Plasma Metabolomic Analysis Of Clonorchis Sinensis Infected Rabbits Based On LC/MS Technique

Clonorchiasis is a very important zoonotic parasitic disease caused by Clonorchis sinensis,which parasitizing in the liver and bile duct of dog,cat,pig and human.In 2009,C.sinensis was classified as a class I biological carcinogen by the World Health Organization(WHO).So,C.sinensis has entered the public view as a current research hotspot.Clonorchiasis is worldwide distribution,but mainly in Asia,such as China,South Korea and Vietnam.It was estimated that13 million people were infected with clonorchiasis in China.Thus,this disease has become an important public health problem.Up to now,the main diagnosis method of clonorchiasis is fecal examination,but there are some limit,such as fail to diagnosis,misdiagnosis or missed diagnosis.In addition,the pathogenesis of C.sinensis is still in the research stage,and the specific pathogenesis is not clear until now.Therefore,to analyze the influence of C.sinensis to biochemical indexes,metabolites and obtain the potential diagnostic biomarkers,the rabbits infection test will conduct.These will provide some technical support for the diagnosis and pathogenesis researches of clonorchiasis in the future.First,the C.sinensis metacercaria were obtained by digesting the positive Pseudorasbora parva.Then,Japanese big-ear rabbits were selected as experimental animals and divided randomly into experimental group and control group with nine rabbits in each group.Each rabbit was kept in a separate metabolic cage,isolated from each other and collected blood.After 15 days of infection,to determine whether rabbits were infected with C.sinensis,the feces of rabbits were collected every day for egg examination,.On the 77 th day after infection,the pathological changes of related tissues were observed,and the liver tissues were stained with HE and Masson method to study the pathological changes of the liver after infected with C.sinensis.The collected blood samples were tested for antibodies and related biochemical indicators,as to understand more information about the host infected by C.sinensis.The plasma metabolites of the 7th,14 th,28th and 63 th days of experimental group and control group were detected,the data of metabolomics were obtained,statistical and biological analysis were carried out.The metabolic pathways involved in differential metabolites were analyzed and the candidate biomarkers with diagnostic significance were screened.Finally,to verify the important candidate diagnostic biomarkers by targeted metabolomics.The results showed that:(1)The first time that detecting eggs of C.sinensis was on the 19 th day and all the experimental group rabbits found eggs were on the 25 th day,and the liver of the experimental group rabbits showed obvious pathological changes;(2)The antibody detection showed that the IgG first significant difference was on the 21 st day,and maintained at a high level from the 21 st to the 77 th day;(3)The biochemical indicators detection showed that the total bile acid,aspartate aminotransferase,alanine aminotransferase and glutamyl transpeptidase were significant differences in most of the infection periods,and most biochemical indexes had no change or irregular change;(4)Metabonomics analysis showed that Different amounts of metabolites were obtained in different infection stages of Clonorchis sinensis,14 th day had the largest quantity;(5)It was found that pyrimidine metabolism,glycine,serine and threonine metabolism,arginine and proline metabolism,purine metabolism and nitrogen metabolism were closely related to Clonorchis sinensis infection by analyzed the pathways of differential metabolites;(6)The verification of targeted metabonomics showed that L-pipecolic Acid,hypoxanthine and D-glucuronate were the biomarkers with diagnostic significance.In conclusion: This study described detail multiple biochemical indexes of C.sinensis infection in rabbits,obtained the plasma metabolic spectrum of host infected C.sinensis for the first time,clarified the features of metabolites in different stages and biological correlation within differential metabolites and obtained three important biomarkers for the diagnosis of clonorchiasis,explored the possible pathogenesis of clonorchiasis for the references of future researches in the diagnosis and pathogenesis.