J Orthop Sci DOI 10.1007/s00776-014-0654-8


Beneficial effect of grape seed proanthocyanidin extract in rabbits with steroid-induced osteonecrosis via protecting against oxidative stress and apoptosis Qichun Song · Zhibin Shi · Wenchao Bi · Ruiyu Liu · Chen Zhang · Kunzheng Wang · Xiaoqian Dang 

Received: 1 August 2014 / Accepted: 17 September 2014 © The Japanese Orthopaedic Association 2014

Abstract  Background  Oxidative damage and apoptosis play dominant roles in the pathogenesis of steroid-induced osteonecrosis (ON). Grape seed proanthocyanidin extract (GSPE) demonstrates antioxidant and antiapoptotic properties. Our aim was to demonstrate the effects of GSPE in preventing steroid-induced ON in rabbits. Methods  Osteonecrosis was induced by high-dose methylprednisolone (40 mg/kg). Rabbits in the preventive medicine group were treated with 100 mg/kg/day GSPE for 14 consecutive days, and the presence or absence of ON was examined histopathologically. Oxidative damage in bone tissue was assessed by immunohistochemical staining of 8-oxo-2’-deoxyguanosine (8-OHdG), malondialdehyde (MDA) levels, and activities of antioxidant enzymes Cu/ Zn superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px). Apoptosis was detected via quantitative terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labelling (TUNEL) staining and activated caspase 3 immunoblotting and activity. Results  GSPE significantly attenuated the changes of immunohistochemical staining of 8-OHdG, MDA levels, and antioxidant enzymes activities, which were caused by methylprednisolone administration. Quantitative TUNEL

Q. Song · Z. Shi · R. Liu · C. Zhang · K. Wang · X. Dang (*)  Department of Orthopaedics, Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an 710004, Shaanxi, People’s Republic of China e-mail: [email protected] W. Bi  Department of Orthopaedics, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi, People’s Republic of China

and caspase 3 assay showed lower apoptosis with GSPE application. Simultaneously, GSPE reduced the incidence of steroid-induced ON in an established rabbit model to 17.6 %, compared with 87.5 % in the steroid-only group. Conclusion  These results reveal that GSPE treatment could inhibit oxidative damage and apoptosis to exert beneficial effects on reducing the incidence of steroid-induced ON in rabbit models.

Introduction Steroid-induced osteonecrosis (ON) of the hip has been reported to occur frequently in patients who receive highdose steroid therapy for diseases such as systemic lupus erythematosus and inflammatory bowel disease, and also for immunosuppression after organ transplants [1]. Although steroid-induced ON is suggested to develop because of ischemic status inside the bone, previous studies have been unable to clarify the detailed pathogenesis of the process by which corticosteroid administration induces the development of ON. It has become increasingly evident that oxidative stress is relevant to the pathogenesis of various diseases, including apoptosis and vascular injury [2, 3]. Recently, it was reported that oxidative injury and apoptosis were present in bone after corticosteroid administration and before the development of ON [4, 5]. Otherwise, prooxidant buthionine sulphoximine could successfully induce ON in rats [6]. Additionally, the presence of oxidation damage was proved to exert oxidative stress in bone marrow vessels, myelocytes, and adipocytes in animal models of steroidinduced ON [7]. Furthermore, excessive reactive oxygen species (ROS) can be induced by steroid administration [8], and apoptosis can be caused by oxidative stress via


