Effects Of Carbon Monoxide Releasing Molecule-3 On The Expression Of Matrix Metalloproteinases And MAPKs Signaling Pathways In Rat Condylar Chondrocytes Induced By Interleukin-1β

Experimental BackgroundTemporomandibular osteoarthritis is a complex condition affecting the entire joint.There are degenerative changes in the bone,cartilage and articular disc in the joint.One of the final outcomes is the loss of articular cartilage.Interleukin-1β is one of the most important cytokines in temporomandibular osteoarthritis.It can up-regulate the expression of several inflammatory mediators and matrix metalloproteinase,such as MMP-3,MMP-9,and MMP-13.Blocking IL-1β or IL-1β-induced inflammation may effectively reduce the progression of osteoarthritis.Various studies showed that CO can inhibit cell proliferation,apoptosis and inflammatory response.Carbon monoxide release molecule-3 is a carbonyl compound composed of transition metals,which can control the release of CO under physiological conditions and simulate CO under physiological conditions.Although carbon monoxide-releasing molecule-3 has been widely studied in anti-inflammatory,it is still not clear whether it can protect against cartilage damage caused by IL-1β.ObjectiveTo investigate the effects of carbon monoxide releasing molecule-3 on the expression of matrix metalloproteinases and MAPKs signaling pathways in interleukin-1β-induced rat condylar chondrocytes.MethodsThe condylar chondrocytes were isolated from the condylar of male Wistar rats aged 3-4 weeks.The morphology of the cultured cells were observed by a microscope.Toluidine blue staining were used for chondrocytes identification.The cytotoxic effects of CORM-3 at different concentration(0,100.200,400,800 μM)were checked by cell proliferation assay.Real-time quantitative PCR was used to detect the mRNA expression of MMP-3,9,13 in each group after stimulation with 0,5,10,20 ng/ml IL-1β for 24 hours.Based on the results,an inflammatory cell model was established by using 10 ng/ml of IL-1β.The mRNA and protein expression of MMP-3,MMP-9 and MMP-13 in IL-1β-induced condylar chondrocytes in the presence of 200μM CORM-3 was detected by qRT-PCR and Western Blot assay.Western Blot was used to detect the expression of P-JNK,P-P38 and P-ERK in each group after stimulation with 10 ng/ml IL-1β for 5 min,10 min and 30 min.Based on the results,10 min was selected as the time for activation of MAPK pathway in subsequent experiments.The mRNA expression of MMP-3,MMP-9 and MMP-13 and protein expression of P-JNK,P-P38,P-ERK,MMP-3,MMP-9 and MMP-13 in IL-1β-induced condylar chondrocytes in the presence of 200p.M CORM-3、30μM SP600125、10 μM SB203580 and 10μM U0126 was detected by qRT-PCR and Western Blot assayResults(1)The primary rat condylar chondrocytes grew adherently.The cells were unevenly distributed.Most of them were polygonal,while a few of these cells were round.The shapes were irregular.On the eighth day,a "paving stone" feature was observed.The toluidine blue staining showed that the cultured cells were metachromatic and the cultured cells were confirmed to be condylar chondrocytes;(2)Cell proliferation experiments showed that 200 μM CORM-3 has no cytotoxicity to condylar chondrocytes;(3)The expression of MMP-3,MMP-9 and MMP-13 was significantly increased by 10ng/ml IL-1β in condylar chondrocytes;(4)200μM CORM-3 significantly inhibited the expression of MMP-3,MMP-9 and MMP-13 in rat condylar chondrocytes induced by IL-1β.(5)10 ng/ml IL-1β significantly activated MAPKs signaling pathway at 10 min.(6)200μM CORM-3 significantly inhibited the activation of P38 and ERK signaling pathways.ConclusionsCORM-3 significantly inhibited the expression level of MMP-3,MMP-9,MMP-13 in IL-1β-induced inflammatory condylar chondrocytes by inhibiting P38 and ERK signaling pathways.It provides a potential theraputic basis for the prevention and treatment of temporomandibular osteoarthritis.