通过体外及体内实验探讨青蒿琥酯对磨损颗粒诱导炎性骨溶解的影响。

体外实验：选取生长良好的小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞株，分为4组进行培养：空白对照组；Ti颗粒组，加入钛颗粒并进行成骨分化培养；青蒿琥酯组，添加10 μmol/L青蒿琥酯并进行成骨分化培养；Ti+青蒿琥酯组同时加入钛颗粒和10 μmol/L青蒿琥酯进行成骨分化培养。使用细胞计数试剂盒-8（CCK-8）测定细胞的活性，同时通过流式细胞技术分析细胞的凋亡情况。体内实验：40只C57BL/6J小鼠按随机数字表法分4组，Ti颗粒组和Ti+青蒿琥酯组切开小鼠头皮植入钛，建立小鼠颅骨骨溶解模型；假手术组（Sham组）、青蒿琥酯组与上述手术步骤相同，但不植入钛。青蒿琥酯组、Ti+青蒿琥酯组建模完成后经灌胃给予青蒿琥酯50 mg/(kg·d)。2周后处死小鼠，苏木素－伊红（HE）染色观察颅骨病理变化；酶联免疫吸附实验（ELISA）检测炎性因子的表达水平，包括肿瘤坏死因子（TNF）α、白细胞介素（IL）1β、IL 6；实时荧光定量聚合酶链式反应（RT-PCR）检测成骨分化相关基因碱性磷酸酶（ALP）、骨钙素（OCN）、矮小相关转录因子-2（Runx-2）、Ⅰ型胶原（COLⅠ）的表达。给药2周后，进行颅骨显微计算机断层扫描（Micro-CT）。计量资料组间比较采用独立样本t检验或单因素方差分析，多重比较采用LSD检验，计数资料组间比较采用卡方检验。

体外实验：CCK-8结果显示，当青蒿琥酯浓度≤10 umol/L时，对成骨细胞增殖无明显影响；当Ti颗粒浓度≤0.5mg/ml时，对MC3T3-E1细胞增殖无明显影响。流式细胞术结果显示，钛颗粒可促进MC3T3-E1细胞的凋亡（t=57.46，P<0.001）；青蒿琥酯可抑制钛颗粒的作用（t=19.64，P<0.01）。体内实验：HE染色结果显示，Ti颗粒组颅骨周围组织中的炎症细胞数量及骨质破坏程度明显高Ti+青蒿琥酯组、青蒿琥酯组和Sham组；经青蒿琥酯干预后炎症反应减轻，骨质破坏得到显著改善。ELISA及PCR结果显示，与Sham组相比，Ti颗粒刺激3种炎性因子浓度显著增加（t=8.872、15.6、18.71，均为P<0.05），成骨相关基因mRNA表达量下降（t=18.31、20.47、23.95、27.22，均为P<0.05）。而与Ti颗粒组相比，Ti+青蒿琥酯组3种炎性因子表达量均有不同程度下调（t=4.672、4.805、3.405，均为P<0.01），成骨相关基因的表达增加（t=12.2、15.15、22.02、16.99，均为P<0.05）。micro-CT扫描显示，Ti颗粒导致骨质破坏增加，促进小鼠颅骨周围骨溶解，青蒿琥酯干预后可缓解钛颗粒引起的骨溶解。

青蒿琥酯可通过抑制炎症因子、上调成骨细胞功能来改善钛颗粒诱导的骨溶解、骨破坏。

To explore the effects of artesunate on wear particle-induced inflammatory osteolysis explored by in vitro and in vivo experiments.

In vitro experiments: healthy mouse embryonic osteoblast precursor cells MC3T3-E1 cells were selected and divided into four groups for culture: blank control group; Ti particle group, where titanium particles were added for osteogenic differentiation culture; artesunate group, where 10 μmol/L artesunate ester was added for osteogenic differentiation culture; combination group, where titanium particles and 10 μmol/L artesunate ester were added simultaneously for osteogenic differentiation culture. Cell activity was determined using the cell counting kit-8 (CCK-8), while apoptosis was analyzed by flow cytometry. In vivo experiments: forty C57BL/6J mice were randomly divided into four groups using a random number table. In the Ti particle group and the Ti + artesunate group, the mice’s scalps were incised and titanium was implanted to establish a mouse skull bone resorption model. In the sham group and the artesunate group, the same surgical procedures were performed without implanting titanium. After establishing the artesunate group and the titanium plus artesunate group, artesunate was administered by gavage at a dose of 50 mg/(kg·d). The mice were executed after two weeks, and hematoxylin-eosin method (HE) staining was used to observe the pathological changes of the skull; enzyme-linked immunosorbent assay (ELISA) was performed to measure the expression levels of inflammatory factors, tumor necrosis factor (TNF) α, interleukin (IL)-1β, and IL -6. Real-time quantitative polymerase chain reaction (RT-PCR) was used to assess the expression of osteogenic differentiation-related genes including alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx-2), and collagen type-Ⅰ (COL I). Two weeks after dosing, Micro-CT of the skull was conducted. For comparisons between groups of measurement data, independent samples t test or one-way analysis of variance was used; for multiple comparisons, LSD test was used; and for comparisons between groups of count data, chi square test was employed.

In vitro experiments: CCK8 results showed that at artesunate concentrations ≤10 μmol/L, there was no significant effect on osteoblast proliferation. Titanium particle concentrations≤0.5 mg/ml did not significantly affect the proliferation of MC3T3-E1 cells. Flow cytometry results showed that titanium particles promoted apoptosis in MC3T3-E1 cells (t=57.46, P<0.001); artesunate inhibited the effect of titanium particles (t=19.64, P<0.01). In vivo experiments: HE staining showed that the number of inflammatory cells and the degree of bone destruction in the peri-cranial tissues of the Ti granules group were significantly higher than those of the Ti+artesunate, artesunate, and Sham groups; the inflammatory response was reduced and bone destruction was significantly improved after artesunate intervention. ELISA and PCR results showed that compared to sham group, the Ti particles stimulated a significant increase in the concentration of the three inflammatory factors (t=8.872, 15.6, 18.71, all P<0.05) and a decrease in the mRNA expression of osteogenesis-related genes (t=18.31, 20.47, 23.95, 27.22, all P<0.05). In contrast, compared with the Ti particles group, the Ti + artesunate group showed different degrees of down-regulation of the expression of the three inflammatory factors (t=4.672, 4.805, 3.405, all P<0.01) and increased expression of osteogenesis-related genes (t=12.2, 15.15, 22.02, 16.99, all P<0.05). Micro-CT scans showed that titanium particles led to an increase in bone destruction and induced osteolysisaround the skull, and that the titanium particle-induced osteolysis could be mitigated by the intervention of artesunate.

Artesunate can improve titanium particles induced osteolysis and bone destruction by inhibiting inflammatory factors and up-regulating the function of osteoblasts.