Wear particle induced periprosthetic osteolysis is the main cause of aseptic loosening of orthopedic implants. The aim of the present study is to determine the protective effect of quercetin (QUE) against titanium (Ti) particle induced endoplasmic reticulum stress (ERS) related apoptosis and osteolysis. In the present study, RAW264.7 cells were pretreated with different concentrations (40, 80, and 160 μmol/l) of QUE for 30 min and then treated with Ti particle (5 mg/ml) for 24 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC Apoptosis Detection Kit, respectively. Protein and mRNA expressions of ERS-related genes were examined by Western blot and real-time PCR, respectively. The release of inflammatory cytokines was detected by ELISA. Then, a mouse calvarial osteolysis model was established. Histological sections of calvaria were stained with Hematoxylin-Eosin (H&E) or tartrate-resistant acid phosphatase (TRAP). The results showed that Ti particle reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of Ti particle were dramatically inhibited by QUE pretreatment. Interestingly, we found that QUE also significantly reduced Ti particle induced up-regulation of the expression levels of protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1), glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and caspase-3 and enhanced the down-regulation of Bcl-2. In addition, QUE decreased Ti particle-induced inflammatory cytokines release from RAW264.7 cells. Moreover, treatment with QUE markedly decreased osteoclast number. In a mouse calvarial osteolysis model, QUE inhibited Ti particle induced osteolysis in vivo by inhibiting osteoclast formation and expressions of ERS-related genes. In conclusion, QUE can protect RAW264.7 cells from Ti particle induced ERS-related apoptosis and suppress calvarial osteolysis in vivo.

Introduction

Total joint replacement (TJR), which is by the implantation of a permanent in-dwelling artificial prosthesis, is a highly successful procedure for promoting the agility in patients with joint dysfunction [1]. It has been proved that titanium (Ti) components have been widely used for joint replacement [2]. However, aseptic loosening due to periprosthetic osteolysis, induced by the adverse biological responses to wear particles, can damage the efficacy and longevity of the prosthetic components [3,4]. The particles generated from the mechanical wear of prosthetic components can be phagocytosed by macrophages and other inflammatory cells [5]. During the pathological process, the macrophages activated by Ti particles release proinflammatory cytokines including interleukin (IL)-6, IL-1β and tumor necrosis factor α (TNF-α). [6,7]. In addition, these cytokines lead to an imbalance in bone metabolism, promote osteoclastogenesis, and stimulate mature osteoclasts to absorb the adjacent bone [8,9]. As no effective drug therapy is currently available to prevent osteolysis, more studies are required.

Recently, endoplasmic reticulum stress (ERS) has attracted particular interest due to its role in inflammatory responses under pathological conditions [10]. Besides, ERS is a novel pathway of cellular apoptosis [11]. Glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone and is used for monitoring ERS [12]. In the early stages, ERS is alleviated by the activation of unfold protein response (UPR). The following signal transduction pathways are involved in the UPR: protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1 (IRE1). If the ERS is not alleviated by the UPR, persistent and severe ERS leads to cellular apoptosis. The apoptosis is initiated by up-regulating the ERS-related proapoptotic marker CCAAT/enhancer-binding protein homologous protein (CHOP) [13].

Quercetin (QUE) is one of the major flavonoids, ubiquitously distributed in plants [14]. QUE has been reported to have anti-inflammatory effects, which are mediated through the suppression of proinflammatory cytokines [15]. Moreover, intense exercise induced ERS and inflammation can be attenuated by QUE [16]. However, the protective effect of QUE against Ti particle induced ERS remains unclear.

We designed the current study to determine the potential protective effect of QUE against Ti particle induced ERS-related apoptosis and osteolysis.

Materials and methods

Cell culture

The murine macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (Manassas, U.S.A.). The cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, U.S.A.) supplemented with 10% FBS (Gibco, U.S.A.), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in a humidified incubator at 37°C in 5% CO2.

Preparation of Ti particles

Titanium alloy particles (Ti-6Al-4V) (mean particle size: 2.3 μm, size range: 0.1–68 μm) were purchased from the Zimmer Corporation (Warsaw, U.S.A.). Ti particles were prepared as previously described [17]. The particles were sterilized at 180°C for 6 h, followed by washing with 70% ethanol for 48 h to remove endotoxin and confirmed endotoxin-free using a commercial detection kit (Sigma, U.S.A.). Ti particles were sonicated and vortexed before treatment.

Cell treatment

Cells (1 × 105/ml) were seeded in culture plates. They were divided into five groups: control group (vehicle DMSO); Ti particle (5 mg/ml) group; Ti + high-dose QUE (160 μmol/l) group; Ti + medium-dose QUE (80 μmol/l) group; Ti + low-dose QUE (40 μmol/l) group. In the Ti + QUE groups, cells were subjected to QUE at a final concentration of 40, 80, or 160 μmol/l for 30 min, followed by treatment with Ti particle (5 mg/ml) for an additional 24 h. Subsequently, cells were harvested for further analysis.

