Remifentanil repairs cartilage damage and reduces the degradation of cartilage matrix in post-traumatic osteoarthritis, and inhibits IL-1β-induced apoptosis of articular chondrocytes via inhibition of PI3K/AKT/NF-κB phosphorylation.

BACKGROUND: Remifentanil (RFT) is an opioid analgesic with a unique pharmacokinetic profile, and plays an important role in the intra- and post-operative periods. Post-traumatic osteoarthritis (PTO) is a particular type of osteoarthritis (OA) that occurs secondary to a traumatic injury. In the present study, we investigated the effects of RFT both in vivo and in vitro.
METHODS: In vivo, 50 Sprague Dawley (SD) rats (7 weeks old) were randomly divided into five groups. Four groups of rats received RFT (0.2, 0.5, and 1 µg) or vehicle (PTO group), while the remaining group served as the control. A PTO model in rats was established using the Hulth method. The cartilage damage, articular cartilage formation, and the degradation of cartilage matrix were evaluated. The effects of RFT on cell proliferation, apoptosis, and nuclear factor (NF)-κB phosphorylation were also examined.
RESULTS: The results indicated that RFT improved cartilage damage, enhanced articular cartilage formation, and inhibited the degradation of cartilage matrix in PTO model rats. Compared with the control group, the protein levels of Osterix (OSX), Collagen type I alpha 1 (COL1A1), and osteocalcin (OC) were down-regulated in PTO model rats. RFT also inhibited the interleukin-1β (IL-1β)-induced apoptosis of chondrocytes in vitro. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/NF-κB pathway was inhibited both in vitro and in vitro.
CONCLUSIONS: RFT has significant potential as a therapeutic intervention to ameliorate PTO and provides a foundation for further clinical studies. 2020 Annals of Translational Medicine. All rights reserved.

Introduction

Osteoarthritis (OA) is a clinical syndrome characterized by pain and disabling degeneration of joint tissues. It is a progressive disease that leads to long-term loss of mobility, loss of independence, and disability (1). Post-traumatic osteoarthritis (PTO) is a particular type of OA that occurs secondary to a traumatic injury (2). Approximately 12% of all OA cases may be attributable to a major joint injury (3). Following a joint injury, surgery is often required to restore function and relieve pain. However, evidence suggests that the OA occurs earlier in patients who have undergone joint surgery than in patients who have not had joint surgery (4). Considering that treatments are limited and there are presently no disease-modifying OA drugs (5), understanding the biological mechanisms is critical in developing more successful treatment options for PTO patients.

Remifentanil (RFT) is one of the most widely used opioid analgesics. It is a highly selective µ-opioid receptor agonist with a strong sedative and analgesic effect (6). RFT is an ultra-short acting opioid that is rapidly degraded in the plasma and tissue by nonspecific esterases (7), and thus, has been used as an adjuvant analgesic for general anesthesia (8). It has also been shown to have a protective effect against hepatic ischemia-reperfusion injury (9). In human keratinocytes, RFT improves hydrogen peroxide-induced oxidative injury (10). Furthermore, it increases osteoblast differentiation in vitro by up-regulation of runt-related transcription factor 2 (RUNX2) and Osterix (OSX) expression. Therefore, RFT has the potential to assist with bone formation and healing (11). RFT also promotes the differentiation of pre-osteoclasts and induces maturation, including the increase in osteoclast size (12). Moreover, RFT preconditioning enhances cell viability and maturation of osteoblasts, and stimulates the expression of proteins including bone morphogenetic protein 2 (BMP-2), osteocalcin (OC), protein kinase B (AKT), type I collagen (CoL1A1), OSX, transforming growth factor (TGF)-β1, hypoxia-inducible factor (HIF)-1α, and RUNX2 in osteoblasts (13). RFT exhibits minimal inhibitory effect on in vitro fresh human articular chondrocyte (hAC) proliferation (14).

