Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF‐κB signaling pathway

Abstract Mechanical stress plays a key role in regulating cartilage degradation in osteoarthritis (OA). The aim of this study was to evaluate the effects and mechanisms of mechanical stress on articular cartilage. A total of 80 male Sprague‐Dawley rats were randomly divided into eight groups (n = 10 for each group): control group (CG), OA group (OAG), and CG or OAG subjected to low‐, moderate‐, or high‐intensity treadmill exercise (CL, CM, CH, OAL, OAM, and OAH, respectively). Chondrocytes were obtained from the knee joints of rats; they were cultured on Bioflex 6‐well culture plates and subjected to different durations of cyclic tensile strain (CTS) with or without exposure to interleukin‐1β (IL‐1β). The results of the histological score, immunohistochemistry, enzyme‐linked immunosorbent assay, and western‐blot analyses indicated that there were no differences between CM and CG, but OAM showed therapeutic effects compared with OAG. However, CH and OAH experienced more cartilage damage than CG and OAG, respectively. CTS had no therapeutic effects on collagen II of normal chondrocytes, which is consistent with findings after treadmill exercise. However, CTS for 4 hr could alleviate the chondrocyte damage induced by IL‐1β by activating AMP‐activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of nuclear factor (NF)‐κB p65. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes; mechanical stress only caused damage with excessive stimulation. Still, moderate biomechanical stress could reduce sensitization to the inflammatory response of articular cartilage and chondrocytes through the AMPK/NF‐κB signaling pathway.

Physical activity is one of the most widely applied nonpharmacological therapies for OA, but the duration and intensity of recommended exercise programs vary widely (Mcalindon et al., 2014). It is well accepted that different types of mechanical loading lead to different biological responses (Grad, Eglin, Alini, & Stoddart, 2011). Adequate exercise has been shown to benefit people with OA by relieving pain and increasing mobility (Barbour et al., 2014), but the pathology of OA is associated with excessive mechanical load. As mechanosensitive cells, chondrocytes synthesize the extracellular matrix and depend on intracellular signals generated in response to biomechanical stress (Harvey, Brosseau, & Herbert, 2014). Increasing evidence suggests that mechanical signaling plays a key role in regulating cartilage damage or repair.
Despite active research in this area, it is still unclear how physical activity affects articular cartilage.
Several therapies aimed at ameliorating inflammatory response are currently being investigated. Molecular studies have revealed that specific biomechanical stimuli generate intracellular signals that are powerful inducers or suppressors of proinflammatory genes in chondrocytes (Knobloch, Madhavan, Nam, Agarwal, & Agarwal, 2008).
Chondrocytes maintain a functional balance between degradation and repair by producing various enzymes, cytokines, and matrix-associated proteins. A loss of this functional balance is associated with changes in the phenotypic characteristics of chondrocytes. Phenotypically, chondrocytes are characterized by their ability to synthesize collagen to withstand changes in their mechanical environment. Thus, the mechanisms by which chondrocytes convert biomechanical signals into intracellular biochemical events need further investigation.
Cyclic tensile strain (CTS) can be applied to cultured chondrocytes in a wide range of strain magnitudes, frequencies, and durations (Agarwal et al., 2004;Huang, Ballou, & Hasty, 2007;Kawakita et al., 2012;Long, Gassner, & Agarwal, 2001;Perera et al., 2010;Xu et al., 2011). The experimental setup is validated, controllable, and allows for the study of cell responses (Colombo, Cahill, & Lally, 2008). CTS also provides new insights into loading and cartilage adaptation. These mechanical signals acting on chondrocytes are critical regulators of tissue adaptation, structure, and function (Ramage, Nuki, & Salter, 2009). It is still unclear how intracellular signals generated by CTS of different durations with or without inflammatory stimulation produce these changes.
AMP-activated protein kinase (AMPK) acts as an intracellular sensor that modulates the energy balance within chondrocytes.
AMPK is exquisitely sensitive to the adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and intracellular calcium (Ca 2+ ) levels. The role of AMPK is not only to regulate protein synthesis related to inflammation but also to modulate mitochondrial biogenesis (Gwinn et al., 2008). Inhibition of AMPK activation significantly impaired mitochondrial function and increased the generation of reactive oxygen species (ROS; Li, Wu, & Tian, 2018;X. Chen et al., 2018;Zhao & Yu, 2018). Further, AMPK responds to energy stress by regulating cell growth and biosynthetic processes, mainly through its inhibition of the nuclear factor (NF)-κB signaling pathway. NF-κB p65 is thought to be a link between tensile loading and the responses of chondrocytes to proinflammatory cytokines (Yang, Wang, Kong, Zhang, & Bai, 2017). Activation of NF-κB p65 is a key event in matrix metalloproteinase (MMP) gene expression (Aupperle et al., 2001).
In this study, we evaluated the potential effects of different durations of treadmill exercise on cartilage with or without monoiodoacetate (MIA) injection. Furthermore, to identify potential contributions of mechanical stress at the cellular level, specifically the AMPK/NF-κB signaling pathway, chondrocytes were subjected to CTS (0.5 Hz, 10%) of different durations with or without interleukin-1β (IL-1β).