Q. Song et al.

both the mitochondria-dependent and mitochondria-independent pathways [3]. These findings demonstrate oxidative stress in the development of ON and suggest that the incidence of ON may be considerably decreased by inhibiting it. Therefore, antioxidants may deter steroid-induced ON by attenuating oxidative stress damage caused by steroid administration. Numerous antioxidative substances including vitamin E and edaravone have been reported to help prevent steroid-induced ON [7, 9]. Proanthocyanidins extracted from grape seeds (GSPE) are the most abundant phenolic and widely used compounts, primarily as nutritional supplements. Besides its free-radical-scavenging and antioxidant activity, GSPE containing procyanidin oligomers and polymers exhibit vasodilatory, immunostimulating, anticarcinogenic, antiallergic, anti-inflammatory, antibacterial, cardioprotective, and antiviral activities. It is also reported that GSPE could diminish lipid peroxidation, platelet aggregation, capillary permeability and fragility, and modulate enzyme activity of phospholipase A2, cyclooxygenase, and lipoxygenase [10]. Accordingly, we undertook our experiment to identify whether the addition of GSPE might provide a prophylactic effect on the development of ON in a steroid-administered rabbit ON model.

groups were treated with vehicle [phosphate-buffered saline (PBS)].


Histopathologic evaluation of ON

Animals, grouping, and treatment

Necrosis of bone and marrow tissues was determined in hematoxylin–eosin (H&E)-stained preparations by light microscopy (Nikon YS100; Nikon Corporation, Tokyo, Japan) in four sections taken from the bilateral femurs of each rabbit. A positive diagnosis of ON was judged to be present when empty lacunae or condensed nuclei of osteocytes within bone trabeculae were noted, accompanied by surrounding bone-marrow-cell or fat-cell necrosis [13]. If ON was found in at least one of the four sections, the rabbit was considered to have ON. The development of ON was judged to be present when ON was identified in either isolated femur. The ON incidence was calculated as the ratio of the number of rabbits with ON to the number of each group.

Fifty male 28-week-old healthy, pathogen-free, adult New Zealand white rabbits, average weight (2.9– 3.5 kg), were used. Animals were obtained from the Experimental Animal Center of Medical College of Xi’an Jiaotong University. All experimental procedures were approved by the Ethics Committee of Medical College of Xi’an Jiaotong University. Animals were randomly divided into steroid group (S, n  = 20), preventive group (P, n  = 20), and control group (C, n  = 10); all rabbits received a standard laboratory diet and water ad libitum. Based on Yamamoto’s established protocol for creating steroid-induced ON [11], all rabbits (except C group) were injected with a single dose of methylprednisolone [(MPSL), Pfizer, Inc., Brussels, Belgium] at 40 mg/kg body weight into the right gluteal muscle. GSPE were provided in the form of capsules containing 100 mg GSPE from General Nutrition Corporation (Pittsburgh, PA, USA), and each capsule was distilled in 6 ml normal saline. During the experiment, the P group received GSPE of 12 ml/kg orally [12] by gavage for 14 consecutive days starting from 1 day before MPSL administration until the rabbits were killed; S and C


Tissue preparation Animals in all groups were sacrificed 2 weeks after MPSL administration with an excessive i.v. dose of sodium pentobarbital, after which the bilateral femurs were rapidly removed and the proximal one third harvested and divided into two parts through the center axis of the femur. Femur halves were immediately fixed in 10 % buffered formalin for 1 week and decalcified with 10 % ethylenediaminetetraacetate (EDTA) for 3 weeks. After decalcification, the femurs were embedded in paraffin, and 4-μm-thick coronal sections were prepared for histomorphology and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labelling (TUNEL) staining. Other halves of the bilateral femurs were homogenized with ice-cold potassium chloride (KCl) (1.15 %) using a homogenizer. Homogenates were then centrifuged at 4,400 g for 10 min at 4 °C to remove cell debris. The supernatant obtained was used to determine superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) levels. Meanwhile, bilateral proximal humeri were collected and stored at −80 °C for Western blot analysis and enzyme activity assay.