In PTO, the initial injury leads to cartilage damage and the rapid death of chondrocytes. In the subsequent days, the increased degradation of cartilage matrix promotes further death of chondrocytes (15). AKT is a serine/threonine protein kinase activated by extracellular factors through a phosphorylation mechanism dependent on phosphatidylinositol 3-kinase (PI3K) (16). The PI3K/AKT pathway is reportedly involved in two key processes (17,18). The degradation of cartilage is induced by matrix metalloproteinases (MMPs) and adamalysin with thrombospondin motif 5 (19). Activation of the PI3K/AKT pathway increases production of MMPs through a variety of downstream target proteins. Nuclear factor (NF)-κB is one of the target proteins of the PI3K/AKT pathway and is also a key regulator of MMP production as a transcriptional factor (20). The PI3K/AKT pathway is also associated with tumor necrosis factor (TNF)-α-induced activation of OA fibroblast-like synoviocytes, which may be involved in the pathogenesis of OA (21). Inhibition of the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway can promote the autophagy of articular chondrocytes and attenuate the inflammatory response in rats with OA (22). Inhibition of the NF-κB signaling pathway affects the incidence and evolution of OA (23). Thus, the inhibition of PI3K/AKT pathway and NF-κB is considered to be an effective treatment for OA (24,25). Allicin has been found to suppress interleukin (IL)-1β-stimulated PI3K/AKT/NF-κB activation in chondrocytes (26), and Nobiletin dramatically suppresses the IL-1β-stimulated phosphorylation of PI3K/AKT and activation of NF-κB in human OA chondrocytes (27). Therefore, evaluation of the PI3K/AKT/NF-κB pathway is important for the treatment of PTO.

In the present study, whether RFT plays a protective role in post-traumatic osteoarthritis has not been reported, the effects of RFT on cartilage damage, degradation of cartilage matrix in PTO model rats, and apoptosis of articular chondrocytes were investigated. Furthermore, the underlying mechanism PI3K/AKT/NF-κB pathway was also demonstrated.

We present the following article in accordance with the ARRIVE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-6000).

Methods

Animals

Fifty Sprague Dawley (SD) rats (7 weeks old; approximately 230–250 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All animal experiments and protocols were performed in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. The Ethics Committee for Experimental Animal Management and Animal Welfare of Beijing Hospital of Traditional Chinese Medicine reviewed and approved the study. Code: No.20190419. Rats were acclimatized for 7 days with free access to water and standard rat nutrients. Before the experiments, the rats were kept in a room under the following conditions: temperature of 23±1 °C; humidity level of 45–55%; and a 12 h light/dark cycle.

PTO model rats

Of the 50 rats,10 were randomly selected as the control group and the remaining 40 were selected as the PTO model group. The PTO model in rats was established using Hulth method (28,29). Rats were anesthetized with a 10% volume fraction chloral hydrate (300 mg/kg). The right knee joints in PTO model group rats were then shaved, and a 2 cm incision was made on the medial side of the knee to expose the knee joint. The anterior cruciate ligament was then cut off and the medial meniscus was completely removed. Meanwhile, the articular cartilage surface was retained. The incision was rinsed with saline and closed layer-by-layer with 3-0 sutures. The rats in the control group underwent sham surgery, which involved cutting open the articular cavity without damaging the cruciate ligament or meniscus. Cefradine 500 mg was injected intraperitoneally once daily for 3 consecutive days (at the same time each day). After surgery, all rats were allowed free exercise out of their cages for 30 min daily (at two separate intervals each day). After 6 weeks, the OA model was achieved.

Administrations of RFT in the PTO model

Rats in the PTO model group were randomly divided into four subgroups: a PTO model group, a PTO + RFT 0.2 µg group, a PTO + RFT 0.5µg group, and aPTO + RFT 1 µg group (n=10 per group). Rats were intraperitoneally injected with RFT for 7 days at doses of 0.2, 0.5, and 1 µg/kg from the day the model was built. The control and PTO model groups were treated with equal volumes of saline once daily until the end of6 weeks. Bone damage, trabecular bone and articular cartilage formation, and cartilage matrix degradation were measured.