| Experimental animals
A total of 80 male Sprague-Dawley (SD) rats (230 ± 10 g; specificpathogen-free) were obtained from HFK Bioscience Co. Ltd. (Beijing, China). This study was carried out in accordance with the recommendations of the Ethics Committee of Shengjing Hospital, China Medical University. The protocol was approved by this committee. Rats were kept in individual plastic cages on sawdust bedding; the environment included a 12 hr:12 hr light: dark cycle with the lights on from 6:00 a.m. to 6:00 p. m., a controlled temperature of 22 ± 2°C, and 70% humidity. The rats had free access to a planned diet. Body weight was recorded at regular intervals. They were adapted to laboratory conditions for 1 week before the experimental procedures. All rats were habituated to ZH-PT treadmill exercise (Zhongshidichuang Science & Technology Development Co. Ltd., Beijing, China) for 1 week at a speed of 10 m/min for 10 min/day to reduce stress. All rats successfully adapted to the treadmill exercise.

| OA model and treadmill running protocols
After the adaptive treadmill exercise, the SD rats were numbered from 1 to 80 and randomly grouped by an Excel function into eight groups (n = 10 for each group): control group (CG); CG subjected to low-, moderate-, or high-intensity treadmill exercise (CL, CM, and CH, respectively); knee OA model group (OAG); and OAG subjected to low-, moderate-, or high-intensity treadmill exercise (OAL, OAM, and OAH, respectively).All rats were anesthetized with 1.5% pentobarbital sodium (30 mg/kg, intraperitoneal injection). Knee joint inflammation was induced by intra-articular injection of MIA (1 mg per cavity in 50 μl sterile saline) by microsyringe through the infrapatellar ligament and into the bilateral knee joint cavity. The rats of CG, CL, CM, and CH received an intra-articular injection of 50 μl sterile saline. CG and OAG rats were kept sedentary, but rats in the other groups began their exercise programs 24 hr after injection. The rats of CL and OAL exercised 30 min once daily, CM and OAM exercised 60 min once daily, and CH and OAH exercised 90 min once daily

| Sampling and tissue preparation
After 4 weeks of treadmill exercise, all rats were anesthetized.
Blood samples were obtained immediately after the animals were anesthetized, and the samples were centrifuged at 3,000 g for 10 min to obtain serum. Intra-articular lavage fluid (IALF) was obtained from the synovial cavity of the right knee of each rat by injection and recovery of 200 μl of phosphate-buffered saline (PBS) three times. All rats were then killed by cervical dislocation.
The left knee joints of all rats were dissected and fixed in 4% paraformaldehyde solution. Articular cartilage was removed from the weight-bearing area of the condyles of the right femur and tibia using a scalpel. All tissues were stored at −80°C until further analysis.

