救必应黄酮类成分抗产ESBLs大肠杆菌作用机理研究

The phenomenon of antibiotic resistance of Escherichia Coli has been serious, It has become urgent problems to be solved to seek safe and effective anti-infection drugs.Cortex Ilecis Rotundae has been a remedy for clearing heat and removing toxicity, hemostasis and antibacterial and the other effects. Prophase researches have proved that this drug had high antibacterial activity. The hypothesis of antibacterial mechanism was put forward in this study by screening out differential gene expression before and after the medicine effect of E.coli with gene chips,and analyzing the gene related to drug effects, the influence of upon bacterial growth, reproduction,and metabolism. It was analyzed that the traditional Chinese medicine influenced on the cell wall, cell membrane and protein through AKP, conductivity, extra-cellular protein content and SDS-PAGE electrophoresis; with the gel retardation assay, external perimysium,circular dichroism and fluorescence spectra, we could analyze the effect of extracts of Cortex Ilicis Rotundae on the structure of bacterial DNA and influence mode, to observe the influence of the extractss of the Chinese medicine on the structure of cell membrane after acting on the bacteria.Obtaining the bacterial DNA by PCR to synthesize the related gene(DNA replication initiators,DNA polymerase III gene,DNA gyrase gene,etc)and verify the combination of the extracts of Chinese medicine and the related genes synthesized by E.coli DNA could inhibit the protein synthesis,elucidate the mechanism of Cortex Ilecis Rotundae against ESBLs-producing E.coli.

大肠杆菌耐药性较为严重，寻求新的有效而安全抗感染药物成为亟待解决的问题。救必应具有清热解毒、止血抗菌等功效，前期研究该药具有较强的抗菌活性。本课题拟通过基因芯片筛选出救必应黄酮类作用大肠杆菌前后差异基因表达,分析与药物作用相关的基因以及药物对细菌生长繁殖、新陈代谢等多方面的影响,提出抗菌机理假设；从碱性磷酸酶（AKP）、电导率、胞外蛋白含量和SDS-PAGE 电泳分析中药对细胞壁、细胞膜和蛋白质的影响；采用凝胶阻滞实验和外光谱、圆二色谱和荧光光谱观察分析救必应提取物对细菌DNA的结构的影响及其方式，采用流式细胞术观察中药提取物作用于菌后对其细胞膜结构的影响。用PCR技术获得细菌DNA合成相关基因（DNA复制起始子基因、DNA聚合酶Ⅲ基因、DNA促旋酶基因等）并通过凝胶阻滞实验验证中药提取物与大肠杆菌DNA合成相关基因的作用及抑制蛋白质的合成，阐明救必应对产ESBLs大肠杆菌耐药抑制机理。

大肠杆菌是引起畜禽养殖经济损失较大的一种病原菌，其耐药性较为严重，对β-内酰胺类药物耐药率达到98%；对多肽类和氨基糖苷类耐药率稍低，分别为18.46%和21.54%。因此寻求新的高效而安全的药物或新的给药方式成为目前迫在眉睫的问题。救必应具有清热解毒、止血抗菌等功效，本实验发现救必应水煮液对大肠杆菌的MIC为0.25g/ml，经分析发挥抗菌作用的主要为黄酮类。本项目采用响应面积法优化并确定了黄酮的提取工艺：乙醇浓度60％，液料比40：1（mL/g），超声功率60%（420W），超声温度55℃的条件下提取40min，平行试验三次，救必应总黄酮的提取率为20.60%。采用二倍稀释法测定救必应总黄酮对链球菌与鲍曼不动杆菌的MIC均为20mg/mL，对粪肠球菌、普通变形杆菌、金黄色葡萄球菌的MIC值均为40mg/mL，对产ESBLs大肠杆菌、铜绿假单胞菌的MIC均为80mg/mL，均具有较好的抑菌作用。采用现代分子生物学技术以及ITRAQ观察了救必应黄酮类作用细菌前后AKP、电导率、细菌胞外蛋白等变化，发现救必应黄酮作用后菌液中的AKP活性、胞外可溶性蛋白含量增多，电导率增加，说明救必应能破坏细菌的细胞壁和细胞膜，使细胞内容物外漏从而引起的各项指标的变化。TEM图像表明救必应总黄酮粗提物作用后可明显引起大肠杆菌细胞壁皱褶、变瘪、菌体缩小等。救必应黄酮作用后可抑制或消除大肠杆菌总蛋白及DNA的合成。采用流式细胞术的Annexin-V和PI双染色法检测凋亡细菌的比例，发现加入救必应总黄酮之后，细菌凋亡的比例增加，并且具有统计学意义，浓度为1/2MIC的救必应总黄酮引起的Annexin-V染色阳性比例小于经1MIC救必应总黄酮作用的试验组。救必应总黄酮可以抑制或消除产ESBLs大肠杆菌总蛋白的合成，主要影响约35-40kDa片段大小的蛋白条带，经基质辅助激光解吸附串联飞行时间质谱仪进行蛋白质谱分析，主要影响果糖磷酸醛缩酶和ADP-甘油-甘露聚糖-6-差向异构酶两种蛋白含量。同时又构建了大肠杆菌败血症模型，验证了救必应的体内治疗效果。本项目发表文章31篇，26篇中文核心，SCI投稿3篇，获得授权专利20项，申请在审的发明专利5项，成果转化5项，申请新兽药1项，已递交农业部。