芪黄汤及其提取成分抗大肠杆菌志贺样毒素Ⅱ型变异体作用及其机理研究
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摘要
猪水肿病(Edema disease of pig,EDP)又称肠毒血症,是由几种特定血清型的致病性大肠杆菌(O138,O139和O141)引发的,而志贺样毒素Ⅱ型变异体(SLT-IIv)是引发EDP的主要毒力因子之一。大肠杆菌菌株大多在消化道和小肠附着繁殖,产生的毒素以特异性机制进入血液循环,引起肠道、皮肤和大脑血管内皮细胞的病变和坏死,进一步引起血管通透性增强,从而导致受影响的器官出现水肿,最终导致动物死亡。这种疾病蔓延迅速,致死率高,给养猪业造成极大的伤害和损失。当前多利用母源抗体预防该病,但效果并不尽如人意。虽然抗生素或抗血清可以用于该病的防治,但是存在耐药性、药物残留和成本高等问题。中兽医学临床实践和现代中药药理学研究表明,许多中药能有效抑制和消除细菌病原体、清除细菌毒素,具有疗效高、毒性低、副作用小、耐药性低及药物残留水平低的优点,并被广泛用于预防和治疗传染病,所以,研究利用中药防治细菌性疾病已成为当今兽医界的热点。前期试验证实,芪黄汤具有很好的治疗SLT-IIv引发的水肿病的作用,本试验即在此基础上进行深入研究。
本研究采用Affymetrix小鼠全基因组表达谱芯片和差异荧光凝胶电泳(DIGE)对芪黄汤抗志贺样毒素Ⅱ型变异体(SLT-IIv)机理进行研究。以小鼠肠微血管内皮细胞(MIMVECs)为研究对象,先后加入SLT-IIv和芪黄汤作用12 h后,提取四个处理组的总RNA和总蛋白。基因芯片及差异荧光凝胶电泳技术检测各组基因、蛋白的变化情况并进行生物学分析。SLT-Ⅱv组与对照组比较共有63个差异表达基因,其中上调基因49个,下调基因14个,73个差异表达蛋白,其中表达下调共25个蛋白,48个蛋白表达上调;芪黄汤与对照组相比较有128个差异表达基因,其中上调基因44个,下调基因84个,26个差异表达蛋白,19个表达上调蛋白,7个蛋白表达下调;SLT-Ⅱv-芪黄汤与对照组比较有108个差异表达基因,其中上调基因72个,下调基因36个,126个差异表达蛋白,上调下调表达各63个蛋白;SLT-Ⅱv-芪黄汤与SLT-Ⅱv组比较有111个差异表达基因,其中上调基因74个,下调基因37个,224个差异表达蛋白,其中139个蛋白表达上调,85个蛋白表达下调。综合各组差异基因、蛋白发现,芪黄汤通过抑制抗凋亡基因的转录,促进受损细胞凋亡,加速机体蛋白的合成,增强细胞能量的代谢,减少不正常肌动蛋白的表达,回复正常细胞骨架排布等方面来改善SLT-Ⅱv所产生的细胞损伤、血管收缩和细胞通透性变化等各项毒理效应。
为进一步研究芪黄汤的作用机理,本研究采用高效液相色谱法分离提纯芪黄汤的水溶性成分。以乙腈和0.5%乙酸水溶液为流动相按一定梯度洗脱条件分离提纯芪黄汤,获得15个可溶于水的提取成分,且通过分析型高效液相色谱和液质连用仪初步鉴定出:7峰为没食子酸,13峰为槲皮素,并进一步研究各成分对MIMVECs的作用。
首先,为测定芪黄汤及其提取成分对经SLT-IIv诱导损伤的MIMVECs的影响,以MIMVECs感染SLT-IIv为细胞模型,芪黄汤及其提取成分分别作用12 h后收集细胞上清液,测定各项指标。结果显示,芪黄汤、醇沉上清组、醇沉沉淀组和2、4、6、11、12、14、15号提取峰部分浓度作用于受损细胞,能极显著(P<0.01)降低LDH的活力,而3、7、8、10和13号提取峰与毒素对照组无显著性差异;除醇沉沉淀组外,各组药物和成分在一定浓度范围内作用于受损细胞后,能极显著(P<0.01)提高受损细胞的SOD活力;芪黄汤组、醇沉上清组、醇沉沉淀和2、3、4、7、8、11、12、14、15号提取峰在部分浓度条件下,能极显著(P<0.01)降低MDA含量,而9、10号提取峰MDA含量与毒素对照组无显著性差异;除5号提取峰外,其余各组药物和成分在一定浓度作用下,GSH-Px活力极显著(P<0.01)高于毒素对照组。以上结果说明,芪黄汤及其成分均可明显提高MIMVECs的SOD、GSH-Px活性,降低MDA含量,从而清除体内大量的对组织、细胞具有严重损害作用的自由基。经3、7、8、11号提取峰作用后,能极显著(P<0.01)降低受损细胞分泌NO的量;各药物成分组与毒素对照组比较,均能显著(P<0.05)或极显著(P<0.01)降低毒素作用后细胞的ET-1含量,而各组间ET-1含量差别不显著。表明,芪黄汤可在由NO和ET-1介导的机体腹泻、水肿等疾病中发挥治疗作用。
其次,从测定BSA通过Transwell小室的OD值变化来探讨芪黄汤及其提取成分对SLT-IIv损伤的MIMVECs通透性的影响,结果表明各种提取成分均能不同程度降低SLT-IIv导致的MIMVECs通透性升高。
最后,应用激光多普勒血流仪来测量芪黄汤及其提取成分导入前后对三阴交穴位区微血管振幅的影响,结果显示,除醇沉沉淀物和5、8、12、14号提取峰作用后微血管振幅变化不显著外,其余芪黄汤部分和提取成分均可不同程度提高微血管振幅,表明芪黄汤和部分提取成分能够改善机体血液微循环。
综上所述,芪黄汤及其提取成分能够有效抗SLT-IIv作用,其机理主要是通过改善MIMVECs损伤、降低微血管通透性和改善微循环,达到治疗猪水肿病的目的。
The Shiga-like toxin type II variant (SLT-IIv) causes pig edema disease (ED) and it may cause vascular endothelial cell lesions in the intestine, subcutis and the brain, leading to edema in affected organs, neurological symptoms and eventually death. The disease spreads quickly and induces high mortality rates, resulting in substantial harm and loss to the pig industry. Many Chinese herbal medicines