| Histology
Left knee joint tissue samples were stored in 4% paraformaldehyde for 7 days. Then, they were washed in water for 5 hr and transferred to 20% EDTA solution (Jianglai Reagent Co., Ltd, Shanghai, China) to decalcify for 21 days; the solution was changed every 3 days. Decalcified samples were dehydrated in an ethanol series and embedded in paraffin. Serial 5-μm sagittal sections were cut from the tibiofemoral joints for histological examination. The sections were stained with hematoxylin and eosin, as well as toluidine blue, to observe the cartilage. Next, the sections were visualized with ScanScope (APERIO CS2, Leica Biosystems Inc., Buffalo Grove, IL). Injuries to the articular cartilage in the femur and tibia were assessed by the Modified Mankin score (scale of 0-14 points; Pritzker et al., 2006) and the Osteoarthritis Research Society International (OARSI) score (scale of 0-24 points; Gerwin, Bendele, Glasson, & Carlson, 2010). Because both the tibial and femoral cartilages were evaluated, the maximum Mankin score was 28 and the maximum OARSI score was 48. Two experienced observers (Yue Yang and Xiaoning Zhang) performed the scoring in a blinded manner.

| Immunohistochemistry
In addition to histomorphological evaluation, serial sections were stained for assessment of collagen II and MMP-13 contents. After deparaffinization and rehydration of the tissue sections, endogenous peroxidase activity was blocked by 3% H 2 O 2 for 20 min.
The slides were washed three times in PBS followed by incubation for 20 min at 37°C with an anti-mouse/rabbit immunoglobulin G (IgG) detection system (PV-9000; Zhongshan Goldenbridge Biotechnology Co., China) and visualized with diaminobenzidine. Nuclei were counterstained with hematoxylin for 5 min. The optical densities of the stained slides were measured using image analysis software (Nikon H600L Microscope and image analysis system, Japan). Collagen II was expressed by relative intensity. MMP-13 was expressed by the percentage of positive cells.

| Western blot analysis
After washing three times with TBST, the membranes were incubated with IgG-horseradish peroxidase-conjugated secondary antibodies (1:10,000; Canlife) at room temperature for 2 hr. After washing with TBST buffer, immunoreactivity was detected with enhanced chemiluminescence and quantified using Quantity ONE (Bio-Rad, Hercules, CA) software. β-actin or histone H2A.X was used as the internal control.

| Isolation and culture of chondrocytes
Chondrocytes were obtained from the articular cartilage of knee joints of male SD rats (150 ± 10 g; specific-pathogen-free). Tissue was collected in sterile PBS. Articular cartilage pieces were incubated by sequential digestion with pronase (2 mg/ml) and collagenase D (1 mg/ ml; Roche, Basel, Switzerland). Cells were cultured in 25-cm 2 cell-culture flasks in Dulbecco's modified Eagle medium (Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (Gibco BRL) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) in a humid atmosphere of 5% CO 2 in air at 37°C. Upon reaching confluence, cells were detached with 0.25% trypsin and split in a 1:3 ratio. The cells were identified by immunohistochemical staining with anti-collagen II antibody (ab34712, 1:100; Abcam; Figure 5a). For all experiments, the fourth through sixth passages were used. Using light microscopy, more than 95% of cells were judged to be chondrocytes.
CTS experiments were performed using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA). To provide uniform radial and circumferential strain on the membranes, the plates were placed on a loading station (located in an incubator with 5% CO 2 ) such that when a vacuum was applied to the loading station, the membrane deformed across the post face, creating uniform biaxial strain. Chondrocytes were subjected to CTS (10%, 0.5 Hz) for different durations (0, 0.5, 1, 2, 4, 8, and 16 hr) with or without IL-1β for 24 hr. The stimulations of CTS and IL-1β on chondrocytes began at the same time. We choose the best condition for further study. Compound C (ab120843; Abcam), a selective and reversible AMPK inhibitor, was used for pretreatment for 1 hr before the stimulation with IL-1β and CTS (Dai et al., 2017

