胰岛素联合利奈唑胺抑制糖尿病细菌性肺炎的机制研究

doi:10.16431/j.cnki.1671-7236.2021.01.039

摘要/Abstract

摘要： 试验旨在研究胰岛素联合利奈唑胺是否同时具有降糖、抗菌以及抑制α-溶血素活性，从而抑制糖尿病细菌性肺炎。本研究通过体外试验探讨了联合用药对高糖和金黄色葡萄球菌α-溶血素共同诱导的巨噬细胞炎症反应的作用，并探讨其机制。通过药物敏感试验测定最小抑菌浓度（MIC）；通过检测D_{600 nm}值绘制生长曲线来考察胰岛素、利奈唑胺单独或联合胰岛素的抗菌活性；通过溶血试验考察药物组合对α-溶血素活性的抑制效果；通过细胞毒性试验判定药物组合的体外安全性；借助Western blotting药物组合对炎症通路蛋白的作用。药物敏感性试验结果显示，胰岛素组未检测到金黄色葡萄球菌8325-4株和DU1090株的MIC值，利奈唑胺和胰岛素+利奈唑胺药物组合对这两种金黄色葡萄球菌的MIC均为0.5 μg/mL；由生长曲线结果可知，胰岛素不抑制正常组和高糖组金黄色葡萄球菌的生长，但是在利奈唑胺组和胰岛素+利奈唑胺药物组合组，0.25~4 μg/mL利奈唑胺可抑制金黄色葡萄球菌的生长；溶血试验结果显示，正常组和高糖组胰岛素均不抑制α-溶血素活性，0.25~4 μg/mL利奈唑胺均可抑制α-溶血素活性；细胞毒性试验显示，正常组和高糖组，细胞存活率均大于88.038%，50 nmol/L胰岛素、0.25 μg/mL利奈唑胺、50 nmol/L胰岛素+0.25 μg/mL利奈唑胺药物组合可提高高糖诱导的MH-S细胞存活率；Western blotting结果显示，50 nmol/L胰岛素单独使用对高糖和α-溶血素共同诱导小鼠肺泡巨噬细胞（MH-S细胞）中TLR2、MAPKs及NLRP3蛋白的表达水平没有抑制作用，而50 nmol/L胰岛素联合0.25 μg/mL利奈唑胺可降低高糖和α-溶血素共同诱导MH-S细胞中上述蛋白的表达水平，其抑制作用比单独使用利奈唑胺时显著。综上所述，胰岛素联合利奈唑胺可通过降糖、抗菌和抑制α-溶血素活性的方式抑制小鼠肺泡巨噬细胞炎症反应。

关键词: 利奈唑胺; 胰岛素; 金黄色葡萄球菌; α-溶血素; 高糖; 小鼠肺泡巨噬细胞

Abstract: The aim of the study was to investigate whether insulin combined with linazolid had both hypoglycemic antibacterial and α-hemolysin activity to inhibit diabetic bacterial pneumonia.The effect of insulin combined with linezolid on macrophage inflammation induced by high glucose and Staphylococcus aureus α-hemolysin was studied in vitro,and its mechanism was discussed.The antibacterial activity of insulin,linezolid alone or in combination with insulin was examined by measuring minimum inhibitory concentration (MIC) in drug sensitivity test and D_{600 nm} to draw growth curve.The inhibitory effect of drug combination on α-hemolysin activity was investigated by hemolysis test.The safety of drug combination in vitro was determined by cytotoxic test.Western blotting was used to analyze the effect of drug combination on inflammatory pathway protein.The results of drug sensitivity test showed that the MIC values of 8325-4 and DU1090 strains of Staphylococcus aureus were not detected in insulin group,and the MIC values of linezolid and insulin+linezolid groups for both strains of Staphylococcus aureus were 0.5 μg/mL.The results of growth curve showed that insulin did not inhibit the growth of Staphylococcus aureus in the normal group and the high glucose group,while in linezolid group and insulin+linezolid groups,0.25 to 4 μg/mL linezolid could inhibit the growth of Staphylococcus aureus.The results of hemolysis test showed that in normal group and high glucose group,insulin did not inhibit α-hemolysin activity,while 0.25 to 4 μg/mL linezolid could inhibit α-hemolysin activity.The cytotoxic test showed that the cell survival rate of normal group and high glucose group was more than 88.038%.The combination of 50 nmol/L insulin,0.25 μg/mL linezolid,50 nmol/L insulin +0.25 μ g/mL linezolid could improve the survival rate of MH-S cells induced by high glucose.The results of Western blotting showed that 50 nmol/L insulin alone did not inhibit the expression of TLR2,MAPKs and NLRP3 in mouse alveolar macrophages (MH-S cells) induced by high glucose and α-hemolysin,while 50 nmol/L insulin combined with 0.25 μg/mL linezolid could reduce the expression level of these proteins in MH-S cells induced by high glucose and α-hemolysin,and the inhibitory effect of linezolid was more significant than that of linezolid alone.In conclusion,insulin combined with linezolid could effectively inhibit the inflammatory response of MH-S cells by lowering high glucose,antibacterial and inhibiting α-hemolysin activity.

Key words: linezolid; insulin; Staphylococcus aureus; α-hemolysin; high glucose; MH-S cells

中图分类号:

R563.1

倪丽慧, 栾文静, 王雪飞, 李媛, 安雅男, 马方雪, 周宏, 王超, 梁俊超, 于录. 胰岛素联合利奈唑胺抑制糖尿病细菌性肺炎的机制研究[J]. 中国畜牧兽医, 2021, 48(1): 356-366.

NI Lihui, LUAN Wenjing, WANG Xuefei, LI Yuan, AN Yanan, MA Fangxue, ZHOU Hong, WANG Chao, LIANG Junchao, YU Lu. Study on the Mechanism of Insulin Combined with Linezolid in Inhibiting Diabetic Bacterial Pneumonia[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 356-366.

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