| Objective To evaluate the effects of artesunate(Artesunate,Art)combined with azithromycin(Azithromycin,Azm)on Toxoplasma gondii(T.gondii)infection in vitro and to observe the effect of drugs on T.gondii infection in macrophages;To investigate the therapeutic effect of artesunate combined with azithromycin on acute T.gondii infection in mice.Methods1.T.gondii tachyzoites stably expressing green fluorescent protein(RH-GFP)were applied to infect mice through enterocoelia.Four groups macrophages were divided in the experiments,namely,(1)the normal saline group(control);(2)the artesunate group with final concentration of 1.5625,3.125,6.25,12.5,25,50,100 and200μg/ml,respectively;(3)the azithromycin group with final concentration of 12.5,25,50,100,200,400,800 and 1600μg/ml,respectively;(4)the combination group with final concentration of Art1.5625+Azm12.5,Art3.125+Azm25,Art6.25+Azm50,Art12.5+Azm100,Art25+Azm200 and Art50+Azm400 μg/ml,respectively.After incubation for 24 h,flow cytometry analysis was used in different parts of extraction to detect the free parasites in the peritoneal macrophages infected by tachyzoites.The ratio of infected macrophages to uninfected cells was counted.Additionally,the fluorescence microscopy was utilized to observe fluorescence of the cells and Giemsa staining was used to study the morphological and structural changes of the parasites.2.The suspension of the macrophages incubated for 24h(in step one)was inoculated into the abdominal cavity of mice for resuscitation,the tachyzoites were detected by the time onset,duration of survival,and the microscopic findings from peritoneal fliuds.Positive or negative results were recorded based on the findings of smear staining.3.With 1×103 RH-GFP tachyzoites inoculated into mice,the model of in vivo Toxoplasma infection was established.The normal saline group(control)and the treatment groups(artesunate group,azithromycin group and combination group)were divided.After inoculation for 2h,the treatment groups were given treatments once a day for 7 days,with the aim of observing the survival time and the smear microscopic examination.Results1.The proportional changes of macrophages in all groups of mice infected with tachyzoites were analyzed 24 h after the drugs incubation:1)The artesunate group(1)Free tachyzoites: Compared with the control group,however,no significant differences(P>0.05)were found at the lowest concentration of 1.5625μg/ml.With the concentration increased to 3.125μg/ml or over,the ratio of free parasites decreased gradually(P<0.05)in the drugs-treated groups.The lowest percentage(0.3%)of free parasites was seen when a high dosage of 50μg/ml artesunate was given.(2)Infected macrophages: The proportion of infected macrophages increased significantly(P<0.05)in the low concentration(1.5625μg/ml and 3.125μg/ml)groups in comparison with the control group.With the concentration of over 12.5μg/ml,the free parasites were found to be significantly reduced(P<0.05).Administration of50μg/ml artisunate to mice resulted in a remarkable reduction of percentage(<0.3%).(3)Uninfected macrophages: Compared with the control group,no significant differences of ratioes were noted at the 1.5625μg/ml and 3.125μg/ml groups(P>0.05).A concentration of over 6.25μg/ml of artisunate notably increased the number of uninfected macrophages(P<0.05).(4)Infection rate of macrophages: The macrophage infection rate showed a downward trend which was higher than the control group at 1.5625μg/ml and 3.125μg/ml groups,and decreased significantly at 6.25μg/ml or over(P<0.05).It was less than 1.6% in 50μg/ml group.2)The azithromycin group(1)Free tachyzoites: With the increase of concentration,the proportion of free parasites decreased significantly compared with the control group(P<0.05).In 400μg/ml group,the proportion of free tachyzoites was less than 0.6%.(2)Infected macrophages: The proportion of 12.5μg/ml group increased compared with the control group,and then decreased gradually.50μg/ml concentration was found to significantly inhibited the growth of parasites(P<0.05).A concentration of 400μg/ml significantly reduced the proportion of infected macrophages to 0.4%.(3)Uninfected macrophages: With the increase of concentration,the proportion of uninfected macrophages increased gradually and reached statistical significance at 25μg/ml(P<0.05).(4)Infection rate of macrophages: Compared with the control group,the infection rate increased at a concentration of 12.5μg/ml and then decreased gradually.It was found significantly less than the control group(P<0.05)as the concentration getting over25μg/ml.The infection rate of macrophages was less than 3.5% at 400μg/ml.3)The combination group(1)Free tachyzoites: With the increase of concentration,the proportion of free tachyzoites decreased significantly compared with the control group(P<0.05).In the Art12.5+Azm100 μg/ml group,the number of free parasites was less than 0.04%.