Cell viability

Cell viability was determined by MTT assay. RAW264.7 macrophage cells were seeded at 5 × 105 cells per well and incubated with QUE at various concentrations (40, 80, and 160 μmol/l) for 24 h at 37°C. After incubation, 20 μl of MTT solution (5 mg/ml) was added to each well and the cells were incubated for 4 h. The formazan crystals were dissolved in 200 μl of DMSO. Absorbance was determined at 570 nm. The results were expressed as a percentage of surviving cells over control cells.

Flow cytometry

To detect apoptotic cells, 1 × 105/ml RAW264.7 macrophage cells were plated in 24-well plates. After the treatment, cells were harvested and analyzed for cell apoptosis by Annexin-V and propidium iodide (PI) staining, using FITC Annexin-V apoptosis detection kit (Life Technology) according to the manufacturer’s instructions. Then, the cells were detected using flow cytometry, and the data were analyzed using FlowJo7.6.1.

Quantitative real-time PCR

RNA was prepared with TRIzol reagent (Invitrogen, U.S.A.). First-strand cDNA was synthesized in a 20-μl reaction volume using SuperScript III reverse transcriptase (Life Technologies). Q-PCR was performed in triplicate to amplify all targets by using a FastStart DNA Master SYBR Green I Kit (Roche, U.S.A.) according to the manufacturer’s instructions and a Roche Light Cycler Thermocycler. Gene expression data were normalized to GAPDH mRNA levels. Primers are listed in Table 1.

Table 1
Primer information for qPCR
GenePrimersProduct size (bp)
PERK F: 5′-GGGTGGAAACAAAGAAGAC-3′ 215 
 R: 5′-CAATCAGCAACGGAAACT-3′  
IRE1 F: 5′-GCGATGGACTGGTGGTAACT-3′ 184 
 R: 5′-GTTTGCTCTTGGCCTCTGTC-3′  
GRP78 F: 5′-GGCGTGAGGTAGAAAAGG-3′ 196 
 R: 5′-ATGGTAGAGCGGAACAGG-3′  
CHOP F: 5′-ACCTTCACTACTCTTGACCCT-3′ 129 
 R: 5′-TCTTCCTCCTCTTCCTCCT-3′  
Caspase-12 F: 5′-CTGGCCCTCATCATCTGCAACAA-3′ 173 
 R: 5′-CGGCCAGCAAACTTCATTAAT-3′  
Caspase-3 F: 5′-GGAGCTGGACTGTGGCATTGA-3′ 94 
 R: 5′-CAGTTCTTTCGTGAGCATGGA-3′  
Bcl-2 F: 5′-ACAGAGGGGCTACGAGTG-3′ 158 
 R: 5′-GGCTGGAAGGAGAAGATG-3′  
TRAP F: 5′-GCTACTTGCGGTTTCACTATGGA-3′ 201 
 R: 5′-TGGTCATTTCTTTGGGGCTTATCT-3′  
RANK F: 5′-AGATGTGGTCTGCAGCTCTTCCAT-3′ 124 
 R: 5′-ACACACTTCTTGCTGACTGGAGGT-3′  
GAPDH F: 5′-GAAGGTGAAGGTCGGAGTC-3′ 166 
 R: 5′-GAAGATGGTGATGGGATTTC-3′  
GenePrimersProduct size (bp)
PERK F: 5′-GGGTGGAAACAAAGAAGAC-3′ 215 
 R: 5′-CAATCAGCAACGGAAACT-3′  
IRE1 F: 5′-GCGATGGACTGGTGGTAACT-3′ 184 
 R: 5′-GTTTGCTCTTGGCCTCTGTC-3′  
GRP78 F: 5′-GGCGTGAGGTAGAAAAGG-3′ 196 
 R: 5′-ATGGTAGAGCGGAACAGG-3′  
CHOP F: 5′-ACCTTCACTACTCTTGACCCT-3′ 129 
 R: 5′-TCTTCCTCCTCTTCCTCCT-3′  
Caspase-12 F: 5′-CTGGCCCTCATCATCTGCAACAA-3′ 173 
 R: 5′-CGGCCAGCAAACTTCATTAAT-3′  
Caspase-3 F: 5′-GGAGCTGGACTGTGGCATTGA-3′ 94 
 R: 5′-CAGTTCTTTCGTGAGCATGGA-3′  
Bcl-2 F: 5′-ACAGAGGGGCTACGAGTG-3′ 158 
 R: 5′-GGCTGGAAGGAGAAGATG-3′  
TRAP F: 5′-GCTACTTGCGGTTTCACTATGGA-3′ 201 
 R: 5′-TGGTCATTTCTTTGGGGCTTATCT-3′  
RANK F: 5′-AGATGTGGTCTGCAGCTCTTCCAT-3′ 124 
 R: 5′-ACACACTTCTTGCTGACTGGAGGT-3′  
GAPDH F: 5′-GAAGGTGAAGGTCGGAGTC-3′ 166 
 R: 5′-GAAGATGGTGATGGGATTTC-3′  

Abbreviations: RANK, receptor activator of NF-κB; TRAP, tartrate-resistant acid phosphatase.

