Int J Clin Exp Pathol 2015;8(4):3602-3612 www.ijcep.com /ISSN:1936-2625/IJCEP0006312
Original Article Effects of nicotine on a rat model of early stage osteoarthritis Qiangrong Gu1,2*, Dong Li1,2*, Bo Wei1,2, Yang Guo1, Junwei Yan1, Fengyong Mao1, Xiang Zhang1, Liming Wang1,2 Department of Orthopedics, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China; Cartilage Regeneration Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China. *Equal contributors. 1 2
Received January 24, 2015; Accepted March 22, 2015; Epub April 1, 2015; Published April 15, 2015 Abstract: The objective of the study was to investigate the effects of nicotine on articular cartilage degeneration and inflammation in a rat model of early stage osteoarthritis (OA), using T2 mapping. In this study, a rat model of early stage OA was established by immobilizing the left knee joints of adult male rats for two weeks. Subsequently, rats were fed with nicotine for two and four weeks. Changes in the articular cartilage from the medial femoral condylar region of the knee were evaluated by gross observation and histological grading with the contents of cartilage matrix detected. T2 values of the articular cartilage were estimated through high-field magnetic resonance imaging (MRI) (7.0T). Levels of serum tumor necrosis factor-α (TNF-α) were assessed by ELISA. The expression of TNF-α and the cholinergic receptor, α7nAChR, in the synovial tissue was measured by RT-PCR. Nicotine treatment ameliorated cartilage destruction, promoted matrix production, reduced the serum level of TNF-α and the expression of TNF-α in the synovial tissue, and increased the expression of α7nAChR in the synovial tissue in the rat model of early stage OA. In conclusion, nicotine prevented cartilage damage and had an anti-inflammatory effect in a rat model of early stage OA. Thus nicotine may have potential as a therapeutic strategy for early stage OA. Keywords: Nicotine, cartilage degeneration, early stage OA, α7nAChR, T2 mapping
Introduction Osteoarthritis (OA) is the most common form of arthritis, affecting millions of people worldwide. It is the major cause of disability in the elderly, affecting about 60% of men and 70% of women above the age of 65 [1]. Currently, the efficacy of disease modifying anti-OA drugs is limited [2]. OA involves the progressive degeneration of articular cartilage including loss of chondrocytes and degradation of the extracellular matrix (ECM) [3]. Proinflammatory cytokines secreted by chondrocytes, including tumor necrosis factor-α (TNF-α), contribute to the progression of OA [4]. Therefore, targeted treatments inhibiting the production of proinflammatory cytokines may have potential as effective therapies in OA [5, 6]. Cholinergic mechanisms are important for regulation of immune function and suppressing
inflammation [7]. T cells, B cells, and macrophages have the ability to produce acetylcholine (Ach). These cells also express various cholinergic receptors on their surface. In particular, the α7 (α7nAChR) subtype is important for immune regulation [8]. Specific stimulation of α7nAChR on monocytes leads to suppression of proinflammatory cytokine production, and this receptor is essential in the cytokine regulating neuro-immune mechanism known as the cholinergic anti-inflammatory pathway [9, 10]. Recent studies have investigated the antiinflammatory and therapeutic effect of the cholinergic mechanism and nicotine on arthritis. Maanen et al. demonstrated that oral nicotine attenuated collagen-induced arthritis in mice and reduced TNF-α expression in the synovial tissue of knee joints [11]. Li et al. reported that treatment with nicotine improved arthritis and reduced serum TNF-α and IL-6 levels in a mouse model of collagen-induced arthritis [12]. Wald-
Effects of nicotine on a rat model of early stage osteoarthritis burger et al. revealed that the α7R subunit is expressed in the intimal lining of rheumatoid arthritis (RA) and OA synovium and in cultured fibroblast-like synoviocytes (FLS); stimulation of FLS by ACh suppressed the expression of several chemokines, including IL-8 and IL-6 [13]. However, research into the effects of nicotine on early stage OA is limited. Magnetic resonance imaging (MRI) has become a common non-invasive method for diagnosis of OA. The recent development of high field strength imaging and functional MRI T2 mapping enables quantitative assessment of early degenerative changes in glycosaminoglycan (GAG) and collagen in articular cartilage [1416]. Many spontaneous and artificially induced (surgery or drug) OA models [17, 18] have been used for testing potential anti-arthritic agents, and disease modifying effects have been reported [19, 20] for agents currently used to treat patients with OA. Joint immobilization is used as a model of OA [21]. The cartilage degeneration induced by this method is reported to match the characteristics of early stage OA and can be reversible [22, 23]. Therefore, in this study, T2 mapping was used to estimate early articular cartilage degeneration in a rat knee joint immobilization model. The objective of the study was to investigate the effects of nicotine on articular cartilage degeneration and inflammation in a rat model of early stage OA using T2 mapping. Materials and methods Experimental design and animals This study included 105 adult male SpragueDawley rats aged 20 weeks (body weight 380400 g) that were randomly assigned to four groups: 1) Sham-operated group (n = 30); 2) Immobilized nicotine-treated group (n = 30); 3) Immobilized saline-treated group (n = 30); and 4) Untreated controls (n = 15). Rats were anesthetized with ketamine (80 mg/ kg) administered intraperitoneally. The left knee joint of each rat was fixed at 150° of flexion with a rigid Kirschner wire (diameter, 0.8 mm) (Fukang Company, Zhangjiagang, China). In immobilized rats, holes were drilled in the femur distal diaphysis and the middle segment of the tiba and the Kirschner wire was clipped
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to create a frame, similarly as previously described [21]. In sham-operated rats, holes were drilled in the femur and tibia and Kirschner wires inserted, but no frames were created. After 2 weeks, the Kirschner wires of immobilized and sham-operated rats were removed, and the rats were allowed to move freely in cages. At this time, nicotine (50 μg/ml; Sigma, St. Louis, MO) was added to the drinking water (normal saline) of the immobilized nicotinetreated rats to stimulate the peripheral part of the cholinergic pathway (nAChR). Immobilized saline-treated rats received normal saline. Drinking water was changed twice weekly. Fifteen rats from each group were killed at two weeks and four weeks after removing immobilization. The protocols for these experiments were approved by the Animal Research Committee of Nanjing Medical University. Tissue preparation Rats were euthanized with an overdose of ketamine. For gross morphological observations, the capsule of the knee was cut with a surgical knife, and the joint was opened and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer, pH 7.4 by perfusion through the aorta. After totally resecting bilateral menisci, the articular cartilage of the femur and tibia was visible. For histological observation and mechanical property testing, knee joints were resected and kept in the same fixative for 24 hours. The fixed specimens were decalcified in 10% EDTA in 0.01 M phosphate-buffer, pH 7.4 for 2 months at 4°C. After dehydration, the specimens were embedded in paraffin. Five-μm serial sections were obtained at the medial condylar region in the sagittal plane. Standardized serial sections were created in the medial condyle region of the knee. Histology and histomorphometry For histological analyses, sections (5 μm in thickness) were stained with hematoxylin-eosin (HE) and safranin O-fast green (SO). HE staining was used to evaluate structures, cells, and tidemarks. SO staining was used to evaluate loss of proteoglycan. Staining results were observed using a light microscope (Ci-L; Nikon, Tokyo, Japan). Semi-quantitative histopathological
