LIU Di,WU Yongli,LI Chun,WANG Minglei,MA Xiaoxiu,LIU Junwei,ZHANG Yanling,YANG Lei

LIU Di,WU Yongli,LI Chun,MA Xiaoxiu,ZHANG Yanling,YANG Lei,Traditional Chinese Medicine and Traumatology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;

WANG Minglei,Radiology Department,General Hospital of Ningxia Medical University,Yinchuan 750004,China;

LIU Junwei,Department of Acu-moxibustion and Tuina,Ningxia Medical University,Yinchuan 750004,China

Abstract OBJECTIVE:To explore the molecular mechanism of warming moxibustion(WM)in knee osteoarthritis(KOA).METHODS:The knee joints of 40 New Zealand rabbits were placed in a plaster cast in an extended position to establish a KOA model.The animals were randomly divided into four groups:the control group,model group,WM group,and diclofenac(DF) group.Hematoxylin-eosin staining and the modified Mankin score were applied to evaluate the histopathological changes.Chondrocyte apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.Western blotting and real-time quantitative polymerase chain reaction were performed to measure the expression of interleukin-1β(IL-1β),prostaglandin E receptor 3 (PTGER3),a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5(ADAMTS-5),matrix metalloproteinase-13 (MMP-13),and C-terminal telopeptides of collagen type Ⅱ(CTX-Ⅱ)in cartilage tissues of the different groups.The concentrations of IL-1β,PTGER3,and CTX-Ⅱin serum were detected by the enzyme-linked immunosorbent assay.RESULTS:Rabbits with KOA in the WM and DF groups showed significantly reduced cartilage erosion and Mankin scores,compared with the untreated rabbits.The number of TUNEL-positive cells observed in the WM group was much fewer than that in the model group.The expression of PTGER3,MMP-13,CTX-Ⅱ,IL-1β,and ADAMTS-5 in cartilage tissues was remarkably downregulated following therapy with WM and DF.Moreover,a marked reduction was observed in the serum levels of IL-1β,PTGER3,and CTX-Ⅱin the WM and DF groups.CONCLUSION:WM exerts favorable therapeutic effects on articular injuries of KOA by regulating the expression of inflammatory and cartilage degradation-related cytokines.

Keywords:warming moxibustion;osteoarthritis,knee;inflammation;cartilage degradation

INTRODUCTION

Moxibustion,a traditional Chinese therapy,delivers thermal stimulation to the human body surface via burning moxa,exerts its effects on acupoints and meridians,and could prevent and alleviate some diseases.4Over the past few years,moxibustion has been adopted in many Asian countries as an efficient approach to relieve pain,and has been gaining popularity in current clinical practice because of its advantages,such as its effectiveness,safety,and absence of side effects.5,6Moxibustion can be classified into scarring moxibustion,warming moxibustion(WM),and herb-partition moxibustion.Among the various types,WM is considered the most practical and convenient,from the perspective of clinical application.The process of WM is as follows:a moxa stick is ignited and then attached to a needle.The needle is immediately penetrated into specific sites on the body surface to stimulate specific acupuncture points.Increasing evidence has shown that WM exerts favorable clinical effects on painful conditions caused by sciatica,arthritis,intervertebral disk herniation,cervical spondylopathy,and osteoarthritis.7-9However,the underlying molecular mechanism by which WM affects KOA remains unclear.

Inflammation plays an essential role in the pathogenesis of KOA.10Generally,some inflammatory factors are overexpressed in the cartilage and synovial fluid of patients with KOA,and these inflammatory cytokines may include interleukin-1 (IL-1),IL-6,and tumor necrosis factor-alpha (TNF-α),among others.11Furthermore,increased levels of inflammatory factors may also lead to a highly catabolic state and chondrocyte apoptosis,contribute to the release of matrix metalloproteinases (MMPs),and may eventually result in progressive cartilage degradation.12Therefore,we speculate that the effect of WM on KOA may be associated with the regulation of inflammatory-related genes and the expression of cartilage catabolic-related genes.

