葫芦素B对IL-1β诱导的软骨细胞损伤及胞外基质代谢紊乱的影响

Effects of cucurbitacin on IL-1β-induced cell injury and metabolism dysfunction in chondrocytes

目的明确葫芦素B对白细胞介素(IL)-1β诱导的软骨细胞损伤及胞外基质代谢紊乱的影响。方法用不同浓度(0.5、1、5、10、20μmol/L)的葫芦素B处理小鼠软骨细胞,随后用IL-1β(10μg/L)处理。采用CCK-8法检测细胞活性,试剂盒检测乳酸脱氢酶(LDH)及Caspase-3的活性,确定葫芦素B最佳处理浓度。采用流式细胞术检测细胞凋亡情况;qRT-PCR检测细胞胞外基质代谢相关的Ⅱ型胶原(CollagenⅡ)及蛋白多糖aggrecan、基质金属蛋白酶(MMP)-3及MMP-13的mRNA水平;酶联免疫吸附测定法(ELISA)检测MMP-3、MMP-13、前列腺素E2(PGE2)的水平,同时检测一氧化氮(NO)的产生;采用Western blot分析葫芦素B对IL-1β介导的软骨细胞中核因子(NF)-κB信号通路活化的影响。结果葫芦素B剂量依赖性地减弱IL-1β对软骨细胞存活的抑制效应(P<0.05),同时抑制IL-1β诱导的LDH释放、细胞凋亡及Caspase-3活性(P<0.05),10μmol/L是其最佳实验剂量。与IL-1β处理组相比,葫芦素B处理可减缓IL-1β对CollagenⅡ、aggrecan的mRNA表达抑制效应(P<0.05)。此外,葫芦素B还可抑制IL-1β诱导的MMP-3及MMP-13的mRNA表达及释放(P<0.05),降低炎性介质NO、PGE2的产生(P<0.05)。同时,IL-1β可明显上调软骨细胞中p-IκB、p-p65 NF-κB的表达,而葫芦素B则可抑制上述分子的表达(P<0.05)。结论葫芦素B可能通过阻断NF-κB信号通路的激活抑制IL-1β诱导的软骨细胞损伤及代谢紊乱,提示其具有潜在的抗骨关节炎的功效。

Objective To explore the function of cucurbitacin B(CuB) in cell injury and metabolism dysfunction in chondrocytes upon interleukin(IL)-1β conditions.Methods Mouse chondrocytes were pre-treated with various doses of CuB(0.5,1,5,10 and 20 μmol/L),prior to expose to IL-1(3(10 μg/L).Cell viability was then determined by CCK-8 assay.The commercial kits were used to detect lactate dehydrogenase(LDH) and Caspase-3 activity.The cell apoptosis was evaluated by flow cytometer.The mRNA levels of metabolism dysfunction-related Collagen Ⅱ,aggrecan,matrix metalloproteinase(MMP)-3 and MMP-13 were analyzed by qRT-PCR.ELISA assay was performed to measure the contents of MMP-3,MMP-13 and PGE-2.The levels of nitrous oxide(NO) were determined by a commercial kit.Western blot assay was conducted to investigate the effects of CuB on the activation of NF-κB signaling in IL-1β-treated chondrocytes.Results CuB dose-dependently ameliorated the suppressive roles of IL-1β on chondrocyte viability(P <0.05).Simultaneously,CuB suppressed IL-1β-induced LDH release,cell apoptosis and Caspase-3 activity(P <0.05).In contrast to IL-1β-treated groups,administration with CuB antagonized the inhibitory effects of IL-1β on the mRNA levels of Collagen Ⅱ and aggrecan(P<0.05).Furthermore,CuB treatment attenuated IL-1β-induced transcripts and releases of MMP-3 and MMP-13(P<0.05),concomitant with reductions in the production of inflammatory mediator NO and PGE2(P<0.05).Additionally,IL-1(3 incubation enhanced the expression of p-κB and p-p65 NF-κB(P<0.05),which were reversed following CuB treatment(P<0.05).Conclusion CuB may suppress IL-1β-induced cell injury and metabolism dysfunction in chondrocytes by blocking the NF-κB signaling,implying a potential function in osteoarthritis therapy.