| Dairy cow endometritis is a common disease of postpartum cows,which affects the milk production of dairy cows to a certain extent,and also causes infertility and even death of dairy cows,causing great economic losses to dairy farms.The main cause is the infection of pathogenic microorganisms,Escherichia coli(E.coli)is the main pathogenic bacteria.At present,the therapeutic effect is mainly achieved through the use of antibiotics.However,the large amount of antibiotics used will make the bacteria resistant and will also bring about drug residues and food safety issues.Therefore,looking for a drug that can replace antibiotics to prevent and treat cow endometritis of great significance.Lactobacillus rhamnosus GR-1(L.rhamnosus GR-1)is a non-toxic,non-side probiotic bacteria isolated from the female urethra,which can stably adhere to the reproductive tract epithelial cells inhibit the adhesion and proliferation of pathogenic bacteria and reduce the occurrence of inflammation,but it is less used in dairy cow endometritis.The cell culture technology has the advantages of high efficiency and economy,and it is commonly used to study large animal diseases.Therefore,this experiment used cell culture technology to isolate and culture primary bovine endometrial epithelial cells(BEECs)in vitro,infection of BEECs through E.coli to construct a model of cellular inflammatory injury in vitro,and then study whether L.rhamnosus GR-1 has a protective effect on it,and determine the added dose and duration of action,to provide a theoretical basis for its clinical application.In this experiment,the uterus of healthy non-pregnant cows was used as the test material,and the improved tissue block digestion and adherence method,combined with immunohistochemical fluorescence identification and cell viability determination method,was successfully isolated and cultured with high purity and good cell characteristics cell viability of BEECs.Different concentrations of E.coli were used to stimulate BEECs,by observing cell morphology changes,CCK-8,ELISA and Western blot techniques,explored the optimal dose and duration of E.coli-induced inflammatory response.The results showed that when the cells were treated with 5 × 105 CFU/mL E.coli for 6 h or with 5 ×104 CFU/mL E.coli for 9 h,IL-1?,IL-6,IL-8 and TNF-? was significantly increased compared with the control group(P<0.05),and the phosphorylation levels of I?B? and p65 protein were significantly increased(P<0.01),indicating that E.coli can stimulate BEECs to produce an inflammatory response and successfully established a model of inflammatory injury of bovine endometrial epithelial cells.Different concentrations of L.rhamnosus GR-1 acted on BEECs,by observing cell morphology changes,CCK-8,ELISA and other techniques,explored whether L.rhamnosus GR-1 has an effect on cells.The results showed that when cells were treated with 5 × 107 CFU/mL L.rhamnosus GR-1 for 9 h,the cell activity was significantly reduced(P<0.05),and only the secretion of TNF-?increased significantly compared with the control group(P<0.05),cells were treated with 5 × 106 CFU/mL L.rhamnosus GR-1 for 12 h,the cell activity was significantly reduced(P<0.05),the secretion of IL-1?,IL-6,IL-8 and TNF-? was no significant difference compared with the control group.cells were treated with 5 × 105 CFU/mL L.rhamnosus GR-1 for 12 h,which had no effect on cell activity and inflammatory factors.It shows that a certain concentration of L.rhamnosus GR-1 has no harmful effect on BEECs,and determined the safe addition concentration and action time of L.rhamnosus GR-1.The method of adding L.rhamnosus GR-1 to pre-treat BEECs 3 h in advance,by observing cell morphology and ELISA technology,explored whether the safe concentration of L.rhamnosus GR-1 could protect the above two inflammatory injury models.The results showed that the secretion of IL-6 and TNF-? in the 5 × 105 CFU/mL L.rhamnosus GR-1 group was significantly lower than that in the E.coli group(P<0.05),and was higher than its own control group,the secretion of IL-6 and TNF-? in the 5 × 106 CFU/mL L.rhamnosus GR-1 group was significantly lower than that of the E.coli group(P<0,01),and was higher than that of its own control group.It showed that adding L.rhamnosus GR-1 for pretreatment 3 h in advance would have a protective effect on cells,and when L.rhamnosus GR-1 was added at a concentration of 5 × 106 CFU/mL,E.coli was added at a concentration of 5 × 105 CFU/mL,combined effect for 6 h,has the best protection effect.Conclusion:the improved tissue block digestion and adherent culture method can shorten the cell culture cycle,improve cell purity and activity,can obtain stable BEECs;Cells treat with 5 × 105 CFU/mL E.coli for 6 h or with 5 × 104 CFU/mL E.coli for 9 h can produce an inflammatory response;Cells treat with 5 × 106 CFU/mL L.rhamnosus GR-1 for 9 h or with 5 × 105 CFU/mL L.rhamnosus GR-1 for 12 h without harmful;Add L.rhamnosus GR-1 3 h in advance has a protective effect on the cells,and when L.rhamnosus GR-1 was added at a concentration of 5 × 106 CFU/mL,E.coli was added at a concentration of 5 × 105 CFU/mL,combined effect for 6 h,has the best protection effect,which is the best add concentration and duration of action. |
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