In the present study,we first established a KOA model by placing the extended knee joint of rabbits in a plaster cast.We then investigated the effects of WM on the damaged cartilage tissues and chondrocyte apoptosis induced by KOA.We also compared the expression of inflammatory biomarkers interleukin-1β (IL-1β) and prostaglandin E receptor 3 (PTGER3),as well as cartilage catabolic biomarkers a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5 (ADAMTS-5),matrix metalloproteinase-13 (MMP-13),and C-terminal telopeptides of collagen type Ⅱ(CTX-Ⅱ) in experimental rabbit knee osteoarthritis under different treatments.Although the expression levels of various genes have been explored in patients with KOA,few studies have investigated the effects of WM on their expressions in a KOA model.This study may lay the theoretical foundation for the wider application of WM in the treatment of KOA.

METHODS

Animals and grouping

Forty healthy New Zealand rabbits (20 male and 20 female rabbits),6 months of age,weighing (2.5 ±3.0) kg,were obtained from the General Hospital of Ningxia Medical University and were used for the establishment of the KOA model.Before formal experiments,all rabbits were kept in plastic cages with a 12-h light-dark cycle at 25 ℃and allowed to acclimatize for 3 d.A plaster cast was applied to the extended left knee joint to create the model of KOA.Thus,the rabbit model of KOA was established according to our previous report.13Successful establishment of rabbit KOA model was determined,based on the results of hematoxylin-eosin(HE)staining and the Mankin score.

Three days later,the animals were randomly divided into four groups (10 in each group):the WM group,diclofenac (DF) group,model group,and control group.Different treatments were administered to the rabbits.The WM group of rabbits were subjected to WM for 15 min each day.The specific procedure was as follows:disposable acupuncture needles were inserted vertically into a depth of about 0.3 cm at the knee acupoints.After routine disinfection,the moxa was cut into strips,0.1 cm in length.The moxa was then ignited from the bottom and the needle was left in place until the moxa burned out.The DF group of rabbits received oral daily doses of diclofenac (15 mg/kg) once daily.The model group of rabbits were placed within strainer for 15 min per day.The control group of rabbits were not subjected to any treatment.

All rabbits were subjected to the respective group protocol,daily for 2 weeks.After 14 days of the various treatments,all rabbits were anesthetized by an intraperitoneal injection of 10% chloral hydrate at a dose of 250 mg/kg,combined with 20 mg/kg CI-634 (tiletamine hydrochloride),and sacrificed by air embolism according to previously described methods with minor modifications.14,15To ensure the animals were fully anesthetized but still alive after being injected with chloral hydrate,we measured the breathing rate(breaths/min),heart rate (beats/min),body temperature (°F),and general condition (breathing,heart rate,and reflexes)of the rabbits pre-anesthesia,post-anesthesia,and on awakening.The corresponding results are presented in Table 1.Cartilage tissues were collected and stored at－80 ℃for subsequent assay.The study was approved by the Ethics Committee of the General Hospital of Ningxia Medical University.

Histopathological studies

Knee samples were washed three times with normal saline,and then fixed in 10%buffered formalin for 24 h.After the samples were decalcified with 10% aqueous formic acid,they were rinsed,dehydrated,embedded in paraffin,and sliced into 4 μm sections.The samples were then stained with HE for microscopic observation.Examination of the histopathological changes were conducted on an Olympus BX-50 light microscope,and evaluated according to the modified Mankin score.16

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Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay

Detection of apoptotic cells was achieved with the TUNEL assay,according to the manufacturer's instructions.After being dehydrated,the knee samples were sliced into 10-μm thick sections.The sections were then fixed in 4% paraformaldehyde for 20 min,washed with phosphate-buffered saline for 30 min,and immersed in 0.1%sodium citrate buffer for 2 min.After the sections were dried,the TUNEL assay was performed using an in situ apoptosis detection kit (Roche Diagnostic,Mannheim,Germany) according to the manufacturer's instructions.The apoptotic cartilage cells were observed using a fluorescent microscope(Leica DM4000B;Leica Microsystems,Wetzlar,Germany).