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Effects of nicotine on a rat model of early stage osteoarthritis grading was performed according to a modified Mankin scoring system established for grading articular cartilage degeneration [24]. Three sections from each sample were scored by two different blinded observers, for a maximum possible score of 14. 7.0 MRI T2 and T2 mapping Knees were kept frozen and intact until MRI was performed. Prior to MRI, 60 knee joints (10 from each group) were thawed and rehydrated in normal saline for 16 hours at 4°C . All MRI images of the knee joints were obtained with a specially designed 7.0-T micro-MR imager (Bruker PharmaScan; Bruker BioSpin, Karlsruhe, Germany) using a volume 60 mm coil. Each joint was placed in a closed atmosphere in a sample tube. All samples were put in the same direction with regard to the magnetic field (anatomic axis parallel to B0) to limit the potential anisotropic effect. For morphological assessment in sagittal planes, T2 weighted images were acquired using a 2D rapid acquisition with refocused echoes RARE sequence (repetition time/echo time [TR/TE] = 2914/36 ms, matrix = 384 × 384, field of view [FOV] = 35 × 35 mm2, section thickness [ST] = 0.5 mm, acquisition time [TA] = 11 min 39 sec 364 ms). T2 maps were reconstructed using an MSME acquisition. The T2 map images were obtained as follows: TR = 3852.4 ms; TE = -11.0 ms, 22.0 ms, 33 ms, 165 ms and 176 ms; matrix = 256 × 256; FOV = 35 × 35 mm; ST = 0.50 mm; TA = 12 min 19 sec 666 ms; four signals acquired; flip angle = 180°. The weight-bearing area of the femoral part of each knee joint was selected as the region of interest (ROI). The average T2 relaxation times were calculated using a Bruker ParaVision 5.0 system, and ROIs were analyzed by a senior musculoskeletal radiologist. Quantitative measurement of GAG and collagen After MRI, 70 femoral condyles (n = 10/each period) were used for biochemical analysis; 35 femoral condyles were used for assessment of proteoglycan content, and 35 femoral condyles were used for assessment of collagen content. Cartilage samples for biochemical assays were
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harvested from the weight-bearing portion of each femoral condyle. Sulfated GAG was measured with a BlyscanTM sGAG assay kit (Biocolor, Newton Abbey, UK) according to the manufacturer’s instructions. Briefly, cartilage samples were cut into small pieces and digested with Papain Extraction Reagent (1 ml) in 1.5 ml labeled microcentrifuge tubes. The papain-digested solution was mixed with Blyscan dye reagent and centrifuged at 12,000 rpm for 10 min. Non-bound GAG dye was removed and dissociation reagent was added to the remaining sample. A microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) was used to measure absorbance at 656 nm. Collagen content in the cartilage was determined with the SircolTM collagen assay (Biocolor, Newton Abbey, UK) according to the manufacturer’s instructions. Briefly, a pepsin-digested cartilage solution was mixed with Sircol dye reagent and centrifuged at 12,000 rpm for 10 min. After removing the supernatant, the remaining sample was washed with acid-salt wash reagent and dissolved in alkali reagent. A microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) was used to measure the absorbance at 555 nm. Cytokine quantification by enzyme-linked immunosorbent assay Two and four weeks after removal from immobilization, five mice in each group were killed, and serum was harvested. Serum levels of TNF-α were measured using a sandwich enzymelinked immunosorbent assay (ELISA) according to manufacturer’s instructions (Keygen Biotech Company, Nanjing, China). Quantitative real-time PCR Synovial tissues around the left knees were obtained from five rats in each group. Samples were stored at -80°C until use. Samples were pulverized in liquid nitrogen, and lysed in Trizol (Invitrogen, California, USA) at room temperature for 10 mins. Total RNA was extracted according to the manufacturer’s instructions. Briefly, total RNA was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed for nACh-7α, TNF-α, and GAPDH
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Effects of nicotine on a rat model of early stage osteoarthritis Table 1. Primer sequences used for RT-PCR Gene GAPDH
Primer sequences Size Forward: 5-GGCCTTCCGTGTTCCTACC-3 103 bp Reverse: 5-CGCCTGCTTCACCACCTTC-3 nACh-7α Forward: 5-GAGCCTGAGACCAAGACTCC-3 128 bp Reverse: 5-TTCACCGAAGAGTCAGCATC-3 TNF-α Forward: 5-TCTTCTCATTCCTGCTCGTGG-3 128 bp Reverse: 5-GGTCTGGGCCATGGAACTGA-3
with an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. GAPDH was used as the housekeeping gene for normalization. The system was programmed to run for 40 cycles. The primer sequences were designed using Primer 5 software and are listed in Table 1. Statistical analysis All statistical analyses were performed using SPSS for Windows, Version 13.0 (Chicago, IL: SPSS Inc). Data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and the Fisher’s least significant difference (LSD) post-hoc test was used to compare differences in Mankin’s scores, GAG, collagen, serum levels of TNF-α, and gene expression. Differences between time points were compared with the Student’s t-test. P values < 0.05 were considered statistically significant. Results Gross morphology Two weeks after removing immobilization, there were no obvious differences in gross morphology between sham-operated, nicotine-treated, saline-treated, and untreated control rats. In all rats, the articular cartilage surfaces were smooth, and no obvious lesions were observed (Figure 1A-C, 1G). Similarly, four weeks after removing immobilization, no apparent degeneration of the articular cartilage was observed in the nicotine-treated and sham-operated groups (Figure 1D, 1F). In the saline-treated group, the surface of the femoral articular cartilage had lost some luster and was not as smooth as the nicotine-treated, sham-operated, and control groups (Figure 1E), but the difference was not significant.
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Histomorphologic analyses Two weeks after removing immobilization, the morphological structure of the articular cartilage in the sham-operated and untreated control groups was normal (Figure 2A, 2D, 2G). In the saline-treated group, there was obvious chondrocyte hypertrophy, cloning of chondrocytes, and bone misalignment (Figure 2B); four weeks after immobilization, hypertrophic chondrocytes with an irregular morphological structure were present (Figure 2E). Apparent fibrillation and cleft formation were not observed. In the nicotine-treated group, mild chondrocyte hypertrophy and bone misalignment was noted 2 weeks after removing immobilization. However, there were more normal chondrocytes compared to the saline-treated group, and more normal chondrocytes in the nicotine-treated group 4 weeks after removing immobilization compared to 2 weeks after removing immobilization (Figure 2C, 2F). Two and four weeks after removing immobilization, safranin-O staining showed a distinct reduction in proteoglycans in the upper and intermediate layers of the cartilage matrix in the saline-treated group (Figure 2I, 2L). Cartilage stating in the nicotine-treated, shamoperated, and untreated control groups was increased compared to the saline-treated group (Figure 2J, 2M). The severity of cartilage destruction was assessed using Mankin’s method (Table 2). Two weeks after removing immobilization, the severity of cartilage destruction in the salinetreated group (3.47 ± 1.19) was significantly greater compared to the untreated control (1.07 ± 0.96) (P < 0.001) and sham-operated (1.33 ± 0.97) (P < 0.001) groups. The nicotinetreated group (2.13 ± 0.92) showed significantly less severe cartilage damage compared to the saline-treated group (P = 0.001), but significantly more severe cartilage damage compared to the untreated control (P = 0.006) and shamoperated (P = 0.035) groups. Four weeks after removing immobilization, the nicotine-treated group (1.73 ± 0.96) still showed significantly less severe cartilage damage compared to the saline-treated group (3.63 ± 1.23) (P < 0.001). The severity of cartilage damage was not significantly different in the nicotine-treated group 4 weeks after removing immobilization com-
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Effects of nicotine on a rat model of early stage osteoarthritis
Figure 1. Gross observation of the left femoral articular cartilage. A. Sham-operated group 2 weeks after removal of immobilization; B. Saline-treated group 2 weeks after removal of immobilization; C. Nicotine-treated group 2 weeks after removal of immobilization; D. Sham-operated group 4 weeks after removal of immobilization; E. Saline-treated group 4 weeks after removal of immobilization; F. Nicotine-treated group 4 weeks after removal of immobilization; G. Untreated control group.
