin and vanadium in OA induced

Figure 2. Insulin and vanadium protected theknee joint against OA in diabetic rats.Scanning electron micrographs (2000�) ofrats’ articular cartilage from the knee joint 8weeks after the induction of diabetes bystreptozotocin. A. Control, control vehicleinjected rats. B. Van., vanadium-treated non-diabetic rats. C. D1, streptozotocin-injectedrats (type 1 diabetic group). D. D1 + Ins.,diabetic group treated with insulin. E.D1 + Van., diabetic group treated with van-adium. F. D1 + Van. + Ins., diabetic grouptreated with vanadium and insulin.Abbreviations: C, chondrocyte; C1, focaldamage of chondrocyte; L, leaving lacunae;N, nuclei; TM, territorial matrix. Note thatarrows point to atrophic chondrocyte shownin (D) and focal damage of leaving lacunaein (E).

DOI: 10.3109/13813455.2016.1159698 Insulin and vanadium protect against OA 151

Figure 3. Insulin and vanadium protectedcollagen fibres in the knee joint against OA indiabetic rats. Scanning electron micrographs(2000�) of rats’ collagen fibres in articularcartilage from the knee joint 8 weeks after theinduction of diabetes by streptozotocin. A.Control, control vehicle injected rats. B. Van.,vanadium-treated non-diabetic rats. C. D1,streptozotocin-injected rats (type 1 diabeticgroup). D. D1 + Ins., diabetic group treatedwith insulin E. D1 + Van., diabetic grouptreated with vanadium. F. D1 + Van. + Ins.,diabetic group treated with vanadium andinsulin. Abbreviations: F, normal or intactcollagen fibrils sheets; F1, damaged collagenfibrils sheets. Note that arrows point to focaldamage of collagen fibril sheets.

Figure 4. Insulin and vanadium inhibit TNF-a and IL-6 in T1DM-treated rats. A, TNF-a and (B) IL-6 levels in blood from the groups (n¼ 6) weremeasured after 8-week treatment by ELISA. Control, control vehicle injected rats; Van., vanadium-treated non-diabetic rats; D1, streptozotocin-injectedrats (type 1 diabetic group); D1 + Ins., diabetic group treated with insulin; D1 + Van., diabetic group treated with vanadium; D1 + Van. + Ins., diabeticgroup treated with vanadium and insulin. All results are the mean (±SD) of three experiments. Significance indicated: p50.05, P1: significant incomparison to control group, P2: significant in comparison to vanadium group, P3: significant in comparison to diabetic group.

152 A. O. El Karib et al. Arch Physiol Biochem, 2016; 122(3): 148–154

by diabetes. These conclusions were supported by the data

indicating that STZ induced T1DM in rats caused a substan-

tial destruction to the articular cartilage of the knee joint that

was protected by insulin and vanadium.

Studies on experimental animals have demonstrated that

administration of STZ, depending on its dose, extensively

reduces beta cells mass and destroys pancreatic islet (Cam

et al., 1997; Yanardag et al., 2005). Our results showed that

vanadium and insulin administration causes decrease glu-

cose level in diabetic group to control level which is in

accordance with previous work that showed that regardless

of the severity of diabetes, vanadium therapy needs a

minimum level of plasma insulin, secreted endogenously, or

received exogenously to assist vanadium to induce normo-

glycemia (Pirmoradi et al., 2014; Yanardag et al., 2005). In

addition, the suppression of the growth body weight seen in

the diabetic group was linked with the impaired carbohy-

drate metabolism as a source of energy (Pires et al., 2014,

Wycherley et al., 2014). However, the suppressive action of

vanadium on the appetite, via anorexigenic stimulation of

the central nervous system and food restriction, was

possibly the cause of reduced growth body weight in the

vanadium-treated group (Marty et al., 2007). This is in

agreement with our findings that showed no significant

changes in the body weight of diabetic or vanadium-treated

groups (data not shown).

We confirmed by electron microscopy examination the

development of OA in the articular cartilage of the knee joint,

which is known to be targeted by diabetes (El Karib, 2014;

Musumeci et al., 2014; Niethard, 1986; Pottie et al., 2006).

Tissue examination revealed a profound destruction of the

cartilage structures such as chondrocytes, territorial matrix

and collagen fibres.

Prolonged hyperglycemia and accumulation of glucose

derived advanced glycation end products (AGEs) were

proposed to contribute to OA development and insulin is

reported to have anabolic effects on bone (Yan & Li, 2013). In

addition, insulin was found to increase proteoglycan synthesis

in primary chondrocytes tissue culture and increased proteo-

glycan and matrix synthesis in articular cartilage explants

obtained from healthy pigs and diabetic human and mice (Cai

et al., 2002). We therefore tested to see if insulin and its

mimicking agent, vanadium is able to prevent articular

cartilage injury in animals following diabetes. Indeed, our

data showed that giving insulin or vanadium partially

protected the knee joint injury. However, when both insulin

and vanadium were given together, they protected the

cartilage and the histological pictures were similar to the

non-diabetic controls. Furthermore, It is important to note that

the relative potencies for these agents in our T1DM-induced

OA animal model were determined to be insulin + van-

adium44insulin4vanadium. Our data also point to the

importance of co-administering insulin and vanadium in

preventing the increase of inflammatory and oxidative

biomarkers levels above the control in diabetic-treated rats

(Figures 4 and 5). It was also interesting to report that the

relative potencies of vanadium were observed here to be well

below that of insulin, which is in agreement with potent

insulin over vanadium that we observed in the healing of the

damaged cartilage.

Our protective approach, by co-administrating insulin and

vanadium, that prevents the diabetic rats to develop OA

concomitant with a complete inhibition of TNF-a, IL-6 and

free radicals points to these cytokines and TBARS oxidative

stress as very important proinflammatory biomarkers to

monitor OA secondary to diabetes, including the healing

progress of the disease. Indeed, our findings are in agreement

with the previously published work on the role of TNF-a and

IL-6 in the pathogenesis of inflammation induced bone loss,

osteoclastic bone resorption and cartilage destruction (Fuller

et al., 2002; Kapoor et al., 2011; Korczowska & Lacki, 2005)

and the reports that proposed IL-6 and TNF-a as significant

predictors of knee OA (Kou & Wu 2014; Livshits et al.,

2009).

Taken together, our data support the conclusion that insulin

protects against development of OA induced by T1DM and

co-administration of vanadium with insulin is more effective

to protect against the diabetic OA complications in STZ-

induced OA in this rat model by lowering biomarkers of

inflammation and levels of oxidative stress.

Figure 5. Different effects of insulin and vanadium on inhibition of TBARS and activation of SOD in T1DM-treated rats. (A) TBARS and (B) SODlevels in blood from the 6 rat groups were measured after 8-week treatment by TBARS assay and ELISA. Control, control vehicle injected rats; Van.,vanadium-treated non-diabetic rats; D1, streptozotocin-injected rats (type 1 diabetic group); D1 + Ins., diabetic group treated with insulin; D1 + Van.,diabetic group treated with vanadium; D1 + Van. + Ins., diabetic group treated with vanadium and insulin. All results are the mean (±SD) of threeexperiments. Significance indicated: p50.05, P1: significant in comparison to control group, P2: significant in comparison to vanadium group, P3:significant in comparison to diabetic group.

DOI: 10.3109/13813455.2016.1159698 Insulin and vanadium protect against OA 153

Declaration of interest

All authors declare that they have no conflict of interest.

This work was supported by grants of King Khalid

University. KKU-Project No 2 (215).

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