The Application And Molecular Mechanism Research Of Macrophage M2 Polarization In Intervertebral Disc Degeneration

Part?:Rapamycin combined with reactive oxygen species response scavenging hydrogel scaffold regulate M2-type polarization of macrophages in the treatment of rat disc degenerationObjectiveInflammation is closely related to Intervertebral Disc Degeneration(IVDD),of which risk factors range from mechanical load to abnormal immune microenvironment,including inflammatory cell infiltration and elevated inflammatory cytokine levels.Imbalance of intervertebral disc immune microenvironment results in degeneration of nucleus pulposus cells and degradation of extracellular matrix,accelerating the progress of IVDD.Macrophages,regarded as the vital cells in intervertebral disc degeneration and inflammatory factors,can be polarized into M1 phenotype that promotes inflammation and M2 phenotype that suppress inflammation and promotes tissue repair.The IVDD microenvironment is characterized by the imbalance of M1/M2 regulation and high levels of reactive oxygen species(ROS),which directly affect the inflammatory environment of the intervertebral disc.Based on this,we have developed a ROS-responsive scavenging hydrogel containing rapamycin Scaffold(Rapa@Gel),which could effectively reduce ROS levels.Rapa slowly released from scaffold can promote macrophage to polarized towards M2 phenotype to improve the local inflammation microenvironment and facilitate tissue repair to delay intervertebral disc degeneration.MethodsScanning electron microscope,rheometer,high performance liquid chromatography(HPLC),animal imager and other detection methods were used to perform physical characterization of ROS-responsive scavenging hydrogel and rapamycin sustained release ability.Photoacoustic methods and titanium sulfate were used to test Rapa@Gel's response to ROS in vivo and in vitro.Histological sections were adopted to detect the histocompatibility of Rapa@Gel.Lipopolysaccharides(LPS)and H₂O₂ were used to simulate the inflammatory environment.Flow cytometry was employed to test the effects of hydrogels on the macrophage polarization.Western blot was applied to detect the expression levels of related proteins.MTT assay was utilized to detect the viability of cells treated with different concentrations of Rapa.Enzyme-linked immunosorbent assay(ELISA)was used to determine the expression levels of related cytokines.Rat tail disc degeneration model was established.After 12 weeks,MRI,Micro-CT,flow cytometry,and histological section staining tests were performed to compare degeneration of the intervertebral disc,body weight and blood indicators.ResultsRapa@Gel has typical hydrogel physical characteristics,evidenced by responding to and eliminating ROS in vitro and in vivo,and has good sustained-release properties.Furthermore,Rapa@Gel possesses good biocompatibility.LPS and H₂O₂ could promote M1 polarization,while Rapa and ROS-responsive hydrogels can promote M2 polarization.Rapa specifically inhibits m TORC1 expression,thereby relieving negative feedback on PI3K-Akt inhibition.Enhancement of Akt expression promoted M2 polarization of macrophages.ROS-responsive hydrogel suppressed M1 polarization by reducing ROS levels,and promoted M2 polarization.ROS level in the degenerated tail disc tissue of rats was significantly higher than control group.In situ injection of Rapa@Gel not only significantly reduced the ROS level in the nucleus pulposus tissue,but also improved the inflammatory environment,delaying the tail disc degeneration in rats.In addition,in situ injection of Rapa can obviously inhibit side effects of Rapa of systemic use.ConclusionROS-responsive hydrogel can release Rapa in the regulated manner and scavenge ROS at the same time.Rapa@Gel exerted the synergistic effect where both this hydrogel scaffold and Rapa can promote the M2 type polarization to effectively improve the inflammatory environment in degenerative intervertebral disc tissue and promote tissue repair,thus delaying intervertebral disc degeneration.In addition,we believe that Rapa can specifically relieve the negative feedback inhibition of PI3K-Akt through inhibiting m TORC1,leading to enhancement of Akt expression which promotes M2 polarization.The study provides us with a theoretical basis about clinical transformation of IVDD treatment strategy.Part ?: Molecular mechanism of M2-type polarization regulation in macrophagesObjective Macrophages are heterogeneous cell population,which can polarize towards M1 or M2 type upon different stimulation.Macrophages with M1 phenotype could promote inflammation while M2 regulates immune microenvironment and promotes tissue repair.Once the interleukin 4 receptor(IL-4R)is activated,M2 polarization could be driven by JAK-STAT6 and PI3K-Akt signaling pathways,but the mechanisms through which IL-4 activates downstream signals remain unclear.Our previous research confirmed that G protein inhibitory ? subunits 1 and 3(G?i1/3)could be recruited to mediate PI3K-Aktm TOR signaling after receptor tyrosine kinases(RTKs)being activated.Here,we focus on the interaction between G?i1 and G?i3(G?i1/3)proteins and the activated Akt signaling pathways mediated by IL-4,as well as the key role of G?i1/3 proteins played on macrophage polarization towards M2 phenotype.Methods Gene knockout,dominant negative mutations,lentiviral transfection,adenoviral transfection,CRISPR/Cas9 and other molecular biological methods were used to regulate the expression of G?i1/3,IL4R?,Gab1 and APPL1 in mouse embryo fibroblasts(MEFs)and mouse bone marrow-derived primary mononuclear macrophages(BMDMs).Western blot and polymerase chain reaction were used to detect the expression level of related proteins and M2 macrophage marker genes.Co-Immunoprecipitation(Co-IP)andImmunofluorescence techniques were used to detect the binding of related target proteins.MTT assay was used to detect cell viability.Finally,we conducted animal experiments in G?i1/3 DKO mice combined with pharmacological intervention,demonstrating that G?i1/3 protein played a crucial role in IL-4 activation downstream signal transduction and macrophage M2 polarization.Results In BMDMs and MEFs,G?i1/3 was gene edited by G?i1/3 DKO/sh RNA and CRISPRCas9.Dominant negative G?i1/3 mutation can effectively inhibit the activation of Akt signaling induced by IL-4.In contrast,the overexpression of G?i1/3 promoted the activation of Akt.Relative studies showed that overexpression of G?i1/3 can contribute to the M2 polarization of macrophages mediated by IL-4.Knock out or inhibition of G?i1/3 hardly led to M2 polarization.In G?i1/3 DKO mice,IL-4/anti-IL-4 complex or chitin injection obviously inhibited M2 macrophage activation.Silencing or knockout of G?i1/3,the dominant negative mutant would suppress IL-4R? endocytosis,endosome transport and Akt activation.In addition,silence of endosome marker protein APPL1 effectively inhibited endothelial transport of IL-4R?,Gab1-Akt activation,and M2 polarization while overexpression of APPL1 promoted the above signal and polarization.Conclusion After the stimulation of IL-4,G?i1/3 mainly binds to the intracellular domain of IL-4R?,interacting with the endosome adaptor protein APPL1 to mediate IL-4R? activating the downstream signal.Binding of G?i1/3 to IL-4R? and APPL1 is necessary for IL-4R? endosome transport and Gab1-Akt activation in BMDMs;G?i1/3 mediates IL-4R? endocytic and endosomal transport,and Akt activation.Activation of Akt promotes M2 polarization of macrophages.In general,G?i1/3 is a key regulatory protein for activating signaling pathways such as downstream Akt and M2 polarization of macrophages by IL-4.