are widely used to prevent and cure infectious diseases, demonstrating high efficacy, lower toxicity, fewer side effects, less drug resistance and lower residual levels than most drugs currently on the market. Therefore, prevention and treatment of bacterial diseases by traditional Chinese medicine has become a research focus. QiHuang Decoction (QHD) has a good effect on edema caused by SLT-IIv, confirmed by the previous experiments in our laboratory. On the basis of it, we carried out this experiment to further investigate the mechanism of QHD.
In these study, an Affymetrix mouse genome array and Two-Dimensional differential gel electrophoresis (2-D DIGE) techniques were used to investigate the mechanisms of QHD on the injury of mouse intestinal microvascular endothelial cells (MIMVECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMVECs were challenged with 10μg/ml SLT-IIv for 12 h and then treated with QHD at a concentration of 200μg/ml for 12 h. Total RNA and proteins were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip? Mouse Genome 430 2.0 microarray and 2-D DIGE. The results showed that 63 genes and 73 proteins were differentially expressed, of which 49 genes were upregulated, 14 genes downregulated, 25 protein downregulated and 48 proteins upregulated in the SLT-IIv group compared to the control group. In the QHD group, 44 genes and 19 proteins were upregulated, 84 genes and 7 proteins were downregulated, compared to the control group. In the SLT-IIv-QHD group, 72 genes were upregulated, 36 genes were downregulated, and the number of either downregulated or upregulated proteins was 63, compared to the control group. When the QHD-treated group was compared to the SLT-IIv group, expression of 74 gene was found to be increased, and all other genes were decreased, with 139 proteins upregulated and 85 downregulated. Analysis of the data demonstrated that QHD specifically and effectively reduced microvascular endothelial cell permeability induced by SLT-IIv in the treatment of pig edema disease. Then, on the basis of these study, we will make a further research about its phamacological actions.
In order to further study the mechanism of QHD, we used high performance liquid chromatography (HPLC) to purify the water-soluble constituents of it. According to a certain condition of gradient elution with acetonitrile and 0.5% acetic acid solution as mobile phase, 15 water-soluble extracts were purified, and peak-7 and peak-13 were initially identified to be gallic acid and quercetin, respectively, by analytical HPLC and LC-MS. Then further study of the effect of the various components on the MIMVECs was conducted.