pared to 2 weeks after removing immobilization (P = 0.253). Quantitative T2 mapping Two weeks after removing immobilization, T2 mapping of the nicotine-treated group demonstrated a lower signal intensity compared to the saline-treated group (Figure 3B, 3C), but a higher signal intensity compared to the untreated control and sham-operated groups (Figure 3A, 3G). Four weeks after removing immobilization, the nicotine-treated group showed a lower signal intensity compared to the saline-treated group, but there was no difference compared to the sham-operated group (Figure 3D-F). Quantifying T2 values showed similar results (Figure 3H). Two weeks after removing immobilization, the mean T2 value of the nicotinetreated group (29.52 ± 4.96 ms) was significantly lower compared to the saline-treated group (38.73 ± 1.58 ms) (P < 0.001), but significantly higher compared to the sham-operated (24.37 ± 4.66 ms) (P = 0.009) and untreated control (23.56 ± 4.65 ms) (P = 0.005 ) groups. Four weeks after removing immobilization, the T2 value of the saline-treated group (39.00 ± 2.11 ms) was significantly higher compared to the untreated control (P < 0.001), the shamoperated (23.82 ± 5.04 ms) (P < 0.001), and nicotine-treated (27.01 ± 4.71 ms) (P < 0.001)
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groups. The difference between the shamoperated group and the nicotine-treated group was not significant (P = 0.105). Quantitative measurement of GAG and collagen Two and four weeks after removing immobilization, there were no significant differences in GAG content of the articular cartilage in the untreated control (83.16 ± 5.21 µg/mg) and sham-operated groups (Figure 4A). Two weeks after removing immobilization, GAG content was significantly decreased in the saline-treated group (69.54 ± 3.09 µg/mg) and nicotinetreated group (74.40 ± 4.22 µg/mg) (P = 0.001) compared to the sham-operated group (82.49 ± 4.46 µg/mg). GAG content was higher in the nicotine-treated group compared to the salinetreated group, but the difference was not significant (P = 0.131). Four weeks after removing immobilization, GAG content of the articular cartilage was significantly decreased in the saline-treated group (68.55 ± 4.86 µg/mg) compared to the sham-operated group (82.06 ± 2.25 µg/mg) (P < 0.001). GAG content was significantly increased in the nicotine-treated group (79.31 ± 4.20 µg/mg) compared to the saline-treated group (P = 0.002). There were no significant differences between the sham-operated group and the nicotine-treated group (P = 0.350).
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Effects of nicotine on a rat model of early stage osteoarthritis
Figure 2. Hematoxylin-eosin (A-G) and Safranin-O staining (H-N). (A and H) Sham-operated group 2 weeks after removal of immobilization; (B and I) Saline-treated group 2 weeks after removal of immobilization; (C and J) Nicotinetreated group 2 weeks after removal of immobilization; (D and K) Sham-operated group 4 weeks after removal of immobilization; (E and L) Saline-treated group 4 weeks after removal of immobilization; (F and M) Nicotine-treated group 4 weeks after removal of immobilization; (G and M) Untreated control group. Magnification × 100.
0.018). Similarly, four weeks after removing immobiliFemoral condyle Sham group Saline group Nicotine group Control group zation, the serum TNF-α 2 weeks 1.33 ± 0.97 3.47 ± 1.19a,b 2.13 ± 0.92a,b,c 1.07 ± 0.96 level in the saline-treated 4 weeks 1.27 ± 1.08 3.63 ± 1.23a,b 1.73 ± 0.96c group (24.57 ± 6.25 pg/ml) Values are the mean ± SD. 2 weeks = 2 weeks after removing immobilization, 4 weeks was significantly increased = 4 weeks after removing immobilization. aP < 0.05 vs. control group; bP < 0.05 vs. compared to the untreated c sham-operated group; P < 0.05 vs. saline-treated group. control (P < 0.001), shamoperated (7.16 ± 1.96 pg/ There were no significant differences in collaml), and nicotine-treated (10.22 ± 4.20 pg/ml) (P < 0.001) groups. The nicotine-treated group gen content of the articular cartilage at any had a lower serum TNF-α level four weeks after time point between any groups (Figure 4B). removing immobilization compared to two Levels of serum TNF-α weeks after removing immobilization, but the difference was not significant (P = 0.347) Two weeks after removing immobilization, the (Figure 5). saline-treated group (23.95 ± 5.62 pg/ml) had a significantly increased mean serum TNF-α Gene expression analysis of the synovial tislevel compared to the untreated control (6.22 ± sues 2.09 pg/ml) (P < 0.001) and sham-operated (6.03 ± 3.72 pg/ml) (P < 0.001) groups. The Two and four weeks after removing immobilizanicotine-treated group (12.83 ± 4.09 pg/ml) tion, the saline-treated group had a significantly had a significantly decreased serum TNF-α increased mean TNF-α RNA level compared to level compared to the saline-treated group (P = the untreated control (P < 0.001) and shamTable 2. Histological score of articular cartilage
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Effects of nicotine on a rat model of early stage osteoarthritis
Figure 3. High-field MRI T2 mapping (A-G) and average T2 values (H) (A) Sham-operated group 2 weeks after removal of immobilization; (B) Saline-treated group 2 weeks after removal of immobilization; (C) Nicotine-treated group 2 weeks after removal of immobilization; (D) Sham-operated group 4 weeks after removal of immobilization; (E) Saline-treated group 4 weeks after removal of immobilization; (F) Nicotine-treated group 4 weeks after removal of immobilization; (G) Untreated control group. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. +P < 0.05, ++P < 0.005 vs. control group; *P < 0.05, **P < 0.005 vs. shamoperated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
operated (P < 0.001) groups. In the nicotinetreated group, TNF-α expression level was significantly decreased compared to the salinetreated group. Two and four weeks after removing immobilization, mean α7nAChR RNA levels were significantly increased in the saline-treated and nicotine-treated groups compared to the untreated control and sham-operated groups (P < 0.001). Four weeks after removing 3608
immobilization, α7nAChR RNA levels were significantly increased in the nicotine-treated and saline-treated groups (P < 0.001) (Figure 6). Discussion In this study, pathological assessments and T2 mapping showed that nicotine ameliorated cartilage destruction and promoted matrix producInt J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis ment with nicotine [11]. Li et al. showed similar results [12]. Wu et al. reported that activating the cholinergic anti-inflammatory pathway with nicotine attenuated collagen-induced arthritis via suppression of the Th17 response [25]. The current study focused on early stage OA. Compared to saline, nicotine increased the number of normal chondrocytes, and the proteoglycan and GAG content in the articular cartilage. The effect seemed to be mediated by decreased serum and expression levels of TNF-α [11, 12].
Figure 4. Biochemical analysis of cartilage. (A) GAG content analysis (B) collagen content analysis. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
Figure 5. Serum TNF-α levels. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
tion in a rat early stage OA model. In addition, nicotine reduced the serum level of TNF-α and the expression of TNF-α in the synovial tissue, and increased the expression of α7nAChR, indicating that nicotine-induced effects in OA are likely due to the peripheral stimulation of α7nAChR. To our knowledge, there is one of the first studies to investigate the effect of nicotine on early stage OA. Van Maanen et al. established a mouse model of clinical arthritis using type II collagen. They observed bone degradation, extensive proteoglycan loss, and inflammatory cell infiltration, which were attenuated by treat3609
The current study showed a strong expression of α7nAChR in salinetreated animals, which was unexpected. Westman et al. used immunohistostaining and found a higher expression of α7nAChR in RA patients’ synovial tissue compared to that of healthy subjects [10], but the difference did not reach statistical significant. Waldburger et al. used quantitative PCR and showed that α7R mRNA levels were similar in both RA and OA patients’ synovium [13]. Schubert and Beckmann measured nicotinic and muscarinic ACh receptor expression in synovial tissue from patients with RA and OA using RT-PCR and immunofluorescence labeling and also found no obvious difference between the samples [26].
