蒙药复方特润舒都乐石油醚提取物及其主要单体抗炎信号通路的研究
|
摘要
炎症（inflammation）作为一种临床常见的症状，普遍发生于机体各个部位。蒙医药是内蒙古特色医药。前期研究已经确立了蒙药复方特润舒都乐“Terunsodolol”，具有很好的抗炎和提高免疫作用，在动物模型以及临床奶牛乳房炎的治疗中取得了显著疗效，但尚存在用药剂量过大、作用机理不清楚等问题，故进一步研究其抗炎作用显著的石油醚提取物及主要单体的抗炎信号转导途径，旨在细胞分子水平上为其二次开发奠定理论基础。
为此，应用天然药物化学、有机化学等知识，利用液相色谱等技术分离鉴定有效活性成分，结合文献指导，确立了石油醚提取物有效单体成分，利用脂多糖（LPS）诱导RAW264.7巨噬细胞体外建立炎症模型，观察石油醚提取物、单体及单体复合物抗炎作用及LPS-TLR4-cytokines/NF-κB信号转导途径。
蒙药复方“Terun sodolol”石油醚提取物体外抗炎作用的研究试验如下：设置不同的LPS浓度（浓度设置为0.5、1、2、5、10、25μg/mL），干预RAW264.7巨噬细胞24h后，收集细胞上清进行ELISA检测，检测标准品绘制标准曲线，计算得出上清液肿瘤坏死因子α（TNF-α）、白介素-6（IL-6）、白介素1β（IL-1β）含量，根据结果确定最显著的LPS作用浓度为25μg/mL。蒙药复方“Terunsodolol”石油醚提取物1、5、10、25、50mg/mL作用于RAW264.7巨噬细胞，检测细胞增殖能力（CCK-8法），结果显示50mg/mL浓度组与空白对照组比较有显著差异（P<0.05），对细胞生长有抑制作用，其余各组均无显著差异(P>0.05)，说明对细胞生长无影响。故后期试验选择5mg/mL、10mg/mL、25mg/mL浓度梯度进行，实验分为空白对照组，炎症模型组，炎症模型+阳性对照组，炎症模型+药物处理组。ELISA检测TNF-α、IL-6、IL-1β含量，Real-time方法检测TNF-α、IL-6、IL-1βmRNA表达，结果显示：LPS模型组比空白对照组TNF-α、IL-6、IL-1β蛋白分泌及mRNA表达均显著升高（P<0.05），炎症模型+药物处理组TNF-α、IL-6、IL-1β蛋白分泌及mRNA表达与LPS模型组比较显著性降低（P<0.05），呈现剂量依赖性。说明蒙药复方“Terun sodolol”石油醚提取物有抗炎和细胞保护效应，与抑制炎性细胞因子的产生有关。
蒙药复方“Terun sodolol”石油醚提取物主要单体及其单体复合物对巨噬细胞RAW264.7细胞作用的有效浓度及时间：蒙药复方“Terun sodolol”主要单体及单体复合物作为试验药物，作用于RAW264.7巨噬细胞，检测细胞增殖能力。细胞增殖能力检测实验（CCK-8）结果显示：蒙药复方主要单体成分胡黄连苷Ⅱ、姜黄素、大黄素、大黄酸，高、中、低剂量组在不同时间点（0、6、12、24、48h）作用于RAW264.7细胞，并没有明显影响细胞生存活力。显示在各单体高浓度组作用时间48h显著地降低了细胞生存率，并发现单体复合物的保护效应较单体略微大一些。因此，根据不同浓度下保护效应明显的原则和药物组合的原则选择了高浓度单体组和中等浓度单体复合物组，作用时间点为24h。
蒙药复方“Terun sodolol”石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞细胞增殖能力的影响：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+吡咯烷二硫代氨基甲酸盐组（PDTC；20μM）、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组（Dex；10μg/mL）。结果显示：与正常细胞对照组比较，LPS的刺激显著降低了巨噬细胞活力，而单体及单体复合物试验药物及PDTC、地塞米松阳性对照药减轻该细胞的死亡，不同程度地恢复了细胞的存活率。
蒙药复方“Terun sodolol”石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞产生细胞因子的影响：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+PDTC、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组。ELISA结果显示LPS模型组较正常细胞对照组TNF-α、IL-6、IL-1β分泌显著升高；各单体及单体复合物试验药物、阳性对照药与模型组比较均显著降低了TNF-α、IL-6、IL-1β分泌。
蒙药复方石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞TLR-4/NF-κB信号通路的研究：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+PDTC、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组。蛋白质水平，用Western blot分析各单体及单体复合物试验药物对TLR4/NF-κB信号通路的影响，检测了磷酸化IκBα（P-IκBα）、P65、P-P65（磷酸化P65）、TLR-4蛋白，结果显示LPS模型组与正常对照组比较TLR-4、P-IκB、NF-κBP-P65蛋白表达显著，说明LPS刺激激活了TLR-4/NF-κB信号通路，而蒙药复方“Terun sodolol”石油醚提取物单体及单体复合物试验药拮抗内毒素LPS的作用显著降低了TLR-4、P-IκB、NF-κBP-P65蛋白的表达；real-time检测TLR-4、NF-κBP65，结果显示在转录水平LPS模型组与正常对照组比较TLR-4、NF-κB-P65mRNA表达显著，而蒙药复方石油醚提取物单体及单体复合物试验药拮抗内毒素LPS的作用显著降低TLR-4、NF-κBP65蛋白的mRNA表达。采用凝胶迁移实验（EMSA）检测DNA结合能力，检测NF-κBP65（核蛋白）活性，结果LPS模型组与正常对照组比较差异显著，NF-κBP65（核蛋白）活性及含量增加，而蒙药复方石油醚提取物单体及单体复合物试验药削弱内毒素LPS的作用，显著降低了NF-κBP65（核蛋白）活性及含量。
以上结果说明蒙药复方石油醚提取物、单体及其单体复合物试验药拮抗内毒素LPS的作用，是通过LPS-TLR4cytokines/NF-κB信号通路实现的。
Inflammation, a common clinical symptom, happened generally in every part oforganism. Mongolian medicine is the ancient and traditional medicine. The previousresearch has already established the Mongolian medicine compound Terun sodololwhich has good function of anti-inflammation and increasing immune. It hassignificant treatment efficacy in animal model and the clinical cow mastitis. However,it still has excessive medication, unclear mechanism of action and other problems.Therefore, this research continues the issue to do more research of anti-inflammatorysignal transduction pathway of its petroleum ether extracts and its main monomer forthe base of the second development in cell molecular level, because its petroleumether extracts has significant function of anti-inflammation.
Therefore, the effective active ingredients can be separated and identified bynature medicinal chemistry, organic chemistry and other knowledge and using liquidchromatography technology to establish the effective monomer components of thepetroleum ether extract. By LPS-induced RAW264.7macrophages, it can establishinflammation model in vitro to observe the function of Mongolian medicine compoundTerun sodolol mainly reflected on petroleum ether extract and monomer and monomercomplex on anti-inflammatory and LPS-TLR4-cytokines/NF-κB signal transductionpathway.
The results of research about Mongolian medicine compound petroleum etherextracts on anti-inflammatory function as following: to interfere RAW264.7macrophages after24hours with different LPS concentration (the concentrations are0.5,1,2,5,10,25μg/mL), and then to collect cell supernatant to do the ELISAdetection. To detect the standard production and to draw the standard curve forcalculating the supernatant of tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6),Interleukine1β(IL-1β)content. According to the results to establish the most effectiveLPS concentration is25μg/mL. Mongolia medicine compound Petroleum ether extract1,5,10,25,50mg/mL act on RAW264.7macrophages, to detect the proliferationability of cell (CCK-8method). The results showed that the obvious differencebetween the group of concentration50mg/mL and blank control group. This is thefunction of suppression to cell growth; other groups have no significant difference. This showed that there are no influences on cell growth, so the later stages ofexperiments choose the concentration steps5mg/mL、10mg/mL、25mg/mL. Theexperiment is divided into blank control group, the inflammation model group, theinflammation model and positive control group, inflammatory model and drugtreatment group. Tumor necrosis factor α (TNF-α）、Interleukin-6(IL-6), Interleukin1β(IL-1β) content were detected with ELISA method, tumor necrosis factor α(TNF-α),Interleukin-6(IL-6), Interleukin1β(IL-1β) mRNA expression were detected withReal-time PCR method. The results showed: LPS model group compare with the blankcontrol group on tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6), Interleukin1β(IL-1β) protein secretion and mRNA expression showed all increase significantly（P<0.05）. When the concentrations of5mg/mL、10mg/mL、25mg/mL petroleumether extract exist, tumor necrosis factor α(TNF-α), Interleukin-6(IL-6), Interleukin1β(IL-1β)protein secretion, mRNA expression and LPS model group compared withLPS model group all decrease significantly（P<0.05）. There is obvious difference andappearing dose-dependent. This demonstrates that anti-inflammatory and cellprotection effect of Mongolian medicine Terun sodolol petroleum ether extract arerelated to the produce of inhibition of inflammatory cytokines.
