利格列汀调控巨噬细胞极化和破骨细胞形成缓解磨损颗粒诱导的骨质溶解

Linagliptin alleviates wear particle-induced inflammatory osteolysis by regulating macrophage polarization and osteoclast formation

背景:研究发现,在利格列汀的干预下,巨噬细胞更多的由M1转向M2极化,降低了相关炎性因子的释放,缓解了局部炎症。目的:探讨利格列汀对巨噬细胞极化、破骨细胞活化及磨损颗粒诱导炎性骨溶解的影响。方法:(1)细胞实验:①巨噬细胞极化:将RAW264.7细胞分4组培养,对照组细胞加入高糖培养基;M1诱导组加入M1诱导培养基(含脂多糖100 ng/mL和干扰素γ20 ng/mL的高糖培养基)模拟炎症环境;利格列汀低、高剂量组分别加入50,200 nmol/L利格列汀处理4 h后加入M1诱导培养基。巨噬细胞极化诱导24 h后,分别进行巨噬细胞极化免疫荧光染色和RT-PCR检测。②破骨细胞活化:将RAW264.7细胞分为4组培养,对照组使用高糖培养基培养,破骨细胞诱导组、利格列汀低剂量组及高剂量组进行破骨细胞诱导,待微小破骨细胞成形后,用利格列汀(50,200 nmol/L)分别干预细胞3 d,进行细胞抗酒石酸酸性磷酸酶染色和RT-PCR检测。(2)动物实验:将24只雄性C57BL/6J小鼠随机分为4组,即假手术组、模型组、利格列汀低剂量组及高剂量组,后3组通过将钛颗粒悬浊液注射至颅骨表面建立颅骨骨溶解模型,从造模后第2天开始,利格列汀低、高剂量组分别灌胃利格列汀(2,10 mg/kg),每天1次,造模3周后,检测血清巨噬细胞极化标志蛋白及炎性因子水平,收集颅骨进行micro-CT扫描、骨参数分析及苏木精-伊红染色评估骨溶解及形态学变化。结果与结论:①细胞实验:与M1诱导组比较,利格列汀低、高剂量组可显著抑制巨噬细胞的M1极化、促进M2极化(P<0.01),且以高剂量组效果更显著(P<0.01)。与破骨诱导组比较,利格列汀低、高剂量组可抑制破骨细胞的活化及骨质吸收,且以高剂量组抑制更显著。与对照组比较,M1诱导组炎性因子mRNA表达升高(P<0.01),而与M1诱导组相比较,利格列汀低、高剂量组炎性因子mRNA表达显著降低(P<0.01)。与对照组比较,破骨诱导组破骨功能标志物的mRNA表达升高(P<0.01);与破骨诱导组比较,利格列汀低、高剂量组破骨功能标志物的mRNA表达降低(P<0.01),且以高剂量组降低更明显。②动物实验:钛颗粒植入导致小鼠颅骨骨溶解破坏,利格列汀可抑制钛颗粒诱导的骨溶解,其中以高剂量组抑制作用更显著。结果表明利格列汀具有调节巨噬细胞极化、抑制破骨细胞活化及对骨骼系统的保护作用。

BACKGROUND:Linagliptin exhibits the capacity to regulate macrophage polarization,shifting them from the pro-inflammatory M1 phenotype towards the antiinflammatory M2 phenotype.This alteration results in a dampened release of inflammatory mediators,thereby mitigating local inflammation.OBJECTIVE:To explore the effects of linagliptin on macrophage polarization,osteoclast activation,and inflammatory osteolysis elicited by wear particles.METHODS:(1)Cell experiments:For macrophage polarization,RAW264.7 cells were cultured and divided into four groups:the control group received highglucose culture medium;the M1-induced group received M1-inducing culture medium(high-glucose culture medium containing 100 ng/mL lipopolysaccharide and 20 ng/mL interferon-γ)to simulate an inflammatory environment;the low-and high-dose linagliptin groups were treated with 50 and 200 nmol/L linagliptin,respectively,for 4 hours before exposure to M1-inducing culture medium.After 24 hours of macrophage polarization induction,immunofluorescence staining and RT-PCR were performed.For osteoclast activation,RAW264.7 cells were cultured and divided into four groups:the control group was cultured with high-glucose culture medium,the osteoclast-induced group and low-and high-dose linagliptin groups were subjected to osteoclast induction.After osteoclast formation,cells were treated with linagliptin(50 and 200 nmol/L)for 3 days.Subsequently,cell tartrate-resistant acid phosphatase staining and RT-PCR were performed.(2)Animal experiments:Twenty-four male C57BL/6J mice were randomly divided into four groups:sham operation group,model group,low-dose linagliptin group,and high-dose linagliptin group.The model group,low-dose linagliptin group,and high-dose linagliptin group were induced to establish a cranial bone resorption model by injecting titanium particle suspension onto the surface of the skull.Starting from the 2nd day after modeling,the low-and high-dose linagliptin groups were orally administered linagliptin(2 and 10 mg/kg,respectively)once daily.After modeling for 3 weeks,serum macrophage polarization marker protein and inflammatory factor levels were detected;skull samples were collected for micro-CT scanning,bone parameter analysis,and hematoxylin-eosin staining to evaluate osteolysis and morphological changes.RESULTS AND CONCLUSION:(1)Cell experiments:Both low and high doses of linagliptin significantly suppressed M1 polarization while promoting M2 polarization compared to the M1-induced group(P<0.01).Notably,the high-dose group exhibited a more pronounced inhibitory effect(P<0.01).Inflammatory factor mRNA expression was elevated in the M1-induced group compared with the control group(P<0.01),whereas inflammatory factor mRNA expression was significantly lower in the low-and high-dose linagliptin groups compared with the M1-induced group(P<0.01).There was a significant upregulation of mRNA expression of osteoclast functional markers in the osteoclast-induced group compared with the control group(P<0.01).Conversely,both low and high doses of linagliptin led to a substantial downregulation of mRNA expression of these markers compared with the osteoclast-induced group(P<0.01),with the high-dose group exhibiting a more pronounced reduction.(2)Animal experiments:Titanium particle implantation induced cranial bone resorption damage in mice.Treatment with linagliptin effectively mitigated this bone resorption,with the high-dose group showing superior efficacy.To conclude,linagliptin has been shown to modulate macrophage polarization,inhibit osteoclast activation,and have a protective effect on the skeletal system.