Isolation and culture of chondrocytes

The 7 weeks old SD rats were sacrificed and soaked in 75% ethanol for 20 min. The articular cartilage was separated and cut up, and 1 mL 0.25% II collagenase type was added. The articular cartilage was then digested in a water bath at 37 °C for 8 hours. When the articular cartilage was almost digested, the supernatant was removed after centrifugation. The cells were mixed with complete culture medium and cultured in an incubator with 5% carbon dioxide (CO2). The chondrocytes were passaged when the density reached 90%. All groups were pretreated with IL-1β for 24 h except for the control group. Chondrocyte experiments involved five groups: the control group, IL-1β group (complete medium with 10 ng/mL IL-1β), RFT 50 ng/mL group (complete medium with 10 ng/mL IL-1β and 50 ng/mL RFT), RFT 100 ng/mL group (complete medium with 10 ng/mL IL-1β and 100 ng/mL RFT), and RFT 200 ng/mL group (complete medium with 10 ng/mL IL-1β and 200 ng/mL RFT). After culturing for 24 h, apoptosis and cartilage matrix degradation was measured. Insulin-like growth factor-1 (IGF-1) was PI3K agonist and were obtained from BiocodeHycel Company, Brussels, Belgium.

Bone morphometry

Rat femurs were dissected free of soft tissue, fixed in 4% paraformaldehyde for 24 h, and scanned under micro-computed tomography (µCT). The morphometry of trabecular bones was assessed by high-resolution Inveon microtomography (Siemens, Munich, Germany). Parameters included bone volume/total volume (BV/TV), trabecular bone surface/bone volume (BSA/BV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone space (Tb.Sp).

Histological examination

Cartilage samples were fixed with 10% neutral buffered formalin. Samples were then decalcified in diethyl-pyrocarbonate treated 0.2 M ethylenediaminetetraacetic acid (EDTA), embedded in paraffin wax, cut into sections (5 µm-thick), and stained with hematoxylin and eosin (H&E), Alcian blue, and Masson trichrome. Immunohistochemical staining was also performed to evaluate the level of NF-κBp65.

Western blot

Eleven primary antibodies, including anti-OSX, anti-COL1A1, anti-OC, anti-MMP-1, anti-MMP-3, andanti-MMP-13, were purchased from Abcam (Cambridge, UK). After culturing for 48 h, the isolated chondrocytes were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer. The total protein concentration was determined using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). Equal amounts of protein were isolated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% skim milk in Tris-buffered saline Tween (TBST) for 1 h, and then incubated with primary antibody anti-OSX (1:1,000, ab22552, Abcam), anti-COL1A1 (1:1,000, ab34710, Abcam), anti-OC (1:500, ab93876, Abcam), anti-MMP-1 (1:1,000, ab137332, Abcam), anti-MMP-3 (1:500, ab53015, Abcam), and anti-MMP-13 (1:3,000, ab39012, Abcam) at 4 °C overnight. The membranes were then washed and subjected to goat anti-rabbit immunoglobulin (Ig)G horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were detected using an enhanced chemiluminescent (ECL) detection kit (Pierce, Rockford, IL, USA). Actin was used as a loading control. The band densities were determined and analyzed with an automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA).

Cell counting Kit-8 (CCK-8) assay

Chondrocytes were inoculated into 96-well plates at a concentration of 2×104 cells/mL and cultured at 37 °C. CCK-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) was added into each well for 30 min at 37 °C and absorbance values were read at 490 nm.

