Study On The Killing Effect Of Artemisinin Combined With Hydroxychloroquine On The Gametophyte Of Plasmodium

Objective:Plasmodium falciparum gametophytes are key to malaria transmission,and reducing gametophyte carriage in patients is an important topic in preventing malaria transmission.In this experiment,we combined artemisinin with hydroxychloroquine（AH,1:1）in two parts,in vivo and in vitro,respectively,to investigate the killing effect of different doses of AH on the gametophytes of Plasmodium falciparum NF54 strain and Plasmodium berghei ANKA strain,aiming to investigate whether there is a correlation between different doses of AH and the inhibition rate of gametophytes,and whether there is a potentiating effect of the combined drug on the killing of gametophytes,and to provide a basis for subsequent exploration of the drug’s effect on The aim was to investigate the correlation between the gametocyte inhibition rate and the gametocyte inhibition rate at different doses of AH,as well as the synergistic effect of the drug combination on gametocyte inhibition.Methods:1.In vitro experiments were conducted to detect the inhibition rate of AH on early and late gametophytes at different concentrations by inducing the production of gametophytes through N-acetyl-D-glucosamine,and to detect whether AH could improve the killing effect on gametophytes by using a blood smear oil microscopy to calculate the infection rate.2.Animal modeling:Three days before Plasmodium inoculation,a solution of phenylhydrazine hydrochloride at a concentration of 25 mg·m L^-1was injected intraperitoneally into female ICR mice at a body mass of 3.5μL·g^-1.On the third day of phenylhydrazine hydrochloride induction,each mouse was inoculated with 5×10⁶i RBC Plasmodium berghei by intraperitoneal injection.At 72 h after inoculation with Plasmodium,a solution of sulfadiazine at a concentration of 0.4 mg·m L^-1was administered by gavage at 15μL·g^-1body mass for 2 days,and sulfadiazine cleared the asexual stage of Plasmodium and yielded more purified gametophytes.3.Grouping and dosing:SPF grade female ICR mice,112,were randomly divided into normal control group（Control group）,infection group（Pb group）,artemisinin combined with hydroxychloroquine low dose group(AHL group,A-32mg·kg^-1,H-41mg·kg^-1),artemisinin combined with hydroxychloroquine medium dose group(AHM group,A-64 mg·kg^-1,H-82 mg·kg^-1),artemisinin combined with hydroxychloroquine high dose group(AHH group,A-128 mg·kg^-1,H-164 mg·kg^-1),artemisinin group(ART group,64 mg·kg^-1),hydroxychloroquine group(HCQ group,82 mg·kg^-1),and ketotifen group(KT group,80 mg·kg^-1).The day of inoculation was recorded as D0,and after successful modeling,the administered group was administered for 3 consecutive days,and the Control and Pb groups were gavaged with equal volume of 0.9%Na Cl solution.body temperature of mice was monitored daily from D1 to D7 for 7 consecutive days;body mass and infection rate of peripheral erythrocytes and gametophytes were monitored daily from D1 to D8 for 8 consecutive days.4.Sampling and index determination:Whole blood was collected from mice by removing eyeballs in D8,and the whole blood was centrifuged and part of the serum was used to detect the expression of glutamate transaminase（ALT）,creatinine（CREA）,urea（UREA）,alkaline phosphatase（ALP）,total protein（TP）,albumin（ALB）,lactate dehydrogenase（LDH）,aspartate aminotransferase（AST）in mice serum levels,and partly for detecting the expression of serum inflammatory factors（IL-1β,IL-6,IL-10,TNF-α）;The dissected liver and spleen were weighed and used for pathological sections.Results:1.In vitro gametophyte culture results showed that the half inhibitory concentrations of AH,artemisinin and hydroxychloroquine on early gametophytes were 8.527,8.802 and16.54 ng·m L^-1,respectively,and on late gametophytes were 16.85,16.27 and 21.59ng·m L^-1,respectively,with the increase of drug concentration,the inhibitory effect of drugs on gametophytes increased.The combination of AH had a synergistic effect in a certain drug range.High concentrations of drugs can completely kill early gametophytes,but equal doses of drugs cannot completely kill late gametes.2.AH low,medium and high doses,artemisinin,hydroxychloroquine and ketotifen all reduced the infection rate of peripheral blood erythrocytes and gametophytes（P<0.01）.There was no statistically significant difference in body mass and temperature between the administered groups（P>0.05）.3.Artemisinin reduced the level of serum CRE expression（P<0.01）.Hydroxychloroquine and ketotifen reduced serum UREA expression（P<0.05）.Medium dose of AH reduced serum IL-1βexpression（P<0.05）.Low,medium and high doses of AH,hydroxychloroquine and ketotifen reduced serum IL-10 expression in mice（P<0.05）.The differences in serum IL-6 and TNF-αbetween the administered groups were not statistically significant（P>0.05）.The combination of medium and high dose could significantly alleviate liver and spleen swelling and malaria pigmentation under pathological sections.Conclusion:1.AH,artemisinin and hydroxychloroquine have obvious killing effect on early gametophytes,and the killing effect on late gametophytes of stage IV gametophytes is fair,but the killing effect on mature gametophytes of stage V is poor.AH has synergistic effect on the killing of early and late gametophytes at certain concentration,and increasing the concentration of the drug can reduce the content of gametophytes,but cannot completely kill the gametophytes of stage V.2.AH,hydroxychloroquine and ketotifen can completely kill the asexual stage Plasmodium and gametophytes in mice,while artemisinin can kill Plasmodium rapidly,but the recurrence of Plasmodium occurs in the later stage.3.AH,artemisinin and hydroxychloroquine all slowed down the decrease of body mass and body temperature,and the medium and high doses of AH could kill Plasmodium faster,reduce the expression of inflammatory factors in vivo,protect the liver and kidney function of mice,protect the organs of infected mice from damage,and reduce the after-effects in mice compared with the use of single drug.