The establishment of effective concentration and time for Mongolian medicinecompound petroleum ether extract main monomer and its monomer complex act onmacrophages RAW264.7cell function: Mongolian medicine compound Terun sodololmain monomer and its monomer complex as the experiment medicine act onmacrophages RAW264.7, the ability of cell proliferation was detected. The result ofexperiment (CCK-8)of the ability of cell proliferation showed: Mongolian medicinecompound main monomer ingredients which are picroside II, curcumin, emodin, rheinin high, medium and low dose groups at different time points (0、6、12、24h) act onRAW264.7cell, there were no obviously influence for the cell exist vitality. But itshowed that at every monomer concentration groups acting time48h can decrease cellexist rate significantly. Additionally, the effect of monomer complex is a little betterthan monomer. Therefore, the later experiments, according to the rule of effectivedefence and medicine composition under different concentrations, to choose highconcentration monomer group and medium concentration monomer complex group,and the time point is24h.
The influence of Mongolia medicine compound petroleum ether extract mainmonomer and monomer complex to LPS introduce RAW264.7on the ability of cellproliferation. The experiment was divided into normal cell control group, the inflammation model group, inflammation model add every monomer administrationgroup, inflammation model add monomer compound group, inflammation model addPyrrolidine dithiocarbamate group (PDTC;20microns), inflammation model addPDTC add the monomer complex group, inflammation model add dexamethasonegroup (Dex;10μg/mL). The results showed that it can more decrease the cell vitalityafter stimulating with LPS comparing with normal cell control groups. Monomer andmonomer complex for testing drugs and PDTC、Dex for positive drug inhibit thedeath of cell, and restore the survive of cells on the different level compared with LPSmodel groups.
The influence of Mongolian medicine compound petroleum ether extract mainmonomer and monomer complex to RAW264.7producing cell factors introduced byLPS: The experiment was divided into normal cell control group, the inflammationmodel group, inflammation model add monomer administration group, inflammationmodel add monomer compound group, inflammation model add PDTC group,inflammation model add PDTC add the monomer complex group, inflammation modeladd dexamethasone group. The result of ELISA showed that TNF-α, IL-6, IL-1βsecretion of LPS model groups increased obviously comparing with normal cellcontrol groups；The secretion of TNF-α, IL-6, IL-1β comparing in each monomer andthe monomer complex test drugs, positive control drug groups with model groupswere decrease significantly.
The study on LPS introduce RAW264.7to cell TLR-4/NF-κB signal pathway wasacted by Mongolian medicine compound petroleum ether extract main monomer andmonomer complex. The experiment was divided into normal cell control group, theinflammation model group, inflammation model add monomer administration group,inflammation model add monomer compound group, inflammation model add PDTCgroup，inflammation model add PDTC add the monomer complex group, inflammationmodel add dexamethasone group. At the protein level, by using Western blot toanalyze the influence of each monomer and the monomer complex test drugs toTLR-4/NF-κB signal pathway. To detect phosphorylation IκBα(P-IκBα)、P65、P-P65(phosphorylation P56), TLR-4protein. The results showed that LPS model groupscompared with normal control groups TLR-4、 P-IκB、 NF-κBP-P65the proteinexpression is obvious which demonstrates that LPS stimulates and activatesTLR-4/NF-κB signal pathway. Mongolia medicine compound petroleum ether extractmain monomer and monomer complex test drugs against the function of endotoxinLPS and decrease the protein expression of TLR-4、P-IKB、NF-κBP-P65. The results of real-time detected TLR-4、NF-κBP65showed that at the transcriptional level, LPSmodel groups compared with normal control groups TLR-4、 NF-κBP65mRNAexpressed significantly. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drugs against the function ofendotoxin LPS and obviously decrease TLR-4、 NF-κBP65mRNA of proteinexpression. The result is the same as Western blotting. Using electrophoretic mobilityshift assays (EMSA) to detect the combine ability of DNA, to detect NF-κBP65(Nucleoprotein) activity. The results showed that LPS model groups compared withnormal control groups express significantly, the activity and content of NF-κBP65(Nucleoprotein) increase. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drμgs weaken the function ofendotoxin LPS, decrease significantly the activity and content of NF-κBP65(Nucleoprotein).
All results above demonstrate that Mongolia medicine compound petroleumether extracts and main monomer and monomer complex test drugs weaken thefunction of endotoxin LPS by TLR4and cytokines/NF-κB signal pathway to realize.
为此，应用天然药物化学、有机化学等知识，利用液相色谱等技术分离鉴定有效活性成分，结合文献指导，确立了石油醚提取物有效单体成分，利用脂多糖（LPS）诱导RAW264.7巨噬细胞体外建立炎症模型，观察石油醚提取物、单体及单体复合物抗炎作用及LPS-TLR4-cytokines/NF-κB信号转导途径。
蒙药复方“Terun sodolol”石油醚提取物体外抗炎作用的研究试验如下：设置不同的LPS浓度（浓度设置为0.5、1、2、5、10、25μg/mL），干预RAW264.7巨噬细胞24h后，收集细胞上清进行ELISA检测，检测标准品绘制标准曲线，计算得出上清液肿瘤坏死因子α（TNF-α）、白介素-6（IL-6）、白介素1β（IL-1β）含量，根据结果确定最显著的LPS作用浓度为25μg/mL。蒙药复方“Terunsodolol”石油醚提取物1、5、10、25、50mg/mL作用于RAW264.7巨噬细胞，检测细胞增殖能力（CCK-8法），结果显示50mg/mL浓度组与空白对照组比较有显著差异（P<0.05），对细胞生长有抑制作用，其余各组均无显著差异(P>0.05)，说明对细胞生长无影响。故后期试验选择5mg/mL、10mg/mL、25mg/mL浓度梯度进行，实验分为空白对照组，炎症模型组，炎症模型+阳性对照组，炎症模型+药物处理组。ELISA检测TNF-α、IL-6、IL-1β含量，Real-time方法检测TNF-α、IL-6、IL-1βmRNA表达，结果显示：LPS模型组比空白对照组TNF-α、IL-6、IL-1β蛋白分泌及mRNA表达均显著升高（P<0.05），炎症模型+药物处理组TNF-α、IL-6、IL-1β蛋白分泌及mRNA表达与LPS模型组比较显著性降低（P<0.05），呈现剂量依赖性。说明蒙药复方“Terun sodolol”石油醚提取物有抗炎和细胞保护效应，与抑制炎性细胞因子的产生有关。
蒙药复方“Terun sodolol”石油醚提取物主要单体及其单体复合物对巨噬细胞RAW264.7细胞作用的有效浓度及时间：蒙药复方“Terun sodolol”主要单体及单体复合物作为试验药物，作用于RAW264.7巨噬细胞，检测细胞增殖能力。细胞增殖能力检测实验（CCK-8）结果显示：蒙药复方主要单体成分胡黄连苷Ⅱ、姜黄素、大黄素、大黄酸，高、中、低剂量组在不同时间点（0、6、12、24、48h）作用于RAW264.7细胞，并没有明显影响细胞生存活力。显示在各单体高浓度组作用时间48h显著地降低了细胞生存率，并发现单体复合物的保护效应较单体略微大一些。因此，根据不同浓度下保护效应明显的原则和药物组合的原则选择了高浓度单体组和中等浓度单体复合物组，作用时间点为24h。
蒙药复方“Terun sodolol”石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞细胞增殖能力的影响：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+吡咯烷二硫代氨基甲酸盐组（PDTC；20μM）、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组（Dex；10μg/mL）。结果显示：与正常细胞对照组比较，LPS的刺激显著降低了巨噬细胞活力，而单体及单体复合物试验药物及PDTC、地塞米松阳性对照药减轻该细胞的死亡，不同程度地恢复了细胞的存活率。
蒙药复方“Terun sodolol”石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞产生细胞因子的影响：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+PDTC、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组。ELISA结果显示LPS模型组较正常细胞对照组TNF-α、IL-6、IL-1β分泌显著升高；各单体及单体复合物试验药物、阳性对照药与模型组比较均显著降低了TNF-α、IL-6、IL-1β分泌。
蒙药复方石油醚提取物主要单体及单体复合物对LPS诱导RAW264.7细胞TLR-4/NF-κB信号通路的研究：实验分为正常细胞对照组、炎症模型组、炎症模型+各单体给药组、炎症模型+单体复合物组、炎症模型+PDTC、炎症模型+PDTC+单体复合物组、炎症模型+地塞米松组。蛋白质水平，用Western blot分析各单体及单体复合物试验药物对TLR4/NF-κB信号通路的影响，检测了磷酸化IκBα（P-IκBα）、P65、P-P65（磷酸化P65）、TLR-4蛋白，结果显示LPS模型组与正常对照组比较TLR-4、P-IκB、NF-κBP-P65蛋白表达显著，说明LPS刺激激活了TLR-4/NF-κB信号通路，而蒙药复方“Terun sodolol”石油醚提取物单体及单体复合物试验药拮抗内毒素LPS的作用显著降低了TLR-4、P-IκB、NF-κBP-P65蛋白的表达；real-time检测TLR-4、NF-κBP65，结果显示在转录水平LPS模型组与正常对照组比较TLR-4、NF-κB-P65mRNA表达显著，而蒙药复方石油醚提取物单体及单体复合物试验药拮抗内毒素LPS的作用显著降低TLR-4、NF-κBP65蛋白的mRNA表达。采用凝胶迁移实验（EMSA）检测DNA结合能力，检测NF-κBP65（核蛋白）活性，结果LPS模型组与正常对照组比较差异显著，NF-κBP65（核蛋白）活性及含量增加，而蒙药复方石油醚提取物单体及单体复合物试验药削弱内毒素LPS的作用，显著降低了NF-κBP65（核蛋白）活性及含量。
以上结果说明蒙药复方石油醚提取物、单体及其单体复合物试验药拮抗内毒素LPS的作用，是通过LPS-TLR4cytokines/NF-κB信号通路实现的。
Inflammation, a common clinical symptom, happened generally in every part oforganism. Mongolian medicine is the ancient and traditional medicine. The previousresearch has already established the Mongolian medicine compound Terun sodololwhich has good function of anti-inflammation and increasing immune. It hassignificant treatment efficacy in animal model and the clinical cow mastitis. However,it still has excessive medication, unclear mechanism of action and other problems.Therefore, this research continues the issue to do more research of anti-inflammatorysignal transduction pathway of its petroleum ether extracts and its main monomer forthe base of the second development in cell molecular level, because its petroleumether extracts has significant function of anti-inflammation.