Flow cytometry

A flow cytometry assay was performed using the annexin V-propidium iodide (PI) apoptosis kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Chondrocytes were seeded in 6-well plates (1×106 cells/well) and cultured for 48 hours. The cells were then harvested by trypsinization and washed with cold phosphate-buffered saline (PBS), and then resuspended in binding buffer. Annexin V-fluorescein isothiocyanate (FITC) and PI were added to the suspension. The suspension was incubated for 15 min in the dark at room temperature, and then the binding buffer was added. Finally, the samples were analyzed by flow cytometry.

BrdU staining

The amount of 5’-bromo-2’-deoxyuridine (BrdU)was measured using the APC-BrdU Flow Kit (Cat Nr.552598, BD Pharmingen™) according to the manufacturer’s instructions. The results were analyzed using the BD FACSDiva software.

Statistical analyses

All experimental data were presented as the mean ± standard deviation (SD). SPSS Statistical analyses were performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Comparisons between two groups were carried out using the Student’s t-test. Differences between the groups were compared by one-way analysis of variance (ANOVA), and Duncan’s method was used as a post-hoctest. Each experiment was performed independently at least in triplicate. Differences were considered statistically significant at P<0.05.

Results

RFT improved the bone damage induced by osteoarthritis

Trabecular bone morphometry was performed to assess the bone damage caused by osteoarthritis. As shown in , osteoarthritis significantly reduced the BV/TV, Tb.Th, and Tb.N values, and increased the BSA/BVand Tb. Sp values compared to the control group. Also, the BV/TV, Tb.Th, and Tb.N in PTO rats treated with 0.5 or 1 µg RFT were increased compared to the PTO model group. Meanwhile, the BSA/BV and Tb.Sp were restored by 0.5 or 1 µg RFT in PTO rats. However, there were no significant differences between the PTO + RFT 0.2 µg group and the PTO model group. Taken together, RFT was found to improve osteoarthritis-induced bone damage in a dose-dependent manner.

Figure 1

Remifentanil (RFT) improved the bone damage induced by osteoarthritis. The statistical analysis of BV/TV (A), BSA/BV (B), Tb.Th (C), Tb. N (D), and Tb. Sp (E). Compared with the control group, *, P<0.05; compared with the post-traumatic osteoarthritis (PTO) model group, #, P<0.05.

RFT enhanced trabecular bone and articular cartilage formation in PTO rats

The results of H&E, Masson, and Alcian staining are shown in . According to these results, severe cartilage loss was observed in the PTO model group; the chondrocytes showed hyperplasia and disorder compared to control group. However, in PTO rats treated with 0.5 and 1 µg RFT, the cartilage loss was restored and the chondrocytes were arranged in neat rows; cartilage structure was clear and with a smooth surface. The contents of trabecular bone () and cartilage () were markedly decreased by osteoarthritis compared to the control group, but were increased by 0.5 and 1 µg RTF compared to the PTO model group. The expression levels of OSX, CoL1A1, and OC were detected by western blot (). In the PTO model group, the expression levels of OSX, CoL1A1, and OC were markedly inhibited compared to the control group. In the PTO + RFT 0.5 µg and PTO + RFT 1 µg groups, the levels of OSX, CoL1A1, and OC were up-regulated compared to the PTO model group. In summary, RFT enhanced trabecular bone and articular cartilage formation in PTO rats in a dose-dependent manner.

Figure 2

Remifentanil (RFT) enhanced trabecular bone and articular cartilage formation in post-traumatic osteoarthritis (PTO) rats. (A) The results of H&E, Masson, and Alcian staining. The relative area of trabecular bone (B) and collagen (C) were analyzed using Image J software. (D) The expressions of osterix (OSX), collagen type I alpha 1 (COL1A1), and osteocalcin (OC) detected by western blot. (E) The protein levels of OSX, CoL1A1, and OC. Compared with the control group, *, P<0.05; compared with the PTO model group, #, P<0.05.