| Immunofluorescence analysis of chondrocytes
After washing with PBS and being fixed with 4% paraformaldehyde for 20 min at room temperature, the cells were permeabilized with 0.5% Triton X-100 for 30 min and incubated in nonspecific binding blocking solution (5% BSA) for 30 min at room temperature. Rabbit polyclonal anti-NF-κB p65 antibody (AB21014, 1:50; Absci) was added to the cells overnight at 4°C followed by staining with Alexa Fluor 488 conjugated anti-rabbit antibody for 60 min at room temperature in darkness. The cytoskeleton was stained with phalloidine for 60 min at 37°C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole for 2 min. After washing, the cells adhered to Bioflex membranes were mounted in PBS with 20% glycerol. The chondrocytes were visualized with a confocal microscope (Olympus, Tokyo, Japan).

| Statistical analysis
Data were analyzed using SPSS statistical software version 16 (SSPS, Inc., Chicago, IL). Results are expressed as means with 95% confidence intervals. Shapiro-Wilk's and Levene's tests were applied to evaluate the normality and homogeneity of the results, respectively. For variables that exhibited a normal distribution, independent samples t test and one-way analysis of variance were used for the statistical analysis of significance. p-values less than 0.05 were considered significant.

| Histological observations and immunohistochemical analysis
Histological assessment (Mankin and OARSI score) and immunohistochemical staining (collagen II and MMP-13) revealed that there were no differences among CL, CM, and CG, but OAM achieved therapeutic effects compared with OAG. CH and OAH showed evidence of potential cartilage damage compared with CG and OAG, respectively (Figures 2 and 3).
F I G U R E 2 Histological evaluation of tibiofemoral joints. Histological features of representative tibiofemoral joints sectioned in the sagittal plane stained with HE (a) and toluidine blue (b). Mankin and OARSI histological scores are shown for each image. F: femur, T: tibia. (c) Mankin score of tibiofemoral joints. Differences between CG and CH (*p < 0.001), CG and OAG ( + p < 0.001), and OAG versus OAM and OAH ( # p < 0.001) were significant. (d) OARSI histological scores for cartilage of tibiofemoral joints. Differences between CG and CH (*p < 0.001), CG and OAG ( + p < 0.001), and OAG versus. OAM and OAH ( # p < 0.001) were significant. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 10 rats in each group. Experimental groups: CG; CL, CM, and CH, control group subjected to different durations of treadmill exercise; OAG; and OAL, OAM, and OAH, OA subjected to different durations of treadmill exercise. CG: control group; OAG: OA group; OARSI: Osteoarthritis Research Society International [Color figure can be viewed at wileyonlinelibrary.com]

| ELISA of TNF-α and IL-1β
There were no significant differences among CG, CL, and CM in the concentrations of TNF-α and IL-1β in serum. The serum concentrations of TNF-α and IL-1β of CH and OAG were higher than those of CG. However, OAM had decreased serum concentrations of TNF-α and IL-1β compared with OAG. The changes in TNF-α and IL-1β concentrations in IALF were similar to those observed in serum ( Figure 4b).

| Western blot analysis
The changes in collagen II, MMP-13, and NF-κB p65 in the cartilage in different groups were similar to those observed in histological observations and immunohistochemical analysis ( Figure 4c).
There was no significant difference in collagen II after 4 hr of CTS (10%, 0.5 Hz), but the content of collagen II in chondrocytes IL-1β induced decreases in the contents of AMPK-α1 (phosphor S487) and IκB-α. CTS (10%, 0.5 Hz, 4 hr) increased the contents of these proteins. Compound C inhibited the increases in levels of these proteins caused by CTS (10%, 0.5 Hz, 4 hr; Figure 7a).