Therefore, the effective active ingredients can be separated and identified bynature medicinal chemistry, organic chemistry and other knowledge and using liquidchromatography technology to establish the effective monomer components of thepetroleum ether extract. By LPS-induced RAW264.7macrophages, it can establishinflammation model in vitro to observe the function of Mongolian medicine compoundTerun sodolol mainly reflected on petroleum ether extract and monomer and monomercomplex on anti-inflammatory and LPS-TLR4-cytokines/NF-κB signal transductionpathway.
The results of research about Mongolian medicine compound petroleum etherextracts on anti-inflammatory function as following: to interfere RAW264.7macrophages after24hours with different LPS concentration (the concentrations are0.5,1,2,5,10,25μg/mL), and then to collect cell supernatant to do the ELISAdetection. To detect the standard production and to draw the standard curve forcalculating the supernatant of tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6),Interleukine1β(IL-1β)content. According to the results to establish the most effectiveLPS concentration is25μg/mL. Mongolia medicine compound Petroleum ether extract1,5,10,25,50mg/mL act on RAW264.7macrophages, to detect the proliferationability of cell (CCK-8method). The results showed that the obvious differencebetween the group of concentration50mg/mL and blank control group. This is thefunction of suppression to cell growth; other groups have no significant difference. This showed that there are no influences on cell growth, so the later stages ofexperiments choose the concentration steps5mg/mL、10mg/mL、25mg/mL. Theexperiment is divided into blank control group, the inflammation model group, theinflammation model and positive control group, inflammatory model and drugtreatment group. Tumor necrosis factor α (TNF-α）、Interleukin-6(IL-6), Interleukin1β(IL-1β) content were detected with ELISA method, tumor necrosis factor α(TNF-α),Interleukin-6(IL-6), Interleukin1β(IL-1β) mRNA expression were detected withReal-time PCR method. The results showed: LPS model group compare with the blankcontrol group on tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6), Interleukin1β(IL-1β) protein secretion and mRNA expression showed all increase significantly（P<0.05）. When the concentrations of5mg/mL、10mg/mL、25mg/mL petroleumether extract exist, tumor necrosis factor α(TNF-α), Interleukin-6(IL-6), Interleukin1β(IL-1β)protein secretion, mRNA expression and LPS model group compared withLPS model group all decrease significantly（P<0.05）. There is obvious difference andappearing dose-dependent. This demonstrates that anti-inflammatory and cellprotection effect of Mongolian medicine Terun sodolol petroleum ether extract arerelated to the produce of inhibition of inflammatory cytokines.
The establishment of effective concentration and time for Mongolian medicinecompound petroleum ether extract main monomer and its monomer complex act onmacrophages RAW264.7cell function: Mongolian medicine compound Terun sodololmain monomer and its monomer complex as the experiment medicine act onmacrophages RAW264.7, the ability of cell proliferation was detected. The result ofexperiment (CCK-8)of the ability of cell proliferation showed: Mongolian medicinecompound main monomer ingredients which are picroside II, curcumin, emodin, rheinin high, medium and low dose groups at different time points (0、6、12、24h) act onRAW264.7cell, there were no obviously influence for the cell exist vitality. But itshowed that at every monomer concentration groups acting time48h can decrease cellexist rate significantly. Additionally, the effect of monomer complex is a little betterthan monomer. Therefore, the later experiments, according to the rule of effectivedefence and medicine composition under different concentrations, to choose highconcentration monomer group and medium concentration monomer complex group,and the time point is24h.
The influence of Mongolia medicine compound petroleum ether extract mainmonomer and monomer complex to LPS introduce RAW264.7on the ability of cellproliferation. The experiment was divided into normal cell control group, the inflammation model group, inflammation model add every monomer administrationgroup, inflammation model add monomer compound group, inflammation model addPyrrolidine dithiocarbamate group (PDTC;20microns), inflammation model addPDTC add the monomer complex group, inflammation model add dexamethasonegroup (Dex;10μg/mL). The results showed that it can more decrease the cell vitalityafter stimulating with LPS comparing with normal cell control groups. Monomer andmonomer complex for testing drugs and PDTC、Dex for positive drug inhibit thedeath of cell, and restore the survive of cells on the different level compared with LPSmodel groups.
The influence of Mongolian medicine compound petroleum ether extract mainmonomer and monomer complex to RAW264.7producing cell factors introduced byLPS: The experiment was divided into normal cell control group, the inflammationmodel group, inflammation model add monomer administration group, inflammationmodel add monomer compound group, inflammation model add PDTC group,inflammation model add PDTC add the monomer complex group, inflammation modeladd dexamethasone group. The result of ELISA showed that TNF-α, IL-6, IL-1βsecretion of LPS model groups increased obviously comparing with normal cellcontrol groups；The secretion of TNF-α, IL-6, IL-1β comparing in each monomer andthe monomer complex test drugs, positive control drug groups with model groupswere decrease significantly.
The study on LPS introduce RAW264.7to cell TLR-4/NF-κB signal pathway wasacted by Mongolian medicine compound petroleum ether extract main monomer andmonomer complex. The experiment was divided into normal cell control group, theinflammation model group, inflammation model add monomer administration group,inflammation model add monomer compound group, inflammation model add PDTCgroup，inflammation model add PDTC add the monomer complex group, inflammationmodel add dexamethasone group. At the protein level, by using Western blot toanalyze the influence of each monomer and the monomer complex test drugs toTLR-4/NF-κB signal pathway. To detect phosphorylation IκBα(P-IκBα)、P65、P-P65(phosphorylation P56), TLR-4protein. The results showed that LPS model groupscompared with normal control groups TLR-4、 P-IκB、 NF-κBP-P65the proteinexpression is obvious which demonstrates that LPS stimulates and activatesTLR-4/NF-κB signal pathway. Mongolia medicine compound petroleum ether extractmain monomer and monomer complex test drugs against the function of endotoxinLPS and decrease the protein expression of TLR-4、P-IKB、NF-κBP-P65. The results of real-time detected TLR-4、NF-κBP65showed that at the transcriptional level, LPSmodel groups compared with normal control groups TLR-4、 NF-κBP65mRNAexpressed significantly. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drugs against the function ofendotoxin LPS and obviously decrease TLR-4、 NF-κBP65mRNA of proteinexpression. The result is the same as Western blotting. Using electrophoretic mobilityshift assays (EMSA) to detect the combine ability of DNA, to detect NF-κBP65(Nucleoprotein) activity. The results showed that LPS model groups compared withnormal control groups express significantly, the activity and content of NF-κBP65(Nucleoprotein) increase. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drμgs weaken the function ofendotoxin LPS, decrease significantly the activity and content of NF-κBP65(Nucleoprotein).