(2)Infected macrophages: Compared with the control group,the proportion decreased significantly with the increase of concentration(P<0.05).In the Art12.5+Azm100μg/ml group,the proportion of infected macrophages was less than 0.2%.(3)Uninfected macrophages: With the increase of concentration,the proportion of uninfected macrophages increased gradually in comparison with the control group (P<0.05).(4)Infection rate of macrophages: Compared with the control group,the infection rate decreased significantly with the increase of concentration(P<0.05).In the Art12.5+Azm100 μg/ml group,the infection rate was less than 1.8%.Compared with the control group,the number and fluorescence intensity of parasites were gradually decreased in a concentration dependent manner under the experimental condition.With the increase of concentration,It was found that tachyzoites showed swelling and deformed,more and bigger vacuoles were formed inside the body,the cell membrane became dissolved and disrupt,the inner structure overflowed and the karyon was darkly stained because of pyknosis.2.Re-inoculation test(1)The artesunate group The mice in 1.5625 to 12.5μg/ml group died one after another 7-12 days after inoculation.The peritoneal fluid smears of dead mice were found positive.Groups from25μg/ml to 200μg/ml remained alive for 15 days.The mice in 25μg/ml group were found to be at the middle stage of the disease,and the number of positive mice in50μg/ml group was 1,while the smears were negative in both 100 and 200μg/ml group.Compared with the control group,no significant differences were found within 1.5625 and 3.125μg/ml groups(P>0.05),but the survival time of mice in 6.25 to 200μg/ml groups was obviously longer(P<0.05).(2)The azithromycin group The groups from 12.5 to 50μg/ml died one after another from 9 to 13 days after inoculation,and the smears were positive.The rest groups remained alive for 15 days,in which 100μg/ml group all had onset,and the number of positive mice in the200μg/ml group was 4 and 1 in 400μg/ml group.Smears in 800 and 1600μg/ml groups displayed negative.Compared with the control group,there were no significant differences in the average survival time of mice in 12.5 and 25μg/ml groups(P>0.05), but significantly prolonged in 50 to 1600μg/ml groups(P<0.05).(3)The combination group The groups from Art1.5625+Azm12.5 to Art6.25+Azm50 μg/ml died on the 8th to 12 th day after inoculation,and the smears were positive.All the other groups survived to the15 th day and the smears were negative.There were no significant differences between the Art1.5625+Azm12.5,Art3.125+Azm25 μg/ml group and the control group(P>0.05),but the mean survival time of groups from Art6.25+Azm50 to Art50+Azm400 μg/ml was significantly longer than the control group(P<0.05).3.Therapeutic effect No mice survived in artesunate group 15 days after incubation,and the abdominal fluid smears of dead mice were positive.Azithromycin group all survived to the 30 th day,smears were negative with a large number of macrophages shown.No mice survived and smears were positive on the 20 th day in the combination group.There was no significant difference in the average survival time between artesunate group and control group(P>0.05),but azithromycin and combination group were significantly prolonged(P<0.05).Conclusions1.Artesunate has a significant anti-Toxoplasma effect in vitro and it is positively correlated with the drug concentration in a certain range.The concentration of50μg/ml can achieve more than 99% of the killing effect by 24 h reaction,inhibiting the proliferation of parasites and reducing the infection rate of macrophages.The treatment of azithromycin could reach similar effects when the concentration is400μg/ml.Advantages are found in the combination group where the inhibitory effect in Art12.5+Azm100 μg/ml group is close to the Art50μg/ml or Azm400μg/ml group: similar effects with lower concentration gradient and fewer drug doses.The change trend of fluorescence is consistent with the results of flow cytometry.The results of giemsa staining showed the killing effect of the drug from morphology.2.The re-inoculation test showed that the results concerning the incidence trend,average survival time,and the swear microscopy supported that in the flow cytometry,which further verified the effect of drugs against T.gondii in vitro.3.Through preliminary drug intervention,artesunate only prolonged the survival time of infected mice to a certain extent,which was remarkably weaker than the effect in vitro.The average survival time of mice in combination group was significantly longer than the control group,but is not better than the azithromycin treatment. |
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