Down-regulation Of Microtubule Acetylation Via αTAT-1 By Nb₂C Nanosheets In The Treatment Of Rat Periodontitis

Periodontitis is a chronic inflammatory disease of the periodontal support tissues caused by bacterial biofilm,and it is one of the main causes of tooth loss in adults.The occurrence,progression,and prognosis of periodontitis vary significantly among individuals,indicating the significant role of host immune response in the disease.Innate immunity is an integral part of the host immune system and serves as the first line of defense against microbial infections.When the homeostasis of the periodontal microbiota is disrupted,macrophages can recognize pathogenic substances,amplify inflammatory signals,and activate the host innate immune system,leading to soft and hard tissue damage in the periodontium.Therefore,regulating macrophage inflammatory responses and inhibiting excessive activation of the host innate immune system have become hot topics in periodontitis-related research.The NOD-like receptor protein 3（NLRP3）inflammasome is a central component of the inflammatory response.It can rapidly upregulate macrophage inflammation by activating a large amount of IL-1β,serving as a crucial"signal amplifier"in periodontitis.The activation of the NLRP3inflammasome relies on microtubule acetylation mediated byα-tubulin acetyltransferase 1（αTAT-1）.Studies have shown that hydrophilic two-dimensional materials can interfere with microtubule assembly and modification through their unique physicochemical properties.Nb₂C nanosheets,composed of two layers of niobium and one layer of carbon,with hydroxyl-terminated end groups,exhibit high hydrophilicity and biocompatibility.Therefore,in this study,focusing on microtubule acetylation as a key regulatory target,we hypothesize that Nb₂C nanosheets can downregulate microtubule acetylation levels throughαTAT-1,thereby treating periodontitis,and explore their underlying mechanisms.The specific research contents are as follows:In chapter 2,we investigated the correlation between microtubule acetylation mediated byαTAT-1 and periodontitis.Firstly,gingival samples were collected from patients with periodontitis and healthy volunteers.Immunohistochemical methods were employed to observe the expression ofαTAT-1,acetylated microtubule protein（Lsy 40α-tubulin）,and NLRP3 in the gingival tissues of periodontitis patients.Subsequently,a rat model of periodontitis was established using the silk ligature method,and immunohistochemical methods were used to observe the expression changes ofαTAT-1,Lsy 40α-tubulin,and NLRP3 in the gingival tissues of rats.The results revealed a significant increase in the expression ofαTAT-1,Lsy 40α-tubulin,and NLRP3 in both clinical periodontitis samples and rat periodontal tissues,indicating a strong correlation between microtubule acetylation mediated byαTAT-1 and periodontitis.In chapter 3,we investigated the response of microtubule acetylation mediated byαTAT-1 in macrophages upon LPS stimulation.Firstly,we stimulated macrophages with LPS and observed the changes in the gene expression ofαTAT-1,α-tubulin,and NLRP3using RT-q PCR.Additionally,we examined the protein-level expression changes ofαTAT-1,Lsy 40α-tubulin,and NLRP3 through Western blot analysis.Furthermore,we utilized si RNA technology to knock downαTAT-1 and evaluated its regulatory effect on the expression of Lsy 40α-tubulin and NLRP3 using RT-q PCR and