Objective: To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1β (IL-1β).
Methods: 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.
Results: 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).
Conclusion: Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.
目的: 观察蜂毒肽（Melittin）对 IL-1β 诱导的大鼠终板软骨细胞（endplate chondrocytes，EPCs）Ⅱ型胶原表达的影响。.
方法: 取 4 周龄 SD 大鼠进行腰椎 EPCs 培养，并行形态学观察、甲苯胺蓝染色及Ⅱ型胶原免疫荧光染色鉴定。取第 3 代 EPCs，采用 MTT 法确定 IL-1β 及 Melittin 对细胞干预的最适浓度。然后将第 3 代 EPCs 随机分为 4 组：A 组为正常组，不加任何药物；B 组加入最适浓度的 IL-1β；C 组加入最适浓度的 Melittin；D 组加入最适浓度的 IL-1β 及 Melittin；干预 48 h 后采用 Western blot 法检测Ⅱ型胶原表达。.
结果: 倒置显微镜观察示，第 1 代细胞多呈多角形，第 5 代后细胞增殖能力逐渐减弱，细胞形态向梭形转变。细胞质中的酸性黏液物质（如蛋白多糖）被甲苯胺蓝染成深蓝色。免疫荧光染色标记Ⅱ型胶原后，可见细胞骨架部分呈阳性表达（呈绿色荧光）。MTT 法检测示，在干预 24、48 h 后，IL-1β 及 Melittin 对 EPCs 均有抑制作用，并呈剂量依赖性，最终确定 IL-1β 及 Melittin 干预的最适浓度分别为 10 ng/mL 和 1.0 μg/mL。Western blot 法检测示，与 A 组比较，B 组 IL-1β 干预后Ⅱ型胶原表达明显减少，C 组 Melittin 干预后Ⅱ型胶原表达明显增加，但 D 组与 A 组比较无明显差异。A、B、C、D 组Ⅱ型胶原相对表达量分别为 0.991±0.024、0.474±0.127、1.913±0.350、1.159±0.297，除 A、D 组间比较差异无统计学意义（ P>0.05）外，其余组间比较差异均有统计学意义（ P<0.05）。.
结论: 研究表明 Melittin 具有保护终板软骨作用，为其用于防治脊柱退变性疾病提供了相关实验依据。.
Keywords: Melittin; collagen type II; endplate chondrocytes; interleukin 1β; rat.