The Role And Mechanism Of CD36/FAK/mTORC1 Pathway In LPS-induced Inflammatory Cytokines Expression In Human Mammary Epithelial Cells

Objective: To study the role and mechanism of CD36/FAK/mTORC1 pathway in LPS-induced inflammatory cytokines expression in human mammary epithelial cells(MCF-10A).Methods:(1)The optimal concertration and action time of LPS were detected in MCF-10 A.(2)LPS was used to infect cells,the expression of CD36,activation of FAK/mTORC1 pathway and secretion of inflammatory cytokines were measured.(3)Cell membrane receptor CD36 and TLR4 were blocked by anti-CD36 and anti-TLR4,activation of CD36/ mTORC1 pathway was explored.(4)After inhibiting the FAK signaling through TAE226,a specific chemical inhibitor of FAK,and RNAi method,combined with LPS infection or not,the expression of inflammatory cytokines and the activation of FAK/mTORC1 pathway were tested.(5)After inhibiting the mTORC1 signaling through rapamycin,a specific chemical inhibitor of mTORC1,and RNAi method,combined with LPS infection or not,the expression of inflammatory cytokines and the activation of mTORC1 pathway were tested.Results:(1)Detecting the influence of LPS on MCF-10 A cell proliferation by MTT assay found that the optimal concentration was 1?g/mL,western blot results showed that action time was 12 h.(2)After LPS was treated with cell,the level of mRNA and protein of which CD36 were significantly increased(p<0.05),the activation of FAK/mTORC1 pathway and related transcription factor were notably heighten by western blot(p<0.05),content of inflammatory cytokine TNF-? and IL-6 were increased by ELISA(p<0.05).(3)When cell member receptor CD36 was blocked,LPS stimulated cell suggested that the phosphorylate level of FAK,S6,4EBP1 were significantly attenuated(p<0.05),compared with control group.At the same time,when CD36 and TLR4 were blocked together,the phosphorylate level of FAK,S6,4EBP1,STAT1 were significantly decreased(p<0.05),compared with control group.(4)TAE226 can remarkably inhibit the activation of FAK/mTORC1 pathway and the induced expression of inflammatory cytokine by LPSstimulation(p<0.05).RNAi method was used to interference FAK,the result were consistent with the above TAE226-treated data,confirming that FAK/mTORC1 pathway was inactive,and the content of TNF-? and IL-6 were attenuated(p<0.05).(5)Rapamycin can obviously inhibit the activation of mTORC1 pathway and the induced expression of inflammatory cytokine by LPS stimulation(p<0.05).RNAi method was used to interference the key component of mTORC1,raptor,the result were consistent with the above Rapamycin-treated data,demonstrating that mTORC1 pathway was weaken,and the content of TNF-? and IL-6were degraded.Conclusion: CD36 is co-receptor of LPS,it could conjugation with mTORC1 by FAK which developing CD36/FAK/mTORC1 pathway.Furthermore,CD36 may be triggers body inflammation by regulate the activation of transcription factor(STAT1 and NF-?B p65)through FAK/ mTORC1 pathway induce LPS in human mammary epithelial cells.