Oleanolic acid prevents cartilage degeneration in diabetic mice via PPARγ associated mitochondrial stabilization

Accepted Manuscript Oleanolic acid prevents cartilage degeneration in diabetic mice via PPARγ associated mitochondrial stabilization Xia Kang, Zhikui Yang, Jun Sheng, Jin-biao Liu, Qing-yun Xie, Wei Zheng, Ken Chen PII:

S0006-291X(17)31262-7

DOI:

10.1016/j.bbrc.2017.06.127

Reference:

YBBRC 38033

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 16 June 2017 Accepted Date: 20 June 2017

Please cite this article as: X. Kang, Z. Yang, J. Sheng, J.-b. Liu, Q.-y. Xie, W. Zheng, K. Chen, Oleanolic acid prevents cartilage degeneration in diabetic mice via PPARγ associated mitochondrial stabilization, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.127. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Oleanolic acid prevents cartilage degeneration in diabetic mice via PPARγγ associated mitochondrial stabilization Xia Kang1, Zhikui Yang2, Jun Sheng1, Jin-biao Liu1, Qing-yun Xie1, Wei Zheng1, Ken Chen4

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Xia Kang and Zhikui Yang are first co-authors in this article. Wei Zheng and Ken Chen contributed equally in this article.

Xia Kang, MD, Jun Sheng, MD, Jin-biao Liu, MD, Qing-yun Xie, MD, Wei Zheng, MD, Ph.D: Department of Orthopedics, Chengdu Military General Hospital, 270 Rongdu Avenue, Jinniu

District, Chengdu, Sichuan, 610083, China.

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1

Zhikui Yang, MD: 2Department of Orthopedics, Xianyang Hospital of Yan'an University, 38

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Wenlin Road, Xianyang, Shaanxi Province, 712000, China

Ken Chen, MD, Ph.D: 4Department of Cardiology, Chengdu Military General Hospital, 270Rongdu Avenue, Jinniu District, Chengdu, Sichuan, 610083, China

Email address: Xia Kang, [email protected]; Zhikui Yang, [email protected]; Jun Sheng, [email protected]; Jin-biao Liu, [email protected]; Qing-yun Xie:

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[email protected]; Wei Zheng, [email protected]; Ken Chen, [email protected]

Address to correspondence to Wei Zheng, MD, Ph.D: Department of Orthopedics, Chengdu Military General Hospital, 270 Rongdu Avenue, Jinniu District, Chengdu, Sichuan, 610083,

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China. E-mail: [email protected].

Funding source: The research reported was supported by National Natural Science Foundation of

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China (Grant no. 81601940) and Foundation of Department of Science and Technology of Sichuan Province (Grants no. 2014JY0009). Competing interests: There are no competing interests to declare.

The authors declare that this manuscript has not been submitted or is not simultaneously being submitted elsewhere, and that no portion of the data has been or will be published in proceedings or transactions of meetings or symposium volumes.

ACCEPTED MANUSCRIPT Oleanolic acid prevents cartilage degeneration in diabetic mice via PPARγγ associated mitochondrial stabilization

Abstract

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Hyperglycemia-induced cartilage degeneration induces osteoarthritis (OA). Since oleanolic acid (OLA) have several pharmacological effects such as anti-inflammatory, anti-oxidant, we hypothesized it possesses protection against high glucose injured cartilage. We now report that OLA decreased type X collagen and reversed the cartilage degeneration in growth plate from

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db/db mice. OLA increased type Ⅱ collagen expression in a concentration-dependent manner (10 to 50 µΜ) in high glucose-treated chondrocytes. OLA prevented the high glucose induced cell

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injury and decreased the level of MMP-13, PGE2 and IL-6 due to decreasing mitochondrial membrane potential and stimulated the ATP production. Moreover, OLA treatment inhibited apoptosis. And the reversed SOD2 expression and activity may be ascribed to decreased SOD2 protein degradation by OLA treatment, via PPPAγ. In conclusion, OLA protected against the high-glucose-induced cartilage injury via PPARγ/SOD2 pathway.