First, to determine the effects of QHD and its extracts on the MIMVECs injury induced by SLT-IIv, the supernatant of MIMVECs, infected with SLT-IIv and then treated with QHD and its extracts for 12 h, were harvested. The results showed that QHD, QHD alcohol precipitation supernatant group(QHD-AP), QHD alcohol precipitation precipitation group(QHD-P) and peak-2, 4, 6, 11, 12, 14, and 15, could significantly (P <0.01) reduced the vitality of LDH at the indicated concentrations, while peak-3, 7, 8, 10 and 13 no significant differences compared to the SLT-IIv group. Except for the QHD-P group, the other groups at the indicated concentrations could significantly increase the SOD activity of damaged cells (P <0.01). QHD group, QHD-AP group, QHD-P group and peak-2, 3, 4, 7, 8, 11, 12, 14, and 15, can significantly decrease the content of MDA at the indicated concentrations (P <0.01), while MDA contents of peak-9 and 10 were not significantly different from the SLT-IIv group. Except for that of peak-5, GSH-Px activities of other groups at the indicated concentrations, were significantly higher than that of the SLT-IIv group (P <0.01). These results showed that almost all the ingredients of QHD can improve the SOD and GSH-Px activities, or decrease the content of MDA in MIMVECs These suggested that QHD and its ingredients can remove a lot of free radicals which damage the tissues and cells. Peak-3, 7, 8, and 11 could significantly reduce the content of NO from SLT-IIv-damaged cells (P <0.01). All indicated drugs can significantly decrease the content of ET-1 from SLT-IIv-damaged cells (P<0.01 or P<0.05 ). These data indicated that QHD can play a important role in the disease like diarrhea or edema caused by NO and ET-1. The results showed that most of the ingredients can markedly enhance the activities of SOD and GSH-Px, and reduce the LDH activity and the content of NO, ET-1, and MDA from SLT-IIv-damaged MIMVECs.
Second, to explore the effects of QHD and its extracts on permeability of MIMVECs damaged by SLT-IIv, the OD values of the BSA through the Transwell were measured. The results showed that the extracts in varying degrees could reduce the MIMVECs permeability induced by SLT-IIv.
Finally, the microvascular amplitude of Sanyinjiao points treated with QHD and its extracts were measured with laser Doppler flowmeter. The results showed that, except for QHD-P and peak-5, 8, 12, and 14, other indicated drugs can increase the microvascular amplitude to some extent, indicating that QHD can play an important role to improve the blood microcirculation.
In conclusion, QHD and its extracts can effectively resist the effect of SLT-IIv, the mechanism of which is to improve the MIMVECs injury, reduce microvascular permeability, and improve the blood microcirculation, further curing the swine edema disease.
