Icariin inhibits titanium particle-induced inflammatory reaction

doi:10.3969/j.issn.2095-4344.2014.16.017

Abstract

Abstract:

BACKGROUND: Studies in vitro have suggested that icariin can attenuate lipopolysaccharide (LPS)-induced acute pneumonia. Is the anti-inflammatory effect of icariin still valid in the presence of wear particles?

OBJECTIVE: With studies in vivo and in vitro, to investigate the regulatory effect of icarrin on titanium particle-induced inflammatory reaction.

METHODS: (1) Studies in vivo: Eighty male C57BL/6 mice aged 6-8 weeks were randomly divided into four groups: control group, icariin group, titanium particle group, and titanium particle+icariin group. Mice in the titanium particle group and titanium particle+icariin group received surgical procedure, and sterile and endotoxin-free titanium particles were implanted on the calvaria surfaces to induce inflammatory reaction. Mice in the control group and icariin group received the same surgery, but no wear particles were implanted. Then icariin was given orally to mice in the titanium particle group and titanium particle+ icariin group with a dose of 200 mg/kg per day for 2 weeks from the day of modeling. Mice in the control group and icariin group were given orally the same dose of placebo. Two weeks later, tumor necrosis factor-α and interleukin-1β at protein and mRNA levels were respectively detected with enzyme-linked immunohistochemical (ELISA) and quantitative real time reverse transcription PCR (qRT-PCR) analysis. (2) Studies in vitro: Mouse monocyte/macrophage RAW264.7 cells were cultured with different conditioned media: control group, nuclear factor receptor ligand кB (RANKL); icariin group, RANKL+icariin; titanium particle group, RANKL+titanium particles; titanium particle+icarrin group, RANKL+icariin+titanium particles. Titanium particles stimulated RAW264.7 cells were co-cultured with RANKL and icariin for 72 hours. Tumor necrosis factor-α and interleukin-1β at protein and mRNA levels in the supernatant were detected with ELISA analysis and qRT-PCR, respectively.

RESULTS AND CONCLUSION: (1) Results in vivo: icariin treatment obviously decreased titanium particle-induced inflammatory cell infiltration and made the thickness of periosteum thinner, down-regulated tumor necrosis factor-α and interleukin-1β expressions at protein and mRNA levels. (2) Results in vitro: when RAW264.7 cells were stimulated with titanium particles for 72 hours, tumor necrosis factor-α and interleukin-1β expressions at protein and mRNA levels in culture media increased obviously; when icariin was administrated, tumor necrosis factor-α and interleukin-1β expressions at protein and mRNA levels down-regulated significantly. These results suggest icariin can obviously suppress titanium particle-induced inflammatory reaction in vivo and in vitro.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: titanium, systemic inflammatory response syndrome, drugs, chinese herbal, tumor necrosis factor-alpha, interleukin-1beta

CLC Number:

R318

Cui Jing-fu, Xu Yao-zeng, Zhu Shi-jun, Zhu Feng, Fu Wen, Shao Hong-guo, Geng De-chun. Icariin inhibits titanium particle-induced inflammatory reaction[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(16): 2563-2569.

