亚硝基铁氰化钠来源的外源性NO体外杀伤旋毛虫肌幼虫作用研究

In vitro killing of Trichinella spiralis muscle larvae by exogenous nitric oxide from SNP

目的探讨亚硝基铁氰化钠(SNP)来源的NO体外杀伤旋毛虫肌幼虫的作用及其相关机制。方法制作浓度为1 000条/ml的旋毛虫肌幼虫悬液。培养板每孔加入0.1 ml肌幼虫悬液,再加入SNP,使其终浓度分别为0.02、0.05、0.10、0.20、0.50 mmol/L和1.00 mmol/L,并设空白对照组,37℃5%CO2培养箱中孵育4 d后收集各孔肌幼虫,镜检计数,计算并比较各组肌幼虫死亡率。另于培养板中每孔加入0.1 ml肌幼虫悬液,分别设A组(对照组,1.00 mmol/L SNP)、B组(0.15 mmol/L Fe SO4+1.00 mmol/L SNP)、C组(1.00 mmol/L L-半胱氨酸+1.00 mmol/L SNP)、D组(0.15 mmol/L Fe SO4+1.00mmol/L L-半胱氨酸+1.00 mmol/L SNP)、E组(0.15 mmol/L Hb+1.00 mmol/L SNP),培养、检测方法同前,计算并比较各组肌幼虫死亡率。结果 0.02 mmol/L SNP组与空白对照组的旋毛虫肌幼虫死亡率分别为(5.50±1.80)%和(4.93±0.25)%(P>0.05)。0.05、0.10、0.20、0.50、1.00 mmol/L SNP组虫体死亡率分别为(20.19±2.71)%、(29.21±2.12)%、(41.81±2.03)%、(47.85±3.79)%和(60.98±5.19)%,与空白对照组比较差异均有统计学意义(P均<0.05)。旋毛虫肌幼虫死亡率与SNP浓度呈正相关(rs=0.875,P<0.05)。B、C、D、E组肌幼虫死亡率分别为(49.48±1.34)%、(47.29±2.79)%、(26.28±1.37)%和(17.93±3.49)%,均较A组(60.98±5.19)%有所下降(P均<0.05)。结论 SNP来源NO对体外培养的旋毛虫肌幼虫具有杀伤作用,而血红蛋白、硫酸亚铁、L-半胱氨酸对该杀伤具有抑制作用,以血红蛋白抑制效果最显著。

Objective To study the lethal effect of exogenous nitric oxide donor sodium nitroprusside（SNP）on the muscle larvae of Trichinella spiralis in vitro cultivation. Methods T. spiralis muscle larvae isolated from the infected BALB/c mice were formulated into a 1 000 larva/ml suspension with RPMI 1640 medium,and 0.1 ml suspension per orifice was cultured with SNP at 37℃ in a humidified 5% CO2 atmosphere. The final concentrations of SNP were 0.02,0.05,0.10,0.20,0.50 and 1.00mmol/L,respectively,and then the experiments were divided into 5 groups：1.00 mmol/L SNP（control group,Group A）,0.15mmol/L Fe SO4＋1.00 mmol/L SNP（Group B）,1.00 mmol/L L-cysteine ＋1.00 mmol/L SNP（Group C）,0.15 mmol/L Fe SO4＋1.00mmol/L L-cysteine ＋ 1.00 mmol/L SNP（Group D）and 0.15 mmol/L Hemoglobin ＋1.00 mmol/L SNP（Group E）. All the groups were incubated with T. spiralis muscle larvae in RPMI 1640 medium. The survivability of the muscle larvae was observed by steromicroscope and the differences of inhibition ratio among these groups were analyzed 4 d after the incubation. Results SNP0.02 mmol/L was not cytotoxic to the muscle larvae with an inhibition of（5.50±1.80）%. The mortality rates of SNP〈0.05,0.10,0.20,0.50,1.00 mmol/L groups were（20.19±2.71）%,（29.21±2.12）%,（41.81±2.03）%,（47.85±3.79）%,（60.98±5.19）%,respectively,significantly higher than that of the control group[（4.93±0.25）%,all P〈0.05]. There was a positive liner correlation between the mortality of muscle larvae and SNP concentrations in the range of 0.02-1.00 mmol/L. Next,Group A,B,C,Dand E led to the mortalities from（60.98±5.19）% to（49.48±1.34）%,（47.29±2.79）%,（26.28±1.37）%,（17.93±3.49）%,respectively,and all the differences between Group A and the other four groups were statistically significant（all P〈0.05）. Con Conclusions Exogenous nitric oxide released from SNP can kill the muscle larvae of T. spiralis. However,hemoglobin,L-cysteine,and FeSO4 can reverse the lethal effect on the parasites. The best inhibitor was hemoglobin.