基于RAW264.7巨噬细胞炎症模型探讨白及抗肺纤维化活性组分及分子机理

发布时间：2018-04-08 17:04

本文选题：白及　切入点：肺纤维化　出处：《浙江中医药大学》2017年硕士论文

【摘要】：目的本实验采用脂多糖(LPS)刺激小鼠单核巨噬细胞白血病细胞(RAW264.7),建立RAW264.7炎症模型,通过该模型指导白及活性组分分离纯化,从分子水平研究白及活性单体成分对LPS刺激RAW264.7炎症模型的抑制作用,并探讨其作用机制。方法体外培养RAW264.7细胞,通过LPS刺激RAW264.7细胞建立巨噬细胞炎症模型,采用RT-PCR方法确定LPS诱导RAW264.7细胞IL-1β mRNA表达升高的最适浓度和最适作用时间。运用醇提-聚酰胺色谱分离制备白及醇提物,用巨噬细胞炎症模型评价药物效应,指导白及活性组分分离纯化,并对单体成分进行结构鉴定。采用MTS法检测细胞存活率,确定白及单体对LPS刺激RAW264.7细胞最适作用浓度。光学显微镜下观察各实验组RAW264.7形态学变化,流式细胞术检测RAW264.7细胞胞外IL-6、TNF-α和MCP-1 的细胞因子含量;RT-PCR检测IL-1β、IL-6、iNOS、COX2、TNF-α、TGF-β的mRNA表达情况;Western Blot检测白及单体对LPS刺激RAW264.7细胞MAPK通路、NF-κB通路、COX2、iNOS、p-IRF3/IRF3的蛋白表达情况。结果1.通过RT-PCR法检测LPS对RAW264.7细胞IL-1β的mRNA相对表达量的影响,IL-1β mRNA相对表达量随着LPS浓度和作用时间增加而升高,且呈剂量依赖性,其中200 ng/mL LPS刺激6 h,即可使IL-1β的mRNA相对表达量提高将近1000倍。2.白及提取物经聚酰胺柱色谱分离所得五个粗分组分经HPLC表征,发现各粗分样品间交叉峰较少,分离效果较好。通过RAW264.7细胞炎症模型,结合RT-PCR分析表明,除80%乙醇洗脱粗分组分(E80)外,其余各粗分组分均可剂量依赖性降低RAW264.7细胞IL-1βmRNA相对表达量,与模型组比较有极显著性差异(P0.01),其中40%乙醇洗脱组分(E40)抑制效果最为显著。3.白及40%乙醇洗脱粗分活性组分(E40)再经硅胶柱细分为E40-(1-6)六个亚组分,并进一步采用RAW264.7细胞炎症模型和RT-PCR分析IL-Iβ mRNA表达的抑制活性。结果表明,不同浓度各亚组分联合LPS作用于RAW264.7细胞6h后,其IL-1βmRNA相对表达量随剂量增加而降低;与模型组相比,除E40-6外,其余各亚组分均有极显著性差异P0.01,其中尤以E40-3、E40-4抑制作用最明显。4.采用半制备液相色谱法对白及E40-3和E40-4两个活性亚组份进行分离、纯化,核磁共振波谱鉴定RT16.6为贝母兰宁(2,7-二羟基-4-甲氧基-9,10-二氢菲),RT21.0为 batatasin Ⅲ(3',3"-二羟基-5'-甲氧基联苄)。5.MTS检测不同浓度白及单体及联合LPS对RAW264.7细胞存活率的影响,未加LPS时,与正常对照组相比,单体组分RT16.6和RT21.0在低剂量时均有一定促进增殖作用,但浓度高于15 μg/mL时,对细胞有一定毒性。不同浓度单体成分联合LPS作用于RAW264.7 24 h后,与正常对照组相比,RT16.6在20 μg/mL、RT21.0在25μg/mL时,对细胞有一定毒性。6.光学显微镜下观察不同浓度白及单体作用于RAW264.7对其细胞形态学的影响,加入不同浓度白及单体后,与正常细胞相比,白及单体RT16.6在1-5μg/mL、RT21.0在2.5-10 μg/mL剂量范围内细胞数目略有增加,形态没有明显变化。7.采用流式细胞术检测不同浓度白及单体成分对LPS刺激RAW264.7细胞相关炎症因子表达影响,结果表明,模型组LPS刺激后可显著增加RAW264.7细胞培养上清中MCP-1、TNF-a、IL-6的含量,与正常组比较具有显著性差异(P0.01)。与模型组相比,白及单体化合物RT16.6可剂量依赖性降低RAW264.7细胞外上清MCP-1和IL-6的含量,具有显著性差异(P0.01);低剂量亦可降低TNF-a分泌量,但高剂量反而促进TNF-a分泌。白及单体RT21.0也可剂量依赖性降低MCP-1、TNF-a、IL-6的分泌,具有显著性差异(P0.01)。8.荧光定量PCR检测不同浓度白及单体对LPS刺激RAW264.7细胞炎症相关基因mRNA表达的影响,与空白对照组相比,模型组加LPS后可增加IL-1β、IL-6、iNOS、COX2、TNF-α mRNA相对表达量,具有显著性差异(P0.01),也增加TGF-βmRNA相对表达量,具有显著性差异(P0.05)。白及单体RT16.6可减少各炎症基因的mRNA相对表达量,具有剂量依赖性。但在低剂量1 μg/mL时,COX2、TNF-α、TGF-β mRNA相对表达量比LPS组高,这可能与低剂量促进细胞增殖有关。白及单体RT21.0也可减少各炎症基因的mRNA相对表达量,除了 TNF-α外,均具有剂量依赖性,与模型组比较具有显著性差异(P0.01)。9.Western Blot检测白及单体对LPS刺激RAW264.7细胞MAPK通路、NF-κB通路、COX2、iNOS、p-IRF3/IRF3的蛋白表达影响。结果表明,与正常对照组相比,模型组 IKK、IKBα、NF-KB、ERK1/2、JNK、P38、IRF3 磷酸化水平均明显升高(P0.01),COX2、iNOS蛋白水平均明显升高(P0.01);而与模型组相比,白及单体RT16.6和RT21.0均能够显著减少IKK、IKBα、NF-κB、JNK、IRF3磷酸化水平(P0.01);在一定程度上也可剂量依赖性地抑制iNOS、COX2蛋白表达(P0.01)。此外,白及单体RT16.6显著增加ERK、P38磷酸化(P0.01);白及单体RT21.0能够显著减少P38磷酸化(P0.01),增加ERK磷酸化;结论1.LPS浓度200 ng/mL刺激6 h即可显著增加RAW264.7细胞相关炎症基因表达水平,可用于相关药物抗炎活性筛选及相关分子机理研究。2.用上述条件建立的RAW264.7巨噬细胞炎症模型评价药物抗炎效应,结合柱色谱技术方法,指导白及活性组分分离纯化,获得两个单体成分,经核磁共振波谱鉴定RT16.6为贝母兰宁(2,7-二羟基-4-甲氧基-9,10-二氢菲),RT21.0为batatasin Ⅲ(3',3"-二羟基-5'-甲氧基联苄)。3.白及药效单体成分治疗肺纤维化疾病的可能分子机制为通过作用于NF-κB、p-IRF3/IRF3、MAPK、iNOS 和 COX2 等多个信号通路及靶点,降低 IL-1β、IL-6、iNOS、COX2、TNF-α和TGF-β等炎症基因表达和抑制MCP-1、TNF-α、IL-6等炎症因子分泌而实现。