Figures/Tables 1

2.1 体内实验结果 2.1.1 实验动物一般情况术后各组小鼠均在1 h内苏醒并自由活动，正常进食，精神状态佳。实验期间小鼠切口愈合佳，无红肿、渗液等不良反应。实验过程中无动物死亡。 2.1.2 淫羊藿苷抑制钛颗粒诱导的炎性细胞浸润苏木精-伊红染色显示对照组和淫羊藿苷组小鼠颅骨骨膜内细胞总量较少，成纤维细胞较多，炎性细胞较少，骨膜较薄；与对照组相比，钛组小鼠颅骨骨膜增厚明显，骨膜内可见大量细胞浸润，炎性细胞居多，成纤维细胞较少，经淫羊藿苷后情况有所好转(图1)。Paint.NET软件测量结果表明对照组、淫羊藿苷组、钛组、钛+淫羊藿苷组颅骨骨膜厚度为分别为(0.07±0.01)，(0.06±0.01)，(0.28±0.04)，(0.12±0.02) mm。钛组与对照组颅骨骨膜厚度比较差异有显著性意义(P < 0.05)，说明钛颗粒的刺激使骨膜炎性增厚明显；钛+淫羊藿苷组与钛组颅骨骨膜厚度相比差异有显著性意义(P < 0.05)，说明淫羊藿苷治疗后炎性增厚的骨膜显著变薄；对照组及淫羊藿苷组骨膜厚度相比差异无显著性意义(P > 0.05)；淫羊藿苷组与钛+淫羊藿苷组颅骨骨膜厚度相比差异有显著性意义(P < 0.05)，这表明淫羊藿苷未能完全抑制钛颗粒诱导的炎症反应。 2.1.3 淫羊藿苷下调肿瘤坏死因子α、白细胞介素1β基因mRNA表达设定对照组肿瘤坏死因子α、白细胞介素1β mRNA表达量为1，钛组中二者表达量分别升至7.6±0.6和9.8±0.9，两组相比差异有显著性意义(P < 0.05)，这表明钛颗粒显著上调肿瘤坏死因子α、白细胞介素1β基因转录水平，钛颗粒的炎症诱导作用在基因层面表现明显；钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量分别降至2.4±0.2和2.1±0.2，与钛组相比差异有显著性意义(P < 0.05)，这表明淫羊藿苷抑制肿瘤坏死因子α、白细胞介素1β释放的机制涉及mRNA水平下调，这从基因层面解释了ELISA检测结果；淫羊藿苷组肿瘤坏死因子α、白细胞介素1β基因mRNA表达量分别为0.8±0.1和0.8±0.1，与对照组相比差异无显著性意义(P > 0.05)，这表明对于正常小鼠淫羊藿苷不能抑制肿瘤坏死因子α、白细胞介素1β基因表达量(图2A)。 2.1.4 淫羊藿苷抑制肿瘤坏死因子α、白细胞介素1β蛋白的释放对照组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度分别为(237.1±37.4) ng/L和(133.8±19.6) ng/L；钛颗粒植入后，肿瘤坏死因子α、白细胞介素1β质量浓度在钛组增长明显，分别达到(1447.8±78.2) ng/L和(889.2±45.9) ng/L，与对照组比较差异有显著性意义(P < 0.05)，这说明植入的钛颗粒刺激骨组织释放过量的炎症因子；钛+淫羊藿苷组经淫羊藿苷口服治疗后肿瘤坏死因子α、白细胞介素1β蛋白质量浓度下降至(568.8±42.3) ng/L和(279.3±19.4) ng/L，与钛组比较差异有显著性意义(P < 0.05)，这表明淫羊藿苷可显著抑制钛颗粒引起的骨组织炎症因子释放；但与淫羊藿苷组相比，钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度仍偏高(P < 0.05)，这表明淫羊藿苷的抑制作用是有限的，只是部分削弱了钛颗粒诱导的炎症因子释放但未能彻底消除，尽管抑制作用是不完全的，但炎症因子浓度的减少有利于局部反应的缓解和炎症细胞浸润减少，这与苏木精-伊红染色结果相一致；淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白质量浓度分别为(99.0±26.2) ng/L和(96.0±13.9) ng/L，与对照组比较差异无显著性意义(P > 0.05)，这表明在无钛颗粒刺激的情况下，淫羊藿苷对正常小鼠肿瘤坏死因子α、白细胞介素1β炎症因子释放无明显抑制作用(图2B)。 2.2 体外细胞培养实验结果 2.2.1 淫羊藿苷下调钛颗粒诱导的肿瘤坏死因子α、白细胞介素1β mRNA表达设定对照组RAW264.7细胞中肿瘤坏死因子α、白细胞介素1β mRNA表达量均为1。钛组肿瘤坏死因子α、白细胞介素1β基因转录量分别为4.1±1.0和10.1±2.0，与对照组相比差异有显著性意义(P < 0.05)，说明钛颗粒显著上调RAW264.7细胞中肿瘤坏死因子α、白细胞介素1β基因表达量；钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量下调至2.1±0.8和7.0±1.2，与钛组相比差异有显著性意义(P < 0.05)，说明淫羊藿苷显著抑制钛颗粒诱导的炎症因子基因表达量；淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA表达量分别为0.9±0.1和0.9±0.1，与对照组相比差异无显著性意义(P > 0.05)，这表明无钛颗粒刺激的环境下，淫羊藿苷对RAW264.7细胞肿瘤坏死因子α、白细胞介素1β基因表达量无明显影响，这与体内PCR结果相一致；与淫羊藿苷组相比，钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β mRNA水平均显著上调(P < 0.05)，这表明钛颗粒刺激环境下淫羊藿苷对炎症因子抑制作用的不完全性(图3A)。 2.2.2 淫羊藿苷抑制钛颗粒诱导的肿瘤坏死因子α、白细胞介素1β蛋白释放对照组肿瘤坏死因子α、白细胞介素1β蛋白水平分别为(79.0±12.8) ng/L和(66.0±7.8) ng/L。钛组肿瘤坏死因子α、白细胞介素1β蛋白水平为(259.4±17.1) ng/L和(265.8±14.2) ng/L，与对照组相比差异有显著性意义(P < 0.05)，说明钛颗粒刺激后炎症因子浓度上升明显；钛+淫羊藿苷组肿瘤坏死因子α、白细胞介素1β水平分别为(147.2±17.1) ng/L和(132.1±9.2) ng/L，与钛组相比差异有显著性意义(P < 0.05)，说明淫羊藿苷干预可显著降低钛颗粒诱导的炎症因子释放；淫羊藿苷组肿瘤坏死因子α、白细胞介素1β蛋白水平分别为(56.0±9.6) ng/L和(47.0±6.3) ng/L，与对照组相比差异无显著性意义(P > 0.05)，这说明淫羊藿苷对正常RAW264.7细胞炎症因子释放无明显抑制作用，这一结果与体内ELISA分析结果一致(图3B)。"

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