Based On NF-κB To Explore The Functional Mechanism Of IPFP In Obese KOA And Sinomenine's Anti-inflammatory Damage Of Chondrocytes

BackgroundIt was well-documented that obesity is a "chronic low-grade inflammation" that can induce knee osteoarthritis(KOA).KOA belongs to the category of "bone impediment" in the theory of traditional Chinese medicine."Elementary Questions" pointed out:"The wind-cold-damp seizes vacuity and invades the bones".Distinction is made between repletion and vacuity patterns.Modern medicine found that obesity could increase the incidence of KOA.Infrapatellar fat pad(IPFP)is related with inflammation,but the internal mechanism is unclear.It was suggested that interleukin-1β(IL-1β)、IL-6、tumor necrosis factor-α(TNF-α)、Leptin and Adiponectin were closely related to the degeneration of the articular cartilage matrix and inflammatory response in IPFP.Furthermore,NF-κB pathway plays a key role in maintaining the inflammatory response in tissuesThe Chinese medicine Orient vine is also known as Dafengteng,Cuifengsan,Qingfangji,etc.Sinomenine(SN),a key monomer of Orient vine,exerts anti-inflammatory,immune regulation,sedative and analgesic functions.Domestic scholars have found that SN can protect cartilage tissue degradation and chondrocyte apoptosis in OA.Based on the above studies,the present study aimed to explore the molecular mechanism of IPFP in obesity KOA and SN against chondrocyte inflammatory injury based on the NF-κB signaling pathway.Experiment 1 Distribution of inflammatory factors and adipokines in synovial fluid and serum of obese KOA patients and correlation between clinical features of obesityObjective:To study the distribution of inflammatory factors and adipokines in synovial fluid and serum of obese KOA patients and the clinical features of obesity.Methods:Synovial fluid and serum were collected from obese(Obese)and non-obese(Lean)KOA patients.The levels of IL-6,IL-1β,TNF-α and Leptin in synovial fluid and serum were measured by enzyme-linked immunosorbent assay(ELISA),and IL-6,IL-10 and TNF-α in synovial fluid and serum were analyzed.The correlation between the levels of Leptin and BMI,age,gender,HDL-C,LDL-C,VLDL-C,CRP,and ESR were tested.Results:BMI in KOA patients was positively correlated with IL-6 in synovial fluid and serum(P<0.05).BMI was positively correlated with Leptin and TNF-α in synovial fluid(P<0.05).VLDL-C in KOA patients was positively correlated with IL-6 in joint fluid(P<0.05);the serum Leptin level in KOA patients was positively correlated with serum IL-6 and IL-1β(P<0.05);TNF-α in serum of KOA patients and IL-6,IL-1β,and Leptin all had significant positive correlations(P<0.05).Summary:The BMI of Obese KOA patients has a positive correlation with Leptin and TNF-a in the synovial fluid,and the level of inflammation in the joint cavity is closely related to obesity。Experiment 2 Distribution of cytokine expression in the Infrapatellar fat pad tissue of obese KOA patientsObjective:To investigate the role of different parts of IPFP(near the synovial side and near the patellar tendon side)in osteoarthritis,and to explore whether the IPFP as a whole participates in the degenerative pathological changes in knee osteoarthritis.Localization and quantitative analysis of cytokine expression closely related to osteoarthritis degeneration.Methods:Subcutaneous adipose tissue,IPFP tissue(near the synovial side Ⅱ and near the patellar tendon side Ⅳ),and suprapatellar fat body Ⅲ were collected from obese(Obese)and non-obese(Lean)KOA patients.Morphological differences in IPFP tissues between Obese and Lean KOA patients were observed using hematoxylin-eosin(H&E)staining.RTqPCR was used to detect the mRNA expression levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α and adipocytokines Leptin,Adiponectin,Visfatin and PPARy in tissues Ⅰ,Ⅱ,Ⅲ and Ⅳof Obese and Lean;The levels of IL-1β,IL-6,TNF-α,Leptin,Adiponectin,Visfatin and PPARy in the Ⅰ,Ⅱ,Ⅲ and Ⅳ tissues of Obese and Lean KOA patients were detected by Western blot.Immunohistochemistry was used to observe the localization of of 1β,IL-6,TNF-α,Leptin and PPARy in tissues Ⅰ,Ⅱ,Ⅲ,and Ⅳ tissues of Obese and Lean KOA patients.Results:In Obese KOA patients,the expression levels of IL-1β in the synovial sides of IPFP were higher than those