| Periodontitis is one of the most prevalent chronic diseases worldwide.If left untreated,it will lead to tooth loss and severely affects masticatory function,esthetics and the life quality of patients.Periodontitis is caused by bacterial infection that also maintains host in an inflammatory state.Therefore,current treatment strategies for periodontitis mainly involve mechanical scaling or root planing.However,scaling or root planing alone has unsatisfactory long-term effects and is easy to replase.Thus,researchers realize that bacterial infection alone may not be sufficient to explain the entire pathogenesis of periodontitis,especially plaque biofilm cannot directly destroy periodontal tissues.What destroys the hard and soft tissue is the the dysregulation of host immune response.After the balance between microorganisms and the host is broken,the destructive immune response begins with infiltration of immune cells,and activation of osteoclast.In the wake of the vicious circle,the destruction of periodontal tissues eventually occurs.In this context,inflammation and host response play a crucial role in the pathogenesis of periodontitis,and the regulation of excessive host immune-inflammatory responses is the key to treating periodontitis.Thus,the treatment strategy for periodontitis has gradually shifted from anti-infective therapy to host modulation therapy(HMT).HMT involves in modulating inflammation,immune response,the production of cytokines,chemokines,or reactive oxygen species(ROS).The balance of pro-inflammation and anti-inflammation will recover in this way.However,there are challenges and an urgent need for the development of HMT drugs to solve the safety and effectiveness of long-term application.In recent years,immunomodulatory nanosystems(IMNs)have developed rapidly in the treatment of inflammatory diseases.The concept of IMNs for treating inflammatory diseases is the same as HMT drugs in periodontitis.Among numerous nanomaterials,carbon dots(CDots)stand out for their extraordinary properties,especially in the field of nanomedicine where they are commonly used to solve the problems of traditional drugs for their poor targeting,low biocompatibility and side effects.It inspired us to develop immunomodulatory CDots to solve the current dilemma of poor safety and efficacy of HMT drugs.In the second chapter,the CDots were successfully synthesized from condensation and carbonization of lipoic acid and polyethyleneimine by hydrothermal method,after which the as-prepared CDots were purified,lyophilized and formulated into solutions for characterization and evaluation of in vitro biocompatibility.The luminescent properties of the as-prepared CDots were obtained by UV-Vis spectrophotometer and fluorescence spectrometer.And it was found that the as-prepared CDots solution exhibits blue photoluminescence(PL)under UV light.The high-resolution transmission electron microscope was carried out to obtain the lattice structure of the as-prepared CDots,which was proved clear and uniform in size with an average diameter of 4.5 nm.Fourier transform infrared and X-ray photoelectron spectroscopy characterizations revealed that the as-prepared CDots are composed of C,N,O and S,and have abundant amino,carboxyl and sulfhydryl groups.Besides,the zeta potential of the CDots in aqueous solution was measured to be 15.7 m V.Moreover,the biological safety of the as-prepared CDots was evaluated by CCK-8,flow cytometry and live-dead cell staining.And the results showed that the morphology,activity,proliferation and apoptosis of RAW 264.7 cells were not significantly affected by the as-prepared CDots at concentrations below 80 μg/m L.In addition,the blue PL emission of CDots was utilized for tracing the distribution of CDots by confocal laser scanning microscopy.And it was found that the as-prepared CDots can be internalized into the cells.In the third chapter,the effect of the as-prepared CDots on pro-inflammatory cytokine was evaluated in an inflammatory model of RAW 264.7 cells.RAW 264.7cells were induced with Porphyromonas gingivalis-derived lipopolysaccharide(Pg-LPS)to establish an in vitro inflammatory cell model.After the addition of the as-prepared CDots,the expression of pro-inflammatory cytokine genes was significantly down-regulated in a dose-dependent manner which was detected by quantitive PCR.And the protein synthesis of pro-inflammatory cytokines was significantly suppressed in a dose-dependent manner which was proved by Western blot.Moreover,the secretion of pro-inflammatory cytokines dose-dependently decreased after being treated with the CDots which was investigated by enzyme-linked immunosorbent assays.Finally,RNA sequencing of cell samples was performed by high-throughput sequencing technology to enrich for differential genes,and a large number of inflammation-related pro-inflammatory cytokines and chemokines were enriched and exhibited down-regulation in the inflammatory state of RAW 264.7 cell samples after CDots treatment.In the fourth chapter,we explored the mechanisms underlying the suppression effect of the as-prepared CDots on pro-inflammatory cytokines by RNA sequencing.The differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,and revealed key nodes located on the same signaling axis,MYC and mTOR signaling pathway.The expression of MYC was significantly up-regulated in the inflammatory cell model,and down-regulated after treatment of the as-prepared CDots,which was also further confirmed by q PCR and Western blot.Moreover,after the addition of MYC gene overexpression vector,the as-prepared CDots lost the inhibitory effect on the expression of pro-inflammatory cytokines genes,demonstrating that the CDots inhibited the expression of pro-inflammatory cytokines by suppressing the expression of MYC.Furthermore,we examined the phosphorylation level of the mTOR signaling pathway.The results showed that CDots inhibited the activation of the mTOR signaling pathway,and the inhibitory effect was lost after the addition of the mTOR agonist MHY1485,suggesting that the as-prepared CDots can inhibit the expression of MYC and thus pro-inflammatory cytokines by suppressing the phosphorylation level of mTOR.In the fifth chapter,we evaluated the in vivo therapeutic effects and biosafety of the as-prepared CDots on periodontitis in an animal model of periodontitis.A rat periodontitis model was established by inserting the maxillary left second molar of rats with silk sutures.The CDots solution was administered in the periodontal pocket.Subsequently,the alveolar bones of the rats were imaged by Micro-CT and analyzed quantitatively.The results indicated that the distance between the cemento-enamal junction and the apex of the alveolar bone crest,and trabecular separation significantly declined,trabecular number and bone volume/tissue volume significantly increased after the treatment of CDots.The inflammatory status of periodontal tissues was assessed by hematoxylin and eosin staining(HE staining),Masson staining and immunohistochemical staining.The results exhibited that the characteristic pathological changes of periodontitis were effectively alleviated,suggesting that CDots have obvious periodontitis treatment effects.Last,to investigate whether the CDots cause side effects in vivo,HE staining of important organs was performed.The results demonstrate that there is no histological abnormity in heart,liver,spleen and kidney of the CDots-treated group,indicating that CDots are safe for in vivo applications.In summary,we synthesized CDots via a one-pot hydrothermal treatment of lipoic acid and polyethyleneimine.The obtained CDots possessed blue photoluminescence,abundant functional groups and excellent biocompatibility.Through transcriptome analysis,Western blot assay,it was demonstrated that our CDots could suppress pro-inflammatory cytokines through the mTOR/MYC signaling axis for the treatment of periodontitis. |
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