PI3K?/AKT Regulating Endotoxin Tolerance Of Kupffer Cells Depends On The Foxo3a-p53/MDM2 Negative Feedback Loop

Objective and Background:The endotoxin tolerance of Kupffer cells(KCs)is closely related to many kinds of clinical infectious diseases such as systemic inflammatory response syndrome.Inhibition of nuclear transcriptional factor ?B(NF-?B)activiation in macrophages is one of the most effective ways to induce endotoxin tolerance.It is reported that phosphatidylinositol 3-kinase ?(PI3K?)makes the toll like receptor 4(TLR4)subcellulary compartmentation,then limiting the signal transduction of TLR4 after being stimulated by lipopolysaccharide(LPS)in macrophage.Subsequently,the secretion of pro-inflammatory factors such as tumor necrosis factor ?(TNF-?)is inhibited,and the secretion of anti-inflammatory factors such as interleukin 10(IL-10)is elevated,then inhibiting the inflammation in induced by LPS.On the other hand,the activation of PI3K?/AKT induced by LPS directly phosphorylates forkhead protein O3a(Foxo3a).The phosphorylated Foxo3 a is exported out nuclear and immediately ubiquitinated and hydrolyzed by p53/murine double mimute(MDM2),relieving the inhibitory effect of Foxo3 a on the activiation of NF-?B.The incompatibility phenomena described above is suggested that the PI3K?/AKT pathway may be negatively regulated by foxo3 a and p53/MDM2 pathways during processing the inflammatory signaling induced by LPS.However,interactions between Foxo3 a and p53/MDM2 in the endotoxin tolerance of KCs,whether foxo3 a and p53/MDM pathways are the balance mechanism of PI3K?/AKT pathways regulating the endotoxin tolerance of KCs are still unclear.Thus,this experiment will study the roles and interactions among P PI3K?/AKT,Foxo3 a,p53/MDM the ndotoxin tolerance models of KCs in vivo and vitro.Methods:1.The endotoxin tolerance(ET)and non-endotoxin tolerance(NET)models of KCs were established after the KCs were isolated and cultivated for 24 h in vitro: The supernatant levels of TNF-? and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA);The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by western blotting assay(WB);The location changes of NF-?B p65 and Foxo3 a in KCs were observed by cell immunofluorescence assay.The ET and NET models of KCs were established after the KCs were respectively transfected with Foxo3 a small interfering RNA plasmid(Foxo3a-siRNA)and Foxo3 a over-expression plasmid(Foxo3a-OE)for 36 h in vitro: The supernatant levels of TNF-? and IL-10 were detected by ELISA;The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by WB;The KCs apoptosis rates were detected by flow cytometry combining with propidium iodide(PI)/annexin ? labeled fluorescein isothiocyanate(FITC).2.The ET and NET models of KCs were established after the KCs were isolated and cultivated for 24 h in vitro.The protein expression and phosphorylation levels of PI3 K and AKT in KCs were were detected by WB.The ET and NET models of KCs were established after the KCs were respectively pretreated with PI3K? agonist recombinant mouse insulin-like growth factor 1 protein(IGF-1)and inhibitors IPI-549 in vitro: The supernatant levels of TNF-? and IL-10 were detected by ELISA;The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by WB;The survival rate of KCs was detected by automatic cell counting instrument.The NET models of KCs were established and pretreated with IGF-1 after the KCs were transfected with Foxo3a-OE for 36 h in vitro or The ET models of KCs were established and pretreated with IPI-549 after the KCs were transfected with Foxo3a-siRNA for 36 h in vitro: The supernatant levels of TNF-? and IL-10 were detected by ELISA;The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by WB;The survival rate of KCs was detected by automatic cell counting instrument.The ET and NET mouse models were established after the mice were respectively pretreated with IGF-1 and IPI-549 in vivo: Haematoxylin-eosin staining(HE)combing with Suzuki evaluation table was used to evaluate the degree of inflammatory response in liver tissue;the mRNA levels of TNF-? and IL-10 in KCs were detected by real-time quantitative polymerase chain