The Intervention Effects And Mechanism Of Regulating The Molecular Function Of Transcription Repressor BCL6 On Endotoxemia

Background: Open-heart surgery under cardiopulmonary bypass can lead to an increase in circulating endotoxin levels,cause endotoxemia,and easily induce the body to produce systemic inflammatory response syndrome,which seriously threatens the prognosis of patients.The oncogene B-cell lymphoma factor 6(BCL6)is a transcriptional repressor that is widely distributed and multifunctional.In diffuse large B lymphoma,the molecular function of BCL6 and the function and mechanism of its BTB domain inhibitor FX1 have been explained in more detail,but in endotoxemia,no related research have been reported.Objective: The molecular function of BCL6 and the mechanism of action of its inhibitors in the mouse endotoxemia model were studied,in order to providing a possible drug reference sought for the prevention and treatment of endotoxemia after the open heart surgery with cardiopulmonary bypass.Methods: I.in vivo animal experiments 1.LPS-induced mouse endotoxemia model was established in groups.*DMSO group: ten C57 mice were used as the negative control group,which received intraperitoneal injection of DMSO alone and then received intraperitoneal injection of PBS buffer alone;*DMSO+LPS group: ten C57 mice were used as the positive control group.Before the preparation of the endotoxemia model of C57 mice,they received intra-abdominal injection of DMSO alone.* FX1+LPS group: ten C57 mice were used as the experimental group.Before establishing the model of endotoxemia in C57 mice,they received intraperitoneal injection of DMSO+FX1(FX1 dissolved in DMSO).2.To observe the effect of FX1 intervention on survival of mouse endotoxemia model.Survival time of all groups of mice was recorded and survival curves were drawn.Paraffin sections and staining were used to evaluate local tissue lesions.3.Effect of FX1 intervention on proinflammatory factor expression in mouse endotoxemia model.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of pro-inflammatory factors in serum.4.Effect of FX1 intervention on macrophage status in mouse endotoxemia model.Immunohistochemistry was used to detect the infiltration degree of macrophages in important organs,and flow cytometry was used to detect the chemotaxis status of macrophages In vitro cell experiments 1.Effects of FX1 intervention on the inflammatory response of macrophages.The effect on the inflammatory response of macrophages after FX1 intervention in the in vitro environment was detected by real-time quantitative PCR and ELISA.2.Effect of knocking down BCL6 on the inflammatory response of macrophages after FX1 intervention.BCL6 was knocked down by adenovirus transfection,and the degree of macrophages inflammatory response after FX1 intervention was detected using real-time quantitative PCR.3.Effects of FX1 intervention on the transcription activity of NF-κB The changes in macrophages NF-κB transcription activity after FX1 intervention were detected by double fluoxerase reporting gene tests.4.The effect of FX1 intervention on the signaling pathways of MAPK and NF-κB after macrophage activation.Changes in the signaling pathways of MAPK and NF-κB in macrophages after FX1 intervention were detected by western blot.5.Effect of FX1 intervention on BCL6 binding to pro-inflammatory target gene promoter The degree of binding of BCL6 to the promoter gene promoter was detected by chromatin immunoprecipitation assay(Ch IP).Results: In vivo experiments: 1.The intervention of BCL6 inhibitor FX1 can significantly improve the survival rate mice endotoxemia models and will not be significantly toxic to C57 mice for individual application;2.The intervention of FX1 was able to significantly reduce the level of IL-1 beta,IL-6,MCP-1 three pro-inflammatory factors in peripheral blood in the endotoxemia model of mice.3.The intervention of FX1 can effectively reduce the macrophages infiltration in the lung and liver of the mice endotoxemia model and the immune damage associated with it.4.Although FX1 did not significantly alter the groups of peritoneal macrophages in the endotoxemia model in mice,it was able to effectively inhibit the activation of part of the peritoneal macrophages.In vitro experiments: 1.After the inflammatory response of macrophages was activated,the expression of BCL6increased gradually,and BCL6 inhibitor FX1 did not alter the expression of BCL6 and its corepressors,nor did it affect the activity of macrophages;2.FX1 as the representative of BCL6 BTB domain inhibitors can significantly inhibited the inflammatory response of macrophages,and its anti-inflammatory effect depended on the expression of BCL6;3.FX1 can significantly enhance the direct inhibition of BCL6 on the NF-κB transcription activity in macrophages.4.FX1 inhibited the activation of the MAPK and NF-κB signaling pathways after macrophages activation.5.FX1 promoted the binding of BCL6 to the promoter of proinflammatory target gene.Conclusion: 1.The application of BCL6 BTB domain inhibitor FX1 can significantly improve the survival rate of mice with endotoxemia and effectively inhibit the inflammatory response of macrophages.2.BCL6 is involved in the inflammatory response of macrophages,and the target of FX1 on macrophages is indeed the BTB domain of BCL6.3.FX1 regulates the molecular functions of BCL6 without affecting the normal expression of BCL6 and its transcriptional corepressors :(1)enhancing the inhibitory effect of BCL6 on MAPK signaling pathway and NF-κB signaling pathway;(2)promoting the direct inhibition effect of BCL6 on the transcriptional activity of NF-κB;(3)the binding force between the c-terminal zinc finger structure and the promoter of the proinflammatory target gene is improved to inhibit the inflammatory response of macrophages.