IRAK-4 On IL-1? Stimulates Osteoarthritis Fibroblast-like Synoviocytes Proliferation And Regulation Of Inflammation

Objective:IL-1? can promote synovial cell proliferation and inflammatory response leading to the occurrence of Osteoarthritis(OA),but the specific mechanism is not clear.Based on the pivotal role of IRAK-4 in the inflammatory TLR signaling pathway,the role and mechanism of IRAK-4 in IL-1? stimulates knee OA joint fibroblast-like synoviocytes proliferation and inflammation regulation were explored in order to provide a new method for the treatment of OA.Method:1?The expression of irak-4,IKK and NF-B in the synovial tissues of 7 patients with osteoarthritis and 6 normal synovial tissues were analyzed by immunohistochemistry to analyze their clinical significance.2?The primary cell types were identified by flow cytometry.3?OA fibroblast synovial cells were cultured with different concentrations of IL-1?(0,1,5,10,15ng/ml)and treated with 10ng/ml IL-1? for different time(0,3,6,12,24h).CCK-8 was used to detect the proliferation of OA fibroblast synovial cells.4?CCK-8 was used to analyze the effect of different interference factors on the proliferation of primary fibroblast-like synoviocytes;Set CON group(without any transfection reagent),IL-1? group(10ng/ml IL-1? was added to OA synovial cells),nc group(scrambled control siRNA sequence transfection),SI-IRAK4 group(IRAK-4-siRNA sequence transfection),IL-1? + nc group(on the basis of IL? group with scrambled control siRNA sequence transfection),and IL-1? + SI-IRAK4 group(on the basis of IL-1? group with IRAK-4-siRNA sequence transfection),CCK-8 experiment was performed after 24 hours,and the effect of different interference factors on the proliferation and growth of synovial cells was reflected by the OD value.5?The extracted OA fibroblast-like synoviocytes were divided into CON group(without any transfection reagents),IL group(10ng/ml IL-1? was added to OA synovial cells),and NC group(on the basis of IL group with scrambled control siRNA sequence transfection)and SI group(on the basis of IL group with IRAK-4-siRNA sequence transfection),Western Blot was used to detect the IRAK-4 protein content in the cells to determine the silencing effect.At the same time,the expression of IKK and NF-?B in the TLR / IL-1 pathway was detected by Western Blot to explore the mechanism of IRAK-4.Results:1?Immunohistochemistry showed that IRAK-4,IKK,and NF-?? were expressed in normal synovial tissue and osteoarthritis synovial tissue,but the expression of related factors in normal synovial tissues was significantly lower than that in synovial tissues of osteoarthritis.2.The primary cells were extracted from synovial tissue,and the positive rate of Vimentin-PE molecular marker protein in synovial cells was 95% by flow cytometry,proving that the extracted cells were fibroblast-like synoviocytes.3?Different concentrations of IL-1? and different interference time points had an effect on the proliferation of OA fibroblast synovial cells,and 10ng/ml of IL-1?had the strongest effect and the most proliferation of OA fibroblast synovial cells interfered with for 24 hours.4 ? CCK-8 detection showed that Si-IRAK4 had no effect on the proliferation of OA fibroblast-like synoviocytes,and the proliferation rate of fibroblast-like synoviocytes in the SI-IRAK4 group was not significantly different from that in the nc group(P> 0.05).There was no statistically significant difference between the CK group and the nc group(P> 0.05).Si-IRAK4 inhibited IL-1?-induced proliferation of OA fibroblast-like synoviocytes.The proliferation rate of fibroblast-like synoviocytes in the IL-1? + SI-IRAK4 group was significantly higher than that of IL-1? + nc.,and there was a statistically significant difference between the two groups(P < 0.0001).There was no significant difference between the IL-1? + nc group and the IL-1? group(P> 0.05).5?Western Blot analysis showed that IL-1? can significantly increase the expression of IRAK-4,NF-?B,and IKK proteins in OA synovial cells,and compared with the CON group,the P values were 0.010,0.022,and 0.043.Compared with the CON group,the expression was statistically significant(P <0.05).The P values of NF-B and IKK protein in NC group and IL group were 0.305 and 0.179,and there was no significant difference between the two groups(P> 0.05).However,the P values of NF-B and IKK in SI group were 0.004 and 0.027,and there were statistically significant differences between the two groups(P < 0.05).Conclusion: IRAK-4 plays an important role in the proliferation of IL-1? stimulates osteoarthritis fibroblast-like synoviocytes,and IRAK-4-siRNA targeting IRAK-4 can inhibit the proliferation and inflammation of fibroblast-like synoviocytes in knee joints.It Provides a new method for the treatment of OA.