Inhibitory Effect Of LIPUS On The Expression Of LPS-induced Inflammatory Factors In U937 Cells

Objective:This study aimed to assess the effect of LIPUS on LPS-induced inflammatory factors in a macrophages cell model(U937)and analysed the underlying mechanisms.Methods:1.After cultured and induced with PMA,U937 cells were stimulated with different concentrations of LPS.The supernatant was collected after 24 h and the content of TNF-α was detected by ELISA.U937 cells were exposed to different intensities of LIPUS.The supernatant was collected after 2 h.The content of TNF-α,IL-8 was detected by ELISA and the gene of IL-8 was assessed by RT-PCR.2.Cells were also divided into 5 groups: U937 cell group,LPS group,LIPUS group,LPS-LIPUS-treated group and Bay-LPS group.U937 cells were seeded in 96-well culture plates.CCK-8 solution was added to each well for testing proliferation and the cells were incubated for another 2 h.The results were assessed at day 3,day 5 and day 7.In addition,U937 cells were cultured in a 10 cm cell culture dish.Apoptosis was determined using a flow cytometer.3.To evaluate the influence of LIPUS on inflammation,cells were assigned to 4 groups: U937 cells group,LPS group,LIPUS group and LPS-LIPUS-treated group.The supernatants were tested using ELISA kits and the gene was assessed using RT-PCR.4.To test the underlying mechanisms of LIPUS on LPS-induced inflammatory factors in U937 cells,U937 cells were also divided into 4 groups: U937 cell group,LPS group,LPS-LIPUS-treated group and Bay-LPS group.RT-PCR was used to test the gene levels of TNF-α,IL-1β,IL-6,IL-8 and TLR4.Western Blotting was used to assess the protein levels of TLR4,p65,p-IκBα and IκBα.The transcription of p65 into nuclei was observed using Immunofluorescence assay.Results:1.U937 cells were matured and inflammatory model was established successfully.The LPS concentration of 1 μg/ml and the intensity of 60 mW/cm2 were used in the following study.2.CCK-8 and apoptosis assay showed that LPS inhibited U937 cell viability and promoted U937 cells apoptosis,but LIPUS inversed this process.3.ELISA and RT-PCR assays showed that LPS increased TNF-α,IL-1β,IL-6 and IL-8 expression,while the opposite role of LIPUS.4.RT-PCR assay showed that LIPUS decreased TNF-α,IL-1β,IL-6,IL-8 and TLR4 expression.WB showed that LIPUS primarily suppressed the degradation and phosphorylation of IκBα and the translocation of p65 into the nuclei.Immunofluorescence assay further confirmed the inhibition of LIPUS on the translocation of p65.Conclusion:LIPUS promoted U937 cell viability and inhibited U937 cells apoptosis significantly.LIPUS alleviated the expression of inflammatory factors induced by LPS in U937 cells.This process was modulated by suppressing the TLR4-NF-κB signalling pathway.