All results above demonstrate that Mongolia medicine compound petroleumether extracts and main monomer and monomer complex test drugs weaken thefunction of endotoxin LPS by TLR4and cytokines/NF-κB signal pathway to realize.
引文
1李虔全，周立社，郭兰萍等.分子生药学在蒙药研究中的应用[J].中国中药杂志，2011，36（19）：2615-2617
2周桂坤，关金凤.蒙药的历史渊源、研究现状及展望[J].中国民族民间医药杂志，2007，84(1)：6-9
3包·照日格图,郑进,海银梅.中药与蒙药药性理论比较研究[J].云南中医学院学报，2005，28（3）：31-33
4巴·吉格木德主编.蒙医基础理论[M].呼和浩特：内蒙古教育出版社，1988.20-56
5何雁,刘勇,罗晓健等.我国民族药发展现状及存在问题[J].中草药，2006，37（12）：1915-1917
6巴图德力根，布图雅等.蒙药清肺汤的抗菌作用及对小鼠非特异性免疫功能影响的实验研究[J].内蒙古民族大学学报，2002，17（8）：353-355
7麻春杰，博·格日勒图，领小.近10年中蒙药多糖的药理作用研究概况[J].中国民族医药杂志，2005，9（5）：46-48
8乌日娜，春花，王纯洁等.15味蒙药和3种蒙方抗大肠埃希菌和金黄色葡萄球菌作用研究[J].动物医学进展，2008，29(2)：52-55
9战永波.治疗奶牛乳房炎蒙药复方的筛选及其抗炎免疫机理的研究[D].内蒙古农业大学博士论文，2010.6
10D.J. Wilson, Y.T. Grohn, G.J. Bennett. Milk Production Change Following ClinicalMastitis and Reproductive Performance Compared Among15Vaccinated and ControlDairy Cattle [J].Journal of Dairy Science，2008，91(10)：3869-3879
11H.W. Barkema, Y.H. Schukken, R.N. Zadoks. Role of Cow, Pathogen, and TreatmentRegimen in the Therapeutic Success of Bovine Staphylococcus aureus Mastitis[J].Journal of Dairy Science,2006,89(6):1877-1895
12聂爱琴，郑明学，古少鹏等.奶牛乳房炎的病因分析[J].科技情报开发与经济，2003，13(3)：183
13Watts J. L.. Etiological agents of bovine mastitis [J]. Veterinary microbiology，1988，16(1)：41-66
14KL Smith. Effect of Vitamin E and Selenium Supplementation on Incidence ofClinical Mastitis and Duration of Clinical Symptoms [J].Dairy Sci1998，71：848-853
15Katholm J.Influence of iron on infection [J].Veterinaertidsskrift，1983(1)：2-6
16Morse， D， M.A.Lorenzo， C.J.Wilcox， R.J.et al. Climatic effects on occurrenceof clinical mastitis [J].Journal of Dairy Science，1988，71(3):848-853
17陈北亭，万一鹤，王建辰等.兽医产科学[M].第二版.北京：农业出版社.1998.353-367
18Pankey RB.Dresch sler PA．Evo lut ion of udderhy—gience：Premilking teatsanitation[J]．Vet．C1in．North Am(Food A nim P ract．)，1993，(9)：519-530
19郭梦尧，王洪海，王国卿等.奶牛乳房炎的病理机制及中药防治研究进展[J].动物医学进展,2010,31(10):93-95
20潘志明，张小荣，焦新安.作为疫苗和疫苗载体的胞内细菌[J].动物兽医进展，2002，23（3）：107
21Yancey R.Vaccines and diagnostic methods for bovine mastitis:fact andfiction[J].Adv Vet Med,1999,41:257-273
22马保臣,杜平，蒋世峰等.奶牛乳腺炎的研究进展[J]．动物科学与动物医学，2002，(9)：21-23
23Blum J W and Dosogene H.Tumor necrosis factor-a and nitrite/nitrate responsesDuring acute mastitis induced by Escherichia coli infection and endotoxin indairy cows [J].Dom Anim Endocrin,2000,19:2230-2235
24杨光华主编.病理学[M].第五版.北京:人民卫生出版社,2001.57
25Hsu HC,Yang WC.alpha-Bulnesene,a novel PAF receptor antagonist isolated fromPogostenon cablin[J]. Biochem Biophys Res Commun,2006,345(3):1033-1038
26Hebert RL,Regoli D.Bradykinin B2type receptor activation regulates fluid andelectrolyte transport in the rabbit kidney[J].Peptides,2005,26(8):1308-1316
27Miller SI,Ernst RK,Bader MW.LPS,TLR4and infectious disease diversity[J].NatRev Microbiol,2005,3(1):36-46
28刘作金，刘长安，龚建平.IRAK-4:TLR/IL-1R家族共同信号转导系统中的关键因子[J].生理科学进展，2005，36（3）：276-279
29Liew FY,Liu H,Xu D.A novel negative regulator for IL-1receptor and Toll-likereceptor4[J].Immunol Lett,2005,96（1）:27-31
30Zuo GQ,Gong JP,Liu CA,et al.Synthesis of Toll-like receptor4in Kupffer cellsand its role in alcohol-induced liver disease[J].Chin Med J,2003,116:297-300
31Alan Aderem, Richard J, Ulecitch, Toll-like receptors in the induction ofthe innate immune response [J]. Nature,2000,406（6797）:782-787
32Sen,R,and Baltimore,D.Multiple nuclear factors interact with the immunoglobulinenhancer sequences[J]. Cell,1986，46(5)：705-716
33Baldwin,A.s,Jr.The NF-κB and I Kb proteins:new discoveriesand insights[J].AnnuRev Immunol,1996，14：649-683
34Han,Y,and Brasier,A.R.Mechanism for biphasic rel A.NF-κBl nuclear translocation in tumornecrosis factor a-stimulate heptaccytes[J].J Biol Chem,1997，272(15)：9825-9832
35Ghosh,S.,May,M.J.et al. NF-κB and Rel proteins:evolutionarily conserved mediators of immuneresponses[J].Annu Rev Immuno,1998,l16：225-260
36Karin,M.,and Ben-NJeriah,Y. Phosphorylation meets ubiquitination:the controlof NF-κB activity[J].Annu Rev Immunol,2000,18：621-663
37Phelps,C.B.,Sengchanthalangsy,L.L.et al. Mechanism of IκBαbinding to NF-κB dimers[J].J Biol Chem,2000,275(38)：29840-29846
38Baeuerle,P.A.,and Baltimore,D. NF-κB:ten years after[J].Cell,199887(1)：13-20
39Beg,A.A.,Sha,W.C.,et al. Constitutive NF-κb activation enhanced granulopoiesis and neonatallethality in IκBα-deficient mice[J].Genes Dev,1995，9(22)：2736-2746
40Karin,M.,and Ben-Neriah,Y. Phosphorylation meets ubiquitination:the control ofNF-κB activity[J].Annu Rev Immunol,2000，18：621-663
41Tak,p.p.,and Firestein,G.S. NF-κB:a key role in inflammatory diseases[J].JClin Invest,2001,107(1):7-11
42Makarov,S.S. NF-κB as a therapeutic target in chronic inflammation:recentadvances[J].Mol Med Today,2000,6(11),441-448
43Yamamoto,Y,and Gaynor,R.B.Therapeutic potential of inhibition of the NF-κBpathway in the treatment of inflammation and cancer[J].Jclin Invest，2001,107：135-142
44包金荣,王纯洁,战永波等.治疗奶牛乳腺炎蒙药复方提取物抗大肠埃希菌和金黄色葡萄球菌作用研究[J].动物医学进展,2010,31(9):55-58
45乌日娜,王纯洁,包金荣等.蒙药特润舒都乐对小鼠特异性免疫机能的影响[J].中国兽医杂志,2009,45(2):57-58
46游晓庆,李厚轩,吴春芳,等.Pg-LPS对兔单核/巨噬细胞炎症因子表达的影响[J].免疫学杂志,2012,28(1):10-14
47潘苗苗，刘学华，佟书娟等.芦荟及其复方提取物对RAW264.7细胞炎性因子的影响[J].中国实验方剂学杂志，2012,18（7）：166-169
48秦爽,贺育兰,杨静等.重组人生长激素对内毒素诱导人巨噬细胞产生TNF-α、IL-1β含量的影响[J].中国感染控制杂志,2008,7(1):9-11.