[Abstract]:The purpose of this experiment using lipopolysaccharide (LPS) stimulation of leukemic cells in mouse macrophage (RAW264.7), a RAW264.7 model of inflammation, purified by the Rhizoma Bletillae model to guide the active component from the inhibition of the molecular level Rhizoma Bletillae active monomer composition on LPS stimulated RAW264.7 inflammation model, and explore its mechanism. Methods RAW264.7 cells were cultured in vitro, establishment of macrophage inflammatory model by LPS stimulation of RAW264.7 cells, using RT-PCR method to determine the expression of the optimal concentration and the best time of RAW264.7 cells induced by LPS. IL-1 beta mRNA with ethanol and separated polyamide chromatography prepared by Rhizoma Bletillae alcohol extract, evaluation of drug effect of macrophage inflammatory model, guide the Rhizoma Bletillae active component separation purification and structural identification of the monomer composition. Cell viability was determined by MTS assay, determine the Rhizoma Bletillae monomer on the LPS optimal stimulation of RAW264.7 cells The concentration of each experimental group. Changes of RAW264.7 morphology observed under light microscope, flow cytometry was used to detect RAW264.7 cell extracellular IL-6, cytokine levels of TNF- alpha and MCP-1; RT-PCR IL-6, detection of IL-1 beta, iNOS, COX2, TNF- alpha, TGF- beta mRNA expression; Western Blot test Rhizoma Bletillae monomer on LPS stimulation of RAW264.7 cells MAPK NF- pathway, B pathway, COX2, iNOS, expression of p-IRF3/IRF3 protein. Results 1. detected by LPS RT-PCR method on RAW264.7 cells of IL-1 beta mRNA expression effects of IL-1 beta mRNA expression increased with LPS concentration and action time increased in a dose-dependent manner, which 200 ng/mL LPS 6 h stimulation, can make the IL-1 beta mRNA expression increased nearly 1000 times the Rhizoma Bletillae.2. extract by polyamide column chromatography separation of the five crude grouping was characterized by HPLC, found that the crude sample cross peak is less, the separation effect Good. By RAW264.7 inflammation model, combined with RT-PCR analysis showed that in 80% ethanol elution coarse grouping (E80), the rest of the points can be roughly divided into groups dose dependently reduced the relative expression of RAW264.7 cells IL-1 beta mRNA, there was a significant difference compared with the model group (P0.01), 40% ethanol fraction (E40) the most significant inhibitory effect of Rhizoma Bletillae.3. 