本研究采用Affymetrix小鼠全基因组表达谱芯片和差异荧光凝胶电泳(DIGE)对芪黄汤抗志贺样毒素Ⅱ型变异体(SLT-IIv)机理进行研究。以小鼠肠微血管内皮细胞(MIMVECs)为研究对象,先后加入SLT-IIv和芪黄汤作用12 h后,提取四个处理组的总RNA和总蛋白。基因芯片及差异荧光凝胶电泳技术检测各组基因、蛋白的变化情况并进行生物学分析。SLT-Ⅱv组与对照组比较共有63个差异表达基因,其中上调基因49个,下调基因14个,73个差异表达蛋白,其中表达下调共25个蛋白,48个蛋白表达上调;芪黄汤与对照组相比较有128个差异表达基因,其中上调基因44个,下调基因84个,26个差异表达蛋白,19个表达上调蛋白,7个蛋白表达下调;SLT-Ⅱv-芪黄汤与对照组比较有108个差异表达基因,其中上调基因72个,下调基因36个,126个差异表达蛋白,上调下调表达各63个蛋白;SLT-Ⅱv-芪黄汤与SLT-Ⅱv组比较有111个差异表达基因,其中上调基因74个,下调基因37个,224个差异表达蛋白,其中139个蛋白表达上调,85个蛋白表达下调。综合各组差异基因、蛋白发现,芪黄汤通过抑制抗凋亡基因的转录,促进受损细胞凋亡,加速机体蛋白的合成,增强细胞能量的代谢,减少不正常肌动蛋白的表达,回复正常细胞骨架排布等方面来改善SLT-Ⅱv所产生的细胞损伤、血管收缩和细胞通透性变化等各项毒理效应。
为进一步研究芪黄汤的作用机理,本研究采用高效液相色谱法分离提纯芪黄汤的水溶性成分。以乙腈和0.5%乙酸水溶液为流动相按一定梯度洗脱条件分离提纯芪黄汤,获得15个可溶于水的提取成分,且通过分析型高效液相色谱和液质连用仪初步鉴定出:7峰为没食子酸,13峰为槲皮素,并进一步研究各成分对MIMVECs的作用。
首先,为测定芪黄汤及其提取成分对经SLT-IIv诱导损伤的MIMVECs的影响,以MIMVECs感染SLT-IIv为细胞模型,芪黄汤及其提取成分分别作用12 h后收集细胞上清液,测定各项指标。结果显示,芪黄汤、醇沉上清组、醇沉沉淀组和2、4、6、11、12、14、15号提取峰部分浓度作用于受损细胞,能极显著(P<0.01)降低LDH的活力,而3、7、8、10和13号提取峰与毒素对照组无显著性差异;除醇沉沉淀组外,各组药物和成分在一定浓度范围内作用于受损细胞后,能极显著(P<0.01)提高受损细胞的SOD活力;芪黄汤组、醇沉上清组、醇沉沉淀和2、3、4、7、8、11、12、14、15号提取峰在部分浓度条件下,能极显著(P<0.01)降低MDA含量,而9、10号提取峰MDA含量与毒素对照组无显著性差异;除5号提取峰外,其余各组药物和成分在一定浓度作用下,GSH-Px活力极显著(P<0.01)高于毒素对照组。以上结果说明,芪黄汤及其成分均可明显提高MIMVECs的SOD、GSH-Px活性,降低MDA含量,从而清除体内大量的对组织、细胞具有严重损害作用的自由基。经3、7、8、11号提取峰作用后,能极显著(P<0.01)降低受损细胞分泌NO的量;各药物成分组与毒素对照组比较,均能显著(P<0.05)或极显著(P<0.01)降低毒素作用后细胞的ET-1含量,而各组间ET-1含量差别不显著。表明,芪黄汤可在由NO和ET-1介导的机体腹泻、水肿等疾病中发挥治疗作用。
其次,从测定BSA通过Transwell小室的OD值变化来探讨芪黄汤及其提取成分对SLT-IIv损伤的MIMVECs通透性的影响,结果表明各种提取成分均能不同程度降低SLT-IIv导致的MIMVECs通透性升高。
最后,应用激光多普勒血流仪来测量芪黄汤及其提取成分导入前后对三阴交穴位区微血管振幅的影响,结果显示,除醇沉沉淀物和5、8、12、14号提取峰作用后微血管振幅变化不显著外,其余芪黄汤部分和提取成分均可不同程度提高微血管振幅,表明芪黄汤和部分提取成分能够改善机体血液微循环。
综上所述,芪黄汤及其提取成分能够有效抗SLT-IIv作用,其机理主要是通过改善MIMVECs损伤、降低微血管通透性和改善微循环,达到治疗猪水肿病的目的。
The Shiga-like toxin type II variant (SLT-IIv) causes pig edema disease (ED) and it may cause vascular endothelial cell lesions in the intestine, subcutis and the brain, leading to edema in affected organs, neurological symptoms and eventually death. The disease spreads quickly and induces high mortality rates, resulting in substantial harm and loss to the pig industry. Many Chinese herbal medicines are widely used to prevent and cure infectious diseases, demonstrating high efficacy, lower toxicity, fewer side effects, less drug resistance and lower residual levels than most drugs currently on the market. Therefore, prevention and treatment of bacterial diseases by traditional Chinese medicine has become a research focus. QiHuang Decoction (QHD) has a good effect on edema caused by SLT-IIv, confirmed by the previous experiments in our laboratory. On the basis of it, we carried out this experiment to further investigate the mechanism of QHD.