RFT alleviated the degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κB p65in PTO rats

MMPs can degrade almost all cartilage matrix, and thus, they can be used as markers of matrix degradation. The levels of MMP-1, MMP-3, and MMP-13 were detected by western blot (), and were found to be greatly increased in the PTO model group compared to the control group. The levels of MMP-1 and MMP-3 were notably reduced by 0.5 and 1 µg RFT in PTO rats, and the level of MMP-13 was reduced by all doses of RFT. The phosphorylation of PI3K, AKT, and NF-κBp65 was also measured by western blot (). The expression of phosphorylated PI3K (p-PI3K)/PI3K was up-regulated in the PTO model group compared to the control group and was down-regulated by 0.5 and 1 µg RFT compared to the PTO model group. The expressions of phosphorylated AKT (p-AKT)/AKT and phosphorylated p65 (p-p65)/p65 were also decreased by RFT compared to the PTO model group. Immunohistochemical staining was performed to evaluate the level of NF-κBp65 (). NF-κBp65-positive cells dramatically increased in the PTO model group compared to the control group and were reduced in the PTO + RFT 0.5 µg and PTO + RFT 1 µg groups compared to the PTO model group. In short, RFT alleviated the degradation cartilage matrix in PTO rats.

Figure 3

Remifentanil (RFT) alleviated the degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κBp65 in post-traumatic osteoarthritis (PTO) rats. (A) The expressions of MMP-1, MMP-3, and MMP-13 were measured by western blot. (B) The expressions of PI3K, AKT, and NF-κBp65 were measured by western blot. (C) The NF-κBp65-positive cells in cartilage matrix detected by immunohistochemical staining. Compared with the control group, *, P<0.05; compared with the PTO model group, #, P<0.05.

RFT inhibited IL-1β induced apoptosis in rats’ articular chondrocytes

The toxicity of RFT to articular chondrocytes was evaluated by cell viability (). A RFT concentration of 500 ng/mL or higher had a significant impact on cell viability, and so, 50, 100, and 200 ng/mL were chosen in the subsequent experiments. The result of BrdU staining showed that IL-1β reduced BrdU-positive cells compared to the control group, while RFT increased BrdU-positive cells in a dose-dependent manner compared to the IL-1β group (). The statistical analysis of BrdU-positive cells was consistent with the above result (). Apoptosis was assessed by flow cytometry assay (). The cell apoptosis rate was substantially increased by IL-1β compared to the control group, however was significantly reduced by RFT compared to the IL-1β group (). The proliferation-related protein Ki67 and apoptosis-related protein caspase-3 were measured by western blot (). The expression of Ki67 was notably inhibited by IL-1β, however it was considerably increased by RFT. The ratio of cleaved caspase-3 to caspase-3 was promoted by IL-1β and then decreased by RFT. These results indicated that RFT inhibited IL-1β-induced apoptosis in the rats’ articular chondrocytes in a dose-dependent manner.

Figure 4

Remifentanil (RFT) inhibited IL-1β-induced apoptosis in rats’ articular chondrocytes. (A) Toxicity of RFT to cells performed by CCK8. The result of BrdU staining (B) and the statistical analysis of BrdU-positive cells (C). The result of flow cytometry assay (D) and the statistical analysis of cell apoptosis rate (E). (F) The protein levels of Ki67 and caspase-3 detected by western blot. Compared with the control group, *, P<0.05; compared with the IL-1β-induced group, #, P<0.05.

RFT alleviated the IL-1β-induced degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κBp65

The expressions of MMPs and PI3K/AKT/NF-κBp65 are shown in and , respectively. In the rats’ articular chondrocytes, the expressions of MMP-1, MMP-3, and MMP-13 were highly increased by IL-1β compared to the control group, however were reduced by RFT compared to the IL-1β group (). The changes in p-PI3K/PI3K, p-AKT/AKT, and p-p65/p65 were similar to MMP-1, MMP-3, and MMP-13 (). The levels of p-PI3K/PI3K and p-AKT/AKT were increased in IGF-1 group compared with IL-1β group. Importantly, the levels of p-PI3K/PI3K and p-AKT/AKT were increased in IGF-1 + RFT 200 ng/mL group compared with IGF-1 group (). Put simply, RFT alleviated the IL-1β-induced degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κBp65 and nuclear translocation of NF-κBp65.

