M2 Macrophage-derived Exosomes Transfer MiR-221-3p To Inhibit Nucleus Pulposus Cell Pyroptosis

Objective:Intervertebral disc degeneration is an age-related disease.It is characterized by the decreased of extracellular matrix,inflammatory activation or cell senescence,which cannot maintain the normal function of the intervertebral disc,resulting in many spinal-related diseases.Several studies have shown that cell pyroptosis is involved in the degeneration of intervertebral disc.In recent years,cell-free therapy with exosomes as a promising tool has been developed rapidly.Therefore,the purpose of this study is to determine whether M2 macrophage-derived exosomes could inhibit the cell pyroptosis of nucleus pulposus and to clarify its related mechanism in cellular,molecular biology and animal in vivo experiments.Methods:Firstly,THP-1 were induced by PMA and IL-4/IL-13 to obtain M2macrophages,and the phenotype of M2 macrophages was determined by flow cytometry and immunofluorescence.The supernatant of M2 macrophage was extracted and M2 macrophage-derived exosomes was obtained.The exosome was verified by transmission electron microscope,particle size analysis and Western blotting experiment.The uptake of fluorescence labeled M2-exos by HNPC was observed by confocal microscope.Secondly,Clinical specimens were collected to verify the expression of Caspase-1 in degenerative nucleus pulposus.The degeneration of HNPCs induced by hydrogen peroxide was used as the model of cell pyroptosis in vitro.The intervention concentration of hydrogen peroxide was detected by CCK-8,and the protective effect of M2-exos on cell viability and the migration of NP cells were observed.TUNEL staining,Annexin V/PI flow apoptosis staining and Hoechst33342/PI staining were used to study the effects of hydrogen peroxide group and M2-exos group on the NP,and the ROS in each group was observed by DCFH-DA.ELISA was performed to observe the expression of IL-1βand IL-18.RT-PCR and Western blotting were used to determine the expression of pyroptosis related Caspase-1,NLRP3and m IL-1βat gene and protein level,respectively.Bioinformatics analysis was used to find the related mi RNA,with low expression in degenerative intervertebral disc and RT-PCR was used to detected whether it was higher expressed in M2-exos.The possible binding target genes of mi R-221-3p were analyzed by bioinformatics,and their possible binding sites were predicted.Dual-luciferase reporter assay was used to verify the binding of mi R-221-3p and PTEN.RNA FISH confirmed the location of mi R-221-3p in HNPC.RT-PCR was used to verify the expression of mi R-221-3p in the HNPC added with M2-exos;Western blotting was used to detect whether mi R-221-3p inhibited PTEN/NLRP3 signal pathway.Thirdly,Extracellular matrix was extracted from bovine nucleus pulposus tissue to prepare decellularized extracellular matrix hydrogel,which was characterized by rheology and electron microscope,and the biocompatibility of hydrogel was detected by incubating with HNPC.Finally,the sustained release effect of hydrogel was detected.Fourth,the rat model of IVD degeneration was established,and the hydrogel loaded with M2-exos was injected,and the disc height and Pfirrmann classification detected by X-ray and MRI scanning respectively.The effect of histological was observed by HE staining,Safranine-O-fast green staining and Alcian blue staining.Protein expression of Col II,Caspase-1 and NLRP3 was determined by Western blotting.Results:Firstly,the CD206/CD163 double positive M2 macrophages was obtained and exosomes were extracted then.Transmission electron microscopy showed that the exosomes were round,and the particle size distribution was 30～150 nm.The results of Western blotting showed that the M2-exos expressed with CD81,TSG101 and Alix.Confocal microscope scanning showed that HNPC absorbed fluorescence-labeled M2-exos.Secondly,the immunohistochemical results showed that the expression of caspase-1 increased with the grade of nucleus pulposus degeneration.The CCK-8showed that the optimal intervention concentration of H₂O₂ was 200μM,and it was observed that M2-exos had a protective effect on cell viability,and increased with the exosomes concentration.At the same time,M2-exos could migrate HNPC.The TUNEL,Annexin V/PI and Hoechst 33342/PI staining showed that M2-exos group could significantly inhibit the pyroptosis of HNPC.The expression of IL-1βand IL-18decreased in the M2-exos group.The RT-PCR and Western blotting showed that M2-exos could inhibit the expression of Caspase-1,NLRP3 and m IL-1β.According to the bioinformatics analysis,the expression of mi R-221-3p was lower in IVDD.RT-PCR showed that compared with M0 macrophage-derived exosomes,M2-exos expressed higher mi R-221-3p.Bioinformatics analysis showed that the possible binding target of mi R-221-3p was PTEN,dual-luciferase reporter gene assay showed that mi R-221-3p could bind to PTEN.RNA FISH confirmed that mi R-221-3p was located in the cytoplasm.Coculture with M2-exos,mi R-221-3p was highly expressed.Western blotting showed that mi R-221-3p inhibited PTEN/NLRP3 signal pathway.Thirdly,the thermosensitive hydrogel of extracellular matrix of decellularized was successfully prepared and detected by rheology.The SEM showed that 1%hydrogel had higher porosity,and the Live/Dead and phalloidin staining showed good biocompatibility.After 30 days of continuous determination,it was found that the sustained release rate of hydrogel was 83.47%±2.25%.Fourth,the rat IVDD model was successfully established by needle punctured.After the injection of hydrogel loaded with/without M2-exos,X-ray and MRI scanning showed that the hydrogel loaded with M2-exos group was significantly better than other groups.HE staining,Safranine-O-fast green staining and Alcian blue staining showed that hydrogel loaded with M2-exos group was better than the others group;Western blotting showed that the expression of Caspase-1and NLRP3 decreased in the hydrogel loaded with M2-exos group.Conclusions:1.M2 macrophage-derived exosomes were obtained from CD206/CD163 double positive-M2 macrophages cellular supernatant;2.M2macrophage-derived exosomes could inhibit the pyroptosis of nucleus pulposus cells and alleviate the degeneration of nucleus pulposus.The molecular mechanism was that M2 macrophage-derived exosomes inhibits PTEN/NLRP3 pathway via transferring mi R-221-3p;3.The decellularized extracellular matrix hydrogel has good biocompatibility and could achieve sustained release of M2-exos.The injection of hydrogel and M2-exos into the rat caudal disc could delay the progress of intervertebral disc degeneration.