RNA extraction and real-time quantitative polymerase chain reaction(RT-qPCR)

Total RNA from the cartilage tissues of the rabbits was extracted using an RNeasy Mini Kit (Qiagen,Hilden,Germany) according to the manufacturer's instructions.The purity and concentration of extracted RNA were determined by the NanoDrop ?ND-1000 spectrophotometer (Thermo Scientific,Waltham,MA,USA).The corresponding primers,including PTGER3,MMP-13,IL-1β,and ADAMTS-5 were purchased from Shanghai Sangon Biotech Co.,Ltd.The cDNA synthesis was performed using a cDNA synthesis kit (TaKaRa Biotechnology,Dalian,China).The cDNA functioned as a template for lincRNA RT-qPCR.The primers are presented in Table 2.The PCR amplification was conducted at 50 ℃(20 min);95 ℃(10 min);40 cycles at 95 ℃(15 s);and 60 ℃(60 s).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH,Bio-Rad,Hercules,CA,USA) was used as an internal control.The relative mRNA levels of target genes were calculated using the 2－ΔΔCtmethod.

Table 1 Physiological parameters of rabbits on pre-anesthesia,post-anesthesia,and awakening stages

Table 2 List of primers for real-time quantitative polymerase chain reaction

Western blot analysis

The protein expression levels of PTGER3,MMP-13,and CTX-Ⅱwere assessed by western blot analysis.Briefly,the frozen cartilage tissues were thawed,washed,weighed,and lysed in radioimmunoprecipitation assay buffer.Total protein was extracted using a Total Protein Sample Kit (Sigma,St Louis,MO,USA)according to the manufacturer's instructions.The bicinchoninic acid method was used to determine the concentration of extracted protein.Subsequently,proteins were resolved by 8%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride transmembrane (Millipore,Bedford,MA,USA).The membrane was blocked with 5%non-fat milk for 1 h and then treated with monoclonal antibodies raised against PTGER3(1∶1000),MMP-13 (1∶1000),CTX-Ⅱ(1∶1000),and GAPDH (1∶2000) in TBST (Tris-buffered saline and Tween-20)at 4 ℃overnight.The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG(1∶1000)at room temperature for 1 h.The signal bands were detected by an enhanced chemiluminescence system (Amersham Biosciences,Piscataway,NJ,USA).

Enzyme-linked immunosorbent assay(ELISA)

Serum levels of inflammatory biomarkers (IL-1β and PTGER3) and cartilage catabolic biomarker (CTX-Ⅱ)were measured by the ELISA.First,the animals were killed and the trunk blood was immediately collected and centrifuged.The levels of IL-1β,PTGER3,and CTX-Ⅱin the supernatants were measured according to a previous report.17The absorbance (optical density)at 450 nm was recorded by a microplate reader(Bio-Rad,Hercules,CA,USA).The concentrations of IL-1β,PTGER3,and CTX-Ⅱwere calculated based on the standard curve.

Statistical analysis

Statistical analysis was performed,using the SPSS 20.0 software (IBM;Chicago,IL,USA).All data were presented as the mean±standard deviation(±s).Comparisons among groups were analyzed using the Student'st-test or Tukey post hoc test,following one-way analysis of variance.Values ofP<0.05 were considered statistically significant.

RESULTS

Warming moxibustion attenuates cartilage damage in rabbits with KOA

To investigate the impact of WM on the damaged cartilage tissues of the experimental rabbits with KOA,H&E staining was performed and the results were evaluated according to the Mankin score.In the normal control group,we observed a glazed cartilage surface with an uneven distribution of cartilage cells (Figure 1A).In the model group,the cartilage tissues showed a chaotic structure of the cartilage layers,irregular aggregation of cartilage cells,some disintegrated necrotic cells,and a partially broken tide line.These changes suggested successful establishment of the rabbit KOA model(Figure 1A).

In the WM and DF groups,minimal degenerative changes were evident.A low number of chondrocytes and a few lymphocytes infiltrated the cartilage.A structured hierarchy of cartilage cells was observed,without signs of aggregation,and a smooth,regular cartilage surface was evident without necrotic cells (Figure 1A).Overall,the degree of cartilage damage was markedly reduced in both the WM and DF-treated groups,as demonstrated by its depth and extent.

According to the evaluation of modified Mankin scores,a significant difference emerged between the control and model groups (aP<0.001,Figure 1B),further indirectly suggesting that the experimental rabbit KOA model was successfully established.The corresponding scores in the WM and DF groups were much lower than those in the model group(bP<0.05,Figure 1B),but no significant difference was observed between the WM and DF groups.Collectively,these results reveal the similar positive effects of WM on the damaged cartilage tissues of rabbits with KOA,compared with DF.

