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目的:研究仙茅提取物对小鼠巨噬细胞株RAW264.7的诱导活化作用。方法:应用水提醇沉方式制备仙茅提取物,其中多糖含量为50%。将不同浓度的仙茅提取物(31.25~500μg/ml)作用于RAW264.7细胞36h后,采用流式细胞术检测膜表面模式识别受体TLR2/4和MR的表达;实时荧光定量PCR(RT-PCR)检测细胞i NOS和TNF-α的mRNA表达水平;收集培养上清液,用ELISA法测定中TNF-α含量,Griess法检测NO的含量。结果:仙茅提取物的剂量在500μg/ml以下时,对细胞增殖影响不明显;与空白对照组相比,浓度高于62.5μg/ml的仙茅提取物刺激RAW264.7细胞可使TNF-a、i NOS mRNA表达量显著升高,促进细胞分泌活性因子TNF-α和NO;仙茅提取物可以有效增加RAW264.7细胞表面的模式识别受体的表达,TLR2由空白组的31.25±5.19上升到39.82±4.53,TLR4由空白组的6.41±2.44上升到24.34±5.22,MR由空白组的7.35±3.54上升到36.24±9.53,且均呈现剂量-反应关系。结论:仙茅提取物可能通过影响巨噬细胞膜表面部分模式识别受体的表达使TNF-α和NO的分泌量增多,诱导巨噬细胞活化,具有潜在的免疫调节价值,此作用与仙茅传统用于治疗虚劳内伤、现代用于免疫调节的临床应用有关。
Objective: To study the inducing and activating effect of Curculigo orchioides extract on mouse macrophage cell line RAW264.7. Method: The extract of Curculigo orchillum was prepared by water-alcohol precipitation method, in which polysaccharide content was 50%. Flow cytometry was used to detect the expression of TLR2 / 4 and MR on the membrane surface of RAW264.7 cells after treated with different concentrations of Curculigo orchids extracts (31.25-500 μg / ml). Real-time fluorescence quantitative PCR -PCR) were used to detect the mRNA expression of iNOS and TNF-α. The culture supernatants were collected and the content of TNF-α was determined by ELISA. The content of NO was detected by Griess method. Results: Compared with the blank control group, the effect of Curculigo orchids extract on the cell proliferation was insignificant when the dose was below 500μg / ml. Compared with the blank control group, the Curculigo orchids extract at a concentration higher than 62.5μg / ml could stimulate the production of TNF- a, i NOS mRNA expression was significantly increased, and promote cell secretion of active factors TNF-α and NO; Curculigo extract can effectively increase the expression of pattern recognition receptors on the surface of RAW264.7 cells, TLR2 from the blank group of 31.25 ± 5.19 Increased to 39.82 ± 4.53, TLR4 increased from 6.41 ± 2.44 in the blank group to 24.34 ± 5.22, and MR increased from 7.35 ± 3.54 in the blank group to 36.24 ± 9.53 in the blank group, showing a dose-response relationship. CONCLUSION: Curculigo koreanum extract may exert potentially immunomodulatory value by affecting the expression of some pattern recognition receptors on the membrane surface of macrophages, increasing the secretion of TNF-α and NO and inducing the activation of macrophages. For the treatment of intractable internal and external injuries, the modern clinical application of immune regulation.