Figure 5

Remifentanil (RFT) alleviated the IL-1β-induced degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κBp65. (A) The expressions of MMP-1, MMP-3, and MMP-13 were measured by western blot. (B) The expressions of PI3K, AKT, and NF-κBp65 were measured by western blot. (C) The expressions of PI3K and AKT were measured by western blot. Compared with the control group, *, P<0.05; compared with the IL-1β-induced group, #, P<0.05; compared with the RFT 200 ng/mL group, $, P<0.05.

Discussion

PTO is a common condition caused by trauma and is disabling in patients who have sustained certain injuries (3). Since PTO typically arises from an injury to the joint, an opportunity exists for early therapeutic, and even prophylactic intervention (30). In this study, RFT was used as a treatment for PTO in rats. The effects of RFT on PTO and the underlying mechanisms were investigated.

In the past, changes in articular cartilage were considered to be the major pathological changes in OA. However, recent studies have revealed that all joint structures including subchondral and trabecular bones will also be affected (31). The BV/TV value reflects the distribution density of trabecular bone and is an important index used to evaluate trabecular bone microstructure. The BV/TV, Tb.Th, and Tb.N values were increased by RFT, and the BSA/BV and Tb.Sp were decreased by RFT.

PTO reduces the content of cartilage and trabecular bone. OSX is a necessary osteoblast-specific transcription factor for bone formation, and regulates the differentiation and proliferation of osteoblasts (32). It is located at chromosome 12q13.13 near the Sp gene. CoL1A1 is an important marker of osteoblast differentiation; studies have shown that the expression of CoL1A1 is closely related to OSX (33). OC is the main non-collagen protein in bone with a molecular weight of 5.8 kD (34). It is a marker of mature osteoblasts and is thought to regulate bone matrix formation (35). The level of OC can directly represent the activity of osteoblasts. Thus, OSX, CoL1A1, and OC are important factors of trabecular bone and articular cartilage formation. In the present study, the expressions of OSX, CoL1A1, and OC were decreased by PTO but increased by RFT in PTO rats. This result indicates that RFT enhances articular cartilage formation by up-regulating the expression of bone formation-related proteins.

MMP is the major enzyme involved in the degeneration of articular cartilage in OA. Articular cartilage consists of chondrocytes and cartilage matrix, and its degeneration typically involves apoptosis of chondrocytes and degradation of cartilage matrix. The main components of cartilage matrix are aggrecan and type II collagen (36). Many MMPs have been found to be increased in OA (37). In the MMP family, MMP-1, MMP-3, and MMP-13 are considered to be the most important in the degeneration of the articular cartilage (38). MMP-1 is one of the principal proteases capable of degrading the bone matrix (39). MMP-13 degrades aggrecan and preferentially digests type II collagen (40). MMP-3 can digest aggrecan and activate other proMMPs including proMMP-13 (41). The intracellular signaling pathway PI3K/AKT has also been shown to be related to the degradation of cartilage matrix. Several studies have reported that the expression of MMPs could be changed via the PI3K/AKT pathway (42). NF-κB is a downstream protein of the PI3K/AKT pathway and can regulate the secretion of MMP (20), with inhibition of NF-κB reducing the secretion of MMP (43). The p-PI3K and p-AKT are phosphorylated activated forms of PI3K and AKT (44). NF-κBp65 is a subunit of NF-κB, and the expression of p-p65 signifies that NF-κBp65 has been activated (45). In this study, the levels of p-PI3K/ PI3K, p-AKT/AKT, and p-p65/p65 were up-regulated by PTO and down-regulated by RFT. Our finding shows that RFT could alleviate the degradation of cartilage matrix by inhibiting the phosphorylation of PI3K/AKT/NF-κBp65 in PTO rats.

