| Toxoplasma gondii is a zoonotic parasite that is distributed worldwide and seriously endangers human health.It is an obligate intracellular parasitic protozoan.Mammals and birds are susceptible to it,and the infection rate of human Toxoplasma gondii is very high.The world average infection rate is about 30%.Pregnant women in the first and second trimesters transmit the worms to the fetus through the placenta,causing miscarriage,stillbirth or malformations.Infections in the third trimester,most of the fetuses show a recessive infection,and symptoms gradually appear only months or even years after birth,such as retinochoroiditis,dyskinesia,cerebral calcification and hydrocephalus.Toxoplasma gondii has been listed as one of the main pathogens of infectious teratogenic(TORCH)syndrome.For immunodeficiency or immunocompromised people,Toxoplasma gondii is also one of the main pathogens causing fatal diseases.In addition,toxoplasmosis can also cause animal abortion,weak births,stillbirths,hindered growth and death,causing huge economic losses to the animal husbandry.Therefore,the prevention and control of Toxoplasma gondii infection has received more and more attention.There are many deficiencies in the existing drugs for the treatment of Toxoplasmosis,and it is urgent to develop a high-efficiency,low-toxicity anti-Toxoplasma drug.Acyl-CoA synthetase(ACS)is a type II fatty acid synthetase that plays an important role in the energy and lipid metabolism of Toxoplasma gondii,a key enzyme that catalyzes the reaction during the synthesis of lipids.At present,ACS has made great progress as a drug target in cryptosporidium and coccidia.However,the research of Toxoplasma gondii,especially as a target for screening and treating drugs,is still blank.Triacsin C is a potent long-chain fatty acyl-CoA synthetase inhibitor.Theoretically,Triacsin C could be able to interfere with the synthesis of tachyzoite lipids of Toxoplasma gondii,thereby inhibiting its proliferation.However,no related reports have been reported.Transcriptomics is an important part of functional genomics research.It is a discipline that studies the laws of gene transcription and transcription regulation in cells or tissues.It can analyze the expression regulation system of all functional genes,understand the expression level and regulatory changes in a comprehensive and in-depth manner,and realize the corresponding relationship between genetic information and the body phenotype,so as to study the regulatory changes of the organism as a whole.Carrying out research on parasite transcriptome lays the foundation for its immunological diagnosis,research on new drug targets,and prevention research.Therefore,in this subject,Triacsin C was applied to T.gondii tachyzoites in vivo under different conditions to test its inhibitory effect on tachyzoites proliferation.At the same time,the damage of drugs to the main organs of experimental animals was observed at different concentrations.By analyzing the transcriptome biological information of Triacsin C in the liver tissue of mice infected with Toxoplasma gondii infection group,infection group with drug,drug group and normal group,it was investigated whether Triacsin C could be used as a new anti-Toxoplasma infection drug.The main research results were as follows:1.Establishment of a standard curve for fluorescence quantitative PCR of Toxoplasma gondiiDesign and screen specific small fragment primers(qTOX-F,qTOX-R)based on the 529 bp repeat sequence of Toxoplasma gondii published by GenBank.The standard curve constructed by the plasmid standard prepared in this experiment was:y=-2.857x+37.769.At these six consecutive points,"108 copies/?L?103 copies/?L" had the best linearity and high correlation,with a value of R2 =0.99,the coefficient of variation of repeated wells was 0.344%?1.038%.2.Triacsin C inhibition of Toxoplasma gondii tachyzoite proliferation in vivo detection and pathological damage to the main organs of target animals120 Kunming female rats were randomly assigned to 12 groups,with 10 mice in each group.Each mouse in groups 1-7 was infected with 2x105RH strain of Toxoplasma gondii tachyzoites.Groups 1-5 were the experimental group and groups 8-12 were the control group.The different doses of Triacsin C were 0 ?g/mL,10 ?g/mL,20 ?g/mL,40 ?g/mL,80 ?g/mL.Take 0.1mL intragastrically once a day for 3 days.In groups 6 and 7,the doses of sulfadiazine and azithromycin were given by intragastric administration of 0.1mL/d at 20 mg/mL and 60 ?g/mL for 3 days.On the 7th day,the mice were