49周晓玲，张海，孟宪丽等.三黄泻心汤对LPS诱导的RAW264.7细胞释放炎性细胞因子影响的研究[J].四川生理科学杂志，2011,33（4）:145-147
50赵丽瑛,罗炳德,张魏巍.巨噬细胞IL-βmRNA在高温和内毒素复合影响下的表达[J].中国工业医学杂志,2008,21(1):7-951BereswillS,Mu ozM,FischerA,PlickertR,HaagLM,OttoB, Kühl AA, Loddenkemper C, G bel UB, Heimesaat MM. Anti-inflammatory effects of resveratrol,curcumin and simvastatin in acute small intestinal inflammation[J]. PLoS One,2010,5(12):e15099
52Deffaud J, Kirchmeyer M, Domagala F, Ficheux H, Netter P, Bianchi A, JouzeauJY. Modulatory effect of rhein on IL-1alpha-induced responses in humanchondrocytes: a comparative study between antibody microarrays and specificELISAs[J].Biorheology,2008,45(3-4):439-455
53Guimar es MR, de Aquino SG, Coimbra LS, Spolidorio LC, Kirkwood KL, Rossa C Jr.Curcumin modulates the immune response associated with LPS-induced periodontaldisease in rats[J]. Innate Immun,2012,18:155-163
54Guo Y, Xu X, Li Q, Li Z, Du F. Anti-inflammation effects of picroside2in cerebralischemic injury rats[J]. Behav Brain Funct,2010,6:43
55Kumar HM, Singh PP, Qazi NA, Srinivas J, Malik F, Sidiq T, Gupta A, KhajuriaA, Suri KA, Satti NK, Qazi GN. Development of novel lipidated analogs of picrosideas vaccine adjuvants: acylated analogs of picroside-II elicit strong Th1andTh2response to ovalbumin in mice[J]. Vaccine,2010,28:8327-8337
56Lopresti AL, Hood SD, Drummond PD. Multiple antidepressant potential modes ofaction of curcumin: a review of its anti-inflammatory, monoaminergic,antioxidant,immune-modulating and neuroprotective effects[J].JPsychopharmacol,2012,26(12):1512-1524
57Ma BL, Ma YM, Yan DM, Zhou H, Shi R, Wang TM, Yang Y, Wang CH, Zhang N. Effectiveconstituents in Xiexin Decoction for anti-inflammation[J]. J Ethnopharmacol,2009,125(1):151-156
58Meng FJ,Jiao SM,Yu B. Picroside II protects cardiomyocytes from hypoxia/reoxygenation-inducedapoptosis by activating the PI3K/Akt and CREB pathways[J]. Int J Mol Med,2012,30(2):263-270
59Zhang W, Li H, Bu H, Chen H, Tong H, Liu D, Guo H, Lin SZ. Emodin inhibits thedifferentiation and maturation of dendritic cells and increases the productionof regulatory T cells[J]. Int J Mol Med,2012,29(2):159-164
60Schreck R, Meier B, M nnel DN, Dr ge W, Baeuerle PA. Dithiocarbamates as potentinhibitors of nuclear factor kappa B activation in intact cells[J]. J ExpMed,1992,175(5):1181-1194
61Walsh S, Jordan GR, Jefferiss C, Stewart K, Beresford JN. High concentrationsof dexamethasone suppress the proliferation but not the differentiation orfurther maturation of human osteoblast precursors in vitro: relevance toglucocorticoid-induced osteoporosis[J].Rheumatology (Oxford),2001,40(1):74－83
62Kenneth，et al. Analysis of relative gene expression data using real-timequantitative PCR and the2(-Delta Delta C(T)) Method[J]. Methods,200111,25(4):402-408
63Noreen M, Shah MA, Mall SM, Choudhary S, Hussain T, Ahmed I, Jalil SF, Raza MI.TLR4polymorphisms and disease susceptibility[J]. Inflamm Res,2012,61(3):177-188
64Peri F, Piazza M. Therapeutic targeting of innate immunity with Toll-likereceptor4(TLR4) antagonists[J]. Biotechnol Adv,2012,30(1):251-260
65Miao EA, Rajan JV, Aderem A. Caspase-1-induced pyroptotic cell death[J]. ImmunolRev,2011,243(1):206-214
66Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages[J].Crit Rev Eukaryot Gene Expr,2010,20:87-103
67Scherer D.C., Brockman J.A., Chen Z., Maniatis T., Ballard D.W. Signal-induceddegradation of IkappaB alpha requires site-specific ubiquitination[J]. ProcNatl Acad Sci USA,1995,92:11259-11263
68Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB tonew insights into transcriptional regulations in innate immunity[J]. BiochemPharmacol,2006,72(9):1102-1113
69Schmalz G, Krifka S, Schweikl H. Toll-like receptors, LPS, and dental monomers[J].Adv Dent Res.2011,23(3):302-306
70H cker H, Karin M. Regulation and function of IKK and IKK-related kinases[J].Sci STKE,2006,2006(357):re13
71Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB tonew insights into transcriptional regulations in innate immunity[J]. BiochemPharmacol,2006,72(9):1102-1113
72Schmalz G, Krifka S, Schweikl H. Toll-like receptors, LPS, and dental monomers[J].Adv Dent Res,2011,23(3):302-306
73Shehzad A, Rehman G, Lee YS. Curcumin in inflammatory diseases. Biofactors.2012Dec22. doi:10.1002/biof.1066.[Epub ahead of print]
74Chang CY, Chan HL, Lin HY, Way TD, Kao MC, Song MZ, Lin YJ, Lin CW. Rhein inducesapoptosis in human breast cancer cells. Evid Based Complement Alternat Med,2012,2012:952504. Epub
75Chang YC, Lai TY, Yu CS, Chen HY, Yang JS, Chueh FS, Lu CC, Chiang JH, HuangWW, Ma CY, Chung JG. Emodin Induces Apoptotic Death in Murine MyelomonocyticLeukemia WEHI-3Cells In Vitro and Enhances Phagocytosis in Leukemia Mice InVivo[J]. Evid Based Complement Alternat Med,2011,2011:523-596.
76Li Y, Xu Y, Lei B, Wang W, Ge X, Li J. Rhein induces apoptosis of human gastriccancer SGC-7901cells via an intrinsic mitochondrial pathway[J]. Braz J Med BiolRes,2012,45:1052-1059
77Gao H, Zhou YW. Inhibitory effect of picroside II on hepatocyte apoptosis[J].Acta Pharmacol Sin,2005,26(6):729-736.