Previous reports indentify a role for the α7nAChR receptor in the antiinflammatory cholinergic pathway [7]. Wang et al. demonstrated that in wild-type mice binding of ACh to α7-nAChR reduced the production of proinflammatory cytokines, including TNF-α, IL-1 [7]. van Maanen et al. found that stimulation of α7nAChR by selective agonists such as AR-R17779 reduced the level of serum TNF-α in a mouse model of collagen-induced arthritis [11] and described an increase in proinflammatory cytokines in α7nAChR knockout mice with experimentally-induced arthritis [27]. Other studies show that TNF-α and IL-1 play an important role in the degradation of the ECM and the destruction of cartilage in OA [28]. Taken together, these data suggest that nicotine Int J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis (dGEMRIC) detected changes in proteoglycan content in knee cartilage among individuals taking collagen hydrolysate and that T2 mapping showed little variability [34]. In the present study, there were higher signal intensities and T2 values in the cartilage of the saline-treated group compared to that of the untreated control and sham-operated groups, as well as a reduced GAG content. In contrast, signal intensities and T2 values were down-regulated in the nicotine group compared to the saline group, which was accompanied by an increased GAG content. The mechanism of nicotine’s antiinflammatory effect on arthritis through α7-nAChR has been studied extensively, but remains controversial. JAK-2/STAT-3 and the NF-κB pathways have been implicated in α7R signaling in mouse macrophages [35, 36]. However, Waldburger et al. suggested that cholinergic signaling does not influence NF-κB-driven promoter activity in FLS from RA patients. Conversely, Zheng showed that nicotine reduced IL-1β-induced cartilage apoptosis through activation of PI3K/ Akt signaling by regulating Bcl-2/Bcl-xl expression. They also observed that nicotine triggered the phosphorylation of S6 through p70S6K or Akt, regulating protein synthesis including the increase of TIMP-1 and the inhibition of MMP13, thereby mediating ECM degradation and synthesis [6].
Figure 6. Gene expression in the synovial tissues. A. TNF-α expression; B. α7nAChR expression. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. salinetreated group.
could reduce the level of TNF-α through α7nAChR in OA. In addition, our study indicated that GAG synthesis was upregulated by nicotine, which promoted the repair of the articular cartilage. Akmal et al. showed that nicotine, at physiological levels found in smokers, increased DNA, GAG, and collagen synthesis of nucleus pulposus cells [29]. However, Gullahorn et al. found that nicotine increased collagen synthesis, but had no effect on GAG synthesis in vitro [30]. Similarly, Ying et al. showed nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and OA patients without increasing GAG synthesis [31]. MRI T2 mapping is a useful noninvasive method for the detection of early degenerative changes of cartilage in OA [14, 32]. In particular, GAG content causes variation in T2 values [33]. Watrin-Pinzano et al. induced rat joint cartilage degradation by hyaluronidase. They observed the signal intensities and T2 values of degraded cartilage increased with loss of GAG content, with no obvious change in collagen content [16]. McAlindon reported that delayed gadolinium enhanced MRI of cartilage
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This study is associated with several limitations. First, we did not attempt to distinguish between different types of collagen. Total collagen content was unchanged, but nicotine is reported to up-regulate synthesis of collagen II; therefore, further investigations are warranted [31]. Second, our data suggested a tendency for a higher expression of α7nAChR in the synovial tissue of the nicotine-treated group compared to the saline-treated group, but no published literature supports these findings. In conclusion, we demonstrated that nicotine treatment attenuated cellular damage, increa-
Int J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis sed the synthesis of the ECM, and reduced the serum TNF-α level in a rat model of early stage OA. These data suggest that nicotine prevented cartilage damage and had an anti-inflammatory effect. In the future, nicotine may have potential as a therapeutic strategy for early OA.
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Acknowledgements
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Financial support for this study was provided by the National Natural Science Foundation of China (81171745). We gratefully acknowledge Mrs. Yuling Peng for her logistical support.
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Disclosure of conflict of interest None. Address correspondence to: Dr. Liming Wang, Department of Orthopedics, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China. Tel: +86-18951670968; Fax: +8625-52269924; E-mail: [email protected]
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Zheng X, Xia C, Chen Z, Huang J, Gao F, Li G, Zhang B. Requirement of the phosphatidylinositol 3-kinase/Akt signaling pathway for the effect of nicotine on interleukin-1beta-induced chondrocyte apoptosis in a rat model of osteoarthritis. Biochem Biophys Res Commun 2012; 423: 606-612. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003; 421: 384-388. Kawashima K, Fujii T. Basic and clinical aspects of non-neuronal acetylcholine: overview of non-neuronal cholinergic systems and their biological significance. J Pharmacol Sci 2008; 106: 167-173. Tracey KJ. The inflammatory reflex. Nature 2002; 420: 853-859. Westman M, Engstrom M, Catrina AI, Lampa J. Cell specific synovial expression of nicotinic alpha 7 acetylcholine receptor in rheumatoid arthritis and psoriatic arthritis. Scand J Immunol 2009; 70: 136-140. van Maanen MA, Lebre MC, van der Poll T, LaRosa GJ, Elbaum D, Vervoordeldonk MJ, Tak PP. Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice. Arthritis Rheum 2009; 60: 114-122. Li T, Zuo X, Zhou Y, Wang Y, Zhuang H, Zhang L, Zhang H, Xiao X. The vagus nerve and nicotinic receptors involve inhibition of HMGB1 release and early pro-inflammatory cytokines function in collagen-induced arthritis. J Clin Immunol 2010; 30: 213-220. Waldburger JM, Boyle DL, Pavlov VA, Tracey KJ, Firestein GS. Acetylcholine regulation of synoviocyte cytokine expression by the alpha7 nicotinic receptor. Arthritis Rheum 2008; 58: 3439-3449. Apprich S, Welsch GH, Mamisch TC, Szomolanyi P, Mayerhoefer M, Pinker K, Trattnig S. Detection of degenerative cartilage disease: comparison of high-resolution morphological MR and quantitative T2 mapping at 3.0 Tesla. Osteoarthritis Cartilage 2010; 18: 1211-1217. Bear DM, Williams A, Chu CT, Coyle CH, Chu CR. Optical coherence tomography grading correlates with MRI T2 mapping and extracellular matrix content. J Orthop Res 2010; 28: 546-552. Watrin-Pinzano A, Ruaud JP, Olivier P, Grossin L, Gonord P, Blum A, Netter P, Guillot G, Gillet P, Loeuille D. Effect of proteoglycan depletion on T2 mapping in rat patellar cartilage. Radiology 2005; 234: 162-170. Bendele AM. Animal models of osteoarthritis. J Musculoskel Neuron 2001; 1: 363-376.