40% ethanol crude active component (E40) by silica gel column E40- (1-6) divided into six subgroups, and further the inhibitory activity of RAW264.7 inflammation model and RT-PCR analysis of IL-I beta mRNA expression. The results showed that different concentrations of each sub group of joint the effect of LPS on RAW264.7 6h cells after the IL-1 beta mRNA relative expression decreased with the dose increased; compared with the model group, except E40-6, the other subfractions showed very significant difference between P0.01, especially E40-3, E40-4 inhibited by the most obvious.4. by semi system Purification by liquid chromatography on E40-3 and E40-4 and two active subfractions were isolated, nuclear magnetic resonance spectroscopy of RT16.6 was identified as Lan Ning Fritillary (2,7- two hydroxy -4- methoxy -9,10- two RT21.0 batatasin hydrogen Philippines), III (3', 3 "- two hydroxy -5'- methoxy bibenzyl).5.MTS detection of different concentrations of monomer and Rhizoma Bletillae combined with LPS on the survival rate of RAW264.7 cells, without LPS, compared with the normal control group, single component RT16.6 and RT21.0 were at a low dose could promote the proliferation, but the concentration is higher than 15 g/mL, have a certain toxicity to the cells. Different concentrations of monomer composition combined with the effect of LPS on RAW264.7 24 h, compared with the normal control group, RT16.6 20 g/mL, RT21.0 25 g/mL,.6. have a certain toxicity under optical microscope observation of different concentrations of Rhizoma Bletillae monomer in RAW264.7 affects the cell morphology of cells with different concentrations of Rhizoma Bletillae The monomer, compared with normal cells, Rhizoma Bletillae monomer RT16.6 1-5 g/mL, RT21.0 cells in the 2.5-10 range of g/mL number increased slightly, morphology did not change significantly.7. influence on the expression of LPS in RAW264.7 cells stimulated by different concentrations of inflammatory cytokines in Rhizoma Bletillae flow cytometry monomer composition. The results showed that the model group after LPS stimulation can significantly increase the RAW264.7 cell culture supernatant of MCP-1, TNF-a, IL-6 content, compared with the normal group with significant difference (P0.01). Compared with the model group, Rhizoma Bletillae monomer compound RT16.6 can dose dependently reduced RAW264.7 content of extracellular supernatant of MCP-1 and IL-6, with significant difference (P0.01) low dose; reduce TNF-a secretion, but high dose but to promote the secretion of TNF-a. Rhizoma Bletillae RT21.0 monomer also dose dependently decreased MCP-1, TNF-a, IL-6 secretion, with significant difference (P0.01).8. fluorescence quantitative PC Effect of R to detect different concentrations of Rhizoma Bletillae monomers on LPS stimulated mRNA gene RAW264.7 expression of inflammatory cells, compared with the control group, the model group after the addition of LPS can increase the IL-1 beta, IL-6, iNOS, COX2, the relative expression of