ELISA for cytokines

TNF-α, IL-6, and IL-1β levels in culture medium were quantitated using monoclonal anti-TNF-α, IL-6, or IL-1β antibodies according to the manufacturer’s instructions (R&D Systems, U.S.A.).

Tartrate-resistant acid phosphatase staining

For differentiation of RAW 264.7 cells into osteoclasts, cells were seeded in 96-well plates (104 cells/well). The tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma) was used to evaluate TRAP expression. TRAP+ cells with more than three nuclei were counted as osteoclasts using optical microscopy, and ImagePro Plus was used to quantitate the data.

Mouse calvarial osteolysis model and staining

The experimental design was approved by the Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University. All animals’ experimental procedures were performed according to the guidelines of the Care and Use of Laboratory Animals by the National Institute of Health, China.

The calvarial osteolysis model was established as published previously [18]. In brief, 24 healthy 6-week-old male BALB/C mice (weighing 18 ± 5 g) were equally randomized into four groups with six rats in each group: control group, Ti group, Ti  +  QUE (50 mg/kg per day) group and Ti  +  QUE (100 mg/kg per day) group. In Ti + QUE groups, mice were fed with a daily dose of 50 or 100 mg  of QUE per kg of body weight, respectively. QUE was fed from the third day before the operation until killed.

To inject Ti particles, mice were anesthetized with an intraperitoneal injection of pentobarbital. The surgical area was manually depilated and disinfected. A 0.5 -cm sagittal incision was made and the periosteum remained intact. Subsequently, a 25-gauge needle was used to inject 100 μl of 30  mg Ti particles resuspended in PBS directly over the calvarial bone and periosteum. Ten days after the operation, mice were killed and the calvaria were excised, fixed, and decalcified in EDTA. Histological sections of calvaria were stained with Hematoxylin-Eosin (H&E) or TRAP according to the manufacturers’ instructions.

Western blot

Proteins were extracted in 100  µl of SDS lysis buffer (10  mM EDTA, 25  mM Tris/HCl, pH 7.4, 95  mM NaCl, 2% SDS) with protease inhibitors (Sigma). Protein content was measured by the BCA method (Pierce) according to the manufacturer’s instructions. Total proteins (40  µg) were separated by SDS/PAGE (10% gel). Then the protein was blotted on to PVDF membrane (Bio–Rad) at 70 V for 65 min. Subsequently, the membranes were incubated with primary antibodies, including anti-PERK antibody (Cell Signaling), anti-IRE1 antibody (R&D Systems), anti-GRP78 antibody (Santa Cruz Biotechnology), anti-CHOP antibody (R&D Systems), anti-cleaved caspase-12 antibody (R&D Systems), anti-cleaved caspase-3 antibody (Cell Signaling), anti-Bcl-2 antibody (R&D Systems), and anti-GAPDH antibody (Sigma–Aldrich) overnight at 4 °C. Then the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (Boster, Wuhan, China) for 1  h at room temperature. The chemiluminescent signal was detected by the ECL Detection Reagents (Amersham).

Statistics analysis

The data are expressed as the mean ± S.D. Statistical analysis was performed with SPSS 16.0. Comparisons between the two groups were evaluated by Student’s t test. A P-value<0.05 was considered statistically significant.

Results

QUE inhibits Ti particle induced cell death

Cells were incubated with various concentrations of QUE (0, 40, 80, 160 μmol/l) for 24 h. As shown in Supplementary Figure S1A, no reduction in cell viability was observed when treated with QUE, demonstrating no detectable cytotoxicity of the drug. However, pretreated with various concentrations of QUE (40, 80, 160 μmol/l) alleviated Ti particle induced reduction in cell viability (Supplementary Figure S1B).

QUE decreases Ti particle induced ERS-related apoptosis of RAW264.7 cells

To evaluate the effect of QUE on cell apoptosis, Annexin V/PI staining was used. As shown in Figure 1A, Ti particle induced cell apoptosis of RAW264.7 cells. However, QUE had a significant anti-apoptotic effect on Ti particle induced cell apoptosis.

