MMEC Apoptosis Regulated By NF-κB Signal Pathway On Mastitis Induced By S.aure

Mastitis is the most prevalent production disease in dairy herds world-wide and is responsible for several production effects. Milk yield and composition can be affected seriously. Additional labor and treatment expense will be increased a lot. One of the most important pathogen caused mastitis in dairy herds is Staphylococcus aureus(S.aureus). Mastitis caused by S.aureus presents persistent infection and the early elimination of the cow.Meanwhile, the S.aureus showed multiple drug resistance and posed a serious threat to public health because the overuse of antibiotics. In recent years, people want to prevent mastitis by improving management of dry period /nutrition and making biological agents,but the results are not satisfactory. It is difficult to prevent mastitis caused by S.aureus because the mechanism of infection is unclear seriously affecting its prevention process.Mammary epithelial cell is the first line of defense, it is contact with the pathogens directly and produce apoptosis. Apoptosis normally do not have a strong inflammatory response. If the cells do apoptosis normally or delay apoptosis can lead to chronic inflammation of the mammary gland. NF-κB is a pleiotropic regulatory role of protein molecules involved in regulation of gene expression in a variety of factors, simultaneously, NF-κB is the intersection of many signal pathways, play a very important role in apoptosis.To study the regulation of NF-κB in apoptosis in mammary epithelial cell incuded by S.aurues mastitis, for mastitis control strategies provide new theoretical basis.In this study, establish the S.aurues mastitis models in vivo and in vitro, To study the regulation of NF-κB MMEC apoptosis induced by S.aurues by measuring the transcription and expression of NF-κB subunits from MMEC after infection and NF-κB pathway associated proteins, providing a new guide in treating mastitis.The first part:mammary epithelial cell infected by S.aureus in vitro. the experimental group will be divided when the cells grow to be 80%. The first part are divided into four groups, PBS control group(group A), PDTC treatment group(group B), S.aureus infection group(group C), PDTC treated before S.aureus1 h induced group(group D). The MOI of mammary epithelial cells infected by S.aureus is 100.The apoptosis rate of mammary epithelial cell was detected by flow cytometry; The apoptotic morphology were observed by Hoechst33342 staining; The m RNA and protein levels NF-κB P65/ P50/IκBɑ were detected by Q-PCR and Western blot; The dose of two cytokines were detected by enzyme-linked immunosorbent assay.The results are as follows: 1.1 The differences in apoptosis of mouse mammary epithelial cell induced by S.aureusThe mammary epithelial cell apoptosis rate was measured with Annex V and PI double staining after the cells were collected infecting 3h.S.sureus can cause mouse mammary epithelial apoptosis cells significanly increased(P<0.01)and the apoptosis rate was significantly lower after the signal path is inhibited(P<0.01).There was no significant difference between PBS group and PDTC group. 1.2 Hoechst33342 staining for apoptotic morphologyThe apoptotic cells was significantly increased in group C after Hoechst33342 staining and group D was significantly reduced. Apoptosis morphological in mammary epithelial show that NF-κB involved in the regulation of apoptosis. The results is same to flow cytometry. 1.3 Real Time PCR to detect the relative expression of NF-κBP65/P50/ IκB ɑThe relative expression of NF-κBP65/P50/ IκB ɑappears the same trend. The relative expression of the three genes in Group C showing increased significant difference compared to group A(P <0.01).whereas the relative expression of the three genes shows reduces significant difference after the NF-κB signaling pathway inhibited. 1.4 Western Blot to detect the expression of NF-κBP65 /IκB ɑThe β-actin is treated as internal control.The expression of P65/IκBα and P-P65/P-IκBα was detected.Any treatment had little effect on the P65 protein levels; IκB degradation occurred in group C and Dɑ; The expression of P-IκB ɑ was increased in group Ccompared to group D. 1.5 ELISA to detect the secretion of pro-inflammatory cytokines IL-6 and TNF- ɑS.aureus can significantly improve the TNF- ɑand secretion of IL-6 compared to group A(P <0.05),TNF- secretion in Group D was significantly decreased ɑ compared to group C(P <0.05). The secretion of IL-6 in Group C shows no significant difference.The establishment of mouse mastitis mode using Kunming mice mastitis delivery over one week, The second part be divided into four groups:PBS control group(group A), PDTC treatment group(group B), S.aureus infection group(group C), PDTC treated before S.aureus1 h induced group(group D). The mammary lesions of different treatment groups was evaluated by pathology tissue sections, pathology anatomic, CBC.Mammary epithelial cell apoptosis was detected to evaluate the NF-κB signal pathway of mastitis by Tunnel, the results are as follows: 2.1 The results of CBC in different groupsAll groups were detected by CBC, platelets and red blood cells system shows no difference between different groups.WBC in group C and D groups were significantly different(P <0.05),W-LCR in group C and D were difference significantly, W-SCC in C and D were significantly different. 2.2 The results of bacteria isolate and anatomical pathology anatomic with different treatmentsThe results used LOG10. It do not isolate bacteria Group A and group B,The CFU of group C is 8.4±0.05,and The CFU of group D is 6.27±0.048.The difference was highly significant(P <0.01).The mammary of group A and group B is healty and milk white. There was no difference between the clinical symptoms in group D and group C. The pathological tissue sections score of group C is3.4±0.54, The pathological tissue sections score of group D is2.4±0.54. The difference was significant(P <0.05). 2.3 Mammary epithelial cell apoptosis from mammary tissue by TunnelGroup A and group B can see the mammary gland structure clearly and several number of apoptotic cells.Group C mammary gland structure is not obvious, there are a large number of mammary epithelial cell apoptosis and reduce the number of apoptotic cells in D group. In vivo animal experiments demonstrated similar regularity with experiments in vitro. Results:1 The apoptosis of MMEC is caused by S.aureus and NF-κB signaling pathway involved in the regulation of mammary epithelial cell apoptosis.2 NF-κB signaling pathway and IκBɑ increased m RNA levels play a role in the apoptosis of MMEC caused by S.aureus.3 The inhibition of NF-κB signaling pathway by inhibiting the phosphorylation level of IκBɑ.4 The secretion of TNF-ɑ and IL-6 is regulated by NF-κB signaling pathway and the secretion of IL-6 may also be regulated by other signaling pathways.5 The mouse mastitis can be caused by S.aureus.The inhibitory of NF-κB signaling pathway can reduce inflammation and can reduce the number of apoptotic cells. consistent with the in vitro results.