in other tissues(P<0.05).The expression of IL-6 in the synovial side of IPFP was lower than that in the patellar tendon side and other tissues(P<0.05),but the mRNA level of IL-6 in IPFP tissues was higher than that in the patellar tendon side of IPFP and other tissues(P<0.05);the expression of TNF-α near the synovial side in IPFP was lower than that in the patellar tendon side(P<0.05),both of which were higher than in other adipose tissues(P<0.05);Leptin protein in synovial side of IPFP was lower than that in patellar tendon side and other tissues(P<0.05),and the mRNA level near the synovial side was higher than that in patellar tendon side of IPFP and other tissues(P<0.05).The expression of Adiponectin near the synovial side in IPFP was lower than that in patellar tendon side of IPFP and other tissues(P<0.05),but both were higher than other adipose tissues(P<0.05).Compared with IPFP,the expression of Visfatin in IPFP was increased(P<0.05).The expression level of PPARy in the IPFP near the synovial side was lower than that in the patellar tendon side and other tissues(P<0.05),but both were higher than other adipose tissues(P<0.05).Compared with the Lean groups,the transcrnption and protein levels of inflammatory factors IL-1β,IL-6 and TNF-α in the IPFP tissues of the Obese groups were higher in the IPFP tissues than in the Lean groups(P<0.05);compared with the Lean groups,the level of Leptin protein was up-regulated in the Obese groups(P<0.05);the levels of Visfatin and PPARy were down-regulated(P<0.05);the difference in the expression level of Adiponectin was not statistically significant(P>0.05).The immunohistochemical results showed that Leptin in group II and IV of Obese KOA patients was higher than that in group Ⅰand Ⅲ.Compared with Lean groups,the expression of Leptin in IPFP tissues was increased in Obese groups.And there was no difference in PPARy among Ⅰ,Ⅱ,Ⅲ and Ⅳ in Obese groups.Summary:1)IL-6,TNF-α,Leptin,and Adiponectin in the synovial side of IPFP in Obese KOA patients were lower than that in the patellar tendon side;2)the levels of IL-1β,IL-6,TNF-a and Leptin in IPFP tissues of Obese KOA patients were higher than those in the Lean groups.Experiment 3 Study of NF-κB signaling pathway in the different parts of the Infrapatellar fat pad tissue of obese KOA patientsObjective:To further investigate the mechanisms of inflammatory activation and articular cartilage degradation,and to detect the activation of NF-κB signaling pathway in different parts of IPFP(near the synovial side and near the patellar tendon side)of obese KOA patients.Methods:Subcutaneous adipose tissue Ⅰ and IPFP tissues(near the synovial side Ⅱ and near the patellar tendon side Ⅳ)and suprapatellar fat body Ⅲ were collected from patients ofObese and Lean KOA.RTqPCR and Western blot were used to detect the expression levels of NF-κB signaling molecules of IκBα,IKKβ,p-P65(S536)and SOCS-3.Immunofluorescence staining was used to detect p-P65 nuclear translocation in the KOA patients in the Ⅰ、Ⅱ、Ⅲ、IV tissue;and also detect SOCS-3 in IPFP tissues.Results:The expression of IκBα in IPFP of Obese KOA patients was down-regulated.Compared with Lean groups,the IκBα expression in the the subcutaneous adipose tissue、the synovial side the of IPFP and the suprapatellar fat body tissue were decreased in the Obese groups(P<0.05).The levels of IKKβ and p-P65(S536)were exactly opposite to IκBα,and the expression of p-P65 and IKKβ were elevated in IPFP tissues,and Obese were higher than Lean groups(P<0.05).Compared with the Lean groups,the SOCS-3 level in the Obese groups decreased(P<0.05),and the expression of SOCS-3 in the IPFP near synovial side of Obese KOA was significantly lower than that in the Lean groups(P<0.05).SOCS-3 in the Obese groups of IPFP were lower than that in the Lean groups.Summary:The activation of NF-κB signaling pathway in IPFP tissues of Obese KOA patients were higher than that of Lean tissues.SOCS-3 in the Obese groups of IPFP were lower than that in the Lean groups.Experiment 4:Interaction mechanism between SOCS-3 and Leptin signals based on NF-κB pathwayObjective:To further investigate the crosstalk between the fat metabolism signal and NF-κB pathway in adipocytes.The mouse embryonic fibroblast preadipocyte cell line 3T3-L1 was used to