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Key words: oleanolic acid, cartilage degeneration, PPPAγ, mitochondrial stabilization

ACCEPTED MANUSCRIPT Introduction Cartilage degeneration is an important pathological process in osteoarthritis, which affects daily living activities and life quality of the elderly and leads to increased morbidity and mortality

1, 2

.

While wearing and tearing on the joint resulting from multi-factors might be initial cause of

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cartilage degeneration and osteoarthritis, metabolic factors (blood glucose, lipemia and/or blood pressure) are considered independent factors involved in the pathological process of osteoarthritis, and cartilage impairment

3-5

. Along this line of consideration, several studies indicated that

diabetes is associated with osteoarthritis

6-9

. Most of the glucose derivatives such as advanced

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glycation end-product (AGEs), sorbitol and diacylglycerol (DAG), are involved in the activation of inflammation processes, and increase the expression of pro-inflammation cytokines10. Those

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glucose derivatives may participate in the increased inflammation in osteoarthritis. Peroxisome proliferator-activated receptor γ (PPARγ) is a member of nuclear hormone receptor superfamily and one kind of ligand-activated transcription factors 11, and plays an important role in peripheral glucose utilization and insulin sensitization

12

. Hyperglycemia down-regulates

PPARγ expression and induces inflammatory and catabolic responses in human chondrocytes and

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diabetic mouse cartilages13, while PPARγ has been found to protect cartilage in osteoarthritis model mice 14. Perhaps, the medicines or plant extracts activating PPARγ might prevent cartilage degeneration.

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Oleanolic acid (OLA, 3β-hydroxyolean-12-en-28-oic acid), one kind of triterpenoid compounds, widely occurs in olive leaves, olive pomace, mistletoe sprouts, clove flowers and

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almost 2000 plant species15, 16, seems to be a natural PPARγ agonist16-18. However, the protective effect of OLA on cartilage is still unclear. Thus, our present study found that OLA protected against the hyperglycemia-induced cartilage degeneration via PPARγ. The study provides a mechanism by which OLA activating PPARγ, stabilizes the mitochondrial, protects against hyperglycemia-induced osteoarthritis.

ACCEPTED MANUSCRIPT Methods Animal Adult db/db mice and C57BL/6J mice (as control), weighing 18 to 24g, were obtained from Model Animal Research Center of Nanjing University, and randomized to the control group and

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oleanolic acid (OLA, 200 mg/kg/day, Sigma-Aldrich, St. Louis, MO)-treated group. Mice were housed in a room with a 12:12h light-dark cycle and a temperature of 25°C, and given free access to tap water. Following death of mice, limbs were dissected for staining. All experiments conformed to the guidelines of the ethical use of animals, and all efforts were made to minimize

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animal suffering and to reduce the number of animals used.

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Primary mouse articular chondrocytes isolation, culture and treatment

Primary cardiomyocytes were isolated from adult mice, weighing 18 to 24g, with a protocol reported previously19. The lower limbs were removed from the mice after euthanasia. After cleaning the bloodstained, the tissue were transferred to digestion solution and incubated 45 min at 37 °C in a 20% O2 and 5% CO2 incubator to isolate the chondrocytes from the cartilage. After

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incubation, serum-free medium was added to the digestion solution containing the residual chondrocytes, and dissociated the cells. After centrifuging for 5 min 200 ×g and washing with 5% PBS, the cells were resuspended in DMEM supplemented with 10% FBS, and plated in culture

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dishes.

For high-glucose treatment, cells were cultured in DMEM containing 33mM glucose for 24h

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followed OLA treatment in a dose-dependent manner (10, 30, 50 mM) for an additional 24h 20, 21. The supernatant or cells were collected for biochemical analysis or further studies.

Histological study

Cartilage tissues were fixed with 4% paraformaldehyde for 24h at 4 °C, decalcified for 2

weeks with 20% EDTA at 4°C, embedded in paraffin, sectioned (4µm), and mounted on slides. The slides were incubated with anti-type X collagen antibody (1:100, Bioss, Beijing, China PR) overnight at 4°C followed immunohistochemistry, or stained with 0.05% Fast Green for 5 min and 0.1% safranin O for 30 min (Sigma Aldrich, St. Louis, MN).