78Meng FJ,Jiao SM,Yu B.Picroside II protects cardiomyocytes from hypoxia/reoxygenation-inducedapoptosis by activating the PI3K/Akt and CREB pathways[J].Int J Mol Med.2012,30(2):263-270
79Flad HD, Grage-Griebenow E, Petersen F, Scheuerer B, Brandt E, Baran J, PryjmaJ,Ernst M. The role of cytokines in monocyte apoptosis[J]. Pathobiology,1999,67(5-6):291-293
80Miao EA, Rajan JV, Aderem A. Caspase-1-induced pyroptotic cell death[J]. ImmunolRev.2011,243(1):206-214
81Nett-Fiordalisi M, Tomaselli K, Russell JH, Chaplin DD. Macrophage apoptosisin the absence of active interleukin-1beta-converting enzyme[J]. J Leukoc Biol,1995,58(6):717-724
82Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages[J].Crit Rev Eukaryot Gene Expr,2010,20(2):87-103
83Suzuki T, Kobayashi M, Isatsu K, Nishihara T, Aiuchi T, Nakaya K, Hasegawa K.Mechanisms involved in apoptosis of human macrophages induced bylipopolysaccharide from Actinobacillus actinomycetemcomitans in the presenceof cycloheximide[J]. Infect Immun,2004,72(4):1856-1865
84Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction by humaninterleukin-6of apoptosis in the promonocytic cell line U937and humanneutrophils[J]. J Biol Chem,1992,267(30):21612-21616
85Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F.Principles of interleukin-6-type cytokine signalling and its regulation[J].Biochem J,2003,374(pt1):1-20
86Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies:integrating mammalian biology[J]. Cell,2001,104(4):487-501
87Regis G, Icardi L, Conti L, Chiarle R, Piva R, Giovarelli M, Poli V, NovelliF. IL-6, but not IFN-gamma, triggers apoptosis and inhibits in vivo growth ofhuman malignant T cells on STAT3silencing[J]. Leukemia,2009,23(11):2102-–2108
88Weber A,Wasiliew P,Kracht M. Interleukin-1(IL-1) pathway[J].Sci Signal,2010,3(105)
89LyeE,MirtsosC,SuzukiN,etal.TheroleofIRAK-4kinaseactivityinIRAK-4-mediatedsignaling[J].JBiolChem,2004，279（39）:40653-40658
90Triantafilou M,Triantafilou k.The dynamics of LPS recognition:complex orchestration of multiplereceptors[J].J Endotoxin Res,2005,11（1）:5-11
91Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction by humaninterleukin-6of apoptosis in the promonocytic cell line U937and humanneutrophils[J]. J Biol Chem，1992，267（30）:21612-21616
92Deffaud J, Kirchmeyer M, Domagala F, Ficheux H, Netter P, Bianchi A, JouzeauJY. Modulatory effect of rhein on IL-1alpha-induced responses in humanchondrocytes: a comparative study between antibody microarrays and specificELISAs[J]. Biorheology，2008，45（3-4）:439-455
93Heo SK, Yun HJ, Park WH, Park SD. Emodin inhibits TNF-alpha-induced human aorticsmooth-muscle cell proliferation via caspase-and mitochondrial-dependentapoptosis[J]. J Cell Biochem，2008，105（1）:70-80
94刘善清.TOll样受体4在信号通路在脂多糖促进肺癌细胞增殖中的机制探讨[J]．肿瘤,2010,30（10）：838-842
95步宏.分子病理学进展[M].四川大学出版社.2003.102-103
96Schaub B, Bellou A, Gibbons FK, Velasco G, Campo M, He H, Liang Y, Gillman MW,Gold D, Weiss ST, Perkins DL, Finn PW. TLR2and TLR4stimulation differentiallyinduce cytokine secretion in human neonatal, adult, and murine mononuclearcells[J]. J Interferon Cytokine Res，2004，24（9）:543－552
97Sharma J, Mishra BB, Li Q, Teale JM. TLR4-dependent activation of inflammatorycytokine response in macrophages by Francisella elongation factor Tu[J]. CellImmunol，2011，269（2）:69-73
98Guo Y, Xu X, Li Q, Li Z, Du F. Anti-inflammation effects of picroside2in cerebralischemic injury rats[J]. Behav Brain Funct，2010，6:43
99Tu CT, Han B, Yao QY, Zhang YA, Liu HC, Zhang SC. Curcumin attenuates ConcanavalinA-induced liver injury in mice by inhibition of Toll-like receptor (TLR)2, TLR4and TLR9expression[J]. Int Immunopharmacol，2012，12（1）:151-157
100Zhu XL, Wang YJ, Yang YΖ, Yang RC, Zhu B, Zhang YΗ, Lin Y, Lu Y, Li XF, O'ByrneKT. Suppression of lipopolysaccharide-induced upregulation of toll-likereceptor4by emodin in mouse proximal tubular epithelial cells[J]. Mol MedReport，2012，6（3）:493-500
101Cabrera-Benitez NE, Pérez-Roth E, Casula M, Ramos-Nuez A, Ríos-Luci C, Rodríguez-Gallego C, Sologuren I, Jakubkiene V, Slutsky AS, Padrón JM, Villar J.Anti-inflammatory activity of a novel family of aryl ureas compounds in anendotoxin-induced airway epithelial cell injury model[J]. PLoS One，2012，7（11）:e48468
102Scherer D.C., Brockman J.A., Chen Z., Maniatis T., Ballard D.W. Signal-induceddegradation of IkappaB alpha requires site-specific ubiquitination[J]. ProcNatl Acad Sci USA，1995，92（24）:11259-11263
103Lawrence T. The nuclear factor NF-B pathway in inflammation[J]. Cold Spring Harb Perspect Biol，2009，1:a001651
104Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB-M, Mueller-Dieckmann C,Farahifar D, Rossi B, Auberger P, Baeuerle PA, Peyron J-F. Tyrosinephosphorylation of IkappaB-alpha activates NF-kappaB without proteolyticdegradation of IkappaB-alpha[J]. Cell，1996，86（5）:787-798
105Peri F, Piazza M, Calabrese V, Damore G, Cighetti R. Exploring the LPS/TLR4signal pathway withsmall molecules[J]. Biochem Soc Trans，2010，38（5）:1390-1395
106JohnsonDA.SyntheticTLR-4activeglycolipidsasvaccineadjuvantsandstand-aloneimmunotherapeutics[J].Curr Topics Med Chem，2008，8（2）:64-79
107Rossignol DP, Lynn M. Antagonism of in vivo and ex vivo response to endotoxinby E5564, a synthetic lipid A analogue[J]. J Endotoxin Res，2002，8（6）:483-488
2周桂坤，关金凤.蒙药的历史渊源、研究现状及展望[J].中国民族民间医药杂志，2007，84(1)：6-9
3包·照日格图,郑进,海银梅.中药与蒙药药性理论比较研究[J].云南中医学院学报，2005，28（3）：31-33
4巴·吉格木德主编.蒙医基础理论[M].呼和浩特：内蒙古教育出版社，1988.20-56
5何雁,刘勇,罗晓健等.我国民族药发展现状及存在问题[J].中草药，2006，37（12）：1915-1917
6巴图德力根，布图雅等.蒙药清肺汤的抗菌作用及对小鼠非特异性免疫功能影响的实验研究[J].内蒙古民族大学学报，2002，17（8）：353-355
7麻春杰，博·格日勒图，领小.近10年中蒙药多糖的药理作用研究概况[J].中国民族医药杂志，2005，9（5）：46-48
8乌日娜，春花，王纯洁等.15味蒙药和3种蒙方抗大肠埃希菌和金黄色葡萄球菌作用研究[J].动物医学进展，2008，29(2)：52-55
9战永波.治疗奶牛乳房炎蒙药复方的筛选及其抗炎免疫机理的研究[D].内蒙古农业大学博士论文，2010.6
10D.J. Wilson, Y.T. Grohn, G.J. Bennett. Milk Production Change Following ClinicalMastitis and Reproductive Performance Compared Among15Vaccinated and ControlDairy Cattle [J].Journal of Dairy Science，2008，91(10)：3869-3879
11H.W. Barkema, Y.H. Schukken, R.N. Zadoks. Role of Cow, Pathogen, and TreatmentRegimen in the Therapeutic Success of Bovine Staphylococcus aureus Mastitis[J].Journal of Dairy Science,2006,89(6):1877-1895
12聂爱琴，郑明学，古少鹏等.奶牛乳房炎的病因分析[J].科技情报开发与经济，2003，13(3)：183
13Watts J. L.. Etiological agents of bovine mastitis [J]. Veterinary microbiology，1988，16(1)：41-66
14KL Smith. Effect of Vitamin E and Selenium Supplementation on Incidence ofClinical Mastitis and Duration of Clinical Symptoms [J].Dairy Sci1998，71：848-853
15Katholm J.Influence of iron on infection [J].Veterinaertidsskrift，1983(1)：2-6
16Morse， D， M.A.Lorenzo， C.J.Wilcox， R.J.et al. Climatic effects on occurrenceof clinical mastitis [J].Journal of Dairy Science，1988，71(3):848-853
17陈北亭，万一鹤，王建辰等.兽医产科学[M].第二版.北京：农业出版社.1998.353-367
18Pankey RB.Dresch sler PA．Evo lut ion of udderhy—gience：Premilking teatsanitation[J]．Vet．C1in．North Am(Food A nim P ract．)，1993，(9)：519-530
19郭梦尧，王洪海，王国卿等.奶牛乳房炎的病理机制及中药防治研究进展[J].动物医学进展,2010,31(10):93-95
20潘志明，张小荣，焦新安.作为疫苗和疫苗载体的胞内细菌[J].动物兽医进展，2002，23（3）：107
21Yancey R.Vaccines and diagnostic methods for bovine mastitis:fact andfiction[J].Adv Vet Med,1999,41:257-273
22马保臣,杜平，蒋世峰等.奶牛乳腺炎的研究进展[J]．动物科学与动物医学，2002，(9)：21-23
23Blum J W and Dosogene H.Tumor necrosis factor-a and nitrite/nitrate responsesDuring acute mastitis induced by Escherichia coli infection and endotoxin indairy cows [J].Dom Anim Endocrin,2000,19:2230-2235
24杨光华主编.病理学[M].第五版.北京:人民卫生出版社,2001.57