Warming moxibustion reduces chondrocyte apoptosis in rabbits with KOA

Chondrocyte death by apoptosis is considered a major characteristic of cartilage damage caused by osteoarthritis (OA).18Therefore,we performed the TUNEL assay to explore the effects of WM on chondrocyte apoptosis.The results showed that the number of TUNEL-positive cells in the superficial layer of articular cartilage in the model group was significantly greater than that in the control group;however,only a few TUNEL-positive cells were observed in the rabbits treated with WM.The apoptotic cells clearly visible in the WM group were much fewer than those in the DF group(Figure 2).These phenomena were partly consistent with the presentations on HE staining.These findings demonstrate that WM can effectively block cartilage injury-induced apoptosis in experimental rabbit KOA.

Figure 1 Histomorphology of cartilage tissues in the knee joints of rabbits with KOA

Figure 2 TUNEL assay for chondrocyte apoptosis in the knee joint of rabbits with KOA in the control,model,WM,and DF groups(×200)

Warming moxibustion decreases PTGER3,MMP-13,and CTX-II protein levels in rabbits with KOA

To explore the impact of WM on inflammation and cartilage degradation in experimental rabbit KOA,the protein expression levels of PTGER3 (inflammatory biomarker),MMP-13 (cartilage catabolic biomarker),and CTX-Ⅱ(cartilage catabolic biomarker) were measured by Western blotting.The corresponding results are presented in Figure 3A.The protein expressions of these three biomarkers of cartilage damage were significantly increased in the model group compared with the control group (aP<0.001,Figure 3B),indicating that KOA is associated with a severe inflammatory response and cartilage degradation.

Figure 3 Protein levels of PTGER3,MMP-13,and CTX-Ⅱin cartilage tissues of the knee joint of rabbits with KOA

After the administration of WM or DF,the high levels of these cartilage damage cytokines were significantly reversed (bP<0.05),demonstrating the positive effects of WM on inflammation and cartilage degradation induced by KOA.No significant differences were observed between the WM and DF groups based in the levels of PTGER3,MMP-13,and CTX-Ⅱ.Taken together,these findings revealed that WM could reduce the protein expression of PTGER3,MMP-13,and CTX-Ⅱin cartilage tissues.

Warming moxibustion reduces IL-1β,PTGER3,ADAMTS-5,and MMP-13 mRNA levels in rabbits with KOA

The RT-qPCR was performed to determine the mRNA levels of inflammatory biomarkers (IL-1β and PTGER3) and cartilage catabolic biomarkers (ADAMTS-5 and MMP-13).In the normal control group,weak expression of IL-1β,PTGER3,ADAMTS-5,and MMP-13 was observed in cartilage tissues;however,these negative signs of cartilage become more severe in the model group (P<0.001,<0.01).After WM treatment,the mRNA expression levels of IL-1β,PTGER3,ADAMTS-5,and MMP-13 were weaker,compared with the model group (P<0.01,<0.05),which further confirmed the protective action of WM on cartilage damage induced by KOA.Compared with the WM group,no significant differences were noted in PTGER3,ADAMTS-5,and MMP-13 mRNA levels,unlike the IL-1β mRNA levels (P<0.05,Table 3).These results support the theory that WM can reduce the expression of IL-1β,PTGER3,ADAMTS-5,and MMP-13 mRNA levels in cartilage tissues.

Warming moxibustion reduces the serum levels of IL-1β,PTGER3,and CTX-II in rabbits with KOA

Based on our previous results,we knew that WM could lead to a significant reduction in the levels of inflammatory factors and expression of cartilage catabolic-related genes at both the protein and mRNA levels in cartilage tissues.However,we did not know whether this effect of WM on these negative signs of cartilage damage also existed in the serum.Thus,ELISA was performed to measure the serum concentrations of IL-1β,PTGER3,and CTX-Ⅱ.As shown in Table 4,the concentrations of IL-1β,PTGER3,and CTX-Ⅱin the control group were (156 ± 7),(246 ± 10),and(346±10)pg/mL,respectively,which reflected increases of about 0.5-fold,1.3-fold,and 0.9-fold in the model group,with some significant differences (P<0.001,<0.01).In the WM group,the high expression levels of IL-1β,PTGER3,and CTX-Ⅱwere significantly reduced,compared with the model group (P<0.01,<0.05).Moreover,a significant difference was noted between the WM group and the DF group inthe expression of PTGER3 and CTX-Ⅱ(P<0.05,Table 4).Based on these results,we concluded that WM can reduce the serum levels of IL-1β,PTGER3,and CTX-Ⅱ.