ACCEPTED MANUSCRIPT Immunoblotting Cells were washed twice with ice-cold PBS and lysed in lysis buffer (Beyotime Biotechnology, Shanghai, China). The homogenates (20 µg of protein) were separated by 8% to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluori (PVDF) membranes.

The blots were then washed with Tris-buffered saline Tween-20 (TBST), blocked

with 1% BSA in TBST buffer for 1 hr, and incubated with the appropriate primary antibody at

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dilutions recommended by the supplier. Transblots were probed with the rabbit anti-type Ⅱ collagen antibody (1:500, Abcam, Cambridge, Massachusetts, US), rabbit anti-PPARγ antibody (1:1000, Abcam, Cambridge, Massachusetts, US), rabbit anti-active caspase 9 antibody (1:1000, Cell Signaling Technology, Massachusetts, US) or anti-SOD2 antibody (1:200, Abcam,

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Cambridge, Massachusetts, US), respectively. Then, the primary antibodies were detected with goat anti-rabbit-IgG (1:5000, Jackson ImmunoResearch Laboratory, PA）conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence (Pierce, MA). The

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amount of protein transferred onto the membranes was verified by immunoblotting for GAPDH (1:500, Santa Cruz, CA).

Measurement of mitochondrial membrane potential

Primary articular chondrocytes were stained with JC-1 dye. Cultured chondrocytes were washed with PBS, trypsinized for 5 min at 37 °C. The cells were tained for 15 min at 37 °C with

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10 µM JC-1 in FBS-free medium. After staining, the cells were washed and re-suspended in PBS, and then the mitochondrial membrane potential was measured using a flow cytometer.

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Small interfering RNA

Small interfering RNA (siRNA) against PPARγ mRNA was synthesized and purified with

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reverse-phase high-performance liquid chromatography as 21-mer phosphorothioate-modified oligodeoxynucleotides22

(PPARγ

CCUCCCUGAUGAAUAAAGATTdTdT-3’,

siRNA

sequence:

scrambled

RNA

5’sequence:

5′-UGGUUUACAUGUUGUGUGAdTdT-3′), obtained from Invitrogen (Carlsbad, CA). The designed sequences showed no homology with other known mammalian sequences deposited in the Genbank database, as screened using the BLAST program. The effects of 50 nM siRNA were compared with scrambled RNA (control). Briefly, 50 nM siRNA or control RNA were mixed with 6 µL of oligofectamine in Optimem medium (Invitrogen Life Technologies) and incubated with

ACCEPTED MANUSCRIPT cells grown in 6-well plates for 24 hr, and, then switched to growth medium and incubated for another 24 hr.

Statistical Analysis

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The data are expressed as mean ± SEM. Comparison within groups was made by one-way

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ANOVA for repeated measures. A value of P<0.05 was considered significant.

ACCEPTED MANUSCRIPT Results The protective effect of OLA on high-glucose impaired cartilage To check the protective effect of OLA on hyperglycemia-induced cartilage damage, db/db mice was treated with OLA. Epiphysis tissues of the proximal epiphysis of the tibia from db/db

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mice with or without OLA administration were measured, and the data showed that more intense of type X collagen staining, a marker of chondrocyte differentiation, was found in growth plate from db/db mice while OLA decreased the high-glucose induced type X collagen expression (Figure 1A). Moreover, high blood glucose disorganized the chondrocytes and increased cartilage

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degeneration in growth plate from db/db mice, OLA reversed the cartilage degeneration (Figure 1B). And the type Ⅱ collagen expression was determined by in vitro study. OLA increased type

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Ⅱ collagen expression in a concentration-dependent manner (10 to 50 µΜ) in high glucose-treated chondrocytes (Figure 1C).