IL-1β is a typical inflammatory cytokine; normal chondrocytes cultured in vitro produce trace amounts of IL-1β. High concentrations of IL-1β stimulate the secretion of MMP, leading to the degradation of cartilage matrix (46). IL-1β can also stimulate chondrocytes to produce nitric oxide, inhibit the proliferation of chondrocytes, and induce the apoptosis of articular chondrocytes (47). Ki67 is a nuclear antigen present in proliferating cells, and is thus an intrinsic marker of cell proliferation (48). Caspase proteins regulate almost all apoptosis-related signaling pathways, in which caspase-3 plays a key role. Cleaved caspase-3 is the active form of caspase-3, and is necessary for many apoptotic nuclear changes (49). So, the increased Ki67 and decreased cleaved caspase-3/caspase-3 indicates enhanced cell proliferation and reduced apoptosis. In this study, the results of BrdU staining and flow cytometry assay revealed greater cell proliferation and decreased apoptosis following treatment with RFT. Taken together, RFT enhances cell proliferation and inhibitsIL-1β-induced apoptosis in articular chondrocytes.

The degradation of cartilage matrix in vitro was also assessed. Changes in the expression of MMP-1, MMP-3, MMP-13, p-PI3K/PI3K, p-AKT/AKT, and p-p65/p65 were consistent with those in PTO rats. NF-κB is a transcription factor widely found in the cytoplasm that remains inactive when cells are motionless (50). The nuclear translocation of NF-κBp65 is an important indicator of NF-κB signaling pathway activation (51), and confirms that gene expression of many cytokines involved in the inflammatory response process of OA were activated by the NF-κB signaling pathway. Once activated, NF-κBp65 mediates various inflammatory factors and enzymes that lead to apoptosis and dysfunction of chondrocytes (52). In the research, the expressions of MMP-1, MMP-3, MMP-13, p-PI3K/PI3K, p-AKT/AKT, and p-p65/p65 were highly increased by IL-1β compared to the control group, however were reduced by RFT compared to the IL-1β group.

Hyperglycemia-induced oxidative stress abrogates remifentanil preconditioning (RPC) cardioprotection by impairing Cav-3-modulated PI3K/Akt and JAK2/STAT3 signaling. Antioxidant treatment with N-acetylcysteine (NAC) restores RPC-induced cardioprotection in diabetes by improving Cav-3-dependent Akt and STAT3 activation and by facilitating the cross talk between PI3K/Akt and JAK2/STAT3 signaling pathways (53). RTF protects adult rat cardiomyocytes from hypoxia-reoxygenation injury and its effects appears to be dependent on the δ-opioid receptor mediated activation of PI3K/Akt and subsequent extracellular signal-regulated kinase (ERK) signaling pathways (54). RTF postconditioning improves global cerebral ischemia-induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway (55). RTF attenuates lipopolysaccharide-induced acute lung injury by downregulating the NF-κB signaling pathway (56). RTF reduces LPS-induced inflammatory response through PARP-1/NF-κB signaling pathway in human aortic endothelial cells (HAECs) (57). In this research, RFT alleviated PTO injury by inhibiting the phosphorylation of PI3K/AKT/NF-κB p65 in vivo and in vitro.

In the present study, we investigated the effects of RFT on cartilage damage and degradation of cartilage matrix in PTO model rats. The in vitro effects of RFT on IL-1β-induced apoptosis and degradation of cartilage matrix were also demonstrated. RFT was found to repair cartilage damage and the reduce degradation of cartilage matrix in PTO in rats, and inhibit apoptosis in IL-1β-induced rats’ articular chondrocytes via inhibition of PI3K/AKT/NF-κB phosphorylation. This study provides a promising approach for the treatment of PTO. However, more studies in this area are needed to further investigate the protective effects of RFT in clinical application.

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