| Background:Peri-implantitis is a plaque related pathological condition characterized by peri-implant mucosal inflammation and progressive loss of supporting bone tissue,which is the most common cause of implant failure.The anatomical defect of the peri-implant area,which lacks the periodontium and the Sabi fibers found in natural teeth,allows for a more rapid development of inflammation.The large-scale production of destructive inflammatory mediators disrupts the immune response,prolongs inflammation,exacerbates tissue loss and ultimately leads to implant failure.omega-3 polyunsaturated fatty acids(ω-3 PUFAs),an essential fatty acid found in cod liver oil and seaweed,play an anti-inflammatory role in a variety of inflammatory diseases.However,their properties such as highly oxidative inactivation and insolubility in water greatly limit their application.Objective:A novel nanostructured lipid carrier(NLC)loaded with docosahexaenoic acid(DHA)was investigated to address the limitations of DHA application and to investigate its anti-inflammatory effect and mechanism on peri-implantitis in rats,in order to provide an objective theoretical basis for the effective prevention and treatment of peri-implantitis and periodontitis in clinical practice.Methods:The experiments were divided into 5 groups: blank control group(Control),inflammation group(LPS),inflammation + blank NLC group(NLC),inflammation +DHA group(DHA),inflammation + DHA-NLC group(DHA-NLC).1.Culture RAW264.7(macrophages)and detect the cytotoxicity of NLC,DHA and DHA-NLC on macrophages by CCK8 assay,respectively.2.A macrophage inflammation model was established by P.gingivalis-LPS induction,and Western blot assay was used to detect pro-inflammatory factors such as IL-1β,IL-6,TNF-α and the protein of P-NF-κB p65,NF-κB p65,P-IκB,IκB in P.gingivalis-LPS-induced macrophages expression levels.3.Immunofluorescence staining was used to observe the effects of NLC,DHA and DHA-NLC groups on the nuclear translocation of NF-κB p65 in macrophages.4.In Wistar rats,micro implants were placed in front of the first molar on the left side of the maxilla.One month later,except for the Control group,the rest of the groups used 4-0 silk ligation combined with P.gingivalis-LPS injection method to establish a rat peri-implantitis model.Later,four sites were selected on the buccal-palatal side of the implant,near and far from the middle,and the same dose of drugs(NLC,DHA,DHA-NLC)was injected locally every three days in the corresponding groups.After two weeks,the intraoral condition of the rats was observed and recorded,and animal specimens were collected.The inflammation status of peri-implant tissues was observed by tissue sectioning,m RNA expression of inflammation-related factors(IL-1β,IL-6,TNF-α)in peri-implant gingival tissues was detected by q PCR,and the establishment of experimental peri-implantitis model and alveolar bone resorption of target teeth in rats was observed by Micro-CT,and finally the differences between groups were statistically analyzed.5.The body weight of the rats was weighed and recorded weekly during the animal experiments.Blood samples of rats were collected for routine blood and blood biochemical analysis at the end of the experiment,while the main internal organs of rats were analyzed histopathologically to evaluate the drug toxicity of NLC,DHA and DHA-NLC.Results:1.CCK-8 assay showed that the NLC and DHA groups had no significant effect on the survival rate of macrophages compared to the Control group at concentrations of 12.5,25,50 and 100 μmol,while the DHA-NLC group showed a pro-proliferative effect.That is,all experimental groups had no significant toxic effects on macrophages at drug concentrations ≤100 μmol,which was much greater than the final drug concentration of 50 μmol used in this experiment.2.After pretreatment of macrophages with 5 μg/m L of P.gingivalis-LPS for 12 h,the corresponding drugs were given according to the group.Western blot results showed that the expression of inflammatory factors IL-1β,IL-6 and TNF-α protein decreased significantly in the cells of DHA-NLC group,suggesting that DHA-NLC had a better inhibitory effect on inflammation.3.Western blot results showed that DHA-NLC better inhibited P.gingivalis-LPS mediated phosphorylation of IκBα and NF-κB p65 compared with other experimental groups,thus inhibiting the activation of NF-κB inflammatory pathway,suppressing macrophage inflammatory factor expression and improving the inflammatory microenvironment.4.Immunofluorescence results further confirmed that DHA-NLC was able to prevent NF-κB p65 from entering the nucleus and reduce the level of NF-κB p65 in the nucleus compared to other experimental groups.5.Gross animal observation showed that the gingiva around the implants in the LPS and NLC groups was dark red,with obvious mucosal swelling,visible food debris accumulation,local probing bleeding,and spontaneous bleeding or pus overflow in individual sites,suggesting that the gingival tissues around the implants were in an obvious inflammatory state;the gingiva in the DHA-NLC group was bright red,and no obvious swelling and bleeding were found,which was not significantly different from the Control group.H&E staining of the tissue sections showed the most obvious peri-implant inflammatory infiltration in the LPS group,with a large number of lymphocytes and plasma cells with darkly stained nuclei and disorganized fibrous connective tissue,showing a typical peri-implant inflammatory state.Inflammatory tissue infiltration was rarely observed in the drug treatment group only in the DHA-NLC group compared to the Control group.The results of q PCR experiments with peri-implant gingival tissues showed that the expression of inflammatory factors was lower in DHA-NLC compared to DHA and NLC alone,and the difference was statistically significant.Micro-CT results showed increased bone resorption in the LPS group compared to the Control group,further confirming the successful construction of the rat peri-implantitis model;Meanwhile,the BV/TV,Tb.N and bone resorption in the DHA-NLC group were not significantly different from those in the Control group,and there was less alveolar bone resorption compared with the NLC group and the DHA alone group.6.Except for the initial period of implant placement,the rats in the experimental group showed a steady increase in body weight for the rest of the time.Histopathological analysis of the blood and major organs of the rats showed that the drugs used in this experiment were not toxic to the rats themselves.Conclusion:1.A combined squalene and DHA-loaded DHA-NLC was successfully prepared by ultrasonic emulsification.2.DHA-NLC inhibited P.gingivalis-LPS-mediated macrophage inflammation by slow release of DHA.3.DHA-NLC inhibits P.gingivalis-LPS-mediated phosphorylation of IκBα,NF-κB p65 and prevents NF-κB p65 from translocating to the nucleus,thereby inhibiting activation of the NF-κB inflammatory pathway.4.DHA-NLC accumulated at high concentrations in the periodontal site by local injection and was slowly released,thus exerting good anti-inflammatory and osteoprotective effects in peri-implantitis in rats. |
PDF Full Text Request