| Contents of AMP, ADP, and ATP in chondrocytes
The effects of CTS (10%, 0.5 Hz) for 4 hr on AMP, ADP, and ATP contents in chondrocytes were measured by HPLC. We noticed a significant decrease in ATP content, increase in AMP content, and an increase in the AMP/ATP ratio after CTS (Figure 6a).

| Intracellular Ca 2+ and ROS analysis of chondrocytes
To evaluate the intracellular Ca 2+ and oxidative stress of chondrocytes, we measured intracellular Ca 2+ detected by Fluo-4AM (Figure 6b) and the production of ROS by DCFH-DA ( Figure 6c). Intracellular Ca 2+ and ROS production were enhanced with IL-1β (1 ng/ml) stimulation. Interestingly, we evaluated the effect of CTS (10%, 0.5 Hz, 4 hr) on ROS generation in chondrocytes exposed to IL-1β (1 ng/ml) and noticed a significant decrease in ROS level. Intracellular Ca 2+ was further increased by CTS (10%, 0.5 Hz, 4 hr) stimulation.

| Immunofluorescence analysis of chondrocytes
A significant nuclear translocation of NF-κB p65 protein was detected in chondrocytes stimulated with IL-1β (1 ng/ml) F I G U R E 3 Immunohistochemical staining in each group. The micrographs show the relative intensities of immunohistochemical staining of collagen II (a) and the percentages of positively stained cells of  in the articular cartilage of each experimental group. Differences between CG and CH (*p < 0.001), CG and OAG ( + p < 0.001), and OAG versus OAL, OAM, and OAH ( # p < 0.001, #a p = 0.019, #b p = 0.006) were significant. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 5 rats in each group. To study the connection between mechanical stress and OA progression, we investigated an animal model using rats subjected to treadmill exercise. Intra-articular injection of MIA induced changes that replicated those observed in humans with OA, including cartilage surface erosion, matrix loss, and inflammation of the synovium (Barve et al., 2007;Cifuentes et al., 2010;Guzman, Evans, Bove, Morenko, & Kilgore, 2003;Schuelert & Mcdougall, 2009). We observed no differences among CG, EL, and EM groups related to the articular cartilage of the knee, including histology (Mankin and OARSI score), protein contents (collagen F I G U R E 4 Comparisons of body weight, enzyme-linked immunosorbent assay, and western blot analyses. (a) The results of body weight comparisons. The differences between CG versus OAL, OAM, and OAH ( + p < 0.001, +a p = 0.004, +b p = 0.001, +c p = 0.003) and the differences between OAG versus OAL, OAM and OAH (*p < 0.001, *a p = 0.001, * b p = 0.012) were significant. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 10 rats in each group. (b) The levels of IL-1β and TNF-α in serum and IALF. Differences between CG and CH (*p < 0.001, * a p = 0.016, * b p = 0.005, * c p = 0.039), CG and OAG ( + p < 0.001, +a p = 0.001), and OAG versus OAL, OAM, and OAH ( # p < 0.001, #a p = 0.028, #b p = 0.012, #c p = 0.003) were significant. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 10 rats in each group. (c) Protein content was determined by western blots of total protein extracted from cartilage. Differences between CG and CH (*p < 0.001, * a p = 0.001), CG and OAG ( + p < 0.001), and OAG versus OAL, OAM, and OAH ( # p < 0.001, #a p = 0.001, #b p = 0.01) were significant. β-actin and histone H2A.X were used as internal standards. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 3 rats in each group. Experimental groups: CG; CL, CM, and CH, control group with different durations of treadmill exercise; OAG; and OAL, OAM, and OAH, OA with different durations of treadmill exercise. CG: control group; IALF: intra-articular lavage fluid; IL-1β: interleukin-1β; OAG: OA group; TNF-α: tumor necrosis factor-α II, MMP-13, and NF-κB p65), or inflammatory mediators (TNF-α and IL-1β). Histological changes were ameliorated in OAM, changes in collagen II content in cartilage were reversed, and levels of inflammatory mediators in serum and IALF were reduced. CH and OAH both showed evidence of potential cartilage damage compared with CG and OAG, respectively.
