The Mechanism Of Resistin On Knee Osteoarthritis Is Regulated Through Its Receptor CAP1 And Activation Of The P38-MAPK/NF-?B Signaling Pathways

Knee osteoarthritis(KOA)is the most common chronic disabling disease in orthopaedics department characterized mainly by degeneration of articular cartilage.The mechanical wear and aging have long been considered to be the risk of KOA.However,new researches recently have found that the underlying mechanism is not just an increase of mechanical stress,but also a low-grade inflammation involving some of adipokines it secretes.Resistin,a 12-k Da cysteine-rich polypeptide hormone protein which is made up of 108 amino acids,is closely related to KOA.Many studies have shown that resistin levels of serum and synovial fluid of local knee joint are significantly higher in patients with KOA than healthy controls.Especially,the resistin level of local synovial fluid in knee joint is closely related to the severity of KOA symptoms.In addition,human chondrocyte experiments in vitro and animal experiments in vivo have confirmed that resistin can promote chondrocyte to release proinflammatory chemokines and matrix-degrading enzymes which take part in pathological process of KOA.According to previous studies,resistin induced inflammatory response through the binding of CAP1 receptors.Besides,CAP1 was identified as a bona fide functional receptor in human,but the exact mechanisms of CAP1 in resistin-induced pathological process of KOA still need to be proved.Activation of p38-MAPK and NF-?B signaling pathways has been shown to significantly promote inflammatory responses and accelerate pathological processes.However,it is unclear whether resistin promotes the release of inflammatory chemokines and matrix-degrading enzymes by chondrocytes through p38-MAPK and NF-?B signaling pathways to induce KOA pathological processes.Hence,the aim of our study is to explore mechanism of resistin in the induction of KOA pathological process and to further update the pathogenesis of KOA,offering a new theoretical basis for prevention of KOA.In our study,we firstly tested the resistin level of serum and synovial fluid in patients with KOA patients and resistin level of serum in healthy controls,and the results confirmed that resistin was higher expression in KOA patients.We furtherly explored the change of CAP1 expression in human KOA cartilage.In vitro chondrocytes experiments,we investigated whether resistin binding to CAP1 receptor,and knockout of CAP1 with adenovirus transfection was used to furtherly verify the role of CAP1 in resistin-induced productions of inflammatory chemokines and matrix-degrading enzymes by chondrocyte.Finally,the role of p38-MAPK and NF-k B signaling pathways were investigated in resistin-CAP1 axis in vitro chondrocytes experiments.This study consists of four parts:1)Expression and clinical correlation analysis of resistin levels of serum and synovial fluid between KOA patients and healthy controls;2)CAP1 expression characteristics in KOA cartilage and its correlation with expression of resistin;3)Stimulatory effects of resistin on KOA chondrocytes and the role of CAP1 in this process;4)The role of p38-MAPK and NF-?B signaling pathways in the productions of inflammatory chemokines and matrix-degrading enzymes induced by Resistin-CAP1 in chondrocytes.Part I: Expression and clinical correlation of resistin levels of serum and synovial fluid between KOA patients and healthy controls.Objective: To detect the expression and clinical correlation of resistin levels of serum and synovial fluid between KOA patients and healthy controlsMethods: Subjects enrolled in this study were included from May 2017 to June 2018.103 patients undergoing primary total knee arthroplasty(TKA)due to KOA were enrolled at Bone and Joint department at our hospital and 86 subjects without KOA symptoms for annual health check-up were enrolled in the health examination center at our hospital.Clinical data,serum samples and synovial fluid samples were collected from KOA patients and healthy control subjects.ELISA assay was used to detect resistin expressions of serum and synovial fluid between the two groups.Clinical characteristics,resistin levels of serum and synovial fluid were compared between two groups,and the correlations with clinical data were also analyzed between the two groups.Results:(1)Compared with the healthy control(Healthy Control,HC)group,KOA group patients were older(KOA vs.HC: 63.67 vs.51.36,p<0.0001),and had greater BMI(KOA vs.HC: 26.42 vs.23.47,p<0.0001)and higher fasting blood glucose(FBG)(KOA vs.HC: 5.54 vs.5.03,p<0.0001).There was no significant difference in gender difference,triglyceride and HDL levels between the two groups.