| Osteoarthritis(OA)is a chronic progressive skeletal system disease characterized by cartilage destruction,and is one of the important factors leading to disability in the elderly population.For a long time,KOA was attributed to the mechanical and aging process – the ?wear? mode.Researchers and clinicians generally believed that long-term joint wear is a key factor in the progress of KOA.However,more and more epidemics and basic research have changed researchers' views of the disease in recent years.More and more studies have shown that metabolic syndrome(Met S)plays an important role in the progression of osteoarthritis,especially in Asian populations.Leptin,the first discovered 16 k Da adipokines,belongs to the class I superfamily of long-chain helical cytokines and is a pro-inflammatory cytokine.Leptin is closely related to Met S and KOA.Kontunen et al.evaluated the concentration of serum leptin in arthritis patients with and without Met S.The results showed that leptin levels were significantly higher in arthritis patients with Met S than those in arthritis patients without Met S.This suggests that Met S may play biological effects in arthritis through leptin.The concentrations of serum and synovial leptin in OA patients usually were higher than those in the normal population.Besides,the concentration of leptin in SF was positively correlated with the severity of imaging KOA and clinical symptoms.Even after adjusting the factors such as age,race and BMI,this correlation still existed.This suggests that the role of leptin in mediating Met S and KOA can be mediated by metabolic pathways,not only mechanical stress.So,leptin may play an important role in the progress of KOA and Met S,and is an important medium for linking KOA and Met S.However,the expression characteristics and clinical characteristics of leptin in Met S-OA patients are unclear.In order to understand the expression characteristics and clinical characteristics of leptin in Met S-OA,this subject was conducted from the following aspects.Firstly,the distribution characteristics of leptin in serum and SF of female KOA patients and healthy controls were measured,and the correlations betweens the distribution of leptin and clinical features were analysed.Secondly,we investigated whether were different in the expressions of leptin in the joint tissues betwe en the two groups of KOA patients.Thirdly,we investigated whether there is a difference in the expressions of leptin receptor,OB-Rb,in the cartilage tissues of two groups of KOA patients.Finally,based on the differences in OB-Rb expressions in cartilage tissue,we continued to study the responses of chondrocytes obtained from KOA patients with and without Met S to different concentrations of leptin.The aim of this study was to investigate the expression characteristics and mechanism of leptin in Met S-OA subtypes,and to provide new evidences for elucidating the role of Met S in OA.Part I: Distribution and clinical relevance of leptin in Met S-OA and non-Met S-OA female patients.Objective: To understand the distribution of leptin in Met S-OA and non-Met S-OA female patients.Methods: Patients who underwent knee surgery for KOA in the Department of Orthopaedic Surgery of the First Hospital of Jilin University from May 2015 to January 2018 were selected.The healthy subjects without KOA-related symptoms and Met S were recruited as the control group.The clinical data of the patients were collected,and the concentration of leptin in serum and joint fluid was measured by ELISA kit.The clinical characteristics of the two groups of patients,as well as the correlation between the clinical characteristics of leptin and KOA in serum and synovial fluid were analyzed.Results:(1)Compared with the control group and the non-Met S-OA group,patients in Met S-OA group had greater BMI(Met S-OA vs.Control: 27.3 vs.24.1,p<0.001;Met S-OA vs.non-Met S-OA: 27.7 vs.24.4,p < 0.001),larger abdominal circumferences(Met S-OA vs.Control: 95.6 vs.83.9,p < 0.001;Met S-OA vs.non-Met S-OA: 95.6 vs.84.3,p<0.001),more blood pressure increase(Met S-OA vs.Control: 45/19 vs.13/47,p<0.001;Met S-OA vs.non-Met S-OA: 45/19 vs.20/36,p<0.001),and higher levels of blood glucose(Met S-OA vs.Control: 5.7 vs.4.9,p<0.001;Met S-OA vs.non-Met S-OA: 5.7 vs.4.9,p < 0.001),total cholesterol(Met S-OA vs.Control: 5.7 vs.4.6,p=0.015;Met S-OA vs.non-Met S-OA: 5.7 vs.4.9,p<0.001),triglycerides(Met S-OA vs.Control: 2.5 vs.1.1,p<0.001;Met S-OA vs.non-Met S-OA: 2.5 vs.1.6,p<0.001),and reduced HDL levels(Met S-OA vs.Control: 1.2 vs.1.5,p<0.001;Met S-OA vs.non-Met S-OA: 1.2 vs.1.5,p<0.001).Compared with the healthy control group,the LDL levels were increased in the Met S-OA and non-Met S-OA groups(Control vs.Met S-OA: 1.2 vs.1.5,p<0.001;Control vs.non-Met S-OA: 1.2 vs.1.5,p<0.001).Compared with the non-Met S-OA group,patients in Met S-OA group had higher ESR(17.5 vs.9.0,p=0.001),C RP(4.8 vs.3.2,p<0.001),and higher WOMAC function scores(54.5 vs.35.3,p=0.004).(2)Compared with the control group,the serum leptin in KOA groups were significantly increased(Met S-OA vs.Control: 19.38 vs.5.63,p<0.001;non-Met S-OA vs.Control: 10.43 vs.5.63,p< 0.001),and serum leptin were significantly higher in the Met S-OA group than those in the non-Met S-OA group(19.38 vs.10.43,p< 0.001).Similar to the distribution in serum,the SF leptin of the Met S-OA group was significantly higher than those in the non-Met S-OA group(19.38 vs.10.43,p<0.001).After adjusting the affecting factors,the above differences still existed.