25Hsu HC,Yang WC.alpha-Bulnesene,a novel PAF receptor antagonist isolated fromPogostenon cablin[J]. Biochem Biophys Res Commun,2006,345(3):1033-1038
26Hebert RL,Regoli D.Bradykinin B2type receptor activation regulates fluid andelectrolyte transport in the rabbit kidney[J].Peptides,2005,26(8):1308-1316
27Miller SI,Ernst RK,Bader MW.LPS,TLR4and infectious disease diversity[J].NatRev Microbiol,2005,3(1):36-46
28刘作金，刘长安，龚建平.IRAK-4:TLR/IL-1R家族共同信号转导系统中的关键因子[J].生理科学进展，2005，36（3）：276-279
29Liew FY,Liu H,Xu D.A novel negative regulator for IL-1receptor and Toll-likereceptor4[J].Immunol Lett,2005,96（1）:27-31
30Zuo GQ,Gong JP,Liu CA,et al.Synthesis of Toll-like receptor4in Kupffer cellsand its role in alcohol-induced liver disease[J].Chin Med J,2003,116:297-300
31Alan Aderem, Richard J, Ulecitch, Toll-like receptors in the induction ofthe innate immune response [J]. Nature,2000,406（6797）:782-787
32Sen,R,and Baltimore,D.Multiple nuclear factors interact with the immunoglobulinenhancer sequences[J]. Cell,1986，46(5)：705-716
33Baldwin,A.s,Jr.The NF-κB and I Kb proteins:new discoveriesand insights[J].AnnuRev Immunol,1996，14：649-683
34Han,Y,and Brasier,A.R.Mechanism for biphasic rel A.NF-κBl nuclear translocation in tumornecrosis factor a-stimulate heptaccytes[J].J Biol Chem,1997，272(15)：9825-9832
35Ghosh,S.,May,M.J.et al. NF-κB and Rel proteins:evolutionarily conserved mediators of immuneresponses[J].Annu Rev Immuno,1998,l16：225-260
36Karin,M.,and Ben-NJeriah,Y. Phosphorylation meets ubiquitination:the controlof NF-κB activity[J].Annu Rev Immunol,2000,18：621-663
37Phelps,C.B.,Sengchanthalangsy,L.L.et al. Mechanism of IκBαbinding to NF-κB dimers[J].J Biol Chem,2000,275(38)：29840-29846
38Baeuerle,P.A.,and Baltimore,D. NF-κB:ten years after[J].Cell,199887(1)：13-20
39Beg,A.A.,Sha,W.C.,et al. Constitutive NF-κb activation enhanced granulopoiesis and neonatallethality in IκBα-deficient mice[J].Genes Dev,1995，9(22)：2736-2746
40Karin,M.,and Ben-Neriah,Y. Phosphorylation meets ubiquitination:the control ofNF-κB activity[J].Annu Rev Immunol,2000，18：621-663
41Tak,p.p.,and Firestein,G.S. NF-κB:a key role in inflammatory diseases[J].JClin Invest,2001,107(1):7-11
42Makarov,S.S. NF-κB as a therapeutic target in chronic inflammation:recentadvances[J].Mol Med Today,2000,6(11),441-448
43Yamamoto,Y,and Gaynor,R.B.Therapeutic potential of inhibition of the NF-κBpathway in the treatment of inflammation and cancer[J].Jclin Invest，2001,107：135-142
44包金荣,王纯洁,战永波等.治疗奶牛乳腺炎蒙药复方提取物抗大肠埃希菌和金黄色葡萄球菌作用研究[J].动物医学进展,2010,31(9):55-58
45乌日娜,王纯洁,包金荣等.蒙药特润舒都乐对小鼠特异性免疫机能的影响[J].中国兽医杂志,2009,45(2):57-58
46游晓庆,李厚轩,吴春芳,等.Pg-LPS对兔单核/巨噬细胞炎症因子表达的影响[J].免疫学杂志,2012,28(1):10-14
47潘苗苗，刘学华，佟书娟等.芦荟及其复方提取物对RAW264.7细胞炎性因子的影响[J].中国实验方剂学杂志，2012,18（7）：166-169
48秦爽,贺育兰,杨静等.重组人生长激素对内毒素诱导人巨噬细胞产生TNF-α、IL-1β含量的影响[J].中国感染控制杂志,2008,7(1):9-11.
49周晓玲，张海，孟宪丽等.三黄泻心汤对LPS诱导的RAW264.7细胞释放炎性细胞因子影响的研究[J].四川生理科学杂志，2011,33（4）:145-147
50赵丽瑛,罗炳德,张魏巍.巨噬细胞IL-βmRNA在高温和内毒素复合影响下的表达[J].中国工业医学杂志,2008,21(1):7-951BereswillS,Mu ozM,FischerA,PlickertR,HaagLM,OttoB, Kühl AA, Loddenkemper C, G bel UB, Heimesaat MM. Anti-inflammatory effects of resveratrol,curcumin and simvastatin in acute small intestinal inflammation[J]. PLoS One,2010,5(12):e15099
52Deffaud J, Kirchmeyer M, Domagala F, Ficheux H, Netter P, Bianchi A, JouzeauJY. Modulatory effect of rhein on IL-1alpha-induced responses in humanchondrocytes: a comparative study between antibody microarrays and specificELISAs[J].Biorheology,2008,45(3-4):439-455
53Guimar es MR, de Aquino SG, Coimbra LS, Spolidorio LC, Kirkwood KL, Rossa C Jr.Curcumin modulates the immune response associated with LPS-induced periodontaldisease in rats[J]. Innate Immun,2012,18:155-163
54Guo Y, Xu X, Li Q, Li Z, Du F. Anti-inflammation effects of picroside2in cerebralischemic injury rats[J]. Behav Brain Funct,2010,6:43
55Kumar HM, Singh PP, Qazi NA, Srinivas J, Malik F, Sidiq T, Gupta A, KhajuriaA, Suri KA, Satti NK, Qazi GN. Development of novel lipidated analogs of picrosideas vaccine adjuvants: acylated analogs of picroside-II elicit strong Th1andTh2response to ovalbumin in mice[J]. Vaccine,2010,28:8327-8337
56Lopresti AL, Hood SD, Drummond PD. Multiple antidepressant potential modes ofaction of curcumin: a review of its anti-inflammatory, monoaminergic,antioxidant,immune-modulating and neuroprotective effects[J].JPsychopharmacol,2012,26(12):1512-1524
57Ma BL, Ma YM, Yan DM, Zhou H, Shi R, Wang TM, Yang Y, Wang CH, Zhang N. Effectiveconstituents in Xiexin Decoction for anti-inflammation[J]. J Ethnopharmacol,2009,125(1):151-156
58Meng FJ,Jiao SM,Yu B. Picroside II protects cardiomyocytes from hypoxia/reoxygenation-inducedapoptosis by activating the PI3K/Akt and CREB pathways[J]. Int J Mol Med,2012,30(2):263-270
59Zhang W, Li H, Bu H, Chen H, Tong H, Liu D, Guo H, Lin SZ. Emodin inhibits thedifferentiation and maturation of dendritic cells and increases the productionof regulatory T cells[J]. Int J Mol Med,2012,29(2):159-164
60Schreck R, Meier B, M nnel DN, Dr ge W, Baeuerle PA. Dithiocarbamates as potentinhibitors of nuclear factor kappa B activation in intact cells[J]. J ExpMed,1992,175(5):1181-1194
61Walsh S, Jordan GR, Jefferiss C, Stewart K, Beresford JN. High concentrationsof dexamethasone suppress the proliferation but not the differentiation orfurther maturation of human osteoblast precursors in vitro: relevance toglucocorticoid-induced osteoporosis[J].Rheumatology (Oxford),2001,40(1):74－83
62Kenneth，et al. Analysis of relative gene expression data using real-timequantitative PCR and the2(-Delta Delta C(T)) Method[J]. Methods,200111,25(4):402-408
63Noreen M, Shah MA, Mall SM, Choudhary S, Hussain T, Ahmed I, Jalil SF, Raza MI.TLR4polymorphisms and disease susceptibility[J]. Inflamm Res,2012,61(3):177-188
64Peri F, Piazza M. Therapeutic targeting of innate immunity with Toll-likereceptor4(TLR4) antagonists[J]. Biotechnol Adv,2012,30(1):251-260
65Miao EA, Rajan JV, Aderem A. Caspase-1-induced pyroptotic cell death[J]. ImmunolRev,2011,243(1):206-214
66Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages[J].Crit Rev Eukaryot Gene Expr,2010,20:87-103
67Scherer D.C., Brockman J.A., Chen Z., Maniatis T., Ballard D.W. Signal-induceddegradation of IkappaB alpha requires site-specific ubiquitination[J]. ProcNatl Acad Sci USA,1995,92:11259-11263
68Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB tonew insights into transcriptional regulations in innate immunity[J]. BiochemPharmacol,2006,72(9):1102-1113
69Schmalz G, Krifka S, Schweikl H. Toll-like receptors, LPS, and dental monomers[J].Adv Dent Res.2011,23(3):302-306
70H cker H, Karin M. Regulation and function of IKK and IKK-related kinases[J].Sci STKE,2006,2006(357):re13
71Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB tonew insights into transcriptional regulations in innate immunity[J]. BiochemPharmacol,2006,72(9):1102-1113
72Schmalz G, Krifka S, Schweikl H. Toll-like receptors, LPS, and dental monomers[J].Adv Dent Res,2011,23(3):302-306
73Shehzad A, Rehman G, Lee YS. Curcumin in inflammatory diseases. Biofactors.2012Dec22. doi:10.1002/biof.1066.[Epub ahead of print]
74Chang CY, Chan HL, Lin HY, Way TD, Kao MC, Song MZ, Lin YJ, Lin CW. Rhein inducesapoptosis in human breast cancer cells. Evid Based Complement Alternat Med,2012,2012:952504. Epub
75Chang YC, Lai TY, Yu CS, Chen HY, Yang JS, Chueh FS, Lu CC, Chiang JH, HuangWW, Ma CY, Chung JG. Emodin Induces Apoptotic Death in Murine MyelomonocyticLeukemia WEHI-3Cells In Vitro and Enhances Phagocytosis in Leukemia Mice InVivo[J]. Evid Based Complement Alternat Med,2011,2011:523-596.