reaction assay(RT-PCR),and the protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a in KCs were detected by WB after separation of liver KCs.3.The protein expression and phosphorylation levels of p53 and MDM2 were detected by WB after the separated KCs were transfected with Foxo3a-OE for 36 h in vitro.The ET and NET models of KCs were established after the KCs were isolated and cultivated for 24 h in vitro.The protein expression and phosphorylation levels of p53 and MDM2 in KCs were detected by WB.The NET models of KCs were established after the KCs were transfected with MDM2-siRNA for 36 h in vitro: The supernatant levels of TNF-? and IL-10 were detected by ELISA;The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by WB;The survival rate of KCs was detected by automatic cell counting instrument.The NET models of KCs were established after the KCs were pretreated with IGF-1 and MDM2 inhibitor Nutlin-3 in vitro: The supernatant levels of TNF-? and IL-10 were detected by ELISA;The protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a were detected by WB;The survival rate of KCs was detected by automatic cell counting instrument.The NET mouse models were established after the mice were pretreated with IGF-1 and Nutlin-3 in vivo: Observe and record the mice survival time;HE combing with Suzuki evaluation table was used to evaluate the degree of inflammatory response in liver tissue;the mRNA levels of TNF-? and IL-10 in KCs were detected by RT-PCR,and the protein expression and phosphorylation levels of NF-?B p65 and Foxo3 a in KCs were detected by WB after separation of liver KCs.Results:1.The activity of NF-?B pathway,phosphorylation levels of Foxo3 a and secretion levels of TNF-? in ET group were significantly lower than those of NET group,while the protein levels of Foxo3 a and secretion levels of IL-10 in ET group were significantly lower than those of NET group.Overexpression of Foxo3 a inhibited the activation of NF-?B pathway and the secretion levels of TNF-?,increased the secretion levels of IL-10,improving the endotoxin tolerance of KCs in NET group.Silence of Foxo3 a increased the activation of NF-?B pathway and the secretion levels of TNF-?,inhibited the secretion levels of IL-10,reducing the endotoxin tolerance of KCs in ET group.2.PI3K? inhibitor suppressed the activation of NF-?B pathway and the secretion levels of TNF-?,increased the secretion levels of IL-10 via decreasing the phosphorylation leveld of Foxo3 a and elevating the protein expression of Foxo3 a by inhibiting the activity of AKT,improving the endotoxin tolerance of KCs and mice in NET group.PI3K? agonist increased the activation of NF-?B pathway and the secretion levels of TNF-?,decreased the secretion levels of IL-10 via elevating the phosphorylation leveld of Foxo3 a and decreasing the protein expression of Foxo3 a by promoting the activity of AKT,reducing the endotoxin tolerance of KCs and mice in ET group.PI3K? agonist counteracted the inhibition effects of Foxo3 a overexpression on NF-?B pathway,reducing the endotoxin tolerance of KCs and mice in NET group.3.Foxo3a-p53/MDM2 formed a negative feedback loop,which maintained the protein stability expression of Foxo3 a in KCs.Silence of MDM2 or functional blockage of MDM2 improved the endotoxin tolerance of KCs and mice in NET group by accumulating protein expression of Foxo3 a.MDM2 inhibitor counteracted the promotion effects of PI3K? agonist on NF-?B pathway,improving the endotoxin tolerance of KCs and mice in NET group.Conclusions:1.The accumulation of Foxo3 a in the nucleus increases the endotoxin tolerance of KCs and mice through inhibiting the activity of NF-?B pathway and the secretion levels of inflammatory cytokines.2.Suppressing the phosphorylated effects of PI3K?/AKT on Foxo3 a or the blocking of ubiquitination degradation effects of p53/MDM2 on Foxo3 a increases the endotoxin tolerance of KCs and mice.3.The negative feedback loop formed by Foxo3a-p53/MDM2 is not only the main mechanism to maintain the protein stability of Foxo3 a in KCs,but also is the main mechanism to balances the regulation effects of PI3K/AKT pathway on endotoxin tolerance of KCs.