Table 3 mRNA levels of IL-1β,PTGER3,ADAMTS-5,and MMP-13 in cartilage tissues of the knee joint of rabbits with KOA(±s)

Table 3 mRNA levels of IL-1β,PTGER3,ADAMTS-5,and MMP-13 in cartilage tissues of the knee joint of rabbits with KOA(±s)

Notes:control group:rabbits with KOA were not subjected to any treatment;model group:rabbits with KOA were placed within strainer for 15 min per day;WM group:rabbits with KOA were subjected to WM for 15 min each day;DF group:rabbits with KOA received oral doses of diclofenac (15 mg/kg) once daily.WM:warming moxibustion;DF:diclofenac;KOA:knee osteoarthritis;IL-1β:interleukin-1β;PTGER3:prostaglandin E receptor 3;ADAMTS-5:a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5;MMP-13:matrix metalloproteinase-13.Compared with the control group,aP <0.001,bP <0.01;compared with the model group,cP <0.01,dP<0.05;compared with the WM group,eP<0.05.

Table 4 Concentration of IL-1β,PTGER3,and CTX-Ⅱin the serum of knee joint of rabbits with KOA(±s)

Table 4 Concentration of IL-1β,PTGER3,and CTX-Ⅱin the serum of knee joint of rabbits with KOA(±s)

Notes:control group:rabbits with KOA were not subjected to any treatment;model group:rabbits with KOA were placed within strainer for 15 min per day;WM group:rabbits with KOA were subjected to WM for 15 min each day;DF group:rabbits with KOA received oral doses of diclofenac (15 mg/kg) once daily.WM:warming moxibustion;DF:diclofenac;KOA:knee osteoarthritis;IL-1β:interleukin-1β;PTGER3:prostaglandin E receptor 3;CTX-Ⅱ:C-terminal telopeptides of collagen type Ⅱ.Compared with the control group,aP<0.01,bP<0.001;compared with the model group,cP<0.05,dP<0.01;compared with the WM group,eP<0.05.

DISCUSSION

Moxibustion is a popular traditional therapy for the treatment of body aches.It is practiced in many Asian countries,4 and typically assumes various forms,including scarring moxibustion,WM,and herb-partition moxibustion.Warming moxibustion is the most practical and convenient method,which has been widely applied to the treat painful conditions induced by various stimulating factors and has shown favorable therapeutic effects.7-9As a common chronic condition,KOA is characterized by its pathological features of insufficient synthesis of the extracellular matrix,as well as biochemical and structural changes of the cartilage and chondrocytes.19Previous studies have confirmed the ability of WM to relieve the symptoms of KOA;8,20however,the molecular mechanism of WM on treatment of KOA remains unclear.In this study,a series of experiments were conducted to preliminarily determine the potential mechanism of WM in KOA.

We first established the experimental rabbit KOA model according to our previous study,13and then administered various treatments.Based on the results of HE staining and the Mankin score,we were able to preliminarily confirm successful establishment of the rabbits KOA model.Compared with the model group,the WM and DF groups showed a smoother,more regular cartilage surface,as well as a more intact cell structure.Moreover,no significant difference was noted between the WM and DF groups in the modified Mankin score,which revealed the similar protective effects of WM to DF on cartilage erosion induced by KOA.

Inflammation is a major inducer of KOA.10As a common negative factor,the inflammatory response can trigger the apoptotic cascade by interrupting intercellular interactions.21Therefore,apoptosis is one of the major,intrinsic mechanisms of OA.The TUNEL assay showed that WM induced a significant reduction in the number of TUNEL-positive cells in the superficial layer of articular cartilage,which was a positive effect of WM on chondrocyte apoptosis that was even superior to the effects of DF.A previous study on patients with KOA has confirmed the anti-inflammatory efficacy of DF,which is widely used in the clinical treatment of KOA.22Based on these findings,the favorable effects of WM on KOA were confirmed,and shown to be no worse than those of DF(Western Medicine).

