摘要
目的：比较青蒿素及其衍生物双氢青蒿素、蒿甲醚和青蒿琥酯对破骨细胞诱导分化能力以及相关通路的调控作用。方法：经核转录因子-κB（NK-κB）受体活化因子配体（receptor activator of nuclear factor-κB ligand,RANKL）诱导小鼠骨髓巨噬细胞（bone marrow-derived macrophages,BMMs）向破骨细胞分化,给予不同浓度的青蒿素、双氢青蒿素、蒿甲醚和青蒿琥酯作用后,通过抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色观察TRAP阳性多核细胞的形成,实时荧光定量PCR法（Real-time PCR）检测TRAP,基质金属蛋白酶-9（MMP-9）,组织蛋白酶-K（cathepsin K,Cts-K）mRNA表达水平;蛋白免疫印迹法（Western blot）检测肿瘤坏死因子受体相关因子6（TRAF6）及p65蛋白表达。结果：RANKL可成功诱导出TRAP阳性的多核细胞,提高破骨细胞主要调控因子TRAP,MMP-9和Cts-K mRNA以及TRAF6和p65蛋白表达水平。青蒿素、双氢青蒿素、蒿甲醚和青蒿琥酯均可显著降低RANKL诱导后异常增加的破骨细胞数、破骨细胞核数和破骨细胞直径（P〈0.05,P〈0.01）,青蒿素可明显降低TRAP mRNA表达水平（P〈0.01）,双氢青蒿素、蒿甲醚和青蒿琥酯可显著下调TRAP,MMP-9和Cts-K mRNA表达水平（均P〈0.01）,以蒿甲醚及青蒿琥酯作用较佳;此外,青蒿素及其衍生物也可以明显抑制RANKL诱导后异常增高的TRAF6和p65蛋白表达,其中青蒿琥酯的作用最显著（P〈0.01）。结论：青蒿素及其衍生物均具有抑制由RANKL诱导的小鼠BMMs向破骨细胞分化的作用,且相关作用可能与下调TRAP,MMP-9和Cts-K基因表达,抑制破骨细胞分化通路主要因子TRAF6/NF-κB的活化有关;相同浓度下蒿甲醚和青蒿琥酯的作用比青蒿素和双氢青蒿素略强。
Objective： To compare the effects of artemisinin,dihydroartemisinin,artesunate and artemether on osteoclasts differentiation,and investigate relevant signaling pathways.Method： Osteoclast differentiation was induced by receptor activator of nuclear factor-κB（NK-κB） ligand（RANKL） in bone marrowderived macrophages（BMMs）.After different concentrations of artemisinin,dihydroartemisinin,artemether and artesunate were treated,tartrate resistant acid phosphatase（TRAP）-positive multinucleated cells were observed by TRAP staining.TRAP,MMP-9 and cathepsin-K（Cts-K） mRNA expressions were detected by Real-time PCR,and TRAF6 and p65 protein expressions were detected by Western blot.Result： TRAP-positive multinucleated cells were successfully induced by RANKL,and TRAP,MMP-9 and Cts-K mRNA expressions,TRAF6 and p65 protein expression were significantly increased after treatment with RANKL.Importantly,artemisinin,dihydroartemisinin,artemether and artesunate could significantly reduce the number,nuclei and diameter of osteoclasts after induction with RANKL（P〈0.05,P〈0.01）,artemisinin obviously reduced TRAP mRNA expression（P〈0.01）,dihydroartemisinin,artemether and artesunate significantly down-regulated the mRNA expressions of TRAP,MMP-9 and Cts-K（P〈0.01）,and artemether and artesunate had a better therapeutic effect.In addition,the abnormal increase of TRAF6 and p65 proteins were obviously suppressed by artemisinin and artemisinin derivatives after induction with RANKL,and the results demonstrated that artesunate was the best one（P〈0.01）.Conclusion： Artemisinin and artemisinin derivatives could inhibit the effect of mouse BMMs in differentiating osteoclasts,which may be related to the down-regulation of TRAP,MMP-9 and Cts-K gene expressions,and the inhibition of TRAF6/NF-κB activation.The effect of artemether and artesunate is better than that of artemisinin and dihydroartemisininat at the same concentration.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2018年第4期84-89,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81703787,81274167)
