枸杞多糖抑制绿脓杆菌毒素引起的小鼠巨噬细胞RAW264.7氧化损伤作用研究
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摘要
目的探讨枸杞多糖减轻绿脓杆菌毒素(Pyocyanin,PCN)对小鼠巨噬细胞RAW264.7氧化损伤机制。方法细胞分为对照组(未处理组),绿脓杆菌毒素处理组,枸杞多糖处理组及枸杞多糖+绿脓杆菌毒素处理组,通过细胞MTT法检测绿脓杆菌毒素处理后枸杞多糖对小鼠巨噬细胞RAW264.7活性的保护作用;采用Western blot检测不同处理组细胞凋亡相关蛋白的表达;采用荧光染色法检测ROS的表达;采用ELASA法检测不同时间点细胞上清中IL-4的表达;采用流式细胞仪检测不同处理组细胞凋亡情况。结果 50μmol/L PCN处理后导致细胞增殖能力下降,凋亡抑制蛋白Bcl-xl表达量下调,ROS表达量上升(t=3.95,P<0.05)。随着处理时间的延长,PCN能够刺激细胞促炎因子IL-4过量表达,促进巨噬细胞细胞凋亡(t=7.15,P<0.05)。100 mg/ml LBP预处理RAW264.7细胞可显著促进Bcl-2(t=1.24,P<0.05)和Bcl-xl的表达(t=1.51,P<0.05),促炎因子IL-4和胞内ROS的表达显著下调(t值分别为8.15和2.57,均P<0.05),并PCN造成的巨噬细胞凋亡减少。结论 LBP可抑制PCN造成的胞内ROS产生,抑制IL-4释放,调控凋亡抑制蛋白Bcl-2、Bcl-xl和凋亡蛋白Caspase3的表达,进而提高PCN作用后RAW264.7细胞的增殖能力,为进一步研究绿脓杆菌致病机制及LBP的免疫保护作用提供了理论基础。
Objective To investigate the effect of Pseudomonas aeruginosa toxin pyocyanin(PCN) metabolite ROS accumulation on the expression of the proteins Bcl-2 and Bcl-xl, the inflammatory factor IL-4, and apoptosis in macrophages. Methods Cells were divided into a control group(untreated group), a P. aeruginosa toxin treatment group, an L. barbarum polysaccharide treatment group, and a P. aeruginosa toxin and L. barbarum polysaccharide treatment group. PCN was detected using a cell MTT assay. Expression of the apoptosis-related protein was detected using Western blotting. Expression of ROS was detected using fluorescence staining. ELASA was used to detect the expression of IL-4 in cell supernatant at different time points. Flow cytometry was used to detect apoptosis in different treatment groups. Results After treatment of cells with PCN, cell proliferation decreased, expression of the apoptosis-inhibiting protein Bcl-xl was down-regulated, and expression of ROS increased. As the duration of treatment progressed, PCN stimulated the overexpression of the pro-inflammatory factor IL-4 and promoted the apoptosis of macrophages. Pretreatment with L. barbarum polysaccharides significantly promoted the expression of Bcl-2(t=1.24, P<0.05), the expression of Bcl-xl(t=1.51, P<0.05), and the expression of pro-inflammatory factor IL-4(t=8.15, P<0.05), which intracellular ROS(t=2.57, P<0.05) was significantly down-regulated, attenuating macrophage apoptosis induced by PCN. Conclusion L. barbarum polysaccharides inhibit the production of intracellular ROS induced by PCN, they inhibit the release of IL-4, regulate the expression of the apoptosis-inhibiting proteins Bcl-2, Bcl-xl, and caspase3, and increase the proliferation of RAW264.7 cells after PCN treatment. The above results provide a theoretical basis for further study of the pathogenesis of P. aeruginosa and the immune protection of L. barbarum polysaccharides.
