| Osteoarthritis(OA)is a chronic and disabling disease that occurs in middle-aged and elderly women.Knee osteoarthritis(KOA)is the most common type[3].In earlier studies related to KOA,the loss of articular cartilage caused by mechanical wear was considered to be the main pathogenic mechanism.With the continuous in-depth research,scholars have gradually discovered the diversity of KOA pathogenic factors and the complexity of the pathogenesis.Many local and systemic factors,such as age,gender,genetic factors,obesity,endocrine,trauma,and joint deformity,have been incorporated into the risk factors of KOA.Among the many pathogenic factors of OA,obesity has become one of the most modifiable risk factors[8].Obese people are not only overweight,but also have systemic metabolic disorders,which can lead to a series of metabolic diseases..As a result,the academic community proposed the concept of metabolic syndrome(Met S).A growing number of studies have shown that Met S plays an important role in KOA progression,especially in Asian populations[11-14].Met S,a syndrome characterized by systemic low-grade inflammation and disturbances in energy metabolism,is a systemic risk factor for KOA.The synovial tissue,the most vascularized tissue in the joint,is more susceptible to systemic factors.Synovitis was considered to be a concomitant manifestation of OA in previous studies and was associated with pain and joint stiffness in OA.In the past 10 years,with the continuous research on the pathogenesis of OA,scholars have found that synovitis is not only an accompanying manifestation of OA,but its role in promoting the pathological process of OA should not be underestimated.The results of animal experiments showed that the joint damage was aggravated after the induction of synovial inflammation in the rabbit knee joint after meniscectomy[32].Synovial components in an inflammatory state can secrete a large number of inflammatory cytokines such as IL-1,IL-6,TNF-α,etc.into the synovial fluid,which will affect the inflammatory state and the balance of synthesis and catabolism of chondrocytes.In addition,it can also secrete enzymes related to matrix degradation,such as matrix metalloproteinases(MMPs),which directly act on cartilage tissue to promote degradation of cartilage matrix.However,the pathogenic mechanism of synovitis is still unclear.Adipokines act as a bridge between Met S and OA.In recent years,there have been a lot of related studies on the involvement of adipokines in the pathological progression of OA.Resistin,one of the classic adipokines,was first known for its role in insulin resistance in mice.A study by Osawa et al.found that serum resistin levels were positively correlated with the amount of Met S components[33].The role of resistin in OA progression has also been reported.Our previous study found that the level of resistin in serum was significantly higher than that in synovial fluid.And the level of resistin in serum of KOA patients was higher than that of healthy controls,which was related to the severity of KOA[29].Higher serum resistin levels were found to be associated with serum markers and structural abnormalities in knee synovitis in a study by Han et al.[20].In a series of studies on Resistin and OA synovitis by Chen and colleagues,it was found that resistin can regulate the inflammation of Fibroblast-like synoviocytes(FLS)through signaling pathways such as MEK,ERK,PKC,p38,JNK,PI3 K,Akt and m TOR[34-36].However,in the Met S population characterized by systemic low-grade inflammation and energy metabolism disorders,is the occurrence of synovitis related to energy metabolism disorders,and what role does resistin play in it? We don’t know yet.Therefore,this study is mainly to explore whether resistin affects the occurrence and development of KOA synovitis by regulating the energy metabolism of FLS,so as to improve the mechanism of Met S promoting the progression of KOA and provide new ideas for the treatment of Met S-related KOA.In this study,we first assessed the severity of Met S using a new metabolic score,and identified the association of systemic inflammation and adipokines with the severity of Met S.Then,we compared the differences in inflammation,catabolism and energy metabolism of FLS in patients with metabolic syndrome-associated knee osteoarthritis(Met S-KOA)and non-Metabolic syndrome-associated knee osteoarthritis(Met S-KOA)in FLS cultured in vitro.The serum of Met S patients was used to stimulate FLS of n Met S-KOA patients to clarify the effect of Met S internal environment on FLS.Further,we stimulated Met S-KOA-FLS with recombinant human resistin to confirm the regulatory effect of resistin on Met S-KOA-FLS inflammation,catabolism and energy metabolism.Finally,we explored the specific mechanism of resistin in regulating the inflammation and catabolism of FLS by detecting the expression of resistin’s receptors and the expression changes of key enzymes of energy metabolism and pathway-related proteins.This study is divided into the following four parts:(1)Metabolic score for insulin resistance is correlated to adipokine disorder and inflammatory activity in female knee osteoarthritis patients in a Chinese population.