QUE decreases Ti particle induced ERS-related apoptosis in RAW264.7 cells

Figure 1
QUE decreases Ti particle induced ERS-related apoptosis in RAW264.7 cells

(A) The ratio of apoptotic cells stained with Annexin V-FITC and PI was detected by flow cytometry analysis (n=3). (B) qRT-PCR analysis of PERK, IRE1, GRP78, CHOP, caspase-12, caspase-3, and Bcl-2 mRNA in the RAW264.7 cells under different treatments (n=3). (C) PERK, IRE1, GRP78, CHOP, cleaved caspase-3, cleaved caspase-12, and Bcl-2 protein levels in RAW 264.7 macrophages were determined by Western blot (n=3). **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: qRT-PCR, quantitative real-time PCR; QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

Figure 1
QUE decreases Ti particle induced ERS-related apoptosis in RAW264.7 cells

(A) The ratio of apoptotic cells stained with Annexin V-FITC and PI was detected by flow cytometry analysis (n=3). (B) qRT-PCR analysis of PERK, IRE1, GRP78, CHOP, caspase-12, caspase-3, and Bcl-2 mRNA in the RAW264.7 cells under different treatments (n=3). (C) PERK, IRE1, GRP78, CHOP, cleaved caspase-3, cleaved caspase-12, and Bcl-2 protein levels in RAW 264.7 macrophages were determined by Western blot (n=3). **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: qRT-PCR, quantitative real-time PCR; QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

To determine whether anti-apoptosis induced by QUE was related to ERS signaling pathways, the changes in apoptosis-related and ERS-related genes were tested by Q-PCR and Western blot. Compared with the control group, the increased PERK, IRE1, GRP78, CHOP, caspase-12, and caspase-3 levels, while decreased Bcl-2 levels were observed in the Ti group (Figure 1B,C). However, QUE dose-dependently decreased PERK, IRE1, GRP78, CHOP, caspase-12, and caspase-3 levels, and increased the Bcl-2 levels in the Ti particle treated RAW264.7 cells (Figure 1B,C).

QUE decreases Ti particle induced inflammatory cytokines release from RAW264.7 cells

To evaluate the effect of QUE on Ti particle induced inflammatory cytokines release, RAW264.7 cells were treated with QUE (40, 80, 160 μmol/l). QUE dramatically inhibited Ti particle induced IL-6, IL-1β, and TNF-α release from RAW264.7 cells (Figure 2A–C). The results demonstrate that QUE has a dose-dependent inhibitory effect on Ti particle induced inflammatory cytokines release in macrophages.

QUE decreases Ti particle induced inflammatory cytokines release from RAW264.7 cells

Figure 2
QUE decreases Ti particle induced inflammatory cytokines release from RAW264.7 cells

(A) IL-6, (B) IL-1β, and (C) TNF-α levels in cell culture supernatants from RAW264.7 cells under different treatments (n=3). **P<0.01 compared with control group; ##P<0.01 compared with Ti group. Abbreviations: QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

Figure 2
QUE decreases Ti particle induced inflammatory cytokines release from RAW264.7 cells

(A) IL-6, (B) IL-1β, and (C) TNF-α levels in cell culture supernatants from RAW264.7 cells under different treatments (n=3). **P<0.01 compared with control group; ##P<0.01 compared with Ti group. Abbreviations: QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

QUE inhibits Ti particle induced differentiation of osteoclasts

To determine whether QUE inhibits osteoclast differentiation, TRAP-positive cells were analyzed by TRAP staining. As shown in Figure 3A, a significant increase in osteoclasts was observed in the RAW264.7 cells treated with Ti particle. Treatment with QUE markedly decreased osteoclast number in a dose-dependent manner. In addition, we analyzed TRAP and receptor activator of NF-κB (RANK) expression in RAW264.7 cells. RANK is present on osteoclast precursors and induces the development and activation of osteoclasts [19]. The mRNA expression levels of TRAP and RANK were up-regulated in Ti group compared with that in control group (Figure 3B). Consistent with its inhibition of osteoclast differentiation, QUE inhibited the mRNA expression levels of TRAP and RANK. These results are the evidence that QUE reduces osteoclast differentiation.

QUE inhibits Ti particle induced differentiation of osteoclasts

Figure 3
QUE inhibits Ti particle induced differentiation of osteoclasts

(A) Representative TRAP staining images (left) and quantitative analysis of the number of TRAP+ osteoclasts (right), magnification ×100 (n=3). (B) qRT-PCR analysis of TRAP and RANK mRNA expression levels in the RAW264.7 cells under different treatments (n=3). **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: qRT-PCR, quantitative real-time PCR; QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

Figure 3
QUE inhibits Ti particle induced differentiation of osteoclasts

(A) Representative TRAP staining images (left) and quantitative analysis of the number of TRAP+ osteoclasts (right), magnification ×100 (n=3). (B) qRT-PCR analysis of TRAP and RANK mRNA expression levels in the RAW264.7 cells under different treatments (n=3). **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: qRT-PCR, quantitative real-time PCR; QUE40, 40 μmol/l QUE; QUE80, 80 μmol/l QUE; QUE160, 160 μmol/l QUE.

