| Background:Knee Osteoarthritis(OA)is a common clinical arthritis,and cartilage degeneration is an important pathological feature of knee OA.Chronic inflammation plays an important role in the early stage of OA disease,and is a key link involved in cartilage degeneration and other OA manifestations.Interleukin-6(IL-6)can affect the proliferation of chondrocytes,the responsiveness of cartilage injury,enhance the inflammatory response of articular joints,and aggravate the degeneration of cartilage.IL-6 binds to soluble IL-6 receptor(sIL-6R)to form a transsignaling pathway,which plays a proinflammatory role by activating JAK1/STAT3.However,the mechanism of trans-signaling pathway in knee OA and the therapeutic effect of sgp130fc,a newly developed specific inhibitor of trans-signaling pathway,in knee OA are still unclear.Purpose:To explore the mechanism of IL-6/sIL-6R trans signaling pathway involved in cartilage degeneration of knee OA through the study and intervention of clinical patients with OA,primary chondrocytes,and OA rats,and to explore the effect of sgp130fc,a specific inhibitor of trans signaling pathway,on knee OA.Methods:1.Knee OA patients and knee non-OA patients(elderly patients with degenerative joints vs young patients with fresh ACL rupture)treated in the Third Xiangya Hospital of Central South University from January 2019 to June 2020 were selected as the research objects.Fat pads and synovial fluid were collected.The expression of sIL-6R in fat pads of OA and non-OA patients was detected by immunohistochemical staining and Western blot WB.The expression levels of IL-6,sIL-6R and sgp130 in synovial fluid of patients with different stages of OA(early vs metaphase vs advanced)were detected by enzyme-linked immunosorbent assay(ELISA)with double antibody,and the activation of IL-6/sIL-6R trans signaling pathway in knee joint was explored.2.Primary chondrocytes of SD rats were extracted and cultured.Hyper-IL-6 and sgp130fc,which can simulate the effect of IL-6/sIL-6R complex,were used to intervene primary chondrocytes,and cell proliferation was detected by CCK8.Real-time Quantitative polymerase chain reaction was performed Q-PCR)and WB were used to detect the main components of chondrocyte matrix,collagen type Ⅱ(COL2)and Aggrecan,and protein metalloenzyme MMP-1,3,9.13 and the expression of genes and proteins related to ADAMTS-4,inflammatory factors IL-1β,COX-2,TNF-α,iNOS and JAK1/STAT3 signaling pathway,and the role of IL-6/sIL-6R trans signaling pathway in the degeneration of rat chondrocytes was clarified.3.Hyper-IL-6 and sgp130fc were used to treat primary chondrocytes of SD rats.Lipid profiles of each group,different lipid molecules between groups and different metabolic pathways were obtained by lipidomics,and the effect of IL-6/sIL-6R trans signaling pathway activation on lipid metabolism of OA chondrocytes was elucidated.4.OA rats were constructed by modified Hulth(cutting the anterior cruciate ligament and removing the medial meniscus)method.Sgp130fc or normal saline were injected into the joints.The cartilage injury score and osteophyte score were used to compare the general changes of articular cartilage between groups.HE staining and saffron solid green staining were used to compare the pathological changes of articular cartilage between groups.Immunohistochemical staining was used to compare the proteins related to cartilage injury and IL-6/sIL-6R trans signaling pathway between groups,so as to clarify the effect of trans-signaling pathway inhibitor sgp130fc on knee OA in rats.Results:1.Compared with non-OA patients,the expression level of sIL-6R in fat pads was increased in OA patients.In addition,the average expression levels of IL-6,sIL-6R and sgp130 in the advanced OA group were 442.8±513.7 pg/ml,478.5±185.7 pg/ml,16817±3151 pg/ml,respectively.They were significantly higher than those in early OA group(43.39±62.58 pg/ml,218.00±111.20 pg/ml,13392±3456.01)(P<0.05);The average expression level of sIL-6R in mid-OA group was 638.10±171.11 pg/ml,which was significantly higher than that in early OA group(218.00±111.20 pg/ml)(P<0.05),the average sgp130 expression level of 16585±354.02 pg/ml was higher than that of 13392±3456.01pg/m in early OA group,but the difference was not statistically significant.The average expression level of IL-6(5.97±6.76 pg/ml)was lower than that of the early OA group(43.39±62.58 pg/ml),and the difference was not statistically significant.2.Hyper-IL-6 can inhibit the proliferation of primary chondrocytes,activate JAK1-STAT3 signaling pathway,inhibit the expression of COL2 and Aggrecan mRNA,and promote the gene and protein expression of protein metalloenzymes MMP-1,3,9,13 and ADAMTS-4.It promoted the expression of IL-1β,COX-2,TNF-α and iNOS,and the high concentration of sgp130fc,a specific inhibitor of IL-6/sIL-6R trans signal,could reverse these changes.3.After Hyper-IL-6 treatment,the lipid concentrations of phosphatidylcholine(PC),phosphatidylethanolamine(PE)and lysophosphatidylcholine(LPC)of chondrocytes increased,and the concentration of sphingomyelin(SM)decreased.However,after sgp130fc intervention,the PC,PE,LPC of chondrocytes decreased,and the concentration of SM increased.4.The gross scores of cartilage injury and osteophyte in Hulth+sgp130fc group were 22.67±2.52 and 6.63±0.57,respectively,which were significantly lower than those in Hulth+NS group(34.67±3.06,10.33±1.53)(P<0.05);The pathological scores of cartilage injury and synovitis in Hulth+sgp130fc group were 3.67±2.08 and 1.33±0.57,respectively,which were significantly lower than 10.33±2.09 and 4.33±0.57 in Hulth+NS group.The expression levels of JAK1,P-JAK1,STAT3,P-STAT3,cartilage degeneration protein MMP-1,3,9,13,ADAMTS-4 and inflammatory factors COX-2,TNF-α and iNOS in Hulth+sgp130fc group were lower than those in Hulth+NS group.The expression level of cartilage matrix protein was higher than that of Hulth+NS group.Conclusions:1.The expression levels of IL-6,sIL-6R and sgp130 in the fat pad of patients with OA and in the knee fluid of patients with advanced OA are significantly increased,suggesting that the IL-6/sIL-6R trans signaling pathway is involved in the occurrence and development of OA.2.It was proved that activation of IL-6/sIL-6R trans signaling pathway can promote inflammation and cartilage degeneration in rats,and sgp130fc can specifically inhibit trans signaling pathway and reduce inflammation and cartilage degeneration.3.Hyper-IL-6 intervention can change the lipid profile of chondrocytes,the concentrations of pro-inflammatory lipid molecules such as PC,PE and LPC are up-regulated,and the concentrations of lipid molecules such as SM,which maintain the cell membrane structure,are down-regulated and the metabolism is accelerated.However,the intervention of sgp130fc can reverse the above changes.4.sgp130fc can alleviate knee OA in rats by inhibiting IL-6/sIL-6R trans signaling pathway. |
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