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Effects of nicotine on a rat model of early stage osteoarthritis [18] Rogart JN, Barrach HJ, Chichester CO. Articular collagen degradation in the Hulth-Telhag model of osteoarthritis. Osteoarthritis Cartilage 1999; 7: 539-547. [19] Smith GN Jr, Myers SL, Brandt KD, Mickler EA, Albrecht ME. Diacerhein treatment reduces the severity of osteoarthritis in the canine cruciate-deficiency model of osteoarthritis. Arthritis Rheum 1999; 42: 545-554. [20] Kobayashi K, Amiel M, Harwood FL, Healey RM, Sonoda M, Moriya H, Amiel D. The longterm effects of hyaluronan during development of osteoarthritis following partial meniscectomy in a rabbit model. Osteoarthritis Cartilage 2000; 8: 359-365. [21] Hagiwara Y, Ando A, Chimoto E, Saijo Y, OhmoriMatsuda K, Itoi E. Changes of articular cartilage after immobilization in a rat knee contracture model. J Orthop Res 2009; 27: 236-242. [22] Setton LA, Mow VC, Muller FJ, Pita JC, Howell DS. Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization. Osteoarthritis Cartilage 1997; 5: 1-16. [23] Haapala J, Arokoski JP, Ronkko S, Agren U, Kosma VM, Lohmander LS, Tammi M, Helminen HJ, Kiviranta I. Decline after immobilisation and recovery after remobilisation of synovial fluid IL1, TIMP, and chondroitin sulphate levels in young beagle dogs. Ann Rheum Dis 2001; 60: 55-60. [24] Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971; 53: 523-537. [25] Wu S, Luo H, Xiao X, Zhang H, Li T, Zuo X. Attenuation of collagen induced arthritis via suppression on Th17 response by activating cholinergic anti-inflammatory pathway with nicotine. Eur J Pharmacol 2014; 735: 97-104. [26] Schubert J, Beckmann J, Hartmann S, Morhenn HG, Szalay G, Heiss C, Schnettler R, Lips KS. Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis. Life Sci 2012; 91: 1048-1052. [27] van Maanen MA, Stoof SP, Larosa GJ, Vervoordeldonk MJ, Tak PP. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice. Ann Rheum Dis 2010; 69: 1717-1723.
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[28] Abramson SB, Yazici Y. Biologics in development for rheumatoid arthritis: relevance to osteoarthritis. Adv Drug Deliv Rev 2006; 58: 212225. [29] Akmal M, Kesani A, Anand B, Singh A, Wiseman M, Goodship A. Effect of nicotine on spinal disc cells: a cellular mechanism for disc degeneration. Spine 2004; 29: 568-575. [30] Gullahorn L, Lippiello L, Karpman R. Smoking and osteoarthritis: differential effect of nicotine on human chondrocyte glycosaminoglycan and collagen synthesis. Osteoarthritis Cartilage 2005; 13: 942-943. [31] Ying X, Cheng S, Shen Y, Cheng X, An Rompis F, Wang W, Lin Z, Chen Q, Zhang W, Kou D, Peng L, Tian XQ, Lu CZ. Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients. Mol Cell Biochem 2012; 359: 263269. [32] Mosher TJ, Dardzinski BJ. Cartilage MRI T2 relaxation time mapping: overview and applications. Semin Musculoskelet Radiol 2004; 8: 355-368. [33] Xia Y. Magic-angle effect in magnetic resonance imaging of articular cartilage: a review. Invest Radiol 2000; 35: 602-621. [34] McAlindon TE, Nuite M, Krishnan N, Ruthazer R, Price LL, Burstein D, Griffith J, Flechsenhar K. Change in knee osteoarthritis cartilage detected by delayed gadolinium enhanced magnetic resonance imaging following treatment with collagen hydrolysate: a pilot randomized controlled trial. Osteoarthritis Cartilage 2011; 19: 399-405. [35] de Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, Berthoud HR, Uematsu S, Akira S, van den Wijngaard RM, Boeckxstaens GE. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immunol 2005; 6: 844-851. [36] Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, Al-Abed Y, Wang H, Metz C, Miller EJ, Tracey KJ, Ulloa L. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004; 10: 1216-1221.
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Original Article Effects of nicotine on a rat model of early stage osteoarthritis Qiangrong Gu1,2*, Dong Li1,2*, Bo Wei1,2, Yang Guo1, Junwei Yan1, Fengyong Mao1, Xiang Zhang1, Liming Wang1,2 Department of Orthopedics, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China; Cartilage Regeneration Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China. *Equal contributors. 1 2
Received January 24, 2015; Accepted March 22, 2015; Epub April 1, 2015; Published April 15, 2015 Abstract: The objective of the study was to investigate the effects of nicotine on articular cartilage degeneration and inflammation in a rat model of early stage osteoarthritis (OA), using T2 mapping. In this study, a rat model of early stage OA was established by immobilizing the left knee joints of adult male rats for two weeks. Subsequently, rats were fed with nicotine for two and four weeks. Changes in the articular cartilage from the medial femoral condylar region of the knee were evaluated by gross observation and histological grading with the contents of cartilage matrix detected. T2 values of the articular cartilage were estimated through high-field magnetic resonance imaging (MRI) (7.0T). Levels of serum tumor necrosis factor-α (TNF-α) were assessed by ELISA. The expression of TNF-α and the cholinergic receptor, α7nAChR, in the synovial tissue was measured by RT-PCR. Nicotine treatment ameliorated cartilage destruction, promoted matrix production, reduced the serum level of TNF-α and the expression of TNF-α in the synovial tissue, and increased the expression of α7nAChR in the synovial tissue in the rat model of early stage OA. In conclusion, nicotine prevented cartilage damage and had an anti-inflammatory effect in a rat model of early stage OA. Thus nicotine may have potential as a therapeutic strategy for early stage OA. Keywords: Nicotine, cartilage degeneration, early stage OA, α7nAChR, T2 mapping
Introduction Osteoarthritis (OA) is the most common form of arthritis, affecting millions of people worldwide. It is the major cause of disability in the elderly, affecting about 60% of men and 70% of women above the age of 65 [1]. Currently, the efficacy of disease modifying anti-OA drugs is limited [2]. OA involves the progressive degeneration of articular cartilage including loss of chondrocytes and degradation of the extracellular matrix (ECM) [3]. Proinflammatory cytokines secreted by chondrocytes, including tumor necrosis factor-α (TNF-α), contribute to the progression of OA [4]. Therefore, targeted treatments inhibiting the production of proinflammatory cytokines may have potential as effective therapies in OA [5, 6]. Cholinergic mechanisms are important for regulation of immune function and suppressing
inflammation [7]. T cells, B cells, and macrophages have the ability to produce acetylcholine (Ach). These cells also express various cholinergic receptors on their surface. In particular, the α7 (α7nAChR) subtype is important for immune regulation [8]. Specific stimulation of α7nAChR on monocytes leads to suppression of proinflammatory cytokine production, and this receptor is essential in the cytokine regulating neuro-immune mechanism known as the cholinergic anti-inflammatory pathway [9, 10]. Recent studies have investigated the antiinflammatory and therapeutic effect of the cholinergic mechanism and nicotine on arthritis. Maanen et al. demonstrated that oral nicotine attenuated collagen-induced arthritis in mice and reduced TNF-α expression in the synovial tissue of knee joints [11]. Li et al. reported that treatment with nicotine improved arthritis and reduced serum TNF-α and IL-6 levels in a mouse model of collagen-induced arthritis [12]. Wald-
Effects of nicotine on a rat model of early stage osteoarthritis burger et al. revealed that the α7R subunit is expressed in the intimal lining of rheumatoid arthritis (RA) and OA synovium and in cultured fibroblast-like synoviocytes (FLS); stimulation of FLS by ACh suppressed the expression of several chemokines, including IL-8 and IL-6 [13]. However, research into the effects of nicotine on early stage OA is limited. Magnetic resonance imaging (MRI) has become a common non-invasive method for diagnosis of OA. The recent development of high field strength imaging and functional MRI T2 mapping enables quantitative assessment of early degenerative changes in glycosaminoglycan (GAG) and collagen in articular cartilage [1416]. Many spontaneous and artificially induced (surgery or drug) OA models [17, 18] have been used for testing potential anti-arthritic agents, and disease modifying effects have been reported [19, 20] for agents currently used to treat patients with OA. Joint immobilization is used as a model of OA [21]. The cartilage degeneration induced by this method is reported to match the characteristics of early stage OA and can be reversible [22, 23]. Therefore, in this study, T2 mapping was used to estimate early articular cartilage degeneration in a rat knee joint immobilization model. The objective of the study was to investigate the effects of nicotine on articular cartilage degeneration and inflammation in a rat model of early stage OA using T2 mapping. Materials and methods Experimental design and animals This study included 105 adult male SpragueDawley rats aged 20 weeks (body weight 380400 g) that were randomly assigned to four groups: 1) Sham-operated group (n = 30); 2) Immobilized nicotine-treated group (n = 30); 3) Immobilized saline-treated group (n = 30); and 4) Untreated controls (n = 15). Rats were anesthetized with ketamine (80 mg/ kg) administered intraperitoneally. The left knee joint of each rat was fixed at 150° of flexion with a rigid Kirschner wire (diameter, 0.8 mm) (Fukang Company, Zhangjiagang, China). In immobilized rats, holes were drilled in the femur distal diaphysis and the middle segment of the tiba and the Kirschner wire was clipped
