| 1.Background and ObjectivePeriodontitis is a multifactorial inflammatory disease that involves various local and systemic factors,from the level of pathogenic microorganisms to the state of the host immune response.Reactive oxygen species(ROS)are oxygen-containing oxidative groups with unpaired electrons.In inflamed periodontal tissue,excessive ROS is known to trigger the inflammatory pathway,destroying the organelle membrane structure and initiating cell apoptosis.To counteract such adverse effects,the Nrf2/HO-1 pathway is the key mechanism to down-regulate ROS levels,closely related to carbon monoxide and serving as an interactional by-product in heme degradation.To utilize the anti-oxidative and anti-inflammatory effects of carbon monoxide,a novel carbon monoxide release molecule,TG-FeCORM,is to be employed in this experiment.TGFeCORM is a metal carbonyl complex composed of a transition metal element core and a carbon monoxide ligand,which is characterized by remarkable biosafety,simple synthesis,rich modification structure,and theoretically has better solubility and controlled release characteristics.The primary objective of this research is to investigate the therapeutic effect of the novel carbon monoxide release molecule.Therefore,the releasing features of TG-FeCORM are to be analyzed in the foremost part.And TG-FeCORM will be introduced in an in-vitro inflammatory HGFs cell culture induced by Porphyromonas gingivalis sonication extract to validify the antioxidative and anti-inflammatory effects of TG-FeCORM.Then,the experimental rat periodontal disease model is to be used to demonstrate the in vivo anti-inflammatory properties of this novel molecule.With the expectation to contribute to balancing the redox state in inflamed periodontal tissue and promoting periodontal lesion healing,this research on TGFeCORM is designed to deepen the understanding of the convoluted mechanism between carbon monoxide and inflammation in periodontal tissue and explore the application of novel carbon monoxide molecule.2.Material and MethodPart Ⅰ In vitro CO release characteristics of TG-FeCORMThe deoxy-myoglobin carbonylation method was used to verify the CO release of TGFeCORM,and the degradation rate of TG-FeCORM in PBS solution was measured by ultraviolet-visible spectrophotometry to evaluate the rate of release in vitro.Part Ⅱ Effect of TG-FeCORM on reactive oxygen species levels and inflammatory factors in human gingival fibroblasts induced by P.gingivalis sonication extractPorphyromonas gingivalis sonication extract(PgSE)was used to induce inflammation in primary human gingival fibroblasts(HGFs,passage 3 to 6)for an inflammatory cell model.The cell-counting kit-8(CCK8)was used to assess cell viability of HGFs under 25,50,100,200,400 μM TG-FeCORM or 0.1,1,10 μg/mL PgSE respectively,and samples were collected after 12 h,24 h and 48 h.DCFH-DA and Amplex Red were used to detect the intracellular and extracellular ROS level respectively.Firstly,the effect of PgSE on the total reactive oxygen species of HGFs was studied.The blank control,0.1,1,and 10 μg/mL of PgSE treatment groups were set up,and the test was performed at 3,6,and 9 hours after PgSE treatment initiated.Then,the effect of TGFeCORM on the ROS level in PgSE stimulated HGFs live cells/culture supernatant was studied.The blank control group,PgSE group,and PgSE+TG-FeCORM group(concentrations at 25 and 50 μM respectively)were set up and treated with PgSE for 3 h.Enzyme-linked immunosorbent assay(ELISA)and quantitative Real-time polymerase chain reaction(qPCR)methods were used to detect the levels of inflammatory cytokines.Blank control group,PgSE stimulation group,and PgSE+TG-FeCORM group(concentrations of 10,25,50,and 100 μM)were set up,and the expression levels of IL-6,IL-8,and IL-1β were studied in ELISA test,and IL-6,IL-8 cytokines were studied in qPCR test.Part Ⅲ Effect of TG-FeCORM on the expression of Nrf2/HO-1 and NF-κB pathwayrelated factors in human gingival fibroblasts induced by P.gingivalis sonication extractWestern Blot and qPCR were used to detect the expression levels of