One prior randomized controlled study revealed that warm needling moxibustion could effectively relieve pain and stiffness,and improve joint function in patients with KOA,with a total effective rate of 88.0%.23Similarly,another randomized controlled study reported a total effective rate of 86.7% in the warming needle moxibustion group of patients with KOA,which was very close to the corresponding value in the western medicine group.24These findings suggest that warm acupuncture is suitable for most,but not all patients with KOA.

Inflammation is considered an important factor that induces the onset of KOA,10 and the abnormal expression of some inflammatory factors has been observed in the cartilage and synovial fluid of patients with KOA.11Furthermore,the structural changes of cartilage tissues in KOA are associated with varying degrees of articular cartilage degradation that is a major characteristic of OA.These alterations are the result of the attack on matrix molecules by inflammatory factors,which then contributes to their release and loss,and results in progressive cartilage degradation.12The importance of inflammation in initiating and inhibiting cartilage degradation in OA has been confirmed by previous studies.25,26Therefore,exploring the molecular mechanisms of WM in the treatment of KOA is necessary to determine the expression of some common inflammatory and cartilage catabolic biomarkers.

Both IL-1β and PTGER3 are inflammatory regulators,and can be indicators of OA.The IL-1β cytokine plays an important role in the process of KOA cartilage degradation induced by inflammation.It can act independently or together with other cytokines to initiate and produce arthritis.In a previous study,the expression level of IL-1β was increased about five-fold in human chondrocytes when induced with the proinflammatory cytokine-TNF-α.27Furthermore,PTGER3 can block matrix synthesis,promote cartilage degradation,and accelerate chondrocyte apoptosis;12therefore,the inhibition of PTGER3 generation is generally associated with relief of OA-related aches and the inflammatory response.28In the present study,the WM and DF groups showed a significant reduction in IL-1β and PTGER3 levels in both cartilage tissues and the serum,as compared with the model group.These findings indicate the favorable anti-inflammatory effects of WM in experimental rabbit KOA.

As common cartilage catabolic biochemical markers,MMP-13,CTX-Ⅱ,and ADAMTS-5 were also evaluated in the present study.As a critical collagenase in the pathogenesis of osteoarthritis,MMP-13 can directly degrade collagen-Ⅱ,which is the most abundant collagen component in the cartilage matrix.29In addition,CTX-Ⅱis a specific biomarker of collagen typeⅡ breakdown and articular cartilage deterioration,which is closely associated with the severity of knee cartilage defects in patients with OA.30Moreover,ADAMTS-5 can induce inflammation and cartilage destruction by facilitating the release of cyclooxygenase 2,prostaglandin E2(encoded by PTGER3),and nitric oxide,and cause increased loss of cartilage at the diarthrodial joint by promoting fibrillation of the cartilage surface.31Further investigations have also revealed a significant downregulation of MMP-13,CTX-Ⅱ,and ADAMTS-5 expression following therapy with WM and DF.Nonetheless,no significant difference was found between the WM and DF groups,indicating that WM may have similar effects to DF in attenuating cartilage degradation induced by KOA.

This study mainly explored the potential molecular mechanisms underlying the effects of warm acupuncture on joint inflammatory responses and articular cartilage degeneration in rabbits with KOA.Our findings confirm that WM exerts favorable therapeutic effects on KOA,by regulating the expression of inflammatory and cartilage degradation-related cytokines.However,the effects of warm acupuncture on chondrocyte apoptosis and inflammation in the rabbit KOA model may involve complex internal molecular mechanisms,such as peptide release,the regulation of signaling pathways,and neurotransmitter delivery,among others.Therefore,more concrete evidence is required from future investigations.

In conclusion,collectively,the present study supports the theory that WM could effectively attenuate articular cartilage injury and chondrocyte apoptosis induced by KOA.These positive effects of WM in the treatment of KOA may be achieved by regulating the expression of inflammatory and cartilage degradation-related cytokines,such as IL-1β,PTGER3,ADAMTS-5,MMP-13,and CTX-Ⅱ.The present study provides a theoretical basis for the application of WM in the treatment of KOA at the molecular level.