Abstract
To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1β (IL-1β). Primary EPCs from the lumbar vertebra of 4-week-old Sprague Dawley rats were cultured in vitro and identified by morphological observation, toluidine blue staining and Col-II immunofluorescence staining. Then, MTT assay was used to determine the optimal concentration of IL-1 and Melittin. Next, EPCs at passage 3 were randomly divided into 4 groups: no treatment was done in group A as control group; the optimal concentration of IL-1β, Melittin, and both IL-1β and Melittin were used in groups B, C, and D respectively. The expression of Col-II was detected by Western blot after 48 hours intervention. Under inverted microscope, the first generation EPCs were polygonal; cell proliferation decreased after fifth generation, and cell morphology changed into fusiform. The acidic mucosubstance in the cytoplasm (such as Aggrecan) was stained dark blue by toluidine blue. After marking Col-II by immunofluorescence, the positive expression of cytoskeleton (green fluorescence) could be observed. MTT assay showed that IL-1β and Melittin could inhibit the EPCs in a dose-dependent manner after intervention of 24 and 48 hours, and the optimal concentrations of IL-1β and Melittin intervention were 10 ng/mL and 1.0 μg/mL respectively. Compared with group A, the expression of Col-II was significantly reduced in group B, and was significantly increased in group C by Western blot assay, but there was no significant difference between group D and group A. The Col-II expression levels of groups A, B, C, and D were 0.991±0.024, 0.474±0.127, 1.913±0.350, and 1.159±0.297 respectively, showing significant difference between the other groups ( P<0.05) except between group A and group D ( P>0.05). Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.
Published Version
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