穿心莲内酯对金黄色葡萄球菌氨基酸及糖代谢的影响研究
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摘要
目的:考察穿心莲内酯对金黄色葡萄球菌氨基酸及糖代谢的影响。方法:将10μL 0.5麦氏浓度(1×10~8 CFU/mL)的金黄色葡萄球菌(ATCC25923)混悬液,分别加入空白或含药MH-B培养基中,使各组细菌终浓度为1×10~6 CFU/mL。各组相同条件下放置于37℃恒温培养箱中连续孵育,并每1小时用酶标仪检测培养体系吸光度,绘制各组细菌24h生长曲线;采用氨基酸衍生化法检测每小时细菌培养体系中细菌培养液及细菌胞内20种氨基酸的含量,分析比较穿心莲内酯对金黄色葡萄球菌24h内氨基酸代谢的影响;每小时取细菌混悬液,采用葡萄糖氧化酶-过氧化物酶法对细菌培养液及细菌胞内葡萄糖进行含量测定,分析比较穿心莲内酯对金黄色葡萄糖球菌24h内葡萄糖代谢的影响。结果:各组细菌24h内生长曲线未见明显变化;给药后金黄色葡萄球菌体外培养基中氨基酸含量各组无差异,但细菌胞体裂解液中氨基酸组成的比例有明显变化,其中以组氨酸、亮氨酸和苯丙氨酸差异较为显著;细菌胞体内葡萄糖含量未见明显差异,但在前10h穿心莲内酯0.5μg/ml、50μg/ml组细菌胞外消耗葡萄糖速率明显增加。结论:穿心莲内酯在体外对金黄色葡萄球菌无直接抑制作用,但能干预金黄色葡萄球菌氨基酸及葡萄糖的代谢,提示其可能通过提高细菌对环境的营养物质摄入从而降低其致病性,从而间接发挥降低细菌毒力的作用。
Objective: To study the effect of Andrographolide on the amino acid metabolism of staphylococcus aureus. Methods: 0. 5 M of staphylococcus aureus( ATCC25923) mixed suspension was added respectively into blank or medicated MH-B medium,making the final bacteria concentration 1 × 10~6 CFU/mL. All groups were placed in 37 ℃ constant temperature incubator,at the same conditions for 24 h incubation.Absorbance was determined by microplate reader each hour,and the growth curve of bacteria within 24 h in each group was plotted. The contents of 20 kinds of amino acids in the bacterial culture medium and the bacterial cell were determined by the method of amino acid derivation,to analyze the effects of andrographolide on amino acid metabolism of staphylococcus aureus in 24 h. Bacterial suspensions were taken every hour to determine the contents of bacterial culture media and glucose in bacterial cells,in order to analyze the effects of andrographolide on glucose metabolism of staphylococcus aureus in 24 h. Results: No difference was observed in bacterial growth curve between groups. No difference was observed in concentration between the amino acid groups in the medium after administration,but the proportion of amino acid composition in the lysate of the bacterial flora changed significantly after administration,while the HIS,LEU and PHE has more significant difference. No significant difference of glucose content was observed in the bacterial cells,while the glucose consumption in the 50 μg/mL and 0. 5 μg/mL drug administration group was significantly increased than that in the blank group for the first 10 hours. Conclusion: Andrographolide had no direct inhibition on staphylococcus aureus in vitro,but has intervention effect on amino acid and glucose metabolism of staphylococcus aureus. It suggests that it may increase the bacteria intake of nutrients to the environment,reduce the pathogenicity of bacteria,in order to reduce the cytotoxicity.
Objective: To study the effect of Andrographolide on the amino acid metabolism of staphylococcus aureus. Methods: 0. 5 M of staphylococcus aureus( ATCC25923) mixed suspension was added respectively into blank or medicated MH-B medium,making the final bacteria concentration 1 × 10~6 CFU/mL. All groups were placed in 37 ℃ constant temperature incubator,at the same conditions for 24 h incubation.Absorbance was determined by microplate reader each hour,and the growth curve of bacteria within 24 h in each group was plotted. The contents of 20 kinds of amino acids in the bacterial culture medium and the bacterial cell were determined by the method of amino acid derivation,to analyze the effects of andrographolide on amino acid metabolism of staphylococcus aureus in 24 h. Bacterial suspensions were taken every hour to determine the contents of bacterial culture media and glucose in bacterial cells,in order to analyze the effects of andrographolide on glucose metabolism of staphylococcus aureus in 24 h. Results: No difference was observed in bacterial growth curve between groups. No difference was observed in concentration between the amino acid groups in the medium after administration,but the proportion of amino acid composition in the lysate of the bacterial flora changed significantly after administration,while the HIS,LEU and PHE has more significant difference. No significant difference of glucose content was observed in the bacterial cells,while the glucose consumption in the 50 μg/mL and 0. 5 μg/mL drug administration group was significantly increased than that in the blank group for the first 10 hours. Conclusion: Andrographolide had no direct inhibition on staphylococcus aureus in vitro,but has intervention effect on amino acid and glucose metabolism of staphylococcus aureus. It suggests that it may increase the bacteria intake of nutrients to the environment,reduce the pathogenicity of bacteria,in order to reduce the cytotoxicity.
引文
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[10]Karina K, Sanchez, Grischa Y Ch, Alexandria M, et al. Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen. Cell,2018,174(16)∶1～13.
[2]王芳,翟文婷,李艳丽,等.穿心莲内酯制剂及药代动力学的研究进展.中国药业,2013,22(9)∶1～4.
[3]王丽娟,谌立巍,何巧,等. 穿心莲内酯的协同抗菌作用及相关机制研究.中药药理与临床,2017,33(6)∶45～49.
[4]中国国家药典委员会.中国药典(一部).中国医药科技出版社,2015∶268～269.
[5]肖敏,杨峰,王旭荣,等. 分光光度法测定金黄色葡萄球菌菌液浓度方法的建立. 动物医学进展,2014,35(11)∶40～43.
[6]杨明明,李国晏,应莲芳,等.细菌培养基中营养物质的代谢及优化方法.微生物学免疫学进展,2016,44(5)∶90～94.
[7]Yan Zhang, Ping Wang, Youdong Xu, et al. Metabolomic Analysis of Biochemical Changes in the Plasma of High-Fat Diet and Streptozotocin-Induced Diabetic Rats after Treatment with Isoflavones Extract of Radix Puerariae. Evidence-Based Complementary and Alternative Medicine,2016,2016(5)∶1～12.
[8]Newgard C B, An J, Bain J R, et al. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metabolism,2009,9(4)∶311～326.
[9]Saha A K, Xu X J, Lawson E, et al. Downregulation of AMPK accompanies leucine- and glucose-induced increases in protein synthesis and insulin resistance in rat skeletal muscle. Diabetes,2010,59(10)∶2426～2434.
[10]Karina K, Sanchez, Grischa Y Ch, Alexandria M, et al. Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen. Cell,2018,174(16)∶1～13.
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