CXCL2 Mediates NF-κb And PI3K/Akt To Regulate LTA-induced Inflammatory Responses In Bovine Mammary Epithelial Cells

Bovine mastitis is a high-incidence disease in dairy farming,which can lead to reduced milk production,milk quality and huge economic losses.Conventional antibiotic therapy not only makes pathogenic bacteria resistant,but also remains in dairy products,posing a threat to human health.Therefore,the use of molecular breeding methods to select dairy cows with high resistance to mastitis and reduce the incidence of mastitis has become a new research entry point to control mastitis.The premise is to screen to obtain candidate genes related to mastitis resistance.The chemokine CXCL2 plays an important role in the body’s immune response,affecting the occurrence and development of various diseases and mediating the occurrence of inflammation and autoimmunity.And some studies have found that CXCL2 is overexpressed in the dairy cow mastitis model,proving that it may be involved in the pathogenesis of dairy cow mastitis,but its anti-inflammatory effect and molecular mechanism in Staphylococcus aureus(S.aureus)infected tissues or cells.uncertain.Therefore,in this study,we first screened and identified healthy cows and cows with clinical mastitis by using somatic cell counting,pathogen isolation,and HE staining of tissue sections,and explored the CXCL2 gene and inflammatory cytokines in breast tissue by RT-q PCR and Western Blot methods.,NF-κB and PI3K/Akt signaling pathways,and the expression changes of upstream key molecules TLR2 and My D88,to preliminarily understand the anti-inflammatory effect of CXCL2 in the mammary gland of cows with mastitis.The results showed that the number of somatic cells in the milk of the cows with mastitis was greater than 5×10~6cells/m L,and Staphylococcus aureus was isolated from it.The acinus swelled and collapsed,and the mammary epithelial cells were loose and shed.Finally,3 healthy cows and 3 sick cows with Staphylococcus aureus mastitis were obtained.The detection results of RT-q PCR and Western Blot showed that the expression level of CXCL2 in the mammary gland tissue of mastitis-affected cows was significantly higher than that of healthy cows(P<0.01).The mammary gland tissue of cows with mastitis was significantly up-regulated(P<0.05);and the expression of p-IκBα/IκBαin the mammary gland tissue of cows with Staphylococcus aureus mastitis was significantly up-regulated(P<0.05),p-p65/p65.The expressions of p-PI3K/PI3K and p-Akt/Akt were significantly up-regulated(P<0.01),and the expression levels of TLR2 and My D88 were also significantly up-regulated(P<0.05);Indicating that the inflammation of the mammary gland of S.aureus causes NF-κB and PI3K/Akt signaling Pathway activation,which may be mediated by TLR2 and My D88 upstream of the pathway under the influence of CXCL2 overexpression.Furthermore,the cow mammary epithelial cell line Mac-T cells were selected as the research object,and different doses of LTA were used to stimulate the Mac-T cells to construct an inflammatory cell model;the CXCL2 gene in the Mac-T cells was silenced by si-RNA method,and the CXCL2 gene in the Mac-T cells was silenced by Western Blot,RT-q PCR,CCK-8,Ed U staining and flow cytometry were used to detect the expression changes of intracellular pro-inflammatory cytokines during LTA-induced Mac-T cell inflammatory response by silencing CXCL2 gene and its effect on cell basic biological properties to clarify the regulatory role of CXCL2 on LTA-induced cellular inflammatory responses in vitro.The results showed that when Mac-T cells were infected with 20μg/m L LTA for 24 h,the m RNA expression levels of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 were significantly up-regulated(P<0.01),and the cows were successfully constructed.Mac-T cell model of mastitis;CXCL2-Bos-103,a specific si-RNA sequence of CXCL2 gene,had the most significant and efficient transfection effect for 24 h,and a CXCL2-silenced Mac-T cell model was successfully constructed;and LTA Stimulation significantly increased the expression levels of CXCL2,TNF-α,IL-6 and IL-1β(P<0.05),while silencing CXCL2 could significantly or inhibit LTA-induced CXCL2 and pro-inflammatory cytokines TNF-α,IL-6 and the release of IL-1β(P<0.05);LTA stimulation can significantly reduce the viability of Mac-T cells(P<0.01),down-regulate the proliferation level,and significantly increase the apoptosis rate(P<0.01).LTA-induced cell damage was slowed down by significantly increasing cell viability(P<0.01),promoting cell proliferation,and significantly reducing apoptosis(P<0.05).Finally,in order to explore the anti-inflammatory molecular mechanism of CXCL2 in the process of inflammatory response in dairy cows mammary gland,immunofluorescence,Western Blot and RT-q PCR were used to detect the key factors and upstream of NF-κB and PI3K/Akt signaling pathway in the process of inflammatory response.Expression levels of TLR2 and My D88.The results showed that LTA stimulation led to the transfer of p65 into the nucleus,the expressions of p-IκBα/IκBα,p-p65/p65 were significantly up-regulated(P<0.01),and the expression levels of p-PI3K/PI3K and p-Akt/Akt were extremely significantly up-regulated(P<0.01),indicating that LTA stimulation led to the activation of NF-κB and PI3K/Akt signaling pathways in Mac-T cells,and compared with the LTA stimulation group,the nuclear transfer of p65 in Mac-T cells treated with silencing of CXCL2 was significantly reduced,p-IκBα/IκBαexpression was significantly decreased(P<0.05),p-p65/p65 expression was significantly decreased(P<0.01),p-PI3K/PI3K and p-Akt/Akt expression levels were significantly down-regulated(P<0.05),It indicated that CXCL2 may participate in the regulation of LTA-induced Mac-T cell inflammatory response through NF-κB and PI3K/Akt signaling pathways;TLR2 and My D88 upstream of the pathway were significantly up-regulated in LTA-induced Mac-T cells(P<0.05).,indicating that the activation of NF-κB and PI3K/Akt pathways may be related to the activation of TLR2 and My D88,and silencing CXCL2can significantly inhibit the high expression of TLR2 and My D88 induced by LTA(P<0.01).and My D88 mediate NF-κB and PI3K/Akt signaling pathways,thereby alleviating LTA-induced inflammatory responses.In conclusion,the expression level of CXCL2 was significantly up-regulated in S.aureus milk cow mammary tissue and LTA-induced inflammatory mammary epithelial cells;Silencing CXCL2 gene could increase cell viability and promote cell proliferation by inhibiting the release of pro-inflammatory cytokines.Reducing apoptosis can slow down LTA-induced inflammatory response;Silencing CXCL2gene can mediate TLR2,My D88,and inhibit the activation of NF-κB and PI3K/Akt signaling pathways,thereby slowing LTA-induced inflammatory response.