In these study, an Affymetrix mouse genome array and Two-Dimensional differential gel electrophoresis (2-D DIGE) techniques were used to investigate the mechanisms of QHD on the injury of mouse intestinal microvascular endothelial cells (MIMVECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMVECs were challenged with 10μg/ml SLT-IIv for 12 h and then treated with QHD at a concentration of 200μg/ml for 12 h. Total RNA and proteins were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip? Mouse Genome 430 2.0 microarray and 2-D DIGE. The results showed that 63 genes and 73 proteins were differentially expressed, of which 49 genes were upregulated, 14 genes downregulated, 25 protein downregulated and 48 proteins upregulated in the SLT-IIv group compared to the control group. In the QHD group, 44 genes and 19 proteins were upregulated, 84 genes and 7 proteins were downregulated, compared to the control group. In the SLT-IIv-QHD group, 72 genes were upregulated, 36 genes were downregulated, and the number of either downregulated or upregulated proteins was 63, compared to the control group. When the QHD-treated group was compared to the SLT-IIv group, expression of 74 gene was found to be increased, and all other genes were decreased, with 139 proteins upregulated and 85 downregulated. Analysis of the data demonstrated that QHD specifically and effectively reduced microvascular endothelial cell permeability induced by SLT-IIv in the treatment of pig edema disease. Then, on the basis of these study, we will make a further research about its phamacological actions.
In order to further study the mechanism of QHD, we used high performance liquid chromatography (HPLC) to purify the water-soluble constituents of it. According to a certain condition of gradient elution with acetonitrile and 0.5% acetic acid solution as mobile phase, 15 water-soluble extracts were purified, and peak-7 and peak-13 were initially identified to be gallic acid and quercetin, respectively, by analytical HPLC and LC-MS. Then further study of the effect of the various components on the MIMVECs was conducted.
First, to determine the effects of QHD and its extracts on the MIMVECs injury induced by SLT-IIv, the supernatant of MIMVECs, infected with SLT-IIv and then treated with QHD and its extracts for 12 h, were harvested. The results showed that QHD, QHD alcohol precipitation supernatant group(QHD-AP), QHD alcohol precipitation precipitation group(QHD-P) and peak-2, 4, 6, 11, 12, 14, and 15, could significantly (P <0.01) reduced the vitality of LDH at the indicated concentrations, while peak-3, 7, 8, 10 and 13 no significant differences compared to the SLT-IIv group. Except for the QHD-P group, the other groups at the indicated concentrations could significantly increase the SOD activity of damaged cells (P <0.01). QHD group, QHD-AP group, QHD-P group and peak-2, 3, 4, 7, 8, 11, 12, 14, and 15, can significantly decrease the content of MDA at the indicated concentrations (P <0.01), while MDA contents of peak-9 and 10 were not significantly different from the SLT-IIv group. Except for that of peak-5, GSH-Px activities of other groups at the indicated concentrations, were significantly higher than that of the SLT-IIv group (P <0.01). These results showed that almost all the ingredients of QHD can improve the SOD and GSH-Px activities, or decrease the content of MDA in MIMVECs These suggested that QHD and its ingredients can remove a lot of free radicals which damage the tissues and cells. Peak-3, 7, 8, and 11 could significantly reduce the content of NO from SLT-IIv-damaged cells (P <0.01). All indicated drugs can significantly decrease the content of ET-1 from SLT-IIv-damaged cells (P<0.01 or P<0.05 ). These data indicated that QHD can play a important role in the disease like diarrhea or edema caused by NO and ET-1. The results showed that most of the ingredients can markedly enhance the activities of SOD and GSH-Px, and reduce the LDH activity and the content of NO, ET-1, and MDA from SLT-IIv-damaged MIMVECs.
Second, to explore the effects of QHD and its extracts on permeability of MIMVECs damaged by SLT-IIv, the OD values of the BSA through the Transwell were measured. The results showed that the extracts in varying degrees could reduce the MIMVECs permeability induced by SLT-IIv.
Finally, the microvascular amplitude of Sanyinjiao points treated with QHD and its extracts were measured with laser Doppler flowmeter. The results showed that, except for QHD-P and peak-5, 8, 12, and 14, other indicated drugs can increase the microvascular amplitude to some extent, indicating that QHD can play an important role to improve the blood microcirculation.
In conclusion, QHD and its extracts can effectively resist the effect of SLT-IIv, the mechanism of which is to improve the MIMVECs injury, reduce microvascular permeability, and improve the blood microcirculation, further curing the swine edema disease.
引文
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