For establishing the benefit of OLA to the high-glucose insult to the chondrocytes, cell viability was check by cell counting kit-8, and OLA prevented the high glucose induced cell injury in a concentration-dependent manner (Figure 2A), while OA decreased the LDH concentration, a

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biomarker of injured cell (Figure 2B). Moreover, some proteins, such as MMP-13, PGE2 and IL-6, play a significant role in cartilage matrix degradation and joint dysfunction23, 24. OLA decreased the level of MMP-13, PGE2 and IL-6 in high glucose-treated chondrocytes (Figure 2C-2E),

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which should be the mechanism of OLA protective effect.

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The protective effect of OLA on mitochondrial function in the high glucose-injured chondrocytes

The mitochondrial dysfunction of chondrocytes results in cartilage degeneration which leads

to osteoarthritis25. Thus, our studies checked the protective effect of OLA on mitochondrial function. OLA reversed the decreasing mitochondrial membrane potential (MMP) of high glucose-injured cells, led to a lower proportion of chondrocytes with mitochondrial depolarization as evidenced through with JC-1 staining (Figure 3A). OLA also stimulated the ATP production (Figure 3B). Our present study found that high glucose induced chondrocytes apoptosis, determined by measurement of active caspase 9 expression, the key signaling protein in mitochondrial-apoptosis pathway. OLA treatment inhibited apoptosis (Figure 3C).

ACCEPTED MANUSCRIPT OLA protects mitochondrial function of high glucose-injured chondrocytes via PPARγγ-SOD2 pathway Previous studies indicated that PPARγ and SOD2 was involved in collagen degradation in diabetic mouse cartilages13, 25. Our present study found that the protein expression of PPARγ and

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SOD2 was increased by OLA treatment in high glucose-injured chondrocytes (Figure 4A and 4B). The PPARγ siRNA blocked the increasing SOD2 activity stimulated by OLA. in high glucose-treated chondrocytes, indicating the role of PPARγ in regulation of OLA on SOD2 (Figure 4C). And the reversed SOD2 expression and activity may be ascribed to decreased SOD2

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protein degradation by OLA treatment (Figure 4D).

ACCEPTED MANUSCRIPT Discussion Some researches indicate that hyperglycemia-induced cartilage degradation plays an important role in the pathologic process of osteoarthritis13, 26, 27. The data presented in our study are consistent with the previous studies. High blood glucose disorganized the chondrocytes and

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increased cartilage degeneration. And the OLA decreased the type X collagen expression and reserved the type Ⅱ collagen degeneration. The increasing type Ⅱ collagen expression may be ascribed to the decreasing effect of MMP-13, PGE2 and IL-6 in high glucose-treated chondrocytes. Tissue remodeling is controlled by matrix metalloproteinases (MMPs) and the MMPs release is an

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important mechanism in cartilage damage28. MMP-13 has been found to cleave the type Ⅱ collagen in OLA articular cartilage 29. And inflammatory cytokines is another collagenase and has

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a predominant role in osteoarthritis 30. OLA, by modulating the T cell immune responses, reduces the expression and production of inflammatory mediators, and suppresses matrix-degrading enzymes31.

Previous studies have identified chondrocyte death following impact to articular cartilage32, 33. We demonstrated high glucose induced chondrocyte death and OLA promoted the cell viability

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and reduced LDH concentration. As response for maintaining the function and hoemostasis of cartilage, apoptosis of chondrocytes lead to articular cartilage disruption33. Furthermore, The chondrocytes mitochondrial dysfunction leads to cartilage degeneration.25 Thus, our study

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identified that exposure of chondrocytes with high glucose resulted in a mitochondria-dependent apoptosis, as determined by the activation of caspase 9, a key signal protein of

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mitochondria-dependent apoptosis, and OLA treatment led to significant decreasing of caspase 9. Additionally, OLA reversed the increasing mitochondrial depolarization and promoted mitochondrial ATP production. OLA, the 3β-hydroxy-olean-12-en-28-oic acid，is widely distributed in the plant kingdom as

free acid or as aglycone of triterpenoid saponins, and is one kind of natural bioactive compounds having antioxidant, anti-inflammatory, and anti-apoptotic potential34, 35. While OLA has been previously shown to enhance PPARγ activity36, 37, we confirmed the ability of OLA to increase PPARγ expression. And PPARγ is involved in the regulation of OLA to SOD2. Mitochondrial superoxide produced by Sod2 loss impaired extracellular matrix homeostasis via mitochondrial dysfunction25. And our data found that OLA decreased protein degradation after high glucose

ACCEPTED MANUSCRIPT treatment, resulting in SOD2 expression and activity increasing, to prevent cartilage degradation, via PPARγ. In conclusion, OLA protected against the high-glucose-induced cartilage injury via PPARγ/SOD2 pathway, which could be a pharmacological agent for the treatment of diabetic

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osteoarthritis.