Immunohistochemistry To examine the development of DNA oxidative injury in bone, sections of femurs were stained immunohistochemically with mouse monoclonal antibody to 8-hydroxy2′-deoxyguanosine (8-OHdG) (N45.1, Abcam, Cambridge, UK) [14]. Briefly, after deparaffinization, sections were treated with 3 % hydrogen peroxide (H2O2) for 10 min, rinsed in PBS (pH 7.6), and pretreated with 3 %

GSPE benefit steroid-induced ON

nonimmune animal serum in PBS for 30 min at room temperature. Then, sections were reacted with 1:20 dilution of 8-OHdG monoclonal antibody overnight at 4 °C. Sections were stained using the avidin–biotin complex method, treated with 3,3’-diaminobenzidine (DAB) for 5 min, and counterstaining was carried out with H&E for 10 min. The immunohistological data of 8-OHdG was quantified using the analysis software Image-Pro Plus (Media Cybernetics, Baltimore, MD, USA) and evaluated by calculating the positive ratio (%PR, the percentage of positive cells/total cells) per appropriate visual field (200× magnification). Specimens from all groups were analyzed, and the mean of data obtained from five independent fields was used as a representative value for each animal. Evaluation of SOD and GSH-Px activities and MDA levels A portion of the supernatant obtained was collected to determine SOD and GSH-Px activities using an ultraviolet and visible (UV–Vis) Recording Spectrophotometer (UV-2100S, Shimadzu Co., Kyoto, Japan), as previously described [15]. The rest of the supernatant obtained was used to measured tissue MDA level using the thiobarbituric acid method, as described by Ohkawa et al. [16]. TUNEL assay We used a standard terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labelling (TUNEL) assay technique to detect the fragmented DNA associated with apoptosis. TUNEL staining was performed using an in situ cell-death detection kit AP (Roche, Mannheim, Germany) in accordance with the manufacturer’s instructions. TUNEL-staining cells show as brown stains, and other cell nuclei show as blue stains. The stained sections were examined under light microscope at 200× magnification. One hundred cells were counted in five microscopic fields per tissue slide. The percentage of TUNEL-positive apoptotic cells was measured, and this represents the apoptotic index (AI), which was accomplished by two blinded researchers.

bicinchoninic acid protein assay reagent (Pierce™, Rockford, IL, USA). Equalized protein samples (3 μg) were used for electrophoresis. Following electrophoresis, proteins were transferred onto 0.45-µm nitrocellulose and polyvinylidene fluoride membranes (Hybond-ECL; Amersham Pharmacia Biotechnology Inc., Piscataway, NJ, USA) and blocked with 3 % bovine serum albumin (BSA) at room temperature for 2 h. Membranes were incubated overnight at 4 °C with anticaspase 3 (Abcam PLC) diluted 1:200. Then, membranes were incubated for 1 h at 4 °C with a 1:2,000 dilution of horse-radish-peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in antibody diluents. Immunoreactive proteins were visualized on a film using an enhanced chemiluminescence kit (NEN Life Science Products Inc., Boston, MA, USA). Relative protein expression was determined using image analysis software (Media Cybernetics). For the quantitative analysis of protein, the intensity of each test band was expressed as the optical density (OD). β-actin was detected using a mouse monoclonal anti-actin antibody (1:3,000; Santa Cruz Biotechnology, Inc.) and was used as an internal control. Caspase 3 activity was accomplished in supernates from homogenates of proximal humeri in a plate reader at 405 nm, and we determined the absorbance of p-nitroaniline obtained from the caspase 3 activity assay kit (Calbiochem, San Diego, CA, USA) according to the manufacturer’s instructions. Statistics Categorical data, i.e., incidence of ON, were analyzed using Fisher’s exact probability test. Numerical data in each group are presented as mean ± standard deviation (SD). Comparisons between multiple groups were performed with one-way analysis of variance (ANOVA), followed by the Student–Newman–Keuls (SNK) multiple comparison procedure. All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant at P 

Beneficial effect of grape seed proanthocyanidin extract in rabbits with steroid-induced osteonecrosis via protecting against oxidative stress and apoptosis.

Oxidative damage and apoptosis play dominant roles in the pathogenesis of steroid-induced osteonecrosis (ON). Grape seed proanthocyanidin extract (GSP...
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