sacrificed,ascites were collected for tachyzoite count,the tachyzoite content in liver tissue was detected,and heart,liver,spleen,lung,kidney and brain tissue were collected for pathological tissue observation.The Toxoplasma tachyzoites count found that compared with the content of tachyzoites in the ascites of mice injected with 0 ?g/mL Triacsin C,the effect of 10?g/mL Triacsin C on the proliferation of tachyzoites was not significant(P>0.05).20?g/mL and 40 ?g/mL Triacsin C had significant differences in the inhibition of tachyzoite proliferation(P<0.05).Injecting 80 ?g/mL Triacsin C,20 mg/mL SD and 60?g/mLL AZ had extremely significant inhibitory effects on tachyzoite proliferation(P<0.01).The count of tachyzoites in the liver was found that compared with the tachyzoites content in the liver of mice injected with 0 ?g/mL Triacsin C,and the injection of 10?g/mL,20 ?g/mL,40 ?g/mL Triacsin C inhibited the proliferation of tachyzoites.The effect was not significant(P>0.05).The injection of 80 ?g/mL Triacsin C,20 mg/mL SD and 60 ?g/mL AZ made tachyzoite proliferation effect was significant(P<0.05).Hi stop athol ogi cal observation,comparing with the drug administration group,found that the heart and brain tissues of the infected group were normal.The damage of tissue structure in the infection groups with different drug concentrations was basically the same.In the infected group,the hepatocyte boundaries were not obvious,the chromatin was marginalized,the hepatocytes were swollen,and the severe manifestation was vacuolar degeneration and necrosis;the spleen tissue structure was destroyed,the splenic cord was arranged disorderly,the white pulp area was bleeding,and there were non-vascular tissues,lymphocyte nuclear enlargement,dissolution,and necrosis;there was viscous substance bleeding in the lung tissue cavity,telangiectasia,and a large number of red blood cells appeared under the mucosa;kidney structure was destroyed,the proximal tubule cone cells were enlarged,and the distal tubule kidney cells were shed,The protein solution oozed out,and a silk-like tissue appeared in the lumen.All tissues of the mice in the administration group were normal.3.Transcriptome bioinformatics analysis of mouse liver tissue under the action of Triacsin CIn this experiment,the next-generation sequencing technology(Next-Generation Sequencing,NGS)was used to perform high-throughput sequencing and bioinformatics analysis on the transcriptome of the liver tissue of the infected group,the infected drug group,the drug group and the normal group.Illumina sequencing results showed that:the infection group,the infection administration group,the administration group and the normal group obtained 52079488± 1161042,5463 03 92±1161042,53578154±2685844,53769402±1173777 Raw reads respectively.Removal of sequencing linker sequences,low-quality reads,sequences with a high N rate,and sequences with too short length,finally obtained high-quality sequencing data clean data,the infection group was 52038760±1158903,and the infection administration group was 54588488±2686113,and the drug group was 53531310±1176376,and the normal group was 53712504±2070001,which accounted for 99.92%,99.92%,99.56%,99.89%of the original data in their respective libraries.And the Q20 of the samples were higher than 95%,and the GC content was between 47%and 51%.There were 14412 differentially expressed genes in the infection administration group and the infection group,and the infection group was used as a control.The expression of 7518 genes was up-regulated and 6894 genes were down-regulated in the infection administration group.There were 77 genes that were significantly differentially expressed,and the number of genes that were significantly up-regulated and down-regulated in the infection-administered group was 27 and 50,respectively.Differentially expressed genes were analyzed by GO function enrichment,and it was found that the differential gene expression was significantly enriched in the process of isoprene biosynthesis,purine nucleotide metabolism,alcohol biosynthesis,secondary alcohol biosynthesis,cholesterol biosynthesis,2'-5'-oligoadenylate synthetase activity,ligand-gated sodium channel activity,steroid dehydrogenase activity,oxidoreductase activity and other biological functions;Pathway enrichment analysis,obtained a total of metabolic pathways,visual 15 enriched signaling pathways including flavonol metabolism,a rachidonic acid,and biosynthesis of terpenoid skeletons. |
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