(2)The internal environment of Met S-KOA patients promotes FLS inflammation,catabolism and fatty acid oxidation(FAO).(3)Resistin promotes inflammation,catabolism and FAO of Met S-KOA-FLS.(4)Resistin up-regulates FAO of Met S-KOA-FLS through CAP1/PKA/CREB pathway to promote inflammation and catabolism.Part Ⅰ: Metabolic Score for Insulin Resistance Is Correlated to Adipokine Disorder and Inflammatory Activity in Female Knee Osteoarthritis Patients in a Chinese Population Objective: This study was to evaluate the metabolic score for insulin resistance(METS-IR)in female knee osteoarthritis(KOA)patients in a Chinese population.The associations between METS-IR and adipokines,erythrocyte sedimentation rate(ESR),and C-reactive protein(CRP)were investigated.Methods:(1)This study included 4686 women from the 2011 China Health and Retirement Longitudinal Study(CHARLS)and 108 women who underwent arthroplasty of KOA at a university hospital.A comparative analysis of the general data of the Met S population and the n Met S population in the sample of the 2011 CHARLS.Using the data of 2011 CHARLS as the national baseline data,METS-IR was calculated,and ROC curve analysis was used to verify the diagnostic performance of METS-IR for Met S.(2)Clinical data of KOA patients,as well as tissue samples such as serum and synovial fluid were collected.METS-IR in KOA patients was calculated and compared to national baseline levels.(3)Serum and synovial fluid adipokines of KOA patients were detected,and the relationship between METS-IR and adipokines,ESR and CRP was analyzed by logistic regression analysis.Results:(1)In the data of 2011 CHARLS,the Met S group had higher levels of age,weight,BMI,waist circumference,blood pressure,fasting blood glucose,triglycerides,and METS-IR than the n Met S group,while the Met S group had lower levels of HDL-C than the n Met S group.The ROC curve analysis of METS-IR for Met S showed that the area under the curve was 0.851,the sensitivity was 0.777,and the specificity was 0.772.(2)The METS-IR of KOA patients was higher than the national baseline level(40.29±6.98 vs 36.20±8.50,P<0.01),and the difference remained even after adjusting for age.Furthermore,Met S-KOA patients had higher METS-IR than n Met S-KOA patients after adjusting for BMI.(3)After adjusting for age and BMI,METS-IR was associated with CRP(OR1.238,95% confidence interval(CI)1.088,1.409,P < 0.01),ESR(OR 1.124,95% CI1.008,1.254,P = 0.036),serum-leptin(OR 1.123,95% CI 1.052,1.199,P < 0.01),serum-resistin(OR 1.134,95% CI 1.011,1.271,P = 0.031),and serum-adiponectin(OR 0.865,95% CI 0.771,0.971,P = 0.014)in KOA patients.Conclusion: METS-IR in female KOA was higher than that of the national baseline.The METS-IR was related to adipokine disorder and inflammatory activity.These findings suggest that METS-IR can be used to evaluate the degree of involvement of Met S in KOA.Part Ⅱ: Internal environment promotes inflammation,catabolism and FAO in XIV FLS in Met S-KOA patients Objective: To analyze the differences in inflammation,catabolism and energy metabolism between Met S-KOA-FLS and n Met S-KOA-FLS,and to explore the effect of Met S internal environment on FLS.Methods:(1)Ten female KOA patients who underwent knee arthroplasty in our hospital were collected,including 5 cases of Met S-KOA and 5 cases of n Met S-KOA.The patients’ synovial tissue were collected and FLS was extracted.The synovitis score of the patients was evaluated by HE staining.ELISA and q PCR were used to detect the differences in the expression of inflammatory-related factors in the two groups of FLS cultured in vitro;and the adhesion experiment was used to verify the adhesion ability of the two groups of FLS to inflammatory cells.(2)ELISA and q PCR were used to detect the differences in the expression of FLS catabolic enzymes between the two groups.And the invasion test were used to compare the migration and invasion abilities of the two groups of FLS.(3)Western blot and q PCR were used to detect the expression levels of key enzymes(HK2,LDHA,CPT1A)of FLS glucose and lipid metabolism in the two groups.