| Objective: To investigate the mechanism of Hippo signaling pathway in sepsis by constructing cell sepsis model of RAW264.7 cells(murine macrophages).Methods: 1.RAW264.7 cells were stimulated with lipopolysaccharide(LPS)to establish a model of sepsis,RAW264.7 cells not stimulated by LPS were divided into normal group,LPS-stimulated RAW264.7cells Cells were divided into sepsis group.The proliferation of normal group and sepsis group was detected by cell counting.YES-associated protein(YAP)and phosphorylated YES-associated protein(p-YAP)were observed by confocal microscopy after immunofluorescence staining(IF)。Western blot was used to detect the expression of YAP and p-YAP in normal group and sepsis group.The expression of TAZ,CD206,NOS2,IL-6,IL-1β and TNF-α in normal group and sepsis group were detected by reverse transcription-polymerase chain reaction(RT-qPCR).2.The RAW26.7 cells was transfected with lentivirus which was constructed RNA interference vector of YAP protein.The transfected cells were divided into RNAi 25 group,RNAi 26 group,RANi27 group and negative group.The efficiency of transfection was evaluated by fluorescence microscope.The expression of YAP protein was detected by western blot,and the effective transfection group was screened.The expression of YAP protein in effective transfection group,no-load group and normal groupwas detected by western blot.3.The LPS-stimulated RAW264.7 cells transfected with lentivirus were divided into YAP interference group(RNAi),and the LPS-stimulated RAW264.7 cells without lentivirus transfected were divided into sepsis group(sepsis).YAP protein was detected by immunofluorescence the expression of the situation.The expression of NOS2,TAZ,CD206,LI-6,IL-1β and TNF-α in sepsis group and RNAi group were detected by RT-qPCR.The proliferation of sepsis group and RNAi group was detected by CCK-8.The apoptosis index of group sepsis and RNAi group was detected by Annexin V/7-AAD staining in flow cytometry.Results:1.LPS can stimulate the proliferation of RAW264.7 cells,YAP protein mainly in the nucleus,p-YAP mainly in the cytoplasm,YAP protein in the sepsis group was higher than the normal group,p-YAP in the sepsis group was lower than The expression of normal group,TAZ and CD206 in sepsis group was lower than that in normal group,and the expression of NOS2,LI-6,IL-1β and TNF-α in sepsis group was higher than that in normal group.2.The lentiviral vector transfected RAW264.7 cells,transfection efficiency can reach more than 80%,RAW264.7 cells were transfected with lentivirus can inhibit the expression of YAP protein.There was no significant difference in YAP protein expression between RAW264.7 cells and empty vector transfected with RAW264.7 cells.3.The expression of TAZ,CD206,NOS2 and LI-6 in RNAi group was higher than that in sepsis group.The expression of IL-1β and TNF-α gene in RNAi group was lower than that in sepsis group.Flow cytometry showed that theapoptotic index of RAW264.7 cells in RNAi group was lower than that of sepsis group.Through CCK-8,there was no effect on cell proliferation between RANi group and sepsis group.Conclusion: 1.LPS can stimulate RAW264.7cells to build sepsis model,YAP protein is mainly expressed in the nucleus,while p-YAP is expressed in cytoplasm,YAP may be involved in the pathogenesis of sepsis through dephosphorylation.In sepsis,the expression of proinflammatory cytokines LI-6,IL-1β and TNF-α were high,the expression of TAZ gene in hippo pathway was low,the type M1 macrophage marker NOS2 gene was high,the type M2 macrophage marker CD206 gene is low expression.2.Lentivirus-mediated YAP interference vector can inhibit the YAP expression in RAW264.7 cells,lentiviral vector can not affect the expression of YAP.3.YAP after RNAi interference,it is found that YAP can promote cell apoptosis and can not promote cell proliferation in sepsis.It can inhibit the transformation of M1 macrophages to M2 macrophages and participate in the release of IL-1β and TNF-α,maybe take part in IL-6 release. |
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