| Objective:To explore the distribution and expression changes of Interleukin-18 (IL-18) in the synovium and articular cartilage of knee osteoarthritis (OA) patients, and its role and mechanism in the pathological processes of osteoarthritis as well; meanwhile, to investigate whether wenjingtongluo decoction treatment could protect synoiocytes and chondrocytes of knee OA patients through affect the expression of IL-18 in vitro, and observe the curative effect.Methods:(1) The synovium and articular cartilage of knee were randomly selected from OA group and healthy group. The morphologic observation of the two groups was measured by H&E staining; The IL-18 mRNA levels of synovium and articular cartilage were measured by real-time PCR; Western blot analysis was used to observe the IL-18 protein levels of synovium and articular cartilage; The distribution and expression of IL-18 in the synovium and articular cartilage were investigated by immunohistochemistry; Immunofluorescence were performed to assess the cellular colocalization of IL-18 and different cell markers in synovium and articular cartilage of OA patients, including ED-1(macrophage marker), MPO(neutrophil marker), THY1(fibroblast marker), CD3 (T cell marker). (2) To investigate the expression of TNF-a in synovium and articular cartilage of OA/healthy group, Western blot was performed. Immunofluorescence and immunoprecipitation were used to assess the specificity of interactions between IL-18 and TNF-a in synovium and articular cartilage of OA patients. (3) Synovium and articular cartilage were isolated from OA group and healthy group, and used to establish cultured primary chondrocytes and synoiocytes. Then cells were observed by fluorescent inverted phase contrast microscope. The cell proteins and culture medium were extracted from the 3rd generation chondrocytes and 2rd generation synoiocytes. Western blot analysis and Elisa were used to study the inflammatory reaction in the cultured chondrocytes and synoiocytes, then cells were randomly divided into two groups:medicated serum group and control group, medicated serum group was added 10% rat wenjingtongluo decoction serum DMEM; control group was added 10% rat serum DMEM; while healthy group did not received any intervention. After 48h, Western blot analysis and Elisa were performed to explore the effect of wenjingtongluo decoction treatment on the expressions of IL-18 and TNF-a in cultured chondrocytes and synoiocytes. (4) Investigating the expression of IL-18 in plasma, synovial fluid and articular cartilage of knee OA patients, and studying their relationship with radiographic severity (Kellgren and Lawrence, KL). IL-18 was measured concurrently in synovial fluid, plasma and articular cartilage samples from 33 patients with knee OA (Ten patients were KL grade 2,12 were KL grade 3 and 11 were KL grade 4) and from 15 controls (KL grade 0). Correlation between plasma, synovial fluid and articular cartilage IL-18 with radiographic severity (K-L classification) was investigated by ELISA and immunofluorescent. (5) According to Kellgren-Lawrance classification,40 patients were divided into four groups:10 patients as control group (KL grade 0),10 patients as mild group (KL grade 1-2),10 patients as moderate group (KL grade 3),10 patients as severe group (KL grade 4), all groups were treated with wenjingtongluo decoction for 1 week, then plasma IL-18 levels were analyzed by Elisa, clinical symptoms and the HSS Functional scores were also investigated.Results:(1) Cell swelling, degrade, mortify and matrix collagen fibrosis were found in OA articular cartilage by HE staining; moreover, marked hyperplasia, inflammatory cells infiltration, fibrosis and vascular proliferation were found in the OA synovium. IL-18 mRNA levels and IL-18 protein expression were increased in OA synovium and articular cartilage compare with healthy group (p<0.05). Immunohistochemistry showed that IL-18 positive cells were increased in OA synovium and articular (p<0.05). Immunofluorescent staining showed that IL-18 expression was widely distributed in macrophag, neutrophil, lymphocyte and fibroblast of OA synovium. Meanwhile, IL-18 expression was widely distributed in OA articular cartilage. (2) Western blot analysis showed that TNF-a protein levels were significantly raised in OA synovium and articular cartilage compare with healthy group (p<0.05). Besides, colocalization of IL-18/TNF-α has been detected in OA synovium and articular cartilage through Double-immunofluorescence staining. Immunoprecipitation showed that IL-18 was correlated with TNF-a in OA synovium and articular cartilage. (3) The 3rd generation chondrocytes and 2rd generation synoiocytes of OA/healty patients were successfully obtained through primary culture and passage culture. Western blot analysis and Elisa indicated that TNF-a and IL-18 protein levels were significantly increased in OA group (p<0.05). It demonstrated that passage cultured chondrocytes and synoiocytes that extracted from OA patients could maintain inflammation at high levels. Wenjingtongluo decoction serum treatment significantly reduced the protein levels of IL-18 and TNF-a in cultured chondrocytes and synoiocytes (p<0.05). (4) In clinical research, IL-18 levels of plasma, synovial fluid and IL-18-positive cells of articular cartilage IL-18 levels were positively correlated with OA severity, P<0.05. Meanwhile, plasma, synovial fluid and articular cartilage IL-18 levels positively correlated with each other, P＜0.05. (5) Wenjingtongluo decoction treatment significantly attenuated plasma IL-18 levels of knee OA patients, increased the HSS scores, and improved clinical symptoms, especially for the mild to moderate patients.Conclusion:(1) In knee OA synovium and articular cartilage, IL-18 expression is significantly increased and correlated with TNF-α. (2) Plasma, synovial fluid and articular cartilage IL-18 levels are positively correlated with OA severity, IL-18 could serve as a biomarker for detemining disease severity. (3) Wenjingtongluo decoction treatment significantly reduces IL-18 expressions in cultured knee OA chondrocytes and synoiocytes, attenuate inflammation levels, offer chondrocytes and synoiocytes protection, and improve clinical symptoms. |
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