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to create a frame, similarly as previously described [21]. In sham-operated rats, holes were drilled in the femur and tibia and Kirschner wires inserted, but no frames were created. After 2 weeks, the Kirschner wires of immobilized and sham-operated rats were removed, and the rats were allowed to move freely in cages. At this time, nicotine (50 μg/ml; Sigma, St. Louis, MO) was added to the drinking water (normal saline) of the immobilized nicotinetreated rats to stimulate the peripheral part of the cholinergic pathway (nAChR). Immobilized saline-treated rats received normal saline. Drinking water was changed twice weekly. Fifteen rats from each group were killed at two weeks and four weeks after removing immobilization. The protocols for these experiments were approved by the Animal Research Committee of Nanjing Medical University. Tissue preparation Rats were euthanized with an overdose of ketamine. For gross morphological observations, the capsule of the knee was cut with a surgical knife, and the joint was opened and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer, pH 7.4 by perfusion through the aorta. After totally resecting bilateral menisci, the articular cartilage of the femur and tibia was visible. For histological observation and mechanical property testing, knee joints were resected and kept in the same fixative for 24 hours. The fixed specimens were decalcified in 10% EDTA in 0.01 M phosphate-buffer, pH 7.4 for 2 months at 4°C. After dehydration, the specimens were embedded in paraffin. Five-μm serial sections were obtained at the medial condylar region in the sagittal plane. Standardized serial sections were created in the medial condyle region of the knee. Histology and histomorphometry For histological analyses, sections (5 μm in thickness) were stained with hematoxylin-eosin (HE) and safranin O-fast green (SO). HE staining was used to evaluate structures, cells, and tidemarks. SO staining was used to evaluate loss of proteoglycan. Staining results were observed using a light microscope (Ci-L; Nikon, Tokyo, Japan). Semi-quantitative histopathological
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Effects of nicotine on a rat model of early stage osteoarthritis grading was performed according to a modified Mankin scoring system established for grading articular cartilage degeneration [24]. Three sections from each sample were scored by two different blinded observers, for a maximum possible score of 14. 7.0 MRI T2 and T2 mapping Knees were kept frozen and intact until MRI was performed. Prior to MRI, 60 knee joints (10 from each group) were thawed and rehydrated in normal saline for 16 hours at 4°C . All MRI images of the knee joints were obtained with a specially designed 7.0-T micro-MR imager (Bruker PharmaScan; Bruker BioSpin, Karlsruhe, Germany) using a volume 60 mm coil. Each joint was placed in a closed atmosphere in a sample tube. All samples were put in the same direction with regard to the magnetic field (anatomic axis parallel to B0) to limit the potential anisotropic effect. For morphological assessment in sagittal planes, T2 weighted images were acquired using a 2D rapid acquisition with refocused echoes RARE sequence (repetition time/echo time [TR/TE] = 2914/36 ms, matrix = 384 × 384, field of view [FOV] = 35 × 35 mm2, section thickness [ST] = 0.5 mm, acquisition time [TA] = 11 min 39 sec 364 ms). T2 maps were reconstructed using an MSME acquisition. The T2 map images were obtained as follows: TR = 3852.4 ms; TE = -11.0 ms, 22.0 ms, 33 ms, 165 ms and 176 ms; matrix = 256 × 256; FOV = 35 × 35 mm; ST = 0.50 mm; TA = 12 min 19 sec 666 ms; four signals acquired; flip angle = 180°. The weight-bearing area of the femoral part of each knee joint was selected as the region of interest (ROI). The average T2 relaxation times were calculated using a Bruker ParaVision 5.0 system, and ROIs were analyzed by a senior musculoskeletal radiologist. Quantitative measurement of GAG and collagen After MRI, 70 femoral condyles (n = 10/each period) were used for biochemical analysis; 35 femoral condyles were used for assessment of proteoglycan content, and 35 femoral condyles were used for assessment of collagen content. Cartilage samples for biochemical assays were
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harvested from the weight-bearing portion of each femoral condyle. Sulfated GAG was measured with a BlyscanTM sGAG assay kit (Biocolor, Newton Abbey, UK) according to the manufacturer’s instructions. Briefly, cartilage samples were cut into small pieces and digested with Papain Extraction Reagent (1 ml) in 1.5 ml labeled microcentrifuge tubes. The papain-digested solution was mixed with Blyscan dye reagent and centrifuged at 12,000 rpm for 10 min. Non-bound GAG dye was removed and dissociation reagent was added to the remaining sample. A microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) was used to measure absorbance at 656 nm. Collagen content in the cartilage was determined with the SircolTM collagen assay (Biocolor, Newton Abbey, UK) according to the manufacturer’s instructions. Briefly, a pepsin-digested cartilage solution was mixed with Sircol dye reagent and centrifuged at 12,000 rpm for 10 min. After removing the supernatant, the remaining sample was washed with acid-salt wash reagent and dissolved in alkali reagent. A microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) was used to measure the absorbance at 555 nm. Cytokine quantification by enzyme-linked immunosorbent assay Two and four weeks after removal from immobilization, five mice in each group were killed, and serum was harvested. Serum levels of TNF-α were measured using a sandwich enzymelinked immunosorbent assay (ELISA) according to manufacturer’s instructions (Keygen Biotech Company, Nanjing, China). Quantitative real-time PCR Synovial tissues around the left knees were obtained from five rats in each group. Samples were stored at -80°C until use. Samples were pulverized in liquid nitrogen, and lysed in Trizol (Invitrogen, California, USA) at room temperature for 10 mins. Total RNA was extracted according to the manufacturer’s instructions. Briefly, total RNA was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed for nACh-7α, TNF-α, and GAPDH
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Effects of nicotine on a rat model of early stage osteoarthritis Table 1. Primer sequences used for RT-PCR Gene GAPDH
Primer sequences Size Forward: 5-GGCCTTCCGTGTTCCTACC-3 103 bp Reverse: 5-CGCCTGCTTCACCACCTTC-3 nACh-7α Forward: 5-GAGCCTGAGACCAAGACTCC-3 128 bp Reverse: 5-TTCACCGAAGAGTCAGCATC-3 TNF-α Forward: 5-TCTTCTCATTCCTGCTCGTGG-3 128 bp Reverse: 5-GGTCTGGGCCATGGAACTGA-3
with an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. GAPDH was used as the housekeeping gene for normalization. The system was programmed to run for 40 cycles. The primer sequences were designed using Primer 5 software and are listed in Table 1. Statistical analysis All statistical analyses were performed using SPSS for Windows, Version 13.0 (Chicago, IL: SPSS Inc). Data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and the Fisher’s least significant difference (LSD) post-hoc test was used to compare differences in Mankin’s scores, GAG, collagen, serum levels of TNF-α, and gene expression. Differences between time points were compared with the Student’s t-test. P values < 0.05 were considered statistically significant. Results Gross morphology Two weeks after removing immobilization, there were no obvious differences in gross morphology between sham-operated, nicotine-treated, saline-treated, and untreated control rats. In all rats, the articular cartilage surfaces were smooth, and no obvious lesions were observed (Figure 1A-C, 1G). Similarly, four weeks after removing immobilization, no apparent degeneration of the articular cartilage was observed in the nicotine-treated and sham-operated groups (Figure 1D, 1F). In the saline-treated group, the surface of the femoral articular cartilage had lost some luster and was not as smooth as the nicotine-treated, sham-operated, and control groups (Figure 1E), but the difference was not significant.