Inhibitory effect of QUE on Ti particle induced osteolysis in a mouse calvaria model in vivo

Next, we analyzed whether QUE suppressed Ti particle induced osteolysis in vivo. Injection of Ti particles into the mouse calvaria notably induced osteolysis compared with control group, while administration of QUE at 50 mg/kg per day or 100 mg/kg per day reduced osteolysis (Figure 4A). Histomorphometric analysis indicated that the average bone area of Ti particle implanted mice was significantly less than that of control group (Figure 4B). In contrast, the bone area of Ti-treated mice was remarkably increased by treatment with QUE (Figure 4B).

Inhibitory effect of QUE on Ti particle induced osteolysis and ERS-related apoptosis in a mouse calvaria model in vivo

Figure 4
Inhibitory effect of QUE on Ti particle induced osteolysis and ERS-related apoptosis in a mouse calvaria model in vivo

(A) Representative photographs of calvarial histology stained with H&E (left), magnification: ×100 (n=3). (B) Bone area was measured using a digitalized image analyzer (IMT i-Solution; Korea) (n=3). (C) Representative TRAP staining images (left) and quantitative analysis of the number of TRAP+ osteoclasts (right), magnification: ×40 (n=3). (D) PERK, IRE1, GRP78, CHOP, cleaved caspase-3, cleaved caspase-12, and Bcl-2 protein levels in the calvaria of mice were determined by Western blot (n=3). *P<0.05 and **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: QUE50: 50 mg/kg per day QUE; QUE100, 100 mg/kg per day QUE.

Figure 4
Inhibitory effect of QUE on Ti particle induced osteolysis and ERS-related apoptosis in a mouse calvaria model in vivo

(A) Representative photographs of calvarial histology stained with H&E (left), magnification: ×100 (n=3). (B) Bone area was measured using a digitalized image analyzer (IMT i-Solution; Korea) (n=3). (C) Representative TRAP staining images (left) and quantitative analysis of the number of TRAP+ osteoclasts (right), magnification: ×40 (n=3). (D) PERK, IRE1, GRP78, CHOP, cleaved caspase-3, cleaved caspase-12, and Bcl-2 protein levels in the calvaria of mice were determined by Western blot (n=3). *P<0.05 and **P<0.01 compared with control group; #P<0.05 and ##P<0.01 compared with Ti group. Abbreviations: QUE50: 50 mg/kg per day QUE; QUE100, 100 mg/kg per day QUE.

To examine whether QUE suppressed Ti particles induced osteoclast formation in the calvaria, TRAP staining was performed. Compared with control group, a significant increase in osteoclasts was observed in the Ti group (Figure 4C). Treatment with QUE at 50 mg/kg per day and 100 mg/kg per day dramatically decreased osteoclasts (Figure 4C).

QUE inhibits Ti particle induced ERS-related apoptosis in vivo

To examine whether ERS-related apoptosis is also decreased by QUE in vivo, Western blot was performed. A significant increase in the PERK, IRE1, GRP78, CHOP, cleaved caspase-12, and cleaved caspase-3 protein levels and a significant decrease in Bcl-2 were found in Ti-implanted calvarium compared with the control group (Figure 4D). QUE significantly reduced the protein expression levels of PERK, IRE1, GRP78, CHOP, cleaved caspase-12, and cleaved caspase-3 and enhanced the levels of Bcl-2 in a dose-dependent manner (Figure 4D). These data demonstrate that QUE treatment reduces Ti particle induced ERS-related apoptosis in vivo.

Discussion

Apoptosis may be an important element in understanding the mechanisms of aseptic loosening and periprosthetic osteolysis [20]. Stea et al. [21] demonstrated that the apoptotic cells in the interface membrane, which were collected from revision surgery for aseptic loosening of hip joint prostheses, were mainly macrophages, and apoptosis was correlated with metal wear. Landgraeber et al. [22] found that in particle-induced osteolysis, apoptosis is pathologically increased. Yang et al. [23] proved that apoptosis in macrophages during particle engulfment is partially responsible for osteolysis in aseptic loosening of joint implants. Our results showed that Ti particle reduced cell viability and induced apoptosis in RAW264.7 macrophages, suggesting that apoptosis may be a crucial factor responsible for wear particle induced osteolysis.

