| The uterine lumen is sterile,and microbes often infect the uterus and particularly the endometrium following dairy cows' parturition,causing non-oestrus,anovulation and dysfunction.Dairy cows would be eliminated as difficulties to conception or failure to conceive,which not only leading to a high cost in treatment and breeding,but also reducing the profitability and sustainability of dairy industry.Meanwhile,due to the big somatotype and high economic cost of the dairy cows,depth investigation on endometrial inflammation was restricted.Therefore,the present study aimed to use pathogenic bacteria which isolated from post-partum dairy cows' uterine mucus to establish rat endometritis model firstly.Secondly,to explore the mechanism of uterine damage and dysfunction caused by pathogen acute and chronic infection,we investigated the endometrial immune barrier and the uterine conception regulates genes expression,providing theoretical reference for the prevention and treatment of clinical endometritis.1 Isolation,Identification of Bacteria in the uterus and its relationship with the mucus characteristics in Postpartum Dairy CowDynamitic changes of bacteria population in diverse time and different uterus states after calving was investigated,and the relationship between bacterial species and the characteristic of uterus mucus was analyzed.Based on the mucus characteristics and the number of polymorphonuclear neutrophils in the uterus,18 Holstein cows were divided into three groups at 21 days following calving.The uterus mucus at postpartum 21 d,35 d,49 d and 56 d were collected and bacteria were isolated.DNA of Single colonies was extracted and sequenced.The results showed that 407 pure bacteria were isolated,and the sequencing result indicated these bacteria belong to 3 phyla(Proteobacteria,Firmicutes,Actinobacteria)and 25 species.The separation rate of E.coli(44.14%),Bacillus sp.(19.31%),Staphylococcus sp.(6.21%),etc.was high,which isolated from both health and sicken cows.Bacillus cereus,Bacillus thuringiensis were only isolated from healthy cows,and Klebsiella pneumonia,Shigella sp,Acinetobacter sp.,Corynebacterium renale,Helcococcus ovis were only isolated from cows with clinical endometritis.Furthermore,the numbers of bacteria isolated from mucus scored 3(98 strains)more than the mucus scored 2(37 strains)or scored 1(10 strains).Bacteria isolated from the mucus scored 3 as well as the thick mucus at the 35 d more than the 21 d after calving,Helcococcus ovis,Shigella sp.Staphylococcus epidermidis,Ainetobcter sp.were only isolated from mucus scored 3.The isolates decreased from 49 days to 56 days after parturition.In conclusion,E.coli was dominated in postpartum uterus with defferent health state,and mucus viscosity increased as more bacterial infection of the uterus.In addition,the occurrence of endometritis may highly related to the appearance of Acinetobacter sp.Shigella sp.,Helcococcus ovis etc.2 Mechanism of endometrial injury and uterine conception dysfunction associate with pathogen acute infection in rat modelIn this study,24 female mature SD rats were chosen and randomly divided into three groups,and rats endometritis model were established by injecting Staphylococcus aureus or Escherichia coli,which isolated from dairy cows' uterus by ourselves,into uterus.Blood and uterine tissue samples of the rats were collected 24 h later of the infection.To confirm if the pathogen had been invidad or the inflammation had been occurred,we detected the TLR receptor and cytokines' expression firstly.To explore the mechanism of uterine damage and dysfunction,we detected the expression of MUC1 and endometrium tight junction genes to investigate the endometrium damage firstly,and then we detected the expression of genes regulated in conception progress to investigate the effect of bacterial acute infection on uterine conception function.The results showed that 24 h after the S.aureus or E.coli infection,the appearance of the uterus became red and swollen,and many neutrophils infiltration into uterine stromal.Compared with the control group,the TLR2(TLR4)/Myd88/NF-KB signal pathway molecule genes expression were significantly up-regulated(P<0.05),the concentration of cytokines(IL-6,IL-1?,IL-8 and TNF-?)in blood were significantly increased(P<0.05).Those indicated a serious inflammation occurred in rat uterus,and the rat model was established successfully.The gene expression of Occludin and Claudin4 were significantly increased after 24 h S.aureus infection(P<0.05),meanwhile the expression of MUC1 and Zol were significantly decreased(P<0.05).After 24 h E.coli infection,the gene expression of MUC1,Occludin and ZOl was significantly decreased(P<0.05),which indicated that acute infection of pathogens can significantly change the genes expression of tight junction protein,weakening the endometrial barrier function.HOXA10 expression was significantly up-regulated after S.aureus infected 24 h,but down-regulated after E.coli infection(P<0.05).LIF expression was significantly down-regulated by S.aureus and E.coli infected 24 h(P<0.05).In addition,the expression of progesterone receptor(PR)and estrogen receptor(ER)were significantly decreased with S.aureus infected 24 h while the expression of ER was significantly increased after 24h of E.coli infection(P<0.05).Indicating that pathogens acute infection significantly changed the expression of uterine conception regulatory genes,interfere with uterine conception.In summary,S.aureus and E.coli infected uterus 24 h could cause inflammation,disrupting the integrity of the endometrial tight junction complex weakening endometrial barrier function and reducing the expression of the implanted regulatory gene interfering uttering conception.3 Mechanism of endometrial injury and uterine conception dysfunction associate with pathogen chronic infection in rat modelSimilar with the second chapter of this study,24 female mature SD rats were randomly divided into three groups and rats endometrial model were established.Blood samples and uterine tissue samples were collected at 5 d later of the treatment.To explore the mechanism of endometrium damage and obstacle of uterine conception caused by chronic uterine inflammation,we detected the MUC1 and tight junction genes expression and uterine conception regulated genes expression.The results showed that,compared with the control group,it's obvious that the uterus were red and swollen in S.aureus or E.coli infected group,numbers of immune cells were observed in uterine tissue sections.Meanwhile,the expression of TLR2 and TLR4,Myd88 and NF-?B as well as the inflammatory factors(IL-6,IL-8,IL-1?,TNF-?)were significantly increased(P<0.05).Indicating that E.coli or S.aureus infected 5 d could lead to chronic inflammation of rat uterus.The expression levels of MUC1,Occludin,ZOI and Claudin4 in infected groups were significantly lower than those in control group(P<0.05).Indicating the endometrial barrier function was weakened at 5 d later of pathogen infection.Moreover,the expression of LIF and HOXA10,even PR and ER,in uterine tissue were significantly down-regulated by S.aureus and E.coli chronic infection.Indicating that with pathogen infection 5 d,the expression of uterine conception regulatory genes were inhibiting.In conclusion,S.aureus and E.coli infected uterus 5 d could cause chronic endometritis,reducing the expression of endometrial MUCI and tight junction complexes delaying the involution of uterus,and inhibiting the expression of blastocyst implantation-related genes(LIF,HOXA10)in uterine tissue disturbing conception. |
PDF Full Text Request