explore the interaction between SOCS-3 and Leptin signaling.We aim to provide a mechanism for regulating NF-κB inflammatory signaling pathway in regulating fat metabolism and joint degeneration in adipose tissue of KOA patients.Methods:The pre-adipocyte cell line 3T3-L1 was purchased from ATCC.3T3-L1 cells were stimulated with 1.5 μg of 10 ng/ml NF-κB activator IL-1β for 0h,1 h,2 h.Recombinant vector(pcDNA\SOCS-3)expressing SOCS-3 was established and transfected into 3T3-L1 cells for 24 h with 1 μg,2 μg,5 μg of pcDNA\SOCS-3 or pcDNA\null.The pcDNA\SOCS-3 vector was combined with Leptin to stimulate the cell 3T3-L1.Western blot was used to analyze the expressed levels of NF-κB signaling molecules,including IκBα,IKKβ,p-P65(S536),RTqPCR and Western blot was used to measure the expression of cytokine signaling inhibitor SOCS-3;the binding of Leptin/Leptin receptor to SOCS-3 was detected by immunoprecipitation.Results:IL-1β stimulated 3T3-L1 cells to activate NF-κB signaling pathway and inhibited the expression of SOCS-3(P<0.05).SOCS-3 in the pcDNA\SOCS-3 group was up-regulated in a time-dose manner(P<0.05),while the expression level of Leptin receptor was decreased(P<0.05).The expression of Leptin receptor was increased in Leptin+pcDNA\SOCS-3 cells(P<0.05),and the expression levels of IKKβ and p-P65 were partially increased(P<0.05),and the expression level of IκBα was partially decreased(P<0.05).We detected higher levels of SOCS-3-Leptin receptor complexes in 3T3-L1 cells(P<0.05)and no SOCS-3-Leptin complex was detected.Summary:IL-1β stimulation in 3T3-L1 cells can activate NF-κB signaling pathway and inhibit SOCS-3;SOCS-3 overexpression inhibits Leptin receptor expression and activation of NF-,κB signaling pathway in 3T3-L1 cells;SOCS-3 regulates the NF-κB signaling pathway by binding directly to the Leptin receptor in 3T3-L1 cells.Experiment 5:The function and mechanism of sinomenine in protecting inflammatory injury of rat knee articular chondrocytesObjective:To explore the protective effect and mechanism of sinokinin(SN),an anti-inflammatory Chinese herbal component,on inflammatory factor-induced chondrocyte injury by regulating NF-κB signaling pathway.Methods:New SD rats were purchased and primary chondrocytes were isolated.Chondrocytes were identified using an Anti-Collagen Ⅱ antibody.SN(0,50,100,150,200,300,400,600,800,1000 μM)stimulated chondrocytes and detected changes in cell proliferation activity using the CCK8 method.RT-PCR and Western blot were used to detect P65 levels in 1.5 μg 10 ng/ml NF-κB activator IL-l0;immunofluorescence staining was used to detect p-P65(S536)nuclear translocation;RTqPCR and Western blot were used to detect MMP-3 and MMP-9 expression.p-P65 levels were detected by RT-PCR and Western blot of 50,100,200,300 μM SN-induced chondrocytes with 1.5 μg of 10 ng/ml NF-κB activator IL-1β;immunofluorescence staining detection of p-P65(S536)nuclear translocation RTqPCR and Western blot were used to detect the expression of MMP-3 and MMP-9Results:The cells isolated after 3 and 7 days of culture were identified as collagenase-positive cells,and the positive rate was close to 100%.SN has an inhibitory effect on the proliferation of chondrocytes.IL-1β induced an increase in the expression of MMP-3 and MMP-9(P<0.05)and were significantly inhibited by SN(P<0.05).The level of p-P65(S536)was increased after treatment withlO ng/ml IL-1β for 2 h.SN had an inhibitory effect on the level of p-P65(S536)(P<0.05).Summary:SN inhibits the proliferation activity of chondrocytes over a wide range of concentrations.MMP-3 and MMP-9 signaling are potential targets for SN to protect against inflammatory damage in chondrocytes.Inhibition of NF-κB signaling pathway activation is one of the downstream molecular mechanisms by which SN protects inflammatory lesions of chondrocytes.By inhibiting the activation of NF-κB signaling pathway,SN regulates the growth activity of chondrocytes and the expression of key proteases MMP-3 and MMP-9,which have important protective effects on inflammatory injured chondrocytes.Conclusions:The activation of NF-κB signaling pathway in IPFP tissues is a key feature in the development of obese KOA.The sinomenine protects chondrocytes from degradation caused by abnormal activation of inflammation.