(2)Higher serum resistin level was found in KOA patients group compared with HC group(KOA vs.HC: 8.26 vs.7.45,ng/ml),however,the difference disappeared after adjusted by age,BMI,FBG,and triglyceride(padj=0.688).Resistin level was significantly higher in serum than in synovial fluid in KOA patients(Serum vs.SF: 8.26 vs.3.26,ng/ml,p<0.0001).(3)For KOA patients,the number of patients with Kellgren-Lawrence(K-L)grade II,III,and IV was 18,38,and 47,respectively.The level of serum resistin in patients with K-L grade IV was significantly higher than that in stage II(K-L ? vs.?: 8.97 vs.7.11,ng/ml,p=0.021).In KOA group patients,synovial fluid resistin levels of grade ? were higher than grade ? and grade ?(K-L ? vs.?: 3.85 vs.2.60,ng/ml,p<0.0001;K-L ? vs.?: 3.85 vs.2.86,ng/ml,p<0.0001).Conclusions: Higher serum resistin level was detected in KOA patients,and expression of resistin in local knee synovial fluid was positively correlated with KOA severity.It showed that resistin level was obviously correlated with KOA.Part II: CAP1 expression characteristics in KOA cartilage and its correlation with expression of resistinObjective: To investigate the expression characteristics of CAP1 in KOA cartilage and its correlation with expression of resistin.Methods: 21 cartilage samples of KOA patients and 10 cartilage samples of fracture of neck of femur patients were collected.Firstly,m RNA and protein expression of CAP1 in cartilage tissues of the two groups were detected by RT-PCR and Western blot assays.Then,immunohistochemical technique was used to detect expressions of CAP1 and resistin in two groups.ASI digital systems(powered by Gen ASIs?)software was used to conduct H-Score for immunohistochemical results in two groups and analyzed their correlation.Results: 21 cases KOA patients(F/M,15/6)and 10 cases fracture of neck of femur patients(F/M,7/3)were enrolled,and patients with femoral neck fracture were older(KOA vs FNF:63.14 vs 76.40,p<0.0001).Compared with cartilage tissues from fracture of the femoral neck,for KOA cartilage tissues,the RT-PCR results showed that m RNA expression of CAP1 was significantly increased(KOA vs.FNF: 2.93 vs.1.03,p<0.0001),and Western blot assays showed that protein expression of CAP1 was also significantly increased(KOA vs.FNF: 2.46 vs.1.00,p=0.010).Immunohistochemical H-Score indicated that CAP1 expression(KOA vs FNF:168.90 vs 66.32,p<0.0001)and resistin expression(KOA vs FNF:152.40 vs 67.45,p<0.0001)were significantly increased,and CAP1 and resistin expression had obviously positive correlation(r=0.631,p=0.002).Conclusion: Expression of CAP1 was upregulated in KOA cartilage tissues and had a positive correlation with resistin expression.Part III: Stimulatory effects of resistin on KOA chondrocytes and the role of CAP1 in this process.Objective: To study stimulatory effects of resistin on KOA chondrocytes and the role of CAP1 in this process.Methods:(1)Nine patients with KOA were selected randomly and divided into 3 groups. Chondrocytes were isolated and cultured from the generally normal KOA cartilage tissues.Chondrocytes were cultured by combination of 3 patients for each group.Human chondrocytes and human recombinant resistin were cultured at concentrations of 0 ng/ml,250 ng/ml,500 ng/ml and 1000 ng/ml for 24 h,48 h,and 72 h,respectively.Compared with chondrocytes cultured without resistin,CCK-8 assay was used to detect the toxic effect of resistin on chondrocyte,and RT-PCR was used to detect the effect of resistin concentration and incubation time on CAP1 expression.(2)Chondrocytes were incubated with human recombinant resistin at dose of 500 ng/ml at a time-dependent manner(24h,48 h,72h).For dose-response effects,human chondrocytes were incubated with human recombinant resistin(0,250,500 or 1000 ng/ml)at cell incubator for 48 h.Adding no resistin was selected as control group.Expressions of CCL3,CCL4,MMP-13,and ADAMTS-4 were tested using RT-PCR after stimulation by resistin.(3)KOA chondrocytes were cultured with human recombinant resistin protein(500 ng/ml)for 48 h,and co-immunoprecipitation was used to detect whether CAP1 protein directly binds to resistin in human chondrocytes.