ACCEPTED MANUSCRIPT Reference 1.

Geusens PP, van den Bergh JP. Osteoporosis and osteoarthritis: Shared mechanisms and epidemiology. Curr Opin Rheumatol. 2016;28:97-103

2.

Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, Kelly-Hayes M, Wolf PA, Kreger BE, Kannel WB. The effects of specific medical conditions on the functional limitations of elders in the framingham study. Am J Public Health. 1994;84:351-358

3.

McClung M. Is altered bone health part of the metabolic syndrome? Menopause.

4.

Hart DJ, Doyle DV, Spector TD. Association between metabolic factors and knee osteoarthritis

RI PT

2010;17:900-901

in women: The chingford study. J Rheumatol. 1995;22:1118-1123 5.

Katz JD, Agrawal S, Velasquez M. Getting to the heart of the matter: Osteoarthritis takes its place as part of the metabolic syndrome. Curr Opin Rheumatol. 2010;22:512-519

Frey MI, Barrett-Connor E, Sledge PA, Schneider DL, Weisman MH. The effect of noninsulin

SC

6.

dependent diabetes mellitus on the prevalence of clinical osteoarthritis. A population based study. J Rheumatol. 1996;23:716-722

Louati K, Vidal C, Berenbaum F, Sellam J. Association between diabetes mellitus and

M AN U

7.

osteoarthritis: Systematic literature review and meta-analysis. RMD Open. 2015;1:e000077 8.

Nieves-Plaza M, Castro-Santana LE, Font YM, Mayor AM, Vila LM. Association of hand or knee osteoarthritis with diabetes mellitus in a population of hispanics from puerto rico. J Clin Rheumatol. 2013;19:1-6

9.

Waine H, Nevinny D, Rosenthal J, Joffe IB. Association of osteoarthritis and diabetes mellitus. Tufts Folia Med. 1961;7:13-19

10.

Aiello FC, Trovato FM, Szychlinska MA, Imbesi R, Castrogiovanni P, Loreto C, Musumeci G.

TE D

Molecular links between diabetes and osteoarthritis: The role of physical activity. Curr Diabetes Rev. 2015 11.

Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W. Differential expression of peroxisome proliferator-activated receptors (ppars): Tissue distribution of ppar-alpha, -beta, and -gamma in the adult rat. Endocrinology. 1996;137:354-366 Cho MC, Lee K, Paik SG, Yoon DY. Peroxisome proliferators-activated receptor (ppar)

EP

12.

modulators and metabolic disorders. PPAR Res. 2008;2008:679137 13.

Chen YJ, Chan DC, Lan KC, Wang CC, Chen CM, Chao SC, Tsai KS, Yang RS, Liu SH. Ppargamma

AC C

is involved in the hyperglycemia-induced inflammatory responses and collagen degradation in human chondrocytes and diabetic mouse cartilages. J Orthop Res. 2015;33:373-381

14.

Vasheghani F, Zhang Y, Li YH, Blati M, Fahmi H, Lussier B, Roughley P, Lagares D, Endisha H, Saffar B, Lajeunesse D, Marshall WK, Rampersaud YR, Mahomed NN, Gandhi R, Pelletier JP, Martel-Pelletier J, Kapoor M. Ppargamma deficiency results in severe, accelerated osteoarthritis associated with aberrant mtor signalling in the articular cartilage. Ann Rheum

Dis. 2015;74:569-578 15.

Liu J. Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol. 1995;49:57-68

16.