Recently, ERS pathway is believed to be a novel apoptotic pathway which is different from mitochondrial pathway and receptor pathway [24]. Increasing evidence suggesting ERS pathway induced apoptosis is related to some pathological processes, such as Alzheimer’s disease [25], diabetes [26], pancreatic cancer [27], and osteoarthritis [28]. However, few studies focussed on whether ERS pathway induced apoptosis was involved in the aseptic loosening. In present study, ERS pathway related markers like PERK, IRE1, GRP78, and CHOP were examined using Western blot and Q-PCR. In mammals, ERS responses can be triggered by activation of three distinct signaling molecules, namely PERK, IRE1, and activating transcription factor 6 (ATF6) [29]. When ERS occurs, PERK and GRP78 dissociate, and activated PERK promotes the phosphorylation of α subunit of translation initiation factor-2 (eIF2α). Phosphorylation of eIF2α decreases protein synthesis, with a few exceptions, such as the activating transcription factor 4 (ATF4) [30]. CHOP is a major target for ATF4, which induces apoptosis in cells [31]. Previous studies have demonstrated that the expression levels of cleaved caspase-3 and GRP78 in macrophages in interface membrane of aseptic loosening were significantly higher than those in the control samples [28]. Our results found that the expressions of PERK, IRE1, GRP78, CHOP, and cleaved caspase-3 were induced, while Bcl-2 was reduced in macrophages in Ti group, indicating that the ERS pathway participated in the process of apoptosis.

The reduction in apoptotic reaction by administration of anti-apoptotic drugs may lead to the prevention of osteolysis. Landgraeber et al. [32] showed that adiponectin decreases wear particle induced osteolysis through its influence on apoptotic and inflammatory pathways. Park et al. [33] found that QUE may suppress the ERS-CHOP pathway induced apoptosis in dopaminergic neurones. Our results demonstrated that QUE inhibited Ti particle induced ERS-related apoptosis and reduced the release of inflammatory cytokines.

The results of the present study also demonstrated that QUE treatment significantly reduced Ti particle induced osteoclast differentiation in a mouse calvaria model. Increasing osteoclastogenesis and the inhibition of bone formation is the primary cause of aseptic loosening and osteolysis [34]. Growing evidence suggests that Ti particle impairs the function of mature osteoblasts as well as inducing osteoclast differentiation [3537], which is consistent with the current study. In addition, our results strongly indicate that QUE inhibits Ti-induced osteolysis in vivo as evident by decreased osteoclast numbers and increased bone area.

In conclusion, QUE inhibits Ti particle induced ERS-related apoptosis and suppresses osteolysis in vivo, and therefore may be a therapeutic drug for the prevention and treatment of osteolysis and loosening after TJA.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

The authors declare that there are no sources of funding to be acknowledged.

Author contribution

L.Z. designed the study. Z.T. and W.L. performed the experiments. X.W. and Z.M. analyzed and explained the data. S.S. drafted the manuscript. All the authors approved the manuscript.