Together, our results corroborated findings that OAM could alleviate cartilage damage in knee OA in a rat model (Galois et al., 2004;Na, Kim, Yong, & Hwangbo, 2014;Qian, Liang, Wang, & Wang, 2014). Accumulated evidence suggests that IL-1β is the pivotal mediator of OA (Bonnelye, Reboul, Duval, Cardelli, & Aubin, 2011). IL-1β has been associated with the presence of joint inflammation and cartilage destruction. In this study, we used IL-1β as an inflammatory agent to induce chondrocyte damage: we found that IL-1β increased in IALF of our OA model. Our Another striking finding of the current study is that the AMPK/NF-κB signal transduction pathway is central to CTS.
AMPK serves as a checkpoint to sustain energy balance by modulating biological responses. AMPK is an evolutionarily conserved serine/threonine kinase that was originally identified as the key player in maintaining cellular energy homeostasis (Jeon, 2016). Binding of AMP or ADP causes conformational changes that enhance net phosphorylation at Thr172 and causes allosteric activation (L. Chen et al., 2012). Moderate CTS could activate the AMPK pathway by increasing the intracellular AMP/ ATP ratio and causing Ca 2+ influx, which has been confirmed.
F I G U R E 5 Western blot analysis of chondrocytes. (a) Representative immunohistochemical image of chondrocytes stained with collagen II. Scale bar, 100 μm. Protein content of chondrocytes was determined in western blots according to different durations of CTS (10%, 0.5 Hz) without (b) and with (c) IL-1β. Differences between normal chondrocytes and chondrocytes subjected to CTS of different durations were significant (*p < 0.001), differences between normal chondrocytes and chondrocytes exposed to IL-1β were significant ( + p < 0.001), differences between IL-1β-induced chondrocytes and CTS of different durations were significant ( # p < 0.001), and differences between IL-1β-induced chondrocytes with CTS for 4 hr and 8 hr were significant ( & p < 0.001). β-actin was used as the internal standard. Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 3 rats in each group. Treatment groups: CG; CL, CM, and CH, control group subjected to different durations of treadmill exercise; OAG, OA group; and OAL, OAM, and OAH, OA subjected to different durations of treadmill exercise. CG: control group; CTS: cyclic tensile strain; IL-1β: interleukin-1β; OAG: OA group [Color figure can be viewed at wileyonlinelibrary.com] Also, the effect of Thr172 in AMPK has been confirmed in many studies (Jeon, 2016). S487 pAMPK antibody also has been used in many studies (Dun, Liu, Zhang, Xie, & Qiu, 2017;Y. Chen et al., 2015). But the phosphorylation at S487 of AMPK in mechanical stress is unknown. And inhibition of AMPK activation significantly impaired mitochondrial function and increased the generation of ROS (Dun et al., 2017;Y. Chen et al., 2015). IL-1β is a classic proinflammatory cytokine, so it is not surprising that it inhibits the expressions of AMPK-α1 (phosphor S487) and IκB (inhibitor of NF-κB)-α (IκB-α). In this study, its actions were mediated by NF-κB p65 nuclear translocation. This is consistent with our observation that CTS (10%, 0.5 Hz) for 4 hr changed the intracellular AMP/ATP ratio and caused Ca 2+ influx, which activated the AMPK pathway. The activation of AMPK reduced the level of ROS, which inhibited the nuclear translocation of NF-κB p65. CTS (10%, 0.5 Hz, 4 hr) inhibited nuclear translocation of NF-κB p65 via the AMPK signal pathway, which was confirmed by analysis of compound C. Hence, the anti-inflammatory actions of CTS (10%, 0.5 Hz) for 4 hr were mediated both by activating the AMPK signal pathway and by inhibiting NF-κB nuclear translocation in IL-1β induced chondrocytes. The activation of the AMPK/ NF-κB pathway nullifies the increased MMP-13 production induced by IL-1β, which inhibits collagen II breakdown in chondrocytes ( Figure 8). Interestingly, the AMPK/NF-κB pathway may explain the reason why moderate CTS had different effects on chondrocytes between models with and without IL-1β.
Because NF-κB p65 exists in the cytoplasm under normal conditions, moderate CTS did not achieve therapeutic effects via the AMPK/NF-κB signaling pathway.
This study has several limitations that must be considered.
First, further study is needed to explore different conditions (such as intensity and frequency) that cause damage to articular cartilage and chondrocytes. Second, CTS is two-dimensional loading: chondrocytes are strained in a monolayer and only one surface is elongated. We will continue to investigate these issues in future studies.

