Study On Exosome/Peptide Hydrogel Scaffold For Relieving Endometritis And Repairing Uterine Damage

Objective: Endometritis is a common disease in gynaecology.Long-term repeated infection of endometritis will cause damage to the endometritis and the internal environment,which in severe cases will cause myositis,and is also the most common cause of abortion and infertility,so attention should be paid to the treatment of endometritis.At present,the clinical treatment of endometritis includes oral antibiotics and intrauterine administration,but the therapeutic effect is not ideal.Exosomes derived from mesenchymal stem cells have been widely used as an effective treatment for inflammatory diseases.However,it is difficult for exosomes to achieve accurate longterm delivery at target locations due to their easy aggregation in organs and short halflife.Therefore,using bioactive scaffolds to deliver exosomes is an effective means to help exosomes play their role.Based on the above requirements and analysis,this paper intends to construct self-assembled polypeptide hydrogels for loading exosomes to achieve in situ and controlled release of exosomes,enhance the therapeutic ability of exosomes through pre-activation or engineering,construct exosome/polypeptide composite hydrogel scaffolds,evaluate the therapeutic effect of scaffolds on endometritis,and explore the therapeutic mechanism.It lays a foundation for further clinical application.Methods:(1)The polypeptide hydrogel scaffolds were prepared and characterized by rheological detection.The biosafety of the scaffolds was evaluated by live staining.MSCderived exosomes were extracted by step-by-step centrifugal method,and exosomes were mixed with peptide solution to prepare exosome/peptide composite hydrogel scaffolds exo@pep.Rheological characteristics,degradability,injectable and self-healing properties of the composite hydrogels were tested,and the biosafety of the composite hydrogels was tested by CCK-8 experiment.The function of the compound hydrogels was evaluated by the proliferation of the cells.(2)MSCs were stimulated by the inflammatory cytokine IL-1β to produce exosomes with enhanced function,and the exosomes were mixed with peptide solution to prepare composite hydrogel scaffolds.At the cellular level,q RT-PCR assay,ELISA assay,scratch assay,invasion assay and tubularization assay were used to evaluate the anti-inflammatory ability,migration promoting ability and angiogenesis effect of hydrogel scaffolds.In animal experiments,rat models of endometritis were established to evaluate the therapeutic effect of hydrogel scaffolds on endometritis,and the therapeutic effect was further verified by HE staining,immunofluorescence assay and q RT-PCR assay.The effect of hydrogel scaffolds on HMGB1/NF-κB signaling pathway was investigated by western blot experiment to study the mechanism of action of hydrogel scaffolds.(3)The engineering exosomes loaded with mi R-21-5p were constructed by electroporation method and mixed with polypeptide solution to prepare composite hydrogel scaffolds.The anti-inflammatory,proliferative,migratory and angiogenic effects of scaffolds were studied at the cellular and animal levels using the methods described above.The effect of hydrogel stent on YAP/JNK signal was studied by western blot experiment to explore the therapeutic mechanism.(4)The composition and content of mi RNAs in the exosomes of human umbilical cord mesenchymal stem cells(UCMSCs)activated by IL-1β were analyzed by the secondgeneration sequencing,and the mi RNAs with the most significant differences in expression were statistically identified.The potential signaling pathways were identified by GO enrichment and KEGG analysis of the target genes of different mi RNAs.Results: The composite hydrogel scaffold loaded with exosomes is in the gel state,has injectable,self-healing and degradability,and can release exosomes.CCK-8experiment showed that the hydrogel scaffold can promote cell proliferation.The composite hydrogel scaffold loaded with IL-1β-activated exosomes can inhibit inflammation,promote cell migration and angiogenesis in vitro,and play a better therapeutic effect on endometritis rats compared with other treatment groups,and inhibit the expression of inflammatory factors in endometritis,and promote tissue repair.The therapeutic function may be realized by inhibiting the HMGB1/NF-κB signaling pathway;Composite hydrogels containing mi R-21-5p engineered exosomes can also play antiinflammatory,cell proliferation and migration,and angiogenesis effects in vivo and in vitro,possibly through the effect of YAP/JNK signaling.Sequencing analysis showed that IL-1β can affect the composition and content of mi RNA in exosomes,which may be the key factor for the enhanced function of exosomes.Conclusions: In this study,polypeptide hydrogel scaffolds were prepared to deliver exosomes for in-situ and long-term release,and engineering exosomes loaded with mi R-21-5p were prepared by pre-activation of inflammatory cytokine IL-1β or electroporation to enhance the function of exosomes.The exosomes/hydrogel composite scaffolds were prepared by mixing these enhanced exosomes with peptide solutions.Experiments at the cell level and animal level have proved that the composite scaffold has stronger antiinflammatory and pro-repair effects,and can promote the generation of blood vessels in vivo and in vitro.Further,through the exploration of the molecular mechanism,the inflammatory composite scaffold can inhibit inflammation by regulating the HMGB1/NF-κB signaling pathway.Moreover,by promoting YAP expression,the downstream signal is regulated to promote cell proliferation and migration,and promote tissue repair anti-inflammatory and repair molecular mechanisms.In addition,the effect of IL-1β on the activation of mi RNA expression in exosomes was demonstrated by second-generation sequencing.The research of this subject is expected to provide a new method for the treatment of endometritis and infertility caused by endometritis,which has certain basic research significance and clinical application value.