Abbreviations

     
  • ATF4

    activating transcription factor 4

  •  
  • Bcl-2

    B-cell lymphoma-2

  •  
  • CHOP

    CCAAT/enhancer-binding protein homologous protein

  •  
  • eIF2α

    α subunit of translation initiation factor-2

  •  
  • ERS

    endoplasmic reticulum stress

  •  
  • GRP78

    glucose-regulated protein

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • H&E

    Hematoxylin-Eosin

  •  
  • IL

    interleukin

  •  
  • IRE1

    inositol-requiring enzyme-1

  •  
  • NF-κB

    nuclear factor-kappa B

  •  
  • PERK

    protein kinase RNA-like ER kinase

  •  
  • PI

    propidium iodide

  •  
  • QUE

    quercetin

  •  
  • Q-PCR

    Quantitative real time polymerase chain reaction

  •  
  • RANK

    receptor activator of NF-κB

  •  
  • TNF-α

    tumor necrosis factor α

  •  
  • TRAP

    tartrate-resistant acid phosphatase

  •  
  • UPR

    unfold protein response

References

1
Rubak
T.S.
,
Svendsen
S.W.
,
Søballe
K.
and
Frost
P.
(
2014
)
Total hip replacement due to primary osteoarthritis in relation to cumulative occupational exposures and lifestyle factors: a nationwide nested case-control study
.
Arthritis Care Res.
66
,
1496
1505
2
Guo
H.H.
,
Yu
C.C.
,
Sun
S.X.
,
Ma
X.J.
,
Yang
X.C.
,
Sun
K.N.
et al
(
2013
)
Adenovirus-mediated siRNA targeting TNF-α andoverexpression of bone morphogenetic protein-2 promotes early osteoblastdifferentiation on a cell model of Ti particle-induced inflammatory response in vitro
.
Braz. J. Med. Biol. Res.
46
,
831
838
3
Noordin
S.
and
Masri
B.
(
2012
)
Periprosthetic osteolysis: genetics, mechanisms and potential therapeutic interventions
.
Can. J. Surg.
55
,
408
417
4
Beck
R.T.
,
Illingworth
K.D.
and
Saleh
K.J.
(
2012
)
Review of periprosthetic osteolysis in total joint arthroplasty: an emphasis on host factors and future directions
.
J. Orthop. Res.
30
,
541
546
5
Chen
Z.
,
Zhan
L.
,
Lu
Z.
,
Ma
Y.
,
Gao
Z.
,
Guo
H.
et al
(
2015
)
Etanercept inhibits pro-inflammatory cytokines expression in titanium particle-stimulated peritoneal macrophages
.
Trop. J. Pharm. Res.
14
,
983
987
6
Liu
F.
,
Zhu
Z.
,
Mao
Y.
,
Liu
M.
,
Tang
T.
and
Qiu
S.
(
2009
)
Inhibition of titanium particle-induced osteoclastogenesis through inactivation of NFATc1 by VIVIT peptide
.
Biomaterials
30
,
1756
1762
7
Suzuki
Y.
,
Nishiyama
T.
,
Hasuda
K.
,
Fujishiro
T.
,
Niikura
T.
,
Hayashi
S.
et al
(
2007
)
Effect of etidronate on COX-2 expression and PGE 2 production in macrophage-like RAW 264.7 cells stimulated by titanium particles
.
J. Orthop. Sci.
12
,
568
577
8
Xu
T.
,
Wang
L.
,
Tao
Y.
,
Ji
Y.
,
Deng
F.
and
Wu
X.H.
(
2016
)
The function of naringin in inducing secretion of osteoprotegerin and inhibiting formation of osteoclasts
.
Evid. Based Complement. Alternat. Med.
2016
,
8981650
9
Boyce
B.F.
,
Li
P.
,
Yao
Z.
,
Zhang
Q.
,
Badell
I.R.
,
Schwarz
E.M.
et al
(
2005
)
TNF-alpha and pathologic bone resorption
.
Keio J. Med.
54
,
127
131
10
Kang
E.B.
,
Kwon
I.S.
,
Koo
J.H.
,
Kim
E.J.
,
Kim
C.H.
,
Lee
J.
et al
(
2013
)
Treadmill exercise represses neuronal cell death and inflammation during Aβ-induced ER stress by regulating unfolded protein response in aged presenilin 2 mutant mice
.
Apoptosis
18
,
1332
1347
11
Park
H.J.
,
Park
S.J.
,
Koo
D.B.
,
Kong
I.K.
,
Kim
M.K.
,
Kim
J.M.
et al
(
2013
)
Unfolding protein response signaling is involved in development, maintenance, and regression of the corpus luteum during the bovine estrous cycle
.
Biochem. Biophys. Res. Commun.
441
,
344
350
12
Lee
A.S.
(
2005
)
The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress
.
Methods
35
,
373
381
13
Kim
I.
,
Xu
W.
and
Reed
J.C.
(
2008
)
Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities
.
Nat. Rev. Drug Discov.
7
,
1013
1030
14
Boeschsaadatmandi
C.
,
Wolffram
S.
,
Minihane
A.M.
and
Rimbach
G.
(
2009
)
Effect of apoE genotype and dietary quercetin on blood lipids and TNF-α levels in apoE3 and apoE4 targeted gene replacement mice
.
Br. J. Nutr.
101
,
1440
1443
15
Bischoff
S.C.
(
2008
)
Quercetin: potentials in the prevention and therapy of disease
.
Curr. Opin. Clin. Nutr. Metab. Care
11
,
733
740
16
Tang
Y.
,
Li
J.
,
Gao
C.
,
Xu
Y.
,
Li
Y.
,
Yu
X.
et al
(
2016
)
Hepatoprotective effect of quercetin on endoplasmic reticulum stress and inflammation after intense exercise in mice through phosphoinositide 3-kinase and nuclear factor-kappa B
