Background
Pyocyanin is a biologically active phenazine pigment produced by the bacterium, Pseudomonas aeruginosa, acting as a nitric oxide (NO) antagonist in various pharmacological preparations and as mediator in biosensors. Pyocyanin can also be used as electron shuttle in microbial fuel cells enabling bacterial electron transfers. Furthermore, Pyocyanin has broad antibiotic activity as well as has been identified as the key molecule produced by Pseudomonas that inhibits growth of pathogenic vibrios in aquaculture systems.[1]
In vitro study was performed to measure the cytotoxicity of Pyocyanin. Results indicated that L-132 cells were prone to Pyocyanin-induced toxicity. The IC50 value of Pyocyanin on inhibition of mitochondrial dehydrogenase activity was 112.01 ± 23.73 mgl-1. The IC50 value of Pyocyanin induced damage of plasma membrane was 21.79 ± 14.23 mg l-1. Moreover, Pyocyanin showed an IC50 of 32.57 ± 16.52 mg l-1?on inhibition of protein synthesis. When Pyocyanin has concentration of 25 mgl-1, 3.9 % inhibition of mitochondrial activity, 47.3 % plasma membrane damage and 26.6 % inhibition of protein synthesis were observed in L-132 cells. Whereas at lower concentration (6.25 mgl-1) the toxicity was negligible, whereas at 200 mg l-1?the values were 64.8, 72.8 and 91.7 %, respectively.[1]
In vivo study demonstrated that Pyocyanin was able to slow the beating of human respiratory tract cilia. The effects of Pyocyanin on tracheal mucus velocity of radiolabeled erythrocytes were tested in anesthetized guinea pigs. The effect of Pyocyanin was slower in onset, 600 ng causing 60% reduction in tracheal mucus velocity at 3 h, and no recovery occurred. Whereas combination of Pyocyanin and 1-hydroxyphenazine produced an initial rapid slowing equivalent to the same dose of 1-hydroxyphenazine given alone, but the later slowing attributed to Pyocyanin was greater than the same dose administered alone. This study demonstrates one mechanism by which products of P. aeruginosa, such as Pyocyanin may facilitate its colonization of the respiratory tract.
参考文献:
[1]. Priyaja P, et al. Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications. Cytotechnology. 2016 Jan;68(1):143-155.
[2]. Arora D, et al. Pyocyanin induces systemic oxidative stress, inflammation and behavioral changes in vivo. Toxicol Mech Methods. 2018 Jul;28(6):410-414.
绿脓菌素是一种具有生物活性的吩嗪色素，由铜绿假单胞菌产生，在各种药理学制剂中充当一氧化氮 (NO) 拮抗剂，并在生物传感器中充当介质。绿脓菌素也可用作微生物燃料电池中的电子穿梭，从而实现细菌电子转移。此外，绿脓菌素具有广泛的抗生素活性，并且已被确定为假单胞菌产生的关键分子，可抑制水产养殖系统中致病性弧菌的生长。[1]
进行了体外研究以测量绿脓素的细胞毒性。结果表明，L-132 细胞容易产生绿脓素诱导的毒性。绿脓素抑制线粒体脱氢酶活性的 IC50 值为 112.01 ± 23.73 mg l-1。绿脓素诱导质膜损伤的 IC50 值为 21.79 ± 14.23 mg l-1。此外，绿脓素抑制蛋白质合成的 IC50 为 32.57 ± 16.52 mg l-1。当绿脓素浓度为 25 mg l-1 时，L-132 细胞线粒体活性抑制 3.9%，质膜损伤 47.3%，蛋白质合成抑制 26.6%。而在较低浓度（6.25 mg l-1）下，毒性可以忽略不计，而在 200 mg l-1 时，该值分别为 64.8、72.8 和 91.7 %。[1]
体内研究表明，绿脓素能够减缓人类呼吸道纤毛的跳动。在麻醉的豚鼠中测试了绿脓素对放射性标记的红细胞气管粘液速度的影响。绿脓素起效较慢，600 ng 导致 3 小时时气管粘液速度降低 60%，并且没有恢复。虽然绿脓素和 1-羟基吩嗪的组合产生了与单独给予相同剂量的 1-羟基吩嗪相当的初始快速减慢，但归因于绿脓素的后期减慢大于单独给予相同剂量。本研究证明了绿脓杆菌的产物（例如绿脓素）可能促进其在呼吸道定植的一种机制。
Protocol
| Cell experiment [1]: |
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Cell lines
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Human embryonic lung epithelial cell line, L-132
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Preparation Method
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Cell cultures in 96 well plates were developed by adding 0.2 ml cell suspension in growth medium containing approximately 5 × 105 cells ml?1 and incubating for 12 h at appropriate temperature.
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Reaction Conditions
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Pyocyanin was prepared with different concentrations in growth medium. Then added to the wells to attain final strength ranging from 6.25 to 200 μg ml?1 for XTT (mitochondrial activity), neutral red up take (plasma membrane damage) and SRB (protein synthesis), 0–200 μg ml?1 for LDH and H2O2, and 25–200 μg ml?1 for glucose consumption in triplicate for each concentration. Incubate for 24 h.
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Applications
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The cytotoxicity of pyocyanin could be assessed by this experiment, facilitating pyocyanin’s safe usage toxicity. Higher concentrations of pyocyanin (175 and 200 mg l?1) only caused significant morphological changes such as clumping, and necrosis as visualized microscopically in L-132 cell line. Human cell membrane was found to be susceptible to oxidative damage induced by pyocyanin.
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| Animal experiment [2]: |
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Animal models
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Male C57BL/6J mice (8-10 weeks, 20-30 g)
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Preparation Method
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Mice were housed under controlled laboratory conditions, maintained on a 12 h day and night cycle. Then mice were acclimatized for 5 days to the lab conditions and handling prior to the actual beginning of the experiments. Mice were administered 0.9% saline as control, and pyocyanin by intranasal route. Another group was treated with LPS (P.aeruginosa) by intraperitoneal route to mimic the effects of ongoing infection on tissue permeability, and pyocyanin was instilled by intranasal route 3h after LPS injection.
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Dosage form
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Pyocyanin (50 μg/50 μL in 0.9% saline); 0.9% saline (50 μL); LPS (3 mg/kg)
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Applications
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Pyocyanin could be diffused into systemic circulation, which was not influenced by the pre-exposure to pseudomonal infestation. This experiment detected the plasma concentration of intranasally administered Pyocyanin. Furthermore, localized administration of Pyocyanin was able to elicit changes to behavior and a systemic pro-inflammatory and pro-oxidant effect.
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参考文献:
[1]. Priyaja P, et al. Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications. Cytotechnology. 2016 Jan;68(1):143-155.
[2]. Arora D, et al. Pyocyanin induces systemic oxidative stress, inflammation and behavioral changes in vivo. Toxicol Mech Methods. 2018 Jul;28(6):410-414.
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