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Histomorphologic analyses Two weeks after removing immobilization, the morphological structure of the articular cartilage in the sham-operated and untreated control groups was normal (Figure 2A, 2D, 2G). In the saline-treated group, there was obvious chondrocyte hypertrophy, cloning of chondrocytes, and bone misalignment (Figure 2B); four weeks after immobilization, hypertrophic chondrocytes with an irregular morphological structure were present (Figure 2E). Apparent fibrillation and cleft formation were not observed. In the nicotine-treated group, mild chondrocyte hypertrophy and bone misalignment was noted 2 weeks after removing immobilization. However, there were more normal chondrocytes compared to the saline-treated group, and more normal chondrocytes in the nicotine-treated group 4 weeks after removing immobilization compared to 2 weeks after removing immobilization (Figure 2C, 2F). Two and four weeks after removing immobilization, safranin-O staining showed a distinct reduction in proteoglycans in the upper and intermediate layers of the cartilage matrix in the saline-treated group (Figure 2I, 2L). Cartilage stating in the nicotine-treated, shamoperated, and untreated control groups was increased compared to the saline-treated group (Figure 2J, 2M). The severity of cartilage destruction was assessed using Mankin’s method (Table 2). Two weeks after removing immobilization, the severity of cartilage destruction in the salinetreated group (3.47 ± 1.19) was significantly greater compared to the untreated control (1.07 ± 0.96) (P < 0.001) and sham-operated (1.33 ± 0.97) (P < 0.001) groups. The nicotinetreated group (2.13 ± 0.92) showed significantly less severe cartilage damage compared to the saline-treated group (P = 0.001), but significantly more severe cartilage damage compared to the untreated control (P = 0.006) and shamoperated (P = 0.035) groups. Four weeks after removing immobilization, the nicotine-treated group (1.73 ± 0.96) still showed significantly less severe cartilage damage compared to the saline-treated group (3.63 ± 1.23) (P < 0.001). The severity of cartilage damage was not significantly different in the nicotine-treated group 4 weeks after removing immobilization com-
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Effects of nicotine on a rat model of early stage osteoarthritis
Figure 1. Gross observation of the left femoral articular cartilage. A. Sham-operated group 2 weeks after removal of immobilization; B. Saline-treated group 2 weeks after removal of immobilization; C. Nicotine-treated group 2 weeks after removal of immobilization; D. Sham-operated group 4 weeks after removal of immobilization; E. Saline-treated group 4 weeks after removal of immobilization; F. Nicotine-treated group 4 weeks after removal of immobilization; G. Untreated control group.
pared to 2 weeks after removing immobilization (P = 0.253). Quantitative T2 mapping Two weeks after removing immobilization, T2 mapping of the nicotine-treated group demonstrated a lower signal intensity compared to the saline-treated group (Figure 3B, 3C), but a higher signal intensity compared to the untreated control and sham-operated groups (Figure 3A, 3G). Four weeks after removing immobilization, the nicotine-treated group showed a lower signal intensity compared to the saline-treated group, but there was no difference compared to the sham-operated group (Figure 3D-F). Quantifying T2 values showed similar results (Figure 3H). Two weeks after removing immobilization, the mean T2 value of the nicotinetreated group (29.52 ± 4.96 ms) was significantly lower compared to the saline-treated group (38.73 ± 1.58 ms) (P < 0.001), but significantly higher compared to the sham-operated (24.37 ± 4.66 ms) (P = 0.009) and untreated control (23.56 ± 4.65 ms) (P = 0.005 ) groups. Four weeks after removing immobilization, the T2 value of the saline-treated group (39.00 ± 2.11 ms) was significantly higher compared to the untreated control (P < 0.001), the shamoperated (23.82 ± 5.04 ms) (P < 0.001), and nicotine-treated (27.01 ± 4.71 ms) (P < 0.001)
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groups. The difference between the shamoperated group and the nicotine-treated group was not significant (P = 0.105). Quantitative measurement of GAG and collagen Two and four weeks after removing immobilization, there were no significant differences in GAG content of the articular cartilage in the untreated control (83.16 ± 5.21 µg/mg) and sham-operated groups (Figure 4A). Two weeks after removing immobilization, GAG content was significantly decreased in the saline-treated group (69.54 ± 3.09 µg/mg) and nicotinetreated group (74.40 ± 4.22 µg/mg) (P = 0.001) compared to the sham-operated group (82.49 ± 4.46 µg/mg). GAG content was higher in the nicotine-treated group compared to the salinetreated group, but the difference was not significant (P = 0.131). Four weeks after removing immobilization, GAG content of the articular cartilage was significantly decreased in the saline-treated group (68.55 ± 4.86 µg/mg) compared to the sham-operated group (82.06 ± 2.25 µg/mg) (P < 0.001). GAG content was significantly increased in the nicotine-treated group (79.31 ± 4.20 µg/mg) compared to the saline-treated group (P = 0.002). There were no significant differences between the sham-operated group and the nicotine-treated group (P = 0.350).
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Effects of nicotine on a rat model of early stage osteoarthritis
Figure 2. Hematoxylin-eosin (A-G) and Safranin-O staining (H-N). (A and H) Sham-operated group 2 weeks after removal of immobilization; (B and I) Saline-treated group 2 weeks after removal of immobilization; (C and J) Nicotinetreated group 2 weeks after removal of immobilization; (D and K) Sham-operated group 4 weeks after removal of immobilization; (E and L) Saline-treated group 4 weeks after removal of immobilization; (F and M) Nicotine-treated group 4 weeks after removal of immobilization; (G and M) Untreated control group. Magnification × 100.