Sporn MB, Liby KT, Yore MM, Fu L, Lopchuk JM, Gribble GW. New synthetic triterpenoids: Potent agents for prevention and treatment of tissue injury caused by inflammatory and oxidative stress. J Nat Prod. 2011;74:537-545

17.

Quang TH, Ngan NT, Minh CV, Kiem PV, Thao NP, Tai BH, Nhiem NX, Song SB, Kim YH. Effect of triterpenes and triterpene saponins from the stem bark of kalopanax pictus on the

ACCEPTED MANUSCRIPT transactivational activities of three ppar subtypes. Carbohydr Res. 2011;346:2567-2575 18.

Wei J, Zhu H, Komura K, Lord G, Tomcik M, Wang W, Doniparthi S, Tamaki Z, Hinchcliff M, Distler JH, Varga J. A synthetic ppar-gamma agonist triterpenoid ameliorates experimental fibrosis: Ppar-gamma-independent suppression of fibrotic responses. Ann Rheum Dis. 2014;73:446-454

19.

Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat Protoc. 2008;3:1253-1260 Mapanga RF, Rajamani U, Dlamini N, Zungu-Edmondson M, Kelly-Laubscher R, Shafiullah M,

RI PT

20.

Wahab A, Hasan MY, Fahim MA, Rondeau P, Bourdon E, Essop MF. Oleanolic acid: A novel cardioprotective agent that blunts hyperglycemia-induced contractile dysfunction. PloS one. 2012;7:e47322 21.

Ge F, Zeng F, Liu S, Guo N, Ye H, Song Y, Fan J, Wu X, Wang X, Deng X, Jin Q, Yu L. In vitro synergistic interactions of oleanolic acid in combination with isoniazid, rifampicin or

SC

ethambutol against mycobacterium tuberculosis. Journal of medical microbiology. 2010;59:567-572 22.

Jiang Y, Li L, Liu B, Zhang Y, Chen Q, Li C. Ppargamma upregulation induced by vagus nerve Monit. 2015;21:268-275

23.

M AN U

stimulation exerts anti-inflammatory effect in cerebral ischemia/reperfusion rats. Med Sci Ansari MY, Haqqi TM. Interleukin-1beta induced stress granules sequester cox-2 mrna and regulates its stability and translation in human oa chondrocytes. Sci Rep. 2016;6:27611 24.

Verma RP, Hansch C. Matrix metalloproteinases (mmps): Chemical-biological functions and (q)sars. Bioorg Med Chem. 2007;15:2223-2268

25.

Koike M, Nojiri H, Ozawa Y, Watanabe K, Muramatsu Y, Kaneko H, Morikawa D, Kobayashi K,

TE D

Saita Y, Sasho T, Shirasawa T, Yokote K, Kaneko K, Shimizu T. Mechanical overloading causes mitochondrial superoxide and sod2 imbalance in chondrocytes resulting in cartilage degeneration. Sci Rep. 2015;5:11722 26.

Laiguillon MC, Courties A, Houard X, Auclair M, Sautet A, Capeau J, Feve B, Berenbaum F, Sellam J. Characterization of diabetic osteoarthritic cartilage and role of high glucose

EP

environment on chondrocyte activation: Toward pathophysiological delineation of diabetes mellitus-related osteoarthritis. Osteoarthritis Cartilage. 2015;23:1513-1522 27.

Cooley J, Broderick TL, Al-Nakkash L, Plochocki JH. Effects of resveratrol treatment on bone

AC C

and cartilage in obese diabetic mice. J Diabetes Metab Disord. 2015;14:10 28.

Fukui T, Tenborg E, Yik JH, Haudenschild DR. In-vitro and in-vivo imaging of mmp activity in cartilage and joint injury. Biochem Biophys Res Commun. 2015;460:741-746

29.

Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, Mitchell P, Hambor J, Diekmann O, Tschesche H, Chen J, Van Wart H, Poole AR. Enhanced cleavage of type ii collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997;99:1534-1545

30.

Mateen S, Zafar A, Moin S, Khan AQ, Zubair S. Understanding the role of cytokines in the pathogenesis of rheumatoid arthritis. Clin Chim Acta. 2016;455:161-171

31.