Objective To investigate the effect of Pseudomonas aeruginosa toxin pyocyanin(PCN) metabolite ROS accumulation on the expression of the proteins Bcl-2 and Bcl-xl, the inflammatory factor IL-4, and apoptosis in macrophages. Methods Cells were divided into a control group(untreated group), a P. aeruginosa toxin treatment group, an L. barbarum polysaccharide treatment group, and a P. aeruginosa toxin and L. barbarum polysaccharide treatment group. PCN was detected using a cell MTT assay. Expression of the apoptosis-related protein was detected using Western blotting. Expression of ROS was detected using fluorescence staining. ELASA was used to detect the expression of IL-4 in cell supernatant at different time points. Flow cytometry was used to detect apoptosis in different treatment groups. Results After treatment of cells with PCN, cell proliferation decreased, expression of the apoptosis-inhibiting protein Bcl-xl was down-regulated, and expression of ROS increased. As the duration of treatment progressed, PCN stimulated the overexpression of the pro-inflammatory factor IL-4 and promoted the apoptosis of macrophages. Pretreatment with L. barbarum polysaccharides significantly promoted the expression of Bcl-2(t=1.24, P<0.05), the expression of Bcl-xl(t=1.51, P<0.05), and the expression of pro-inflammatory factor IL-4(t=8.15, P<0.05), which intracellular ROS(t=2.57, P<0.05) was significantly down-regulated, attenuating macrophage apoptosis induced by PCN. Conclusion L. barbarum polysaccharides inhibit the production of intracellular ROS induced by PCN, they inhibit the release of IL-4, regulate the expression of the apoptosis-inhibiting proteins Bcl-2, Bcl-xl, and caspase3, and increase the proliferation of RAW264.7 cells after PCN treatment. The above results provide a theoretical basis for further study of the pathogenesis of P. aeruginosa and the immune protection of L. barbarum polysaccharides.
引文
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[2] Lau GW,Hassett DJ,Ran H,et al.The role of Pyocyanin in Pseudomonas aeruginosa infection[J].Trends Mol Med,2004,10(12):599-606.
[3] Zowalaty ME,Thani A,Webster TJ,et al.Ashour,Pseudomonas aeruginosa:arsenal of resistance mechanisms,decades of changing resistance profiles,and future antimicrobial therapies[J].Future Microbiol,2015,10(10):1683-706.
[4] Kovacic P,Somanathan R.Toxicity of imine-iminium dyes and pigments:electron transfer,radicals,oxidative stress and other physiological effects[J].J Appl Toxicol,2014,34(8):825-34.
[5] Lovewell RR,Patankar YR.Berwin B.Mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa [J].Cell Mol Physiol ,2014,306(7):L591-603.
[6] Vincent JL.Vaccine development and passive immunization for Pseudomonas aeruginosa in critically ill patients:a clinical update[J].Future Microbiol,2014,9(4):457-63.
[7] Rada B,Leto TL.Pyocyanin effects on respiratory epithelium:relevance in Pseudomonas aeruginosa airway infections[J].Trends Microbiol,2013,21(2):73-81.
[8] Jin M,Huang Q,Zhao K,et al.Biological activities and potential health benefit effects of polysaccharides isolated from Lycium barbarum L[J].Int J Biol Macromol,2013,(54) :16-23.
[9] Masci A,Carradori S,Casadei MA,et al.Lycium barbarum polysaccharides:Extraction,purification,structural characterisation and evidence about hypoglycaemic and hypolipidaemic effects,A review [J].Food Chem,2018,9(4):457-63.
[10] Potterat O.Goji (Lycium barbarum and L.chinense):phytochemistry,pharmacology and safety in the perspective of traditional uses and recent popularity[J].Planta Med,2010,76(1) :7-19.
[11] Cheng J,Zhou ZW,Sheng HP,et al.An evidence-based update on the pharmacological activities and possible molecular targets of Lycium barbarum polysaccharides[J].Drug des Devel Ther,2015,(9):33-78.
[12] Liu Y,Cao L,Du J,et al.Protective effects of Lycium barbarum polysaccharides against carbon tetrachloride-induced hepatotoxicity in precision-cut liver slices in vitro and in vivo in common carp (Cyprinus carpio L.) [J].Comp Biochem Physiol C Toxicol Pharmacol ,2015,169:65-72.
[13] Tang WM,Chan E,Kwok CY,et al.A review of the anticancer and immunomodulatory effects of Lycium barbarum fruit[J].Inflammopharmacology,2012,20(6):307-14.
[14] Karaiskos I,Giamarellou H.Multidrug-resistant and extensively drug-resistant Gram-negative pathogens:current and emerging therapeutic approaches[J].Expert Opin Pharmacother,2014,15(10):1351-70.
[15] Pitt T.Biology of Pseudomonas aeruginosa in relation to pulmonary infection in cystic fibrosis[J].J R Soci Med ,1986,79(Suppl 12):13-8.
[16] Martis N,Leroy S,Blanc V.Colistin in multi-drug resistant Pseudomonas aeruginosa blood-stream infections:a narrative review for the clinician[J].J Infect ,2014,69(1):1-12.