Effect Of Calcitonin On The Inflammtory In IL-1β-induced Chondrocytes Of Rat

Obiective To investigate the effect of the calcitonin (CT) on matrix metalloproteinase-13(MMP-13), collagen type II (Col II) and P38kinase (P38) and phosphorylated protein(p-P38) expression in interleukin-1β (IL-1β) induced articular cartilage chondrocytes,thusto explore the possible mechanism of CT on osteoarthritis(OA).Methods1Chondrocytes were isolated and cultured with trypsin plus two doublecollagenase.2The chondrocytes were identified by immunohistochemical staining of ColII and Toluidin Blue staining.3The second-generation chondrocytes were diveded intothree groups:normal control group: DMEM/F12complete medium24h15min; IL-1β-induced group: with10ng/ml IL-1β of DMEM/F12complete medium24h15min;CTtreated group:after adding the first containing10ng/ml IL-1β in DMEM/F12completemedium15min, plus50ng/ml CT further flasks and cultured24h. The ultrastructuralchanges was observed by transmission electron microscopy scan,we also use Westernblot to detect the expression of MMP-13, Col II, P38and p-P38, Real-time PCR was usedto detect MMP-13, Col II mRNA expression.Results1Toluidine blue staining: normal chondrocytes secrete acid glycosaminoglycan,which is a characteristic that can be stained with toluidine blue purple, thus to identify theChondrocytes.2Col II immunohistochemical staining: Col II is Chondrocytes’ specificsecretions, so the use of Col II immunohistochemical staining proved positivechondrocytes Col II expression, brown granules appear in the cytoplasm.3transmissionelectron microscopy analysis: Compared with the control group, the projections andfolds of cell surface in IL-1β-induced group almost disappeared, a large cytoplasmicvacuoles, the number of mitochondria reduced swelling of endoplasmic reticulum; thesechanges in the CT treatment group reduce the extent than of IL-1β-induced group, butheavier than the control group.4the expression of MMP-13situation: Compared withControl, IL-1β induced the expression of MMP-13were significantly increased (P<0.05);CT treatment group, The expression of MMP-13also increased significantly (P<0.05),but compared with IL-1β-induced group, the expression of MMP-13had significantlydecreased after adding CT (P<0.05).5the situation Col II expression: Compared withControl, the expression of Col II in IL-1β-induced group and CT treatment group were significantly lower (P<0.05); However, the CT treatment group compared with IL-1β-induced group expression of Col II, the difference was not statistically significant.6theprotein expression of P38and p-P38: P38expression in each group had no significantchanges in total protein. The expression levels of p-P38in IL-1β-induced group wassignificantly higher than Control group and CT treatment group, a statistically significantdifference; the expression of p-P38in CT treatment group is significantly higher than theControl group (P<0.05).Conclusion Calcitonin significant improvement IL-1β-induced injury in chondrocytesfor OA rat model, may be one of the mechanisms by inhibition the activating of the P38signaling pathway to reduce the expression of MMP-13, thereby reducing the degradationof Col II, which play a role in delaying the OA process.