A Preliminary Study On The Effect Of Calcitonin On The MAPKs Signaling Pathway Kinases In IL-1β-induced Chondrocyte Of Rat Vitro Models

Objective:To investigate the effect of calcitonin (CT) on extracellular sigal-regulated protein kinase (ERK) and p38 kinase(P38) of MAPK signal pathway and matrix metalloproteinase-13(MMP-13) in IL-1β-induced chondrocytes in the early phase of osteoarthritis(OA).Methods:1.Culture and identification of chondrocytes:The articular chondrocytes obtained from one month old Sprague-Dawley rats were cultured,then the chondrocytes were identified by Toluidin Blue staining and immunocytochemistry staining of collagen typeⅡ(ColⅡ).2.The second passaged chondrocytes were divided into six groups:the blank group(A group) was cultured with DMEM for 24h15min;the IL-1β(10ng/ml) group(B group) was cultured with DMEM for 24h and the other 15min with IL-1β(10ng/ml);the low dosage CT prevention group(C group) was cultured with 50 ng/ml CT for 24h and the other 15min with IL-1β;the high dosage CT prevention group(D group) was cultured with 500 ng/ml CT for 24h and the other 15min with IL-1β;the low dosage CT control group(E group) was cultured with 50 ng/ml CT for 24h15min;and the high dosage CT control group(F group) was cultured with 500 ng/ml CT for 24h15min.Immunocytochemistry and Western blot were applied to determine protein expression levels of ERK, p38 and their phosphorylation level and MMP-13.Results were analyzed statistically by ANOVA.Results:1. The Phaenotype of chondrocytes was metachromasia by Toluidin Blue staining. 2.The Phaenotype of chondrocytes was identified with immunocytochemistry of ColⅡ.3.The results of immunocytochemistry:(1)The IOD of ERK1/2 and P38:No significant difference was observed between any two groups (P>0.05).(2)The IOD of p-ERK1/2,p-p38 and MMP-13:In contrast with other groups, the IOD of p-ERK1/2,p-p38 and MMP-13 in group B remarkably upregulated,showing significant difference (p<0.05);the IOD of p-ERK1/2,p-p38 and MMP-13 in group C and D were lower than that in group B,but higher than that in group A, E and F respectively,showing significant difference(p<0.05);between group D and group C,the difference of the IOD of p-ERK1/2,p-p38 and MMP-13 was not statistically significant(p>0.05);among group A ,E and F, the difference of the IOD of p-ERK1/2,p-p38 and MMP-13 was not statistically significant (p>0.05).4. The results of Western blot:(1) The OD of ERK1/2 and P38: No significant difference was observed between any two groups (P>0.05).(2) The OD of p-ERK1/2,p-p38 and MMP-13: In contrast with other groups,the OD of p-ERK1/2,p-p38 and MMP-13 in group B remarkably upregulated, showing significant difference(p<0.05);the OD of p-ERK1/2, p-p38 and MMP-13 in group C and D were lower than group B,but higher than group A, E and F respectively, showing significant difference (p<0.05);between group D and C group, the difference of the OD of p-ERK1/2, p-p38 and MMP-13 was not statistically significant (p>0.05);among group A , E and F, the difference of the OD of p-ERK1/2, p-p38 and MMP-13 was not statistically significant (p>0.05).Conclusion:These results indicate that CT can protect rat chondrocyte from OA induced by IL-1βthrough decreasing the expression of MMP-13 and inhibition of mitogen-activated protein kinase (ERK and p38) phosphorylation in rat chonrocytes in vitro.