氢氧机在抗真菌辅助治疗中的效果评价及机制初探

Effect and mechanism of oxyhydrogen machine in antifungal adjuvant therapy

目的基于体外试验研究氢氧机单独应用或联用抗真菌药对真菌的作用效果及作用机制。方法选择临床分离获得的12株病原真菌作为试验菌株,使用氢氧机按固定的流速和档位(气流量3 L/min,雾化档5档)处理菌悬液,以通气0 min时(未通气时)和通气处理菌悬液5、10 min时为3个观察时点,各时点分别取菌悬液100μL涂布于沙氏葡萄糖琼脂培养基,于37℃温箱内培养2 d后进行菌落计数,计算存活率和杀伤率。采用微量液基稀释法测定4种抗真菌药伏立康唑(VOR)、泊沙康唑(POS)、氟康唑(FLU)、伊曲康唑(ITR)的最低抑菌浓度(MIC)。应用流式细胞仪测定细胞内活性氧(ROS)活性变化。结果氢氧机5 min通气处理对12株菌株的杀伤率与通气0 min比较,差异均有统计学意义(P<0.05),整体杀伤率范围为6.3%~76.2%;与5 min通气处理相比,氢氧机10 min通气处理对12株菌株的杀伤率增高,差异均有统计学意义(P<0.05),整体杀伤率范围为19.6%~93.5%。菌悬液体外药敏试验结果显示,氢氧机通气处理10 min后,ITR、POS、VOR对菌株的MIC值下降了1~4倍,FLU对菌株的MIC值降低了1~5倍;高浓度氢气处理后,抗真菌药对其中11株菌株的MIC值明显降低。对经过不同时间通气处理的ATCC00279菌株进行细胞内ROS检测发现,与通气0 min比较,通气10 min后菌株对ITR、VOR的ROS百分比增加,差异有统计学意义(P<0.05)。结论单独氢氧机通气处理即可实现高达93.5%的菌株杀伤效果,将其与抗真菌药联用还可使菌株对药物的敏感性增强,进一步降低抗真菌药对真菌的MIC,其机制可能与氢氧机能增强真菌细胞内ROS活性有关。

Objective To explore antifungal effect and mechanism of oxyhydrogen machine alone oRcombined with antifungal agents based on in vitro study.Methods Twelve strains of clinically isolated pathogenic fungi were selected as study strains,and the bacterial suspension was treated with a hydrogen-oxygen machine at a fixed flow rate and geaR(gas flow rate of 3 L/min at the fifth atomization geaRposition).100μL of bacterial suspension was respectively coated on Sarge glucose AGARmedium at 0 min of ventilation(without ventilation)and 5 and 10 min of ventilation foRbacterial suspension,and colony counts were performed afteRthe medium was cultured at 35℃foR2 days.Survival rate and kill rate were calculated.The minimal inhibitory concentrations(MICs)were evaluated before oRafteRoxyhydrogen treatment foRfouRantifungal drugs:voriconazole(VOR),posaconazole(POS),fluconazole(FLU),itraconazole(ITR).The intracellulaRreactive oxygen species(ROS)activity was measured by flow cytometry.Results The killing rate of 12 strains afteR5 min ventilation by hydrogen oxygen machine showed significant difference compared with that at 0 min of ventilation(P<0.05).The overall killing rate ranged from 6.3%to 76.2%.The killing rate of 12 strains at 10 min of ventilation by hydrogen oxygen machine increased compared with 5 min of ventilation(P<0.05),with the overall killing rate of 19.6%to 93.5%.The results of external drug sensitivity test of bacterial suspension liquid showed that the MIC values of ITR,VOR,and POS against the strains afteR10 min treatment were decreased 1 time to 4 times,and the MIC value of FLU against the strains was reduced 1 time to 5 times.AfteRtreatment with high concentration of hydrogen,the MICs of antifungal agents against 11 strains were significantly decreased.The intracellulaRROS detection of ATCC00279 strain afteRdifferent time of ventilation showed that the percentage of ROS in strains by ITRand VORincreased afteR10 min of ventilation compared with 0 min of ventilation(P<0.05).Conclusion Directly ventilation by oxyhydrogen machine enables to obtain direct killing of up to 93.5%of the pathogenic fungi.Oxyhydrogen treatment combined with antifungal drugs can increase the susceptibility of the strain to the drugs to furtheRdecrease MIC of antifungal agents against fungi.The mechanisms might be associated with the increased intracellulaRROS activity of fungal cells by oxyhydrogen machine.