the antioxidant pathway Nrf2/HO-l and the inflammatory pathway NF-κB related factors.Blank control group,PgSE stimulation group,PgSE+inactivated TG-FeCORM group(iTG-FeCORM,25 μM)and TG-FeCROM group(25 μM,50 μM)were set up.Firstly,the expression of Nrf2,HO-1,SOD1 and Catalase at the protein and mRNA levels were studied.Subsequently,the protein and mRNA expression levels of p65 and IκB subunits,and the phosphorylation ratios of p65 and IκB were studied.Part IV In vivo effect of TG-FeCORM on periodontal tissue inflammatory response and bone destruction in experimental rat periodontitis modelThe effect of TG-FeCORM on periodontal tissue destruction and remodeling was studied on experimental rat periodontitis model.Control group,experimental periodontitis group,and TG-FeCORM treatment group were set up.Experimental rat periodontitis model was established by ligation of the maxillary first molar,and TG-FeCORM carboxymethylcellulose paste was applied locally.Animals were euthanized 4 weeks after ligature.HE and Masson staining were performed after neutral EDTA decalcification and embedding process.3.ResultsPart Ⅰ In vitro CO release characteristics of TG-FeCORMDeoxy-myoglobin carbonylation results indicated TG-FeCORM has the characteristic of releasing CO.The UV-Vis spectrophotometry results showed that the degradation half-life of TG-FeCORM was approximately 50 min,which is a significantly longer half-life of TGFeCORM than CORM-3 molecule(approximately 10 min).Part Ⅱ Effect of TG-FeCORM on reactive oxygen species levels and inflammatory factors in human gingival fibroblasts induced by P.gingivalis sonication extractCCK-8 results showed that TG-FeCORM at 25,50 and 100 μM had no significant effect on the viability of HGFs after 12 h,24 h and 48 h incubation.And 0.1 μg/mL of PgSE had no significant effect on the cell viability of HGFs after 12 h and 24 h of incubation.The ROS assay by DCFH-DA and Amplex Red showed that 0.1 μg/mL,I μg/mL and 10 μg/mL of PgSE could significantly up-regulate the total intracellular ROS levels of HGFs at 3 h and 6 h,which is reversed by 25,50 μM of TG-FeCORM.ELISA results showed that the cytokine concentrations of IL-6 and IL-8 in supernatant significantly increased after PgSE stimulation,which is reversed by TG-FeCORM at 25,50 and 100 μM.The qPCR results showed concomitant effects in the level of IL-6,IL-8 and IL-1β.Part III Effect of TG-FeCORM on the expression of Nrf2/HO-1 and NF-κB pathwayrelated factors in human gingival fibroblasts induced by P.gingivalis sonication extractWestern blot results showed that for 25,50 μM TG-FeCORM treatment group,the protein expression of Nrf2 and HO-1 enhanced significantly compared to PgSE stimulation group,with the elevated expression of Nrf2 in the nucleus.Also,TG-FeCORM treatment significantly down-regulated the phosphorylation levels of p65,IL-1β and IκB compared to PgSE stimulation group.qPCR results showed that 25,50 μM TG-FeCORM treatment significantly elevated the mRNA expressions of Nrf2,HO-1,SOD1,Catalase and inhibited the expression of p65 subunit compared to the PgSE stimulation group,.Part IV In vivo effect of TG-FeCORM on periodontal tissue inflammatory response and bone destruction in experimental rat periodontitis modelHE and Masson staining results showed that TG-FeCORM treatment significantly attenuated tissue destruction in experimental rat periodontitis model,indicated by reduced attachment loss,reduced inflammatory cell infiltration in connective tissue,reduced collagen degeneration and alveolar bone resorption.4.ConclusionThe results showed that TG-FeCORM inhibited the increase of reactive oxygen species in HGFs stimulated by PgSE with the elevation of the expression of Nrf2/HO-1 pathway and inhibition of the expression of NF-κB signaling pathway and downstream inflammatory factors.TG-FeCORM can effectively alleviate the periodontal lesions in experimental rat periodontitis model.The existing results provide a theoretical basis for the application of this new CORM molecule in the periodontal direction. |
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