The Pro-inflammatory Adipokine Chemrin Involved In Chondrocyte Proliferation And Metabolism By Increased Generation Of NO

BACKGROUND: OA is that inflammation leads to cartilage degeneration,and chondrocytes are the only cells in cartilage,which bear the brunt of damage.Some studies have shown that Chemerin can stimulate leukocytes to migrate to inflammatory sites and increase the inflammatory signal of chondrocytes,suggesting that Chemerin may play a role in joint inflammation.In the previous clinical study,our group found that the level of serum Chemerin in patients with OA was significantly increased,and the level of Chemerin in synovial fluid was related to the severity of OA based on KL(Kellgren-Lawrence)grading.In addition,NO as a key inflammatory mediator of OA,we speculated whether Chemerin is similar to IL-1β can caused the increase of NO secretion and played an important role as a pro-inflammatory factor in OA.OBJECTIVE: To investigate the effect of Chemerin on the proliferation and metabolism of chondrocytes and its possible mechanism.METHODS: The chondrocytes of neonatal mice were isolated by collagenase type Ⅱ digestion then cultured.Cell growth curves were established and the range of concentrations of chemerin that exhibited toxicity to normal cartilage cells screened using an MTT assay.Subsequently,interleukin-1 beta(IL-1β(10ng/mL))was used to stimulate the chondrocytes in order to establish an in vitro model of OA induction and explore then effect of chemerin treatment of the cells.After the chondrocytes had been cultured in the presence of the drug for 2 days,cell morphology,proliferation and metabolism were evaluated by HE staining and diacetate fluorescein(FDA)/propidium iodide(PI)staining.In addition,the expression of induced nitric oxide polymerase(i NOS)was analyzed by measuring the secretion of NO.Furthermore,qRT-PCR was used to quantify gene expression of the cartilage protective gene proteoglycan(Acan),type Ⅱ collagen(Col2a1),arthritis-related gene matrix metalloprotease-13(MMP13)and nitric oxide synthase 2(Nos 2,or i NOS).RESULTS:(1)The chondrocytes cultured in vitro exhibited healthy activity and morphology under microscope.(2)MTT test showed that Chemerin could inhibit the proliferation of neonatal rat chondrocytes in vitro at the concentration of 50ng/mL(P<0.001).(3)Under HE staining,chondrocytes were still mainly round-like and polygonal.It is worth mentioning that there was no significant difference in cell morphology between each drug treatment group and Control group.(4)The red staining area and proteoglycan staining were the most in Control group,while those in drug group were lighter,which could reduce the synthesis of ECM,and the staining degree decreased most in Chemerin(50ng/mL)+ IL-1 β(10ng/mL)group(P=0.002<0.001).(5)FDA/PI staining showed that all the drug groups could promote the apoptosis of chondrocytes,and the survival chondrocytes in Chemerin+IL-1 β group were the least(P=0.001<0.001).(6)The amount of NO secreted by chondrocytes in each group was significantly higher than that in Control group,especially in Chemerin+IL-1 β group(P<0.001),and however the secretion of NO in Chemerin group in the presence of L-NMMA was significantly lower than that in the absence of L-NMMA(P < 0.008).(7)qRT-PCR results further showed that Chemerin and IL-1 β had similar effects,which could down-regulate the expression of chondrogenesis marker genes Acan and Col2a1 and up-regulate the expression of chondrocytes catabolism genes MMP13 and Nos2.At the same time,the expression of MMP13 in Chemerin group with L-NMMA was significantly lower than that without L-NMMA(P=0.034<0.05).CONCLUSION AND SIGNIFICANCE:(1)Chemerin is closely related to chondrocyte apoptosis in vitro.(2)The effect of Chemerin on the proliferation and metabolism of chondrocytes may be due to the high secretion of NO caused by increasing the expression of Nos2,which leads to the increase of the expression of catabolism gene MMP13 in chondrocytes.(3)Chemerin is synergistic with IL-1 β to some extent.(4)Chemerin may play an important role in obesity-induced OA,and is a new hub between two.It can be used as a novel and reliable biomarker of disease severity,and is expected to contribute to the clinical monitoring and intervention of early OA in obese patients.