(4)The recombinant adenovirus vector was transfected into human chondrocytes to knockdown expression of CAP1.The chondrocytes transfecting with recombinant adenovirus vector were divided into control group(Control-sh RNA1)and CAP1 knockout group(CAP1-sh RNA).RT-PCR and Western blot were performed to detect expression of CAP1 in chondrocytes after knockdown of CAP1.Two groups of chondrocytes were treated with resistin(500ng/ml)for 48 h and 72 h respectively.The m RNA expressions of CCL3,CCL4,MMP-13,and ADAMTS-4 were detected by RT-PCR,and ELISA assays were conducted to test secreted protein levels of CCL3,CCL4,MMP-13 and ADAMTS-4 in supernatant of chondrocytes.Results:(1)Compared with the control group incubating without resistin,the results of CCK-8 assay showed that no significant inhibition of chondrocyte growth was observed at any concentration of resistin within 24 h;only a significant inhibition on the growth of chondrocytes was found after incubation with resistin at dose of 1000 ng/ml for 48 h(p<0.01)and 72 h(p<0.01).(2)After resistin stimulated KOA chondrocytes at time-dependent manner,the results showed that expressions of CCL3 and MMP13 upregulated gradually and reached peak at 48 h with declining after the remaining time course.Expressions of CCL4 and ADAMTS-4 rapidly increased to peak level at 24 h with gradually declining after that.The results of stimulatory effects of resistin on chondrocytes at a dose-response manner indicated that m RNA expression of CCL3 increased gradually with the increase of concentration of resistin,but increased slowly after resistin was higher than 500ng/ml.The m RNA expression of CCL4 and ADAMTS-4 upregulated as the concentration of resistin increased.The m RNA expression of MMP-13 peaked at 500ng/ml and then decreased.(3)Results of Co-immunoprecipitation proved that CAP1 can directly bind to exogenous resistin in chondrocytes to exert a role of receptor for resistin.(4)RT-PCR and Western blot showed that CAP1 expression was silenced more than 90% in chondrocytes after chondrocytes were transfected with CAP1-sh RNA.Compared with control-sh RNA group that chondrocytes were stimulated with human recombinant resistin at 48 h and 72 h,m RNA levels of CCL3,CCL4,MMP-13,and ADAMTS-4 were significantly decreased at 48 h and 72 h by RT-PCR assays.ELISA indicated that the secreted protein levels in chondrocyte supernatant were significantly decreased for CCL4 at 48 h and for MMP-13 at 72 h.Conclusion: Higher concentration of resistin can cause cytotoxicity for chondrocytes and inhibit their growth.Resistin induced KOA chondrocytes to upregulate expressions of CCL3,CCL4,MMP-13 and ADAMTS-4 which promoted inflammation and matrix degradation,and CAP1 as a direct receptor of resistin played a key role in this process.Knockdown of CAP1 expression in chondrocytes can significantly downregulate expressions of CCL3,CCL4,MMP-13,and ADAMTS-4.Part IV: The role of p38-MAPK and NF-?B signaling pathway in regulation of productions of inflammatory chemokines and matrixdegrading enzymes by chondrocyte when induced by resistin-CAP1.Objective: To investigate the role of p38-MAPK and NF-k B signaling pathways on resistin-CAP1 axis in inflammatory response and matrix degradation involved in chondrocytes.Methods: KOA chondrocytes were cultured and divided into five groups : resistin(500ng/ml)was added for 30 min,60min,6h,24 h and adding no resistin as blank control group.Four groups were designed by knockdown of CAP1 or not and adding with or without resistin(500ng/ml): CAP1-sh RNA group,CAP1-sh RNA plus resistin group,Control-sh RNA group,Control-sh RNA group plus resistin group.Protein expressions of p-38,phospho-p38,p65,phospho-p65 were investigated by western blot assays to analyzed the role of p38-MAPK and NF-k B signaling pathways in KOA chondrocytes stimulated by resistin and the role of CAP1 in the process.Results: After chondrocytes stimulated by resistin,western blot assays showed that protein expressions of p38 and p65 had no obvious change,and protein level of phospho-p38 increased at 6h and peaked at 24 h,and protein level of phospho-p65 peaked at 60 min and then decreased slightly.By contrast with control-sh RNA group,in CAP1-sh RNA group,protein expressions of phospho-p38 and phospho-p65 significantly decreased,and protein expressions of p38 and p65 also decreased lightly.Conclusion: The p38-MAPK and NF-?B signaling pathways were activated by resistin-CAP1 to regulate inflammation response and matrix degradation of KOA.