Proteomics-based Discovery Of A Novel Target For The Treatment Of Paragonimus Proliferus Infection

Objective:Paragonimus is a foodborne parasite.Human infection leads to paragonimiasis.Paragonimiasis has hidden onset,various clinical manifestations and high misdiagnosis rate,which often brings great mental and economic pressure to patients.In recent years,reports on the misdiagnosis rate of paragonimiasis have increased year by year,which poses a great threat to social and public security.Paragonimus is an important zoonotic pathogen.At present,the research on Paragonimus mainly focuses on morphological classification,life history and epidemiological investigation,but there are few reports on the signal pathway and molecular mechanism of the interaction between Paragonimus and its host.In this study,the rat model of Paragonimus proliferus infection was used to explore the signal pathway and molecular mechanism of rat lungs in response to Paragonimus proliferus infection,as well as the infection and development process of Paragonimus proliferus,so as to reveal the interaction mechanism between Paragonimus proliferus and the host.Methods:This study established an animal model of rats infected with Paragonimus proliferus,and explored the interaction mechanism between Paragonimus proliferus and rats through two parts.In the first part,rats were observed.The rats were divided into normal group,infection group and treatment group with trichlorobendazole combined with gentiopicrin.Firstly,HE staining and Masson staining were used to clarify the lung injury caused by paragonimiasis and the therapeutic effect of trichlorobendazole combined with gentiopicrin on lung injury.Secondly,proteomic analysis of rat lung tissue was carried out.The second part takes Paragonimus proliferus as the observation object.Proteomic analysis of three representative developmental stages of Paragonimus proliferus(cysticercus,child and adult)was carried out.Verify the analysis results.Results:Part Ⅰ:proteomic study of three groups of rat lung tissues.A total of 32549 peptide sequences and 5365 proteins were identified(3889 of which were unique peptides≥ 2).Compared with the normal group,there were 181 differentially expressed proteins in the infection group,including 107 up-regulated proteins and 74 down-regulated proteins.Compared with the infection group,there were 290 differentially expressed proteins in the treatment group,including 94 up-regulated proteins and 196 down-regulated proteins.Through the bioinformatics analysis and experimental verification of the identified differential proteins,it was found that:1)the superoxide production pathway and lysosomal pathway mediated by the activation of NADPH oxidase in the lung tissue of infected rats were activated.Further experimental results confirmed that the level of ROS and the number of lysosomes in the lung tissue of infected rats increased significantly.2)The endoplasmic reticulum pathway was enriched in the lung tissue of rats treated with trichlorobendazole combined with gentiopicrin.Western blot experiment confirmed that the endoplasmic reticulum stress response of lung tissue in treated rats decreased.Part Ⅱ:through the proteomic study of the three developmental stages of Paragonimus proliferus,the protein expression changes in the infection process of Paragonimus proliferus were systematically revealed.A total of 2781 proteins were identified,and 137,155 and 22 differentially expressed proteins were found in each comparison group.Proteomic data were submitted using iprox to improve the protein mass spectrum of Paragonimus proliferus.Through bioinformatics analysis of the identified differential proteins,it is found that the differential proteins are mainly involved in biological regulation,metabolism,cellular processes,protein binding and different enzyme activities,and are secreted by peroxisome and cell.It is enriched in ECM receptor interaction and lysosome pathway.Among them,7 belong to the increase of lysosomal acid hydrolase protein level in the development stage of Paragonimus proliferus from child to adult.At the same time,lysosome fluorescence probe staining confirmed that lysosome increased significantly in Paragonimus proliferus adults.In addition,our experiments show that MG-101 protease can inhibit the lysosomal expression of Paragonimus proliferus and play an effective role in the treatment of Paragonimus proliferus infection.Conclusions:1)The large increase of ROS level and lysosome number in rat lung tissue may be the main means for the host to resist Paragonimus infection.2)Reducing endoplasmic reticulum stress response can effectively improve the impact of Paragonimus proliferus infection.3)The increase of lysosomes is very important for the development of Paragonimus proliferus from child to adult.4)The use of lysosomal inhibitor cysteine protease MG-101 can effectively inhibit the development of Paragonimus proliferus.The above conclusions will help us to clarify the mechanism of Paragonimus proliferus parasitism on the host,and help us to find potential targets to block parasitism.