The Effective Chemicals And Pathogenic Gene Screening In Leptospira Interrogans Induced Uterine Infection

Leptospirosis, a zoonosis of worldwide distribution and recognized as anemerging natural focus infectious diseases, is caused by Leptospira interrogans(L.interrogans). Leptospirosis is acquired through contact with non-intact skin ormucous membranes of human and animals. A serious infection of leptospirosis canlead to multiple organ hemorrhage, failure or even death, while chronic infectionoften cause the chronic damage of liver and kidney, the abortion of pregnancy humansand animals. In animal, reproductive disorder is the most common clinical symptomscaused by leptospirosis, such as the abortion, stillbirth, premature birth, which inflictpain on animals while generating huge economic. Therefore, prevention and treatmentfor the causes of animal leptospirosis are very important. Considering the paucity ofresearch on animal leptospirosis, this thesis makes study in two aspects. On the onehand, we focus on the reproductive disorder caused by animal leptospirosis throughobserving the influence of L.interrogans on uterine function, and screening effectivedrug to provide possible solutions on clinical treatment of animal leptospirosis. On theother hand, we explore the pathogenic mechanism of animal leptospirosis by using thetranscriptome sequencing technology (RNA-Seq) to provide fundamentally theoreticaland experimental basis on the study of leptospirosis.L.interrogans has been confirmed can enter animal uterus, and multiple factorsexpressed by uterus are closely related to embryonic implantation and fetaldevelopment. Therefore, we put extracellular matrix (ECM) and proinflammatorycytokines expressed by uterus which have a relationship with pregnancy as the keypoint, dogs which has been proved cause abortion by leptospirosis as an experimentmodel, uterine infection by L.interrogans, collect samples of uterus in different pointsin time, then preliminary analysis the influence of L.interrogans on uterine functionby observing the histopathological changes, determining the expression ofproinflammatory cytokines (such as TNF-α, IL-1β and IL-6) and extracellular matrix(such as CL IV, FN and LN) expression. The results showed that L.interrogans cancause uterine tissue damage of dogs, such as inflammatory cell infiltration, unclearendometrial boundary and incomplete edge of endometrium, and a remarkable rise inthe expression of proinflammatory cytokines, and a notable decline in the expressionof ECM protein. Moreover, proinflammatory cytokines play an important role in eachphase of pregnancy as well as in tissue damage. Therefore, anti-inflammatorytreatment should be consider to alleviate the inflammatory response, while antibiotic treatment to should be consider to remove L.interrogans from host.Anti-inflammatory therapy by traditional Chinese medicine has been a hot area forseveral years, and our lab has been focused on the study of cow mastitis andendometritis, screened traditional Chinese medicine which has anti-inflammatoryeffect by using mouse models. In this study, we establish mice uterine infection byL.interrogans, and choose emodin, thymol and astragalin which has been proved byour laboratory have anti-inflammatory effect as therapeutics, to evaluate theanti-inflammatory effect of this three medicines by intraperitoneal injection of emodin(4mg/kg), the thymol (20mg/kg) and astragalin (50mg/kg) before and afterinfection,and to determine expression of TNF-α, IL-1β and IL-6. Consideringproinflammatory cytokines secreted by endometrial epithelial cells have a closerelationship with fetus, we focus on the NF-κB and MAPKs signaling pathway ofmouse endometrial epithelial cells(EECs). Emodin (40μg/mL), thymol (40μg/mL) andastragalin (100μg/mL) are used to treat with the EECs before infection withL.interrogans, then the expression of proinflammatory cytokines and thephosphorylation of NF-κB and MAPKs is measuring to analysis the effect and themechanism of this three medicines. According to the results, the medicines cansignificantly reduce the expression of proinflammatory cytokines through inhibitingthe phosphorylation of NF-κB and MAPKs signaling pathways. Therefore, emodin,thymol and astragalin should be consider to prevention and treatment uterineinflammation caused by leptospirosis. Antibiotics are used for treating leptospirosis,after some antibiotic treatment, L.interrogans still can be detected in the urine ofhuman or animals, such as penicillin. This suggested that L.interrogans can escape byurine, cause chronic tissue damage and environmental pollution. In this study, weassess the therapeutic effect of cefepime (2,5,10and20mg/kg),ertapenem (2.5,5,and10mg/kg) and norfloxacin (20,40, and80mg/kg) by determining the survivalrate, pathogen clearance and tissue damage situation in golden hamsters model whichare sensitive to L.interrogans. The results showed that cefepime in three treatmentdose (5,10and20mg/kg) can significantly improve the survival rate, removeL.interrogans and reduce tissue damage in hamsters, which suggested that cefepimecan be used in the clinical treatment of leptospirosis.It was helpful to treatment with leptospirosis by screening effective drugs, but theprevention and control of leptospirosis is depending on the study of pathogenicmechanism. At present, the pathogenic mechanism of leptospirosis has not beenclarified, the poor knowledge of pathogenic gene in L.interrogans is one of the mostimportant reason. Based on this cognition, gene expression of and screen potentialpathogenic factor in virulent, moderate virulent and avirulent strains are compared byRNA-Seq, which used to study the pathogenic mechanism in gene level. The resultsshowed that compared with avirulent strain, hundreds of differentially expressed genes were found in virulent and moderate virulent strains. Some of them has beenproved relate to virulence genes such as katE, hemO and htpG, potential virulencegenes sph1and sph2, adhesion and chemotaxis gene fliC, fliE, fliK, fliE, fliS, flgG,flgC, ligB and lipL32which has been proved interactions with the ECM of host. Theresults suggested that the data of different virulence L.interrogans can help thescreening of virulence genes. In addition, the differentially expressed genes wereinvolved in various biochemical pathways such as carbon metabolism, lipidmetabolism, energy production, the combination of compounds and antioxidant. Thegenes which involved in the above processes may be play key roles in the alleviationof virulent. Thus, the transcriptome research of different virulence L.interrogans willsupply theoretical and experimental basis in further clarify the pathogenic mechanismof leptospirosis.