76Li Y, Xu Y, Lei B, Wang W, Ge X, Li J. Rhein induces apoptosis of human gastriccancer SGC-7901cells via an intrinsic mitochondrial pathway[J]. Braz J Med BiolRes,2012,45:1052-1059
77Gao H, Zhou YW. Inhibitory effect of picroside II on hepatocyte apoptosis[J].Acta Pharmacol Sin,2005,26(6):729-736.
78Meng FJ,Jiao SM,Yu B.Picroside II protects cardiomyocytes from hypoxia/reoxygenation-inducedapoptosis by activating the PI3K/Akt and CREB pathways[J].Int J Mol Med.2012,30(2):263-270
79Flad HD, Grage-Griebenow E, Petersen F, Scheuerer B, Brandt E, Baran J, PryjmaJ,Ernst M. The role of cytokines in monocyte apoptosis[J]. Pathobiology,1999,67(5-6):291-293
80Miao EA, Rajan JV, Aderem A. Caspase-1-induced pyroptotic cell death[J]. ImmunolRev.2011,243(1):206-214
81Nett-Fiordalisi M, Tomaselli K, Russell JH, Chaplin DD. Macrophage apoptosisin the absence of active interleukin-1beta-converting enzyme[J]. J Leukoc Biol,1995,58(6):717-724
82Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages[J].Crit Rev Eukaryot Gene Expr,2010,20(2):87-103
83Suzuki T, Kobayashi M, Isatsu K, Nishihara T, Aiuchi T, Nakaya K, Hasegawa K.Mechanisms involved in apoptosis of human macrophages induced bylipopolysaccharide from Actinobacillus actinomycetemcomitans in the presenceof cycloheximide[J]. Infect Immun,2004,72(4):1856-1865
84Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction by humaninterleukin-6of apoptosis in the promonocytic cell line U937and humanneutrophils[J]. J Biol Chem,1992,267(30):21612-21616
85Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F.Principles of interleukin-6-type cytokine signalling and its regulation[J].Biochem J,2003,374(pt1):1-20
86Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies:integrating mammalian biology[J]. Cell,2001,104(4):487-501
87Regis G, Icardi L, Conti L, Chiarle R, Piva R, Giovarelli M, Poli V, NovelliF. IL-6, but not IFN-gamma, triggers apoptosis and inhibits in vivo growth ofhuman malignant T cells on STAT3silencing[J]. Leukemia,2009,23(11):2102-–2108
88Weber A,Wasiliew P,Kracht M. Interleukin-1(IL-1) pathway[J].Sci Signal,2010,3(105)
89LyeE,MirtsosC,SuzukiN,etal.TheroleofIRAK-4kinaseactivityinIRAK-4-mediatedsignaling[J].JBiolChem,2004，279（39）:40653-40658
90Triantafilou M,Triantafilou k.The dynamics of LPS recognition:complex orchestration of multiplereceptors[J].J Endotoxin Res,2005,11（1）:5-11
91Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction by humaninterleukin-6of apoptosis in the promonocytic cell line U937and humanneutrophils[J]. J Biol Chem，1992，267（30）:21612-21616
92Deffaud J, Kirchmeyer M, Domagala F, Ficheux H, Netter P, Bianchi A, JouzeauJY. Modulatory effect of rhein on IL-1alpha-induced responses in humanchondrocytes: a comparative study between antibody microarrays and specificELISAs[J]. Biorheology，2008，45（3-4）:439-455
93Heo SK, Yun HJ, Park WH, Park SD. Emodin inhibits TNF-alpha-induced human aorticsmooth-muscle cell proliferation via caspase-and mitochondrial-dependentapoptosis[J]. J Cell Biochem，2008，105（1）:70-80
94刘善清.TOll样受体4在信号通路在脂多糖促进肺癌细胞增殖中的机制探讨[J]．肿瘤,2010,30（10）：838-842
95步宏.分子病理学进展[M].四川大学出版社.2003.102-103
96Schaub B, Bellou A, Gibbons FK, Velasco G, Campo M, He H, Liang Y, Gillman MW,Gold D, Weiss ST, Perkins DL, Finn PW. TLR2and TLR4stimulation differentiallyinduce cytokine secretion in human neonatal, adult, and murine mononuclearcells[J]. J Interferon Cytokine Res，2004，24（9）:543－552
97Sharma J, Mishra BB, Li Q, Teale JM. TLR4-dependent activation of inflammatorycytokine response in macrophages by Francisella elongation factor Tu[J]. CellImmunol，2011，269（2）:69-73
98Guo Y, Xu X, Li Q, Li Z, Du F. Anti-inflammation effects of picroside2in cerebralischemic injury rats[J]. Behav Brain Funct，2010，6:43
99Tu CT, Han B, Yao QY, Zhang YA, Liu HC, Zhang SC. Curcumin attenuates ConcanavalinA-induced liver injury in mice by inhibition of Toll-like receptor (TLR)2, TLR4and TLR9expression[J]. Int Immunopharmacol，2012，12（1）:151-157
100Zhu XL, Wang YJ, Yang YΖ, Yang RC, Zhu B, Zhang YΗ, Lin Y, Lu Y, Li XF, O'ByrneKT. Suppression of lipopolysaccharide-induced upregulation of toll-likereceptor4by emodin in mouse proximal tubular epithelial cells[J]. Mol MedReport，2012，6（3）:493-500
101Cabrera-Benitez NE, Pérez-Roth E, Casula M, Ramos-Nuez A, Ríos-Luci C, Rodríguez-Gallego C, Sologuren I, Jakubkiene V, Slutsky AS, Padrón JM, Villar J.Anti-inflammatory activity of a novel family of aryl ureas compounds in anendotoxin-induced airway epithelial cell injury model[J]. PLoS One，2012，7（11）:e48468
102Scherer D.C., Brockman J.A., Chen Z., Maniatis T., Ballard D.W. Signal-induceddegradation of IkappaB alpha requires site-specific ubiquitination[J]. ProcNatl Acad Sci USA，1995，92（24）:11259-11263
103Lawrence T. The nuclear factor NF-B pathway in inflammation[J]. Cold Spring Harb Perspect Biol，2009，1:a001651
104Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB-M, Mueller-Dieckmann C,Farahifar D, Rossi B, Auberger P, Baeuerle PA, Peyron J-F. Tyrosinephosphorylation of IkappaB-alpha activates NF-kappaB without proteolyticdegradation of IkappaB-alpha[J]. Cell，1996，86（5）:787-798
105Peri F, Piazza M, Calabrese V, Damore G, Cighetti R. Exploring the LPS/TLR4signal pathway withsmall molecules[J]. Biochem Soc Trans，2010，38（5）:1390-1395
106JohnsonDA.SyntheticTLR-4activeglycolipidsasvaccineadjuvantsandstand-aloneimmunotherapeutics[J].Curr Topics Med Chem，2008，8（2）:64-79
107Rossignol DP, Lynn M. Antagonism of in vivo and ex vivo response to endotoxinby E5564, a synthetic lipid A analogue[J]. J Endotoxin Res，2002，8（6）:483-488