Choi JK, Kim SW, Kim DS, Lee JY, Lee S, Oh HM, Ha YS, Yoo J, Park PH, Shin TY, Kwon TK, Rho MC, Kim SH. Oleanolic acid acetate inhibits rheumatoid arthritis by modulating t cell immune responses and matrix-degrading enzymes. Toxicol Appl Pharmacol. 2016;290:1-9

32.

Thomas CM, Fuller CJ, Whittles CE, Sharif M. Chondrocyte death by apoptosis is associated with cartilage matrix degradation. Osteoarthritis Cartilage. 2007;15:27-34

ACCEPTED MANUSCRIPT 33.

Thomas CM, Fuller CJ, Whittles CE, Sharif M. Chondrocyte death by apoptosis is associated with the initiation and severity of articular cartilage degradation. Int J Rheum Dis. 2011;14:191-198

34.

Long C, Yang J, Yang H, Li X, Wang G. Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant, antiinflammatory, and antiapoptotic activities. Mol Med Rep. 2016;13:4697-4704

35.

Wang X, Liu R, Zhang W, Zhang X, Liao N, Wang Z, Li W, Qin X, Hai C. Oleanolic acid improves Cell Endocrinol. 2013;376:70-80

36.

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hepatic insulin resistance via antioxidant, hypolipidemic and anti-inflammatory effects. Mol Wang Y, Porter WW, Suh N, Honda T, Gribble GW, Leesnitzer LM, Plunket KD, Mangelsdorf DJ, Blanchard

SG,

Willson

TM,

Sporn

MB.

A

synthetic

triterpenoid,

2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (cddo), is a ligand for the peroxisome proliferator-activated receptor gamma. Mol Endocrinol. 2000;14:1550-1556

Bradshaw AD, Puolakkainen P, Dasgupta J, Davidson JM, Wight TN, Helene Sage E. Sparc-null

SC

37.

mice display abnormalities in the dermis characterized by decreased collagen fibril diameter

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EP

TE D

M AN U

and reduced tensile strength. J Invest Dermatol. 2003;120:949-955

ACCEPTED MANUSCRIPT Figure legends Figure 1. The protective effect of OLA on high-glucose induced cartilage degradation. A: OLA improved type X collagen expression in cartilage. The type X collagen expression was assessed by immuohistochemisty staining. (Scale bar=100µm)

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B: OLA reduced cartilage degradation. Fast Green and safranin O staining was performed to assess cartilage degradation. (Scale bar=50µm)

C: type Ⅱ collagen expression in high glucose and OLA treated chondrocytes. Results are expressed as the ratio of type Ⅱ collagen and GAPDH. (*P<0.05 vs. the others, n=4 in each

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group)

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Figure 2. The protective effect of OLA on high-glucose induced chondrocytes injury. A: OLA improved cell viability of chondrocytes. The cell viability was assessed by cell counting kit-8. (*P<0.05 vs. the others, n=8 in each group)

B: Effect of OLA on LDH level of cell supernatant after high-glucose treatment. (*P<0.05 vs. others, n=8).

n=8).

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C: Effect of OLA on the level of PGE2 after high-glucose treatment. (*P<0.05 vs. others,

n=8).

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D: Effect of OLA on the level of MMP-13 after high-glucose treatment. (*P<0.05 vs. others,

E: Effect of OLA on the level of IL-6 after high-glucose treatment. (*P<0.05 vs. others,

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n=8).

Figure 3. The protective effect of OLA on mitochondrial function A: Mitochondrial membrane potential (MMP, ∆ψm) was measured with JC-1 staining.

Fluorescent intensity of JC-1 was determined via flow cytometry. These experiments were performed in five times with similar results. (*P<0.05 vs. control, n=4). B: Effect of OLA on ATP production in chondrocytes cells after high-glucose treatment. (*P<0.05 vs. others, n=4). C: Effect of OLA on active caspase 9 expression in chondrocytes cells after high-glucose treatment. Results are expressed as the ratio of active caspase 9 and GAPDH. (*P<0.05 vs. others,

ACCEPTED MANUSCRIPT n=4).