.
Oxid. Med. Cell Longev.
2016
,
8696587
17
Rakshit
D.S.
,
Ly
K.
,
Sengupta
T.K.
,
Nestor
B.J.
,
Sculco
T.P.
,
Ivashkiv
L.B.
et al
(
2006
)
Wear debris inhibition of anti-osteoclastogenic signaling by interleukin-6 and interferon-gamma. Mechanistic insights and implications for periprosthetic osteolysis
.
J. Bone Joint Surg. Am.
88
,
788
799
18
Liu
X.
,
Qu
X.
,
Wu
C.
,
Zhai
Z.
,
Tian
B.
,
Li
H.
et al
(
2014
)
The effect of enoxacin on osteoclastogenesis and reduction of titanium particle-induced osteolysis via suppression of JNK signaling pathway
.
Biomaterials
35
,
5721
5730
19
Aoyama
E.
,
Kubota
S.
,
Khattab
H.M.
,
Nishida
T.
and
Takigawa
M.
(
2015
)
CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG
.
Bone
73
,
242
248
20
Gallo
J.
,
Vaculova
J.
,
Goodman
S.B.
,
Konttinen
Y.T.
and
Thyssen
J.P.
(
2014
)
Contributions of human tissue analysis to understanding the mechanisms of loosening and osteolysis in total hip replacement
.
Acta Biomater.
10
,
2354
2366
21
Stea
S.
,
Visentin
M.
,
Granchi
D.
,
Cenni
E.
,
Ciapetti
G.
,
Sudanese
A.
et al
(
2000
)
Apoptosis in peri-implant tissue
.
Biomaterials
21
,
1393
1398
22
Landgraeber
S.
,
Jaeckel
S.
,
Löer
F.
,
Wedemeyer
C.
,
Hilken
G.
,
Canbay
A.
et al
(
2009
)
Pan-caspase inhibition suppresses polyethylene particle-induced osteolysis
.
Apoptosis
14
,
173
181
23
Yang
F.
,
Wu
W.
,
Cao
L.
,
Huang
Y.
,
Zhu
Z.
,
Tang
T.
et al
(
2011
)
Pathways of macrophage apoptosis within the interface membrane in aseptic loosening of prostheses
.
Biomaterials
32
,
9159
9167
24
Xiang
J.
,
Gu
X.
,
Qian
S.
and
Chen
Z.
(
2010
)
Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival
.
J. Biomed. Biotechnol.
2010
,
705431
25
Hitomi
J.
,
Katayama
T.
,
Eguchi
Y.
,
Kudo
T.
,
Taniguchi
M.
,
Koyama
Y.
et al
(
2004
)
Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death
.
J. Cell Biol.
165
,
347
356
26
Laybutt
D.R.
,
Preston
A.M.
,
Åkerfeldt
M.C.
,
Kench
J.G.
,
Busch
A.K.
,
Biankin
A.V.
et al
(
2007
)
Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes
.
Diabetologia
50
,
752
763
27
Nawrocki
S.T.
,
Carew
J.S.
,
Pino
M.S.
,
Highshaw
R.A.
,
Dunner
K.
Jr
,
Huang
P.
et al
(
2006
)
Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis
.
Cancer Res.
65
,
11658
11666
28
Yang
L.
,
Carlson
S.G.
,
Mcburney
D.
and
Horton
W.E.
Jr
(
2005
)
Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis
.
J. Biol. Chem.
280
,
31156
31165
29
Wolfson
J.J.
,
May
K.L.
,
Thorpe
C.M.
,
Jandhyala
D.M.
,
Paton
J.C.
and
Paton
A.W.
(
2008
)
Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways
.
Cell. Microbiol.
10
,
1775
1786
30
Harding
H.P.
,
Novoa
I.
,
Zhang
Y.
,
Zeng
H.
,
Wek
R.
,
Schapira
M.
et al
(
2000
)
Regulated translation initiation controls stress-induced gene expression in mammalian cells
.
Mol. Cell.
6
,
1099
1108
31
Ling
H.
,
Jing
Y.
,
Xu
Q.
,
Chen
R.
,
Chen
L.
and
Fang
M.
(
2016
)
miRNA-1283 regulates the PERK/ATF4 pathway in vascular injury by targeting ATF4
.
PLoS ONE
11
,
e0159171
32
Landgraeber
S.
,
Putz
S.
,
Schlattjan
M.
,
Bechmann
L.P.
,
Totsch
M.
,
Grabellus
F.
et al
(
2014
)
Adiponectin attenuates osteolysis in aseptic loosening of total hip replacements
.
Acta Biomater.
10
,
384
393
33
Park
E.
and
Chun
H.S.
(
2016
)
Protective effects of quercetin on dieldrin-induced endoplasmic reticulum stress and apoptosis in dopaminergic neuronal cells
.
Neuroreport
27
,
1140
1146
34
Sun
S.
,
Guo
H.
,
Zhang
J.
,
Yu
B.
,
Sun
K.
and
Jin
Q.
(
2013
)
Adenovirus-mediated expression of bone morphogenetic protein-2 activates titanium particle-induced osteoclastogenesis and this effect occurs in spite of the suppression of TNF-α expression by siRNA
.
Int. J. Mol. Med.
32
,
403
409
35
Nakano
M.
,
Tsuboi
T.
,
Kato
M.
,
Kurita
K.
and
Togari
A.
(
2003
)
Inhibitory effect of titanium particles on osteoclast formation generated by treatment of mouse bone marrow cells with PGE 2
.
Oral Dis.
9
,
77
83
36
Lee
Y.E.
,
Park
K.S.
,
Park
E.K.
,
Im
S.U.
,
Choi
Y.H.
and
Song
K.B.
(
2015
)
Polycan suppresses osteoclast differentiation and titanium particle‐induced osteolysis in mice
.
J. Biomed. Mater. Res. B Appl. Biomater.
104
,
1170
1175
37
Cui
J.
,
Zhu
M.
,
Zhu
S.
,
Wang
G.
,
Xu
Y.
and
Geng
D.
(
2014
)
Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis
.
J. Surg. Res.
192
,
447
453
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

Supplementary data