The Research Of Specific Deubiquitination Enzyme 25 Regulating Polarization Of M2 Type Macrophages

ObjectiveTo investigate the effect of specific deubiquitination enzyme USP-25 on the polarization of macrophages.To observe the trend of changes in the polarization of M2-type macrophages after knockout or increase of USP-25 expression,so as to determine whether USP-25 plays a pro-inflammatory role or an anti-inflammatory role in the polarization of macrophages.At the same time,the expression level changes of related cell products and the level changes of signal molecules in related pathways were observed to explore the mechanism of USP-25 affecting macrophage polarization,so as to further clar ify the influence of USP-25 and deubiquitination on immune regulation and its mechanism of action.Methods1.M2-type polarizat ion induct ion of C57 mouse bone marrow-der ived macrophages(BMDM)Previous references and prel iminary experiments were carried out to extract primary macrophages from mouse bone marrow and polarizat ion induct ion was conducted in the direction of M2-type macrophages.In the experiment,the most suitable conditions for the polarization induction of BMDM to M2-type macrophages were explored,and the appropriate concentrations and time of M-CSF and IL-4 or IL-13 were determined,so as to determine an induction condition that could steadily induce sufficient M2-type macrophages.At the same time,I have mastered the method of extracting primary macrophages from bone marrow,laying a good foundation for subsequent exper iments.2.Comparing the induction efficiency and marker expression levels of M2-type macrophages in wild-type mice and USP25-/as well as USP25 overexpression mice.Primary macrophages from bone marrow of wild-type C57 mice and USP25-/-C57 mice were extracted,respectively,and induced into M2-type macrophages under the same experimental conditions.F4/80 and CD206 were detected by flow cytometry to observe the transformation efficiency of M2-type macrophages.Total cell proteins and RNA were extracted for Western blot,reverse transcription and RT-PCR to detect the expression levels of markers Arg-1 and Fizzle in M2-type macrophages,to determine whether USP25 would affect polarization induction and marker expression in M2-type macrophages.Meanwhi le,the effect of USP25 on the polarization of M2-type macrophages was verified by compar ing the wild-type mice and the M2-type macrophages transfected with USP25.3.Detecting expression of STAT6 and PPAR-γ pathway in wild-type mice and USP25-/-miceOn the precondition of the results obtained in the above experimental steps,RNA and total proteins were extracted respectively for RT-PCR and Western-blot detection of STAT6,STAT3,C/EBP β and other related pathway molecules,and the changes i n the express i on levels of key molecules i n the pathway were observed.The ubiquitination at K48 and K63 sites of the molecules with related changes in expression was detected to observe whether the ubiquitination levels of the molecules with changes in expression levels were affected,so as to further clarify the action site and mechanism of USP25 and narrow the experimental scope.4.To find the deubiquitination site of USP25 and clarify its mechanismAfter find ing the protein with changed expression and changed ubiquitination level at K63 or K48 sites,immunoprecipitation was performed by constructing carrier containing labeled protein to determine whether USP25 interacts with this molecule to determine whether USP25 regulates the expression of this molecule by affecting the ubiquitination level of K48 or K63 of this molecule.After verifying the interaction,pull-down or yeast two-hybrid experiments were carried out to determine whether USP25 could directly bind to the molecule to reverse the ubiquitination process and reduce the level of ubiquitination,thus affecting the degradation of related molecules or signal transduction as well as affecting the pathway expression,so as to clarify the specific mechanism of USP25’s influence on macrophage polarization.ResultsSpecific deubiquitination enzyme 25(USP25)promoted the polarization of mouse type M2 macrophages,and it was positively correlated with the expression of Arg-1,a marker of type M2 macrophages.When USP25 was knocked out or inhibited,the expression of type M2 macrophages was inhibited,and the mechan i sm was related to the regulation of Stat6 ubiquitination level and the change of expression level.