Figure 5. The PPARγ/SOD2 signal pathway is involved in the protection of OLA on high-glucose injured chondrocytes

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A and B: Effect of OLA on PPARγ and SOD2 expression in chondrocytes cells after high-glucose treatment. Results are expressed as the ratio of PPARγ and SOD2 and GAPDH. (*P<0.05 vs. others, n=4).

C: Effect of OLA and PPARγ siRNA on SOD2 activity in chondrocytes after high-glucose

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treatment. (*P<0.05 vs. others except the siRNA group, n=4).

D: SOD2 protein degradation in high-glucose treated chondrocytes with or without OLA.

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The cells were incubated with cycloheximide (10-5 M) for the indicated times. Results are

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expressed as percent change of control in each group (n = 8, *P < 0.05 vs. WT).

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Fig. 1A

C57BL/6 mice + OLA

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db/db mice

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C57BL/6 mice

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Fig. 1B

C57BL/6 mice + OLA

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C57BL/6 mice

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db/db mice

Scale bar=5um

db/db mice + OLA

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Fig. 1C HG 0

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collagen Ⅱ

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GAPDH

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800

control

AC C

PGE2 concetration (pg/ml)

1000

0

10

HG

30

50

OA (mM)

ACCEPTED MANUSCRIPT

350

RI PT

*

300

SC

250

M AN U

200 150

0

control

EP

50

TE D

100

AC C

MMP-13 concentration (ng/ml)

Fig. 2D

0

10

30

HG

50

OA (mM)

ACCEPTED MANUSCRIPT

RI PT

500

*

M AN U

SC

400 300

TE D

200

0

EP

100

control

AC C

IL-6 concentration (pg/ml)

Fig. 2E

0

10

30

HG

50

OA (mM)

ACCEPTED MANUSCRIPT

.8 .6

M AN U EP

1.0

TE D

1.2

*

AC C

(Fold change of control)

Relative MMP

SC

RI PT

Fig. 3A jc-1

.4 .2

0.0 control

HG

OA

ACCEPTED MANUSCRIPT

RI PT

Fig. 3B ATP

SC

(Fold change of control)

1.2

M AN U

.8

*

.2

0.0

EP

.4

TE D

.6

AC C

ATP production

1.0

control

HG

OA

ACCEPTED MANUSCRIPT

Control

HG

OA

M AN U

SC

Active caspase-9

RI PT

Fig. 3C

1.0

TE D

*

.6 .4 .2

EP

.8

AC C

Active caspase-9 / GAPDH

GAPDH

0.0 control

HG

OA

ACCEPTED MANUSCRIPT

Fig. 4A Control

HG

OA

RI PT

PPARγ

M AN U

SC

GAPDH

TE D

.8 .6

*

.2 0.0

EP

.4

AC C

PPARγ / GAPDH

1.0

control

HG

OA

ACCEPTED MANUSCRIPT

Fig. 4B Control

HG

OA

RI PT

SOD2

M AN U

SC

GAPDH .7

TE D

.5 .4

EP

.3

*

.2 .1 0.0

AC C

SOD2 / GAPDH

.6

control

HG

OA

ACCEPTED MANUSCRIPT

RI PT

Fig. 4C

SC M AN U

1.0

*

.6

.2 0.0

EP

.4

TE D

.8

AC C

(Fold change of control)

SOD2 activity

1.2

Control

HG

OA

Scramble siRNA siRNA OA HG

ACCEPTED MANUSCRIPT

Fig. 4D HG 1

2

3

0

1

2

3 Time of CHX treatment (h)

RI PT

0

HG+OLA

SC

SOD2

M AN U

GAPDH

TE D

1.0

*

.6 .4

EP

.8

HG HG+OA

* *

AC C

SOD2 / GAPDH

1.2

.2 0.0 0

1

2

3

Time of CHX treatment (h)

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Highlights: 1. Oleanolic acid prevents cartilage degeneration secondary to diabetes 2. The mechanism relies on mitochondrial stabilization 3. Oleanolic acid can be a potential therapy in diabetes associated osteoarthritis