TNF- alpha mRNA, with significant difference (P0.01), also increased the relative expression of TGF- beta mRNA, with a significant difference (P0.05). Rhizoma Bletillae RT16.6 monomer can reduce the inflammatory gene relative expression of mRNA, in a dose dependent manner. But in the low dose 1 g/mL, COX2, TNF- alpha, TGF- beta mRNA relative expression quantity is higher than the LPS group, which may promote cell proliferation associated with low dose Rhizoma Bletillae RT21.0 monomer can also reduce the inflammatory gene relative expression of mRNA, TNF- in addition to alpha, both in a dose dependent manner. Compared with the model group with significant difference (P0.01).9.Western Blot detection of Rhizoma Bletillae monomer on LPS RAW264.7 cells stimulated MAPK pathway, NF- B pathway, COX2, iNOS, The expression of p-IRF3/IRF3 protein. Results show that, compared with the normal control group, model group, IKK, NF-KB, ERK1/2, IKB alpha, JNK, P38, IRF3 phosphorylation levels were significantly increased (P0.01), COX2, iNOS protein levels were significantly increased (P0.01); compared with the model group, Rhizoma Bletillae and RT16.6 monomer RT21.0 could significantly reduce IKK, IKB alpha, NF- kappa B, JNK, phosphorylation of IRF3 (P0.01); to a certain extent also dose dependently inhibited the expression of COX2 (iNOS, P0.01). In addition, Rhizoma Bletillae monomer RT16.6 significantly increased ERK, phosphorylation of P38 (P0.01); Rhizoma Bletillae monomer RT21.0 can significantly reduce the phosphorylation of P38 (P0.01), increased the phosphorylation of ERK; conclusion 1.LPS concentration of 200 ng/mL 6 h stimulation significantly increased RAW264.7 gene expression level of inflammatory cells, can be used to study the anti-inflammatory activity of related drug screening and related molecular mechanism of.2. with RAW264.7 macrophage inflammatory conditions above established The evaluation model of drug anti-inflammatory effect, combined with column chromatography method, guiding Rhizoma Bletillae active component separation and purification, two monomer composition, the NMR spectrum of RT16.6 was identified as Lan Ning Fritillary (2,7- two hydroxy -4- methoxy -9,10- two RT21.0 batatasin hydrogen Philippines), III (3', 3 "- two hydroxy -5'- methyl oxy bibenzyl) possible molecular mechanism of.3. Rhizoma Bletillae efficacy monomer composition for treatment of diseases of pulmonary fibrosis by acting on NF- kappa B, p-IRF3/IRF3, MAPK, iNOS and COX2 multiple signaling pathways and targets, reduce IL-1 beta, IL-6, iNOS, COX2, expression of TNF- alpha and TGF- beta gene and inhibiting inflammation MCP-1, TNF- alpha, IL-6 and other inflammatory cytokines secretion.

【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563

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