(3)Correlation analysis showed that BMI was positively correlated with the concentration of leptin in serum and joint fluid(rserum=0.517,p<0.001;r SF=0.472,p<0.001).The abdominal circumference was positively correlated with serum leptin(r=0.538,p<0.0011)and SF leptin(r=0.475,p<0.001).The K-L stage of KOA was positively correlated with the concentration of leptin in serum and SF(rserum= 0.233,p=0.011;r SF= 0.190,p=0.040).The WOMAC functional score of the patients was positively correlated with the concentration of leptin in serum and synovial fluid(rserum=0.449,p<0.001;r SF=0.423,p<0.001).In serum,leptin was positively correlated with FBS and triglyceride(r FBS=0.197,p=0.041;rtriglyceride=0.190,p=0.048),and negatively correlated with HDL level(r=-0.216,p=0.024).In SF,leptin was only positively correlated with triglyceride levels significant ly(r=0.220,p=0.021).Analysis of the indicators of inflammation found that the level of leptin in serum and joint fluid was positively correlated with the concentration of CRP(rserum = 0.279,p= 0.003;r SF=0.254,p=0.008).Conclusions: The differential distribution of Leptin in KOA patients suggests that leptin may be an important mediator of Met S associated with KOA.Part II: Expression of leptin in the periarticular tissues of KOA patientsObjective: To understand the expression characteristics of leptin in the periarticular tissues in KOA patients,including synovial membrane,and infrapatellar fat pad.Methods: According to age,BMI and K-L classification,10 cases of Met S-OA and non-Met S-OA K-L 4 women were included.Synovial membrane and infrapatellar fat pad tissues of patients were collected.Immunohistochemistry and Western blot were used to understand the expression of leptin protein in the periarticular tissues,and the expression of leptin m RNA in the tissues around the joint was detected by RT-PCR.RESULTS: Immunohistochemistry and Western blot showed that the leptin protein was expressed in the synovial membrane,and infrapatellar fat pad tissues of the two groups.And the leptin protein expression in the Met S-OA group was more than that in the non-Met S-OA patients.RT-PCR further confirmed that the expression of leptin in the synovial membrane,and infrapatellar fat pad tissues of the Met S-OA group was higher than that of non-Met S-OA patients(synovial membrane 2.38 vs.4.61,p=0.011;infrapatellar fat pad 2.32 vs.4.14,p=0.046).Conclusion: Met S-OA patients with high expression of leptin in the synovial membrane and infrapatellar fat pad tissues suggest that Met S can participate in the KOA process by regulating the level of leptin secreted by the periarticular tissues.Part III: Expression of OB-Rb in cartilage tissues of KOA patientsObjective: To understand the expression characteristics of OB-Rb in cartilage tissues of two groups of KOA patients.Methods: According to age,BMI and KL classification,10 cases of Met S-OA and n Met S-OA KL stage 3 women were included.5 patients with femoral neck fracture and without Met S were selected as controls.Cartilages with little affected was collected.Immunohistochemistry and Western blot were used to understand the expression of OB-Rb protein in cartilage tissues,and the expression of OB-Rb m RNA in was detected by RT-PCR.Results: Immunohistochemistry and Western blot showed that,in the protein level,the expressions of OB-Rb in cartilage tissues of KOA group were significantly higher than that of cartilage of patients with femoral neck fracture,and the expressions of OB-Rb in cartilage of Met S-OA group were also higher than of non-Met S-OA.RT-PCR further confirmed that the expressions of OB-Rb in cartilage tissue of patients with KOA group were significantly higher than that of patients with femoral neck fracture(Met S group: 6.27 vs.11.13,p=0.004;Met S group: 8.27 vs.11.13,p= 0.033),and O B-Rb expressions in cartilage in patients with Met S-OA were also higher than in non-Met S-OA group(6.27 vs.8.2 78,p=0.029).Conclusion: OB-Rb was differentially expressed in cartilage of Met S-OA and non-Met S-OA patients,suggesting that there may be differences in the response of chondrocytes to leptin between the two kinds of KOA patietns.Part IV: Met S affects the chondrocyte responsiveness to leptin in patients with osteoarthritis.Objective: To investigate whether Met S affects the chondrocyte responsiveness to leptin in patients with osteoarthritis.Methods: According to age,BMI and K-L classification,10 cases of Met S-OA and 10 non-Met S-OA K-L 3 women were included,and chondrocytes were extracted from cartilage tissue without obvious injury.C hondrocytes were stimulated with 0 ng/ml,10 ng/ml,20 ng/ml,100 ng/ml,and 500 ng/ml leptin for 48 hours.RNA was extracted and the expression of catabolic enzymes(MMP-13,ADAMTS-4 and ADAMTS-5)and pro-inflammatory factors(TNF-?,IL-1?,i NOS)genes were detected.Results: In addition to MMP-13 in Met S-OA patients,20 ng/ml leptin did not activate other genes in the chondrocytes of both groups.At the same time,100 ng/ml leptin can up-regulate i NOS and IL-1? m RNA levels in the two groups of chondrocytes.After treatment with 100 ng/ml leptin,ADAMTS-5 m RN A expression was up-regulated in chondrocytes collected from Met S-OA patients.However,this stimulation did not exist at 500 ng/ml level.After treatment with only 500 ng/ml leptin,ADAMTS-4 m RNA was up-regulated in Met S-OA chondrocytes.Conclusion: Met S participates in the progression of KOA by regulating the differential response of chondrocytes to leptin stimulation. |
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