F I G U R E 6
The results of HPLC, Ca 2+ , and ROS analyses in chondrocytes. (a) The contents of AMP, ADP, and ATP of chondrocytes subjected to CTS for 4 hr. Differences between normal chondrocytes and chondrocytes subjected to CTS were significant (*p < 0.001, *p = 0.009). Results according to independent sample t test, presented as means with 95% confidence intervals; n = 3 rats in each group. The fluorescence microscopy of Ca 2+ (b) and ROS (c) in chondrocytes. Differences between normal chondrocytes and chondrocytes exposed to IL-1β were significant (*p < 0.001), and differences between IL-1β-induced chondrocytes and those subjected to CTS for 4 hr were significant ( + p < 0.001). Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 3 rats in each group. ADP: adenosine diphosphate; AMP: adenosine monophosphate; ATP: adenosine triphosphate; CTS: cyclic tensile strain; HPLC: high-performance liquid chromatography; IL-1β: interleukin-1β; ROS: reactive oxygen species [Color figure can be viewed at wileyonlinelibrary.com] In summary, we used treadmill exercise in rats as an animal model and CTS applied to chondrocytes as a cellular model to explore the effects of mechanical stress in OA. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes: mechanical stress only caused damage under excessive stimulation. However, moderate mechanical stress could reduce sensitization to inflammatory responses of articular cartilage and chondrocytes through the AMPK/NF-κB pathway. Our results not only provide crucial leads to unveiling the effects of mechanical stress on articular cartilage and chondrocytes but also provide molecular evidence for biochemical signals generated by mechanicalstress.
F I G U R E 7 Western blot and immunofluorescence analysis results of NF-κB p65 in chondrocytes. (a) The results of western blot analysis of AMPK-α1 (phosphor S487) and IκB-α. Differences between normal chondrocytes and chondrocytes exposed to IL-1β were significant (*p < 0.001), differences between IL-1β-induced chondrocytes and those subjected to CTS for 4 hr were significant ( + p < 0.001), and differences between IL-1β-induced chondrocytes subjected to CTS for 4 hr and compound C were significant ( # p < 0.001). Results according to one-way analysis of variance, presented as means with 95% confidence intervals; n = 3 rats in each group. (b) Effects of CTS for 4 hr on nuclear translocation of NF-κB p65 in IL-1β-induced chondrocytes. The chondrocytes were immunostained using anti-NF-κB p65 rabbit antibody (green) and visualized by confocal microcopy. The cytoskeleton was defined by phalloidine (red) and the cell nucleus was defined by DAPI (blue). Scale bar, 50 μm. AMPK: AMP-activated protein kinase; CTS: cyclic tensile strain; IκB-α: IκB (inhibitor of NF-κB)-α; IL-1β: interleukin-1β; NF-κB: nuclear factor-κB [Color figure can be viewed at wileyonlinelibrary.com]