Dynamicsof Intestinal Cytokines And Screening Of Differentially Expressed MicroRNAs In Mice Infected With Cryptosporidium Parvum

Cryptosporidium parvum is a worldwide-distributed parasitic protozoan that parasitizes the vacuoles of the epithelial cells of the host digestive tract,causing diarrhea in humans and many animals,especially it can cause Fatal diarrhea in immunodeficiency or immunosuppressors is a serious threat to the health of people and livestock.The main causes of cryptosporidiosis in dairy cows are Cryptosporidium parvum,Cryptosporidium bovis,Cryptosporidium andersoni.Among them,the diarrhea,weight loss,and slow growth of the calves before weaning are mainly Cryptosporidium parvum.The disease is widespread and can be transmitted through sewage,feces and food.The sporulated oocysts enter the digestive tract to establish colonization.The minimally invasive damage to the digestive tract causes the immunity of the diseased cattle to decline,providing opportunities for other pathogens to invade.In the epidemiological survey of various cattle farms in Hebei Province,the average infection rate of Cryptosporidium was 5.8%,which caused serious economic losses to the dairy industry,and there is currently no effective treatment.Studying the invasion and pathogenic mechanism of Cryptosporidium parvum provides reference and theoretical value for the prevention and treatment of micro-cryptosporidiosis.In this experiment,a mouse model of Cryptosporidium parvum infection was established.The cryptosporidium oocysts were isolated and stored from cow dung by the laboratory,identified by genotyping,passaged by mice,purified by saturated sucrose floating method and stored at 4 °C.For less than 1 month,the test animals were given three-week-old BALB/c mice,dexamethasone acetate 15g/ml,immunosuppressed for 1 week,and infected with Cryptosporidium parvum.After challenge,at 1d,3d,7d,respectively.Mouse intestinal epithelial cells were isolated on 14 days.Each mouse was isolated from the small intestine,and RNA was extracted and reverse transcribed into cDNA.The relative expression levels of IFN-?,TNF-?,IL-2,IL-4 and IL-6 were detected by real-time PCR.The expression of IFN-? and TNF-? in the jejunum after infection was significantly increased(P<0.05),and the expression of IL-2 was increased 14 days after infection.The expression level of IL-2 increased 14 days after infection(P<0.01).IL-4 showed a slow upward trend at 1d,3d and 7d after infection,but slightly increased compared with the control group,but the difference was not obvious(P>0.05).The expression level increased at 14 days after infection,and the expression level was the highest,which was 5.04 times that of the control group.The difference was significant compared with the control group(P<0.01).The expression of IL-6 showed a stable trend at the initial stage of infection.The expression of IL-6 was significantly different from that of the control group at 1d,3d and 7d after infection(P<0.05),which was between 2 and 3 times of the control group.The expression level decreased slightly 14 days after infection,which was not significantly different from the control group(P>0.05)but still higher than the control group.The results indicate that the Th1-type immune response is related to the host's immune defense against the infection of Cryptosporidium parvum.IFN-? can positively respond to the invasion of Cryptosporidium parvum by inducing a series of chemokines,and TNF-? induces macrophage apoptosis.Increases the permeability of endothelial cells,activates neutrophils and lymphocytes,promotes the synthesis and release of other cytokines,and plays an important role in the process of worming.The Th2-type response mechanism represented by IL-4 and IL-6 did not participate in the main role.One week after infection with Cryptosporidium parvum,the total RNA of mouse small intestine was extracted according to the method of Trizol(Invitrogen,USA),and the quality of RNA was detected by formaldehyde denaturing agarose gel and Bioanalyzer 2100.After the sample was tested,Small RNA was used.The Sample Pre Kit is used to construct the library.After the library is constructed,the Qubit2.0 is used for preliminary quantification to ensure the library quality.The generated library was sent to Beijing Nuohe Zhiyuan Technology Co.,Ltd.for HiSeq sequencing,and the raw data was processed in the following aspects,namely,removal of joints,contamination sequences and low-quality reads,and bioinformatics analysis.There were 204 differentially expressed microRNAs,of which 126 were up-regulated and 78 were down-regulated;differential microRNA target genes were analyzed by Geneontology(GO)enrichment analysis and KEGG Pathways enrichment analysis.It indicates that differential microRNAs are involved in Ras signaling pathway,microbial infection,cancer pathway,receptor interaction pathway,and interaction of chemokines and cytokine receptors.Partially differentially expressed microRNAs were verified by real-time quantitative PCR((mmu-miR-10,mmu-miR-196,mmu-miR-27,mmu-miR-146,mmu-miR-145,mmu-miR-21,mmu-)miR-1981,let-7i and mmu-miR-101).Consistent with the sequencing results.The effect of the differentially expressed microRNA miR-196 on the target gene TLR-11 was verified by a dual luciferase reporter assay.A portion of the 3' UTR region of the TLR-11 gene was ligated into the pmir-GLO vector,and the mmu-miR-196 mimetic and the vector were transfected together into 293 T cells for 48 hours.According to the dual luciferase reporter detection system kit instructions,the luminescence value was detected by a multi-function microplate reader.Compared with the control group,the firefly luminescence value of the test group was relatively significant compared with the sea kidney(P<0.05).It was demonstrated that miR-196 inhibits the expression of TLR-11.Thus,it was judged that TLR-11 is a target gene of miR-196.