0.018). Similarly, four weeks after removing immobiliFemoral condyle Sham group Saline group Nicotine group Control group zation, the serum TNF-α 2 weeks 1.33 ± 0.97 3.47 ± 1.19a,b 2.13 ± 0.92a,b,c 1.07 ± 0.96 level in the saline-treated 4 weeks 1.27 ± 1.08 3.63 ± 1.23a,b 1.73 ± 0.96c group (24.57 ± 6.25 pg/ml) Values are the mean ± SD. 2 weeks = 2 weeks after removing immobilization, 4 weeks was significantly increased = 4 weeks after removing immobilization. aP < 0.05 vs. control group; bP < 0.05 vs. compared to the untreated c sham-operated group; P < 0.05 vs. saline-treated group. control (P < 0.001), shamoperated (7.16 ± 1.96 pg/ There were no significant differences in collaml), and nicotine-treated (10.22 ± 4.20 pg/ml) (P < 0.001) groups. The nicotine-treated group gen content of the articular cartilage at any had a lower serum TNF-α level four weeks after time point between any groups (Figure 4B). removing immobilization compared to two Levels of serum TNF-α weeks after removing immobilization, but the difference was not significant (P = 0.347) Two weeks after removing immobilization, the (Figure 5). saline-treated group (23.95 ± 5.62 pg/ml) had a significantly increased mean serum TNF-α Gene expression analysis of the synovial tislevel compared to the untreated control (6.22 ± sues 2.09 pg/ml) (P < 0.001) and sham-operated (6.03 ± 3.72 pg/ml) (P < 0.001) groups. The Two and four weeks after removing immobilizanicotine-treated group (12.83 ± 4.09 pg/ml) tion, the saline-treated group had a significantly had a significantly decreased serum TNF-α increased mean TNF-α RNA level compared to level compared to the saline-treated group (P = the untreated control (P < 0.001) and shamTable 2. Histological score of articular cartilage
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Figure 3. High-field MRI T2 mapping (A-G) and average T2 values (H) (A) Sham-operated group 2 weeks after removal of immobilization; (B) Saline-treated group 2 weeks after removal of immobilization; (C) Nicotine-treated group 2 weeks after removal of immobilization; (D) Sham-operated group 4 weeks after removal of immobilization; (E) Saline-treated group 4 weeks after removal of immobilization; (F) Nicotine-treated group 4 weeks after removal of immobilization; (G) Untreated control group. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. +P < 0.05, ++P < 0.005 vs. control group; *P < 0.05, **P < 0.005 vs. shamoperated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
operated (P < 0.001) groups. In the nicotinetreated group, TNF-α expression level was significantly decreased compared to the salinetreated group. Two and four weeks after removing immobilization, mean α7nAChR RNA levels were significantly increased in the saline-treated and nicotine-treated groups compared to the untreated control and sham-operated groups (P < 0.001). Four weeks after removing 3608
immobilization, α7nAChR RNA levels were significantly increased in the nicotine-treated and saline-treated groups (P < 0.001) (Figure 6). Discussion In this study, pathological assessments and T2 mapping showed that nicotine ameliorated cartilage destruction and promoted matrix producInt J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis ment with nicotine [11]. Li et al. showed similar results [12]. Wu et al. reported that activating the cholinergic anti-inflammatory pathway with nicotine attenuated collagen-induced arthritis via suppression of the Th17 response [25]. The current study focused on early stage OA. Compared to saline, nicotine increased the number of normal chondrocytes, and the proteoglycan and GAG content in the articular cartilage. The effect seemed to be mediated by decreased serum and expression levels of TNF-α [11, 12].
Figure 4. Biochemical analysis of cartilage. (A) GAG content analysis (B) collagen content analysis. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
Figure 5. Serum TNF-α levels. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. saline-treated group.
tion in a rat early stage OA model. In addition, nicotine reduced the serum level of TNF-α and the expression of TNF-α in the synovial tissue, and increased the expression of α7nAChR, indicating that nicotine-induced effects in OA are likely due to the peripheral stimulation of α7nAChR. To our knowledge, there is one of the first studies to investigate the effect of nicotine on early stage OA. Van Maanen et al. established a mouse model of clinical arthritis using type II collagen. They observed bone degradation, extensive proteoglycan loss, and inflammatory cell infiltration, which were attenuated by treat3609
The current study showed a strong expression of α7nAChR in salinetreated animals, which was unexpected. Westman et al. used immunohistostaining and found a higher expression of α7nAChR in RA patients’ synovial tissue compared to that of healthy subjects [10], but the difference did not reach statistical significant. Waldburger et al. used quantitative PCR and showed that α7R mRNA levels were similar in both RA and OA patients’ synovium [13]. Schubert and Beckmann measured nicotinic and muscarinic ACh receptor expression in synovial tissue from patients with RA and OA using RT-PCR and immunofluorescence labeling and also found no obvious difference between the samples [26].
Previous reports indentify a role for the α7nAChR receptor in the antiinflammatory cholinergic pathway [7]. Wang et al. demonstrated that in wild-type mice binding of ACh to α7-nAChR reduced the production of proinflammatory cytokines, including TNF-α, IL-1 [7]. van Maanen et al. found that stimulation of α7nAChR by selective agonists such as AR-R17779 reduced the level of serum TNF-α in a mouse model of collagen-induced arthritis [11] and described an increase in proinflammatory cytokines in α7nAChR knockout mice with experimentally-induced arthritis [27]. Other studies show that TNF-α and IL-1 play an important role in the degradation of the ECM and the destruction of cartilage in OA [28]. Taken together, these data suggest that nicotine Int J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis (dGEMRIC) detected changes in proteoglycan content in knee cartilage among individuals taking collagen hydrolysate and that T2 mapping showed little variability [34]. In the present study, there were higher signal intensities and T2 values in the cartilage of the saline-treated group compared to that of the untreated control and sham-operated groups, as well as a reduced GAG content. In contrast, signal intensities and T2 values were down-regulated in the nicotine group compared to the saline group, which was accompanied by an increased GAG content. The mechanism of nicotine’s antiinflammatory effect on arthritis through α7-nAChR has been studied extensively, but remains controversial. JAK-2/STAT-3 and the NF-κB pathways have been implicated in α7R signaling in mouse macrophages [35, 36]. However, Waldburger et al. suggested that cholinergic signaling does not influence NF-κB-driven promoter activity in FLS from RA patients. Conversely, Zheng showed that nicotine reduced IL-1β-induced cartilage apoptosis through activation of PI3K/ Akt signaling by regulating Bcl-2/Bcl-xl expression. They also observed that nicotine triggered the phosphorylation of S6 through p70S6K or Akt, regulating protein synthesis including the increase of TIMP-1 and the inhibition of MMP13, thereby mediating ECM degradation and synthesis [6].
Figure 6. Gene expression in the synovial tissues. A. TNF-α expression; B. α7nAChR expression. 2 weeks = 2 weeks after removing immobilization, 4 weeks = 4 weeks after removing immobilization. *P < 0.05, **P < 0.005 vs. sham-operated group; #P < 0.05, ##P < 0.005 vs. salinetreated group.
could reduce the level of TNF-α through α7nAChR in OA. In addition, our study indicated that GAG synthesis was upregulated by nicotine, which promoted the repair of the articular cartilage. Akmal et al. showed that nicotine, at physiological levels found in smokers, increased DNA, GAG, and collagen synthesis of nucleus pulposus cells [29]. However, Gullahorn et al. found that nicotine increased collagen synthesis, but had no effect on GAG synthesis in vitro [30]. Similarly, Ying et al. showed nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and OA patients without increasing GAG synthesis [31]. MRI T2 mapping is a useful noninvasive method for the detection of early degenerative changes of cartilage in OA [14, 32]. In particular, GAG content causes variation in T2 values [33]. Watrin-Pinzano et al. induced rat joint cartilage degradation by hyaluronidase. They observed the signal intensities and T2 values of degraded cartilage increased with loss of GAG content, with no obvious change in collagen content [16]. McAlindon reported that delayed gadolinium enhanced MRI of cartilage
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This study is associated with several limitations. First, we did not attempt to distinguish between different types of collagen. Total collagen content was unchanged, but nicotine is reported to up-regulate synthesis of collagen II; therefore, further investigations are warranted [31]. Second, our data suggested a tendency for a higher expression of α7nAChR in the synovial tissue of the nicotine-treated group compared to the saline-treated group, but no published literature supports these findings. In conclusion, we demonstrated that nicotine treatment attenuated cellular damage, increa-
Int J Clin Exp Pathol 2015;8(4):3602-3612
Effects of nicotine on a rat model of early stage osteoarthritis sed the synthesis of the ECM, and reduced the serum TNF-α level in a rat model of early stage OA. These data suggest that nicotine prevented cartilage damage and had an anti-inflammatory effect. In the future, nicotine may have potential as a therapeutic strategy for early OA.
[6]
Acknowledgements
[7]
Financial support for this study was provided by the National Natural Science Foundation of China (81171745). We gratefully acknowledge Mrs. Yuling Peng for her logistical support.
[8]
Disclosure of conflict of interest None. Address correspondence to: Dr. Liming Wang, Department of Orthopedics, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, Jiangsu, China. Tel: +86-18951670968; Fax: +8625-52269924; E-mail: [email protected]
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