为研究红曲黄素C(monascinol,MC)、红曲素(monascin,MS)和红曲黄素(ankaflavin,AK)的抗炎机理,从红曲菌CP-1固态发酵后得到的红曲米中提取分离出红曲黄色素MC,并使用薄层层析法(thin-layer chromatography,TLC)和HPLC进行纯度分析。构建2 μg/mL的脂多糖(lipopolysaccharide,LPS)诱导人THP-1巨噬细胞和小鼠RAW264.7巨噬细胞炎症模型,并进行形态学观察,利用噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法测定细胞活力,以MS和AK作为参比,实时荧光定量法(quantitative real-time polymerase chain reaction,RT-qPCR)和酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)测定红曲黄色素MC对脂多糖诱导的THP-1细胞和RAW264.7细胞中白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的影响。结果表明,提取出的红曲黄色素MC纯度能达到98%以上,可用于细胞实验。6 μg/mL的MC、MS和AK诱导THP-1细胞24 h后,细胞存活率均能达到90％以上,符合实验要求。6 μg/mL的MC、MS和AK处理THP-1细胞和RAW264.7细胞24 h后,在转录水平和翻译水平上均显著抑制了IL-6的表达(P<0.01)。MC在转录水平和翻译水平上显著抑制了THP-1细胞和RAW264.7细胞中TNF-α的表达量(P<0.01),MS和AK仅在翻译水平上显著抑制了RAW264.7细胞TNF-α的分泌(P<0.01)。
To investigate the anti-inflammatory mechanisms of monascinol (MC), monascin (MS), and ankaflavin (AK), the Monascus yellow pigment MC was extracted and separated from the red yeast rice obtained by the solid-state fermentation of Monascus CP-1 and then its purity was analyzed using thin-layer chromatography (TLC) and HPLC. Inflammatory models of human THP-1 macrophages and mouse RAW264.7 macrophages were established using 2 μg/mL lipopolysaccharide (LPS), and morphological observations were conducted. Cell viability was determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. The effect of Monascus yellow pigment MC on the expression of IL-6 and TNF-α in LPS-induced THP-1 and RAW264.7 cells was measured using quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), with MS and AK as references. The results indicated that the purity of MC reached over 98%, making it suitable for cellular experiments. After 24 hours of treatment with 6 μg/mL of MC, MS, and AK, the cell viability of THP-1 cells exceeded 90%, meeting the experimental requirements. After treating THP-1 cells and RAW264.7 cells with 6 μg/mL of MC, MS, and AK for 24 hours, the expression of interleukin-6 (IL-6) was significantly suppressed at both the transcriptional and translational levels (P<0.01). MC inhibited the expression of tumor necrosis factor-α (TNF-α) in THP-1 and RAW264.7 cells at both the transcriptional and translational levels significantly (P<0.01), whereas MS and AK only suppressed the secretion of TNF-α in RAW264.7 cells at the translational level significantly (P<0.01).
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