| Mastitis in dairy cows is one of the three most common diseases in the world’s dairy farms,seriously affecting milk production and economic efficiency.The occurrence of mastitis is influenced by many factors,including pathogens,genetics and the environment.An inflammatory response occurs when mammary tissue is exposed to chemical and physical damage,pathogens and a variety of other factors.G protein coupled receptor 120(GPR120),which is widely distributed in tissue cells,has been shown to play an important regulatory role in this process,but the regulatory network and mechanisms of action related to GPR120-mediated immune protection in mammary tissue of dairy cows are not yet well understood.Therefore,in order to clarify the downstream regulatory genes and transcription factors mediated by GPR120 and the molecular mechanism of their anti-inflammatory effects,we firstly constructed a model of LTA-induced inflammation in mammary epithelial cells,and then clarified the regulatory effects of GPR120 on LTA-induced inflammatory responses in mammary epithelial cells of cows by using the GPR120 activator TUG-891 and the inhibitor AH7614.The next step was to screen and identify GPR120-mediated downstream functional genes,transcription factors and inflammatory pathways that were significantly enriched by transcriptome co-analysis;on the basis of this,we used the candidate genes obtained from the screening as targets and functional techniques to investigate the mechanism of GPR120-mediated inflammatory response in dairy epithelial cells.The results showed that(1)treatment with the inhibitor AH7614 in the inflammatory cell model significantly reduced GPR120 gene expression levels(P <0.05),and the use of the activator TUG-891 resulted in highly significant upregulation of GPR120 gene expression levels(P < 0.01);stimulation of LTA significantly promoted Mac-T cell TNF-α,IL-1β,IL-6 secretion(P < 0.05),while treatment with activator TUG-891 significantly reduced the expression of the above three pro-inflammatory cytokines(P < 0.05),and treatment with inhibitor AH7614 further significantly upregulated the expression of the three pro-inflammatory cytokines(P < 0.05);meanwhile,compared with the control group,the GPR120-mediated inhibitor treatment group further stimulated cell viability,promoted The GPR120 mediator inhibitor group further stimulated cell viability,promoted cell proliferation and decreased apoptosis compared with the control group(P < 0.05),while cell viability,cell proliferation and apoptosis were significantly increased in the activator group(P < 0.05).(2)Transcriptome co-analysis showed that,compared with the control group,147 differentially expressed genes were screened in the inhibitor group,of which 118 genes were significantly up-regulated(P < 0.05)and 29 genes were significantly down-regulated(P < 0.05);1660differentially expressed genes were screened in the activator group,of which 667 genes were up-regulated(P < 0.05)and 993 In the activator group,1660 differentially expressed genes were screened,667 genes were up-regulated(P < 0.05)and 993 genes were down-regulated(P < 0.05).Among them,171 differential genes were associated with immune traits.They were enriched to functional pathways such as immune system and signal transduction.In addition,210 nuclear transcription factors were screened in the activator group,of which 24 were associated with inflammatory response;36transcription factors were screened in the suppressor group,of which 7 were associated with inflammatory response;CRYAB genes were significantly up-regulated in the activator group and closely associated with the occurrence of inflammatory response,and were selected as candidate genes for subsequent functional mechanism studies.(3)CRYAB protein expression level was highly significantly down-regulated in the LTA+AH7614 group(P < 0.01);CRYAB expression could be significantly up-regulated in the LTA+TUG891 group(P < 0.05);activation of GPR120 may exert anti-inflammatory effects by promoting the expression of CRYAB.While protein blotting results showed that p-IκB/IκBα,p-p65/p65,p-ERK/ERK,p-JNK/JNK,and p-p38/p38 expression would be significantly upregulated upon LTA stimulation(P < 0.05),activation of GPR120 significantly decreased p-IκB/IκBα,p-p65/p65,p-ERK/ERK,p JNK/JNK,and p-p38/p38 expression levels(P < 0.05),and inhibition of GPR120 significantly upregulated the expression of these five proteins(P < 0.05);meanwhile,TLR4 and My D88 expression levels were significantly upregulated upstream of the LTA-induced inflammatory intracellular detection pathway(P < 0.05),and activation of GPR120 significantly inhibited the high expression of TLR4 and My D88(P < 0.05).Activation of GPR120 significantly suppressed the high expression of TLR4 and My D88(P < 0.01),while inhibition of GPR120 significantly upregulated the expression of TLR4 and My D88(P <0.05).Together,these results suggest that GPR120 may mediate NF-κB and MAPK signaling pathways through CRYAB,TLR4 and My D88,thereby alleviating the LTA-induced inflammatory response.As shown above,activation of GPR120 gene can increase cell viability,promote cell proliferation,reduce apoptosis,and inhibit the expression of pro-inflammatory cytokines,thus exerting certain protective effects against LTA-induced inflammatory damage in Mac-T cells;A total of 171 GPR120-mediated downstream differentially expressed genes and 25 differentially expressed intranuclear transcription factors were screened for immune traits;among them,the CRYAB gene could be activated by GPR120 and inhibit the activation of NF-κB/ MAPK inflammatory pathway by downregulating the expression of TLR4 and My D88,thus regulating the inflammatory response and exerting an anti-inflammatory effect.anti-inflammatory effect. |
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