(4)5% serum of Met S patient was used to stimulate n Met S-KOA-FLS,and 5%FBS was used as a control to detect the differences of inflammation-related factors and catabolic enzymes in FLS by q PCR.Western blot and q PCR were used to detect the differences in the expression of key enzymes of glucose and FAO in FLS after Met S’ serum stimulation.Results:(1)HE staining results showed that compared with n Met S-KOA patients,Met S-KOA patients had more severe synovial inflammation.q PCR results showed that the expression levels of CCL2,CCL3,CXCL-8,ICAM1,TNF-α and IL-6 in Met S-KOA-FLS were significantly higher than those in n Met S-KOA-FLS.The ELISA results showed that the protein expression level of CCL3 was consistent with the q PCR.The adhesion experiment results showed that Met S-KOA-FLS had stronger adhesion ability than n Met S-KOA-FLS.(2)The results of q PCR and ELISA showed that compared with nMet S-KOA-FLS,the expressions of MMP13 and ADAMTS4 were increased in Met S-KOA-FLS.The results of nvasion assay showed that Met S-KOA-FLS had stronger migration and invasion ability than n Met S-KOA-FLS.(3)The detection results of key enzymes of glucose metabolism showed that the gene expressions of LDHA was significantly up-regulated in Met S-KOA-FLS.In the detection of key enzymes of FAO,compared with n Met S-KOA-FLS,the expression of CPT1 A in Met S-KOA-FLS was significantly up-regulated.(4)After stimulating n Met S-KOA-FLS with 5% serum from Met S patients,the results of q PCR showed that the expression levels of CCL3,ICAM1,TNF-α,MMP13 and ADAMTS4 were significantly up-regulated.The western blot results showed that the protein levels of HK2 and LDHA were not significantly different before and after serum stimulation.The gene and protein levels of CPT1 A,the key enzyme of FAO,were significantly up-regulated after Met S’ serum stimulation.Conclusion: Compared with n Met S-KOA-FLS,Met S-KOA-FLS has up-regulated expression of inflammation-related factors and catabolic enzymes,and increased FAO.The internal environment of Met S patients promotes FAO and phenotypic shifts in inflammation and catabolism in FLS.Part Ⅲ : Resistin promotes inflammation,catabolism and FAO of Met S-KOA-FLS Objective: To explore the regulatory effect of resistin on inflammation,catabolism and FAO in Met S-KOA-FLS.Methods:(1)Met S-KOA-FLS was stimulated with the recombinant human resistin for 24 h,and the gene expression changes of CCL2,CCL3,ICAM1,VCAM1,TNF-α,IL-6,VEGF and b FGF were detected by q PCR.The protein expression level of CCL3 was detected by ELISA.The adhesion ability of Met S-KOA-FLS after stimulation was detected by adhesion assay.(2)Met S-KOA-FLS was stimulated with the recombinant human resistin for 24 h,and the gene expression levels of MMP1,MMP3,MMP13,ADAMTS4 and ADAMTS5 were detected by q PCR.The protein expression levels of MMP13 and ADAMTS4 were detected by ELISA.The invasion ability of Met S-KOA-FLS after stimulation was verified by invasion test.(3)Met S-KOA-FLS was stimulated with the recombinant human resistin for 24 h,and the expression levels of key enzymes of glycolipid metabolism(HK2,LDHA,CPT1A)were detected by q PCR and western blot.Glucose uptake and lactate production experiments were used to detect the changes of Met S-KOA-FLS in glucose uptake and lactate production after stimulation.Results:(1)Using resistin to stimulate Met S-KOA-FLS,compared with the control,the expression levels of CCL2,CCL3,ICAM1,VCAM1 and TNF-α were significantly up-regulated.The results of ELISA showed that the protein expression level of CCL3 was consistent with the results of q PCR.The results of adhesion experiments showed that the adhesion ability of FLS was significantly enhanced after resistin stimulation.(2)After stimulation with resistin,the expression levels of MMP13 and ADAMTS4 in Met S-KOA-FLS were significantly up-regulated.The scratch experiment results showed that the migration ability of Met S-KOA-FLS was significantly increased at both 12 h and 24 h after resistin stimulation.The invasion assay confirmed that the invasion ability of Met S-KOA-FLS was significantly enhanced after stimulation of resistin.(3)After resistin stimulated Met S-KOA-FLS,compared with the control,the expression levels of HK2 and LDHA were no significantly difference.Both the gene and protein expression levels of CPT1 A were up-regulated after resistin stimulation.Conclusion: Resistin has no significant effect on the proliferation and apoptosis of Met S-KOA-FLS.Resistin promotes inflammation,catabolism,and FAO in Met S-KOA-FLS.Resistin may be the main active substance regulating FLS phenotype in Met S’ serum.Part Ⅳ: Resistin promotes inflammation and catabolism by up-regulating FAO in Met S-KOA-FLS via CAP1/PKA/CREB pathway Objective: To investigate whether resistin regulates inflammation and catabolism by promoting FAO in Met S-KOA-FLS.To explore the specific mechanism by which resistin promotes FAO of Met S-KOA-FLS.Methods:(1)Met S-KOA-FLS was divided into 4 groups by applying etomoxir(Eto),an inhibitor of CPT1 A,which were control group,Resistin group,Eto group and Resistin + Eto group.The gene expression levels of CCL3,ICAM1,TNF-α,MMP13 and ADAMTS4 were detected by q PCR.The protein expression levels of CCL3,MMP13 and ADAMTS4 were detected by ELISA.Adhesion experiments were used to verify the adhesion ability of Met S-KOA-FLS in different treatment groups.The invasion ability of Met S-KOA-FLS in different treatment groups was verified by invasion assay.(2)Applying 2-DG,a glycolysis inhibitor,Met S-KOA-FLS was divided into 4groups,namely control group,resistin group,2-DG group and resistin+2-DG group.The gene expression levels of CCL3,ICAM1,TNF-α,MMP13 and ADAMTS4 were detected by q PCR.The protein expression levels of CCL3,MMP13 and ADAMTS4 were detected by ELISA.Adhesion experiments were used to verify the adhesion ability of Met S-KOA-FLS in different treatment groups.The invasion ability of Met S-KOA-FLS in different treatment groups was verified by invasion assay.(3)Comparative analysis of the expression of TLR4 and CAP1 in Met S-KOA-FLS and n Met S-KOA-FLS by q PCR and western blot.Differential expression of CAP1 in synovial tissue of Met S-KOA and n Met S-KOA patients by immunohistochemistry.Using TLR4 inhibitor TAK-242,Met S-KOA-FLS was divided into 4 groups(control group,resistin group,TAK-242 group,resistin+TAK-242 group),and q PCR and western blot were used to detect CPT1 A in different treatment groups to validate the role of TLR4 in the regulation of CPT1 A by Resistin.CAP1-small interfering RNA(si RNA)was applied to inhibit the expression of CAP1,and Met S-KOA-FLS was divided into 4 groups(control group,resistin group,CAP1-si RNA group,resistin+CAP1-si RNA group),and q PCR and western blot were used to detect CPT1 A in different treatment groups to validate the role of CAP1 in the regulation of CPT1 A by resistin.(4)CAP1-small interfering RNA(si RNA)was applied to inhibit the expression of CAP1,and Met S-KOA-FLS was divided into 4 groups(control group,resistin group,CAP1-si RNA group,resistin+CAP1-si RNA group),and q PCR and western blot were used to detect CREB and p CREBser133 in different treatment groups.Then the PKA inhibitor H-89 and the p CREBser133 inhibitor KG-501 were applied respectively,and Met S-KOA-FLS was divided into control group,resistin group,H-89 group,resistin+H-89 group,KG-501 group and resistin+ KG-501 group.The gene and protein expression levels of CPT1 A and the protein expression levels of CREB and p CREBser133 were detected by q PCR and western blot.Results:(1)Compared with the control group,the gene and protein levels of inflammation-related factors(CCL3,ICAM1,TNFα)and matrix degrading enzymes(MMP13,ADAMTS4)were significantly up-regulated after resistin stimulation.The application of Eto inhibited the expression of resistin-induced inflammatory markers and the enzymes of matrix-degrading.The increase in resistin-induced adhesion and invasion was significantly inhibited by Eto.(2)Compared with the control,the gene and protein levels of inflammation-related factors(CCL3,ICAM1,TNFα)and matrix degrading enzymes(MMP13,ADAMTS4)were significantly up-regulated after resistin stimulation.The expression of inflammatory markers and matrix-degrading enzymes were also up-regulated in FLS with 2-DG alone.Co-incubation of FLS with resistin and 2-DG enhanced resistin-induced inflammation and catabolism.The results of adhesion and invasion experiments showed that the application of Resistin and 2-DG both led to the increase of the adhesion and invasion ability of FLS,and the combined application of the two led to further enhancement of the above-mentioned FLS’ functions.(3)The results of q PCR and western blot showed that compared with n Met S-KOA-FLS,the gene and protein levels of CAP1 were up-regulated in Met S-KOA-FLS,while the protein level of TLR4 was no significant difference.The results of immunohistochemistry of synovial tissue showed that there was indeed an increased expression of CAP1 in Met S-KOA synovial tissue.Tthe results of q PCR and western blot showed that TAK-242 could not inhibit the up-regulation of CPT1 A expression in resistin-induced FLS.After applying si RNA to interfere with the expression of CAP1,the expression of CPT1 A induced by resistin was abolished.(4)The expression of p CREBser133 was increased after the application of resistin,and the phosphorylation of CREB induced by resistin was inhibited after the application of si RNA to inhibit the expression of CAP1.After application of H-89,resistin-induced CPT1 A expression and CREB phosphorylation were inhibited.The application of KG-501 did not affect the expression level of p CREBser133,but significantly inhibited the expression of CPT1 A.Conclusion: Resistin promotes inflammation and catabolism by up-regulating the FAO of Met S-KOA-FLS.Upregulation of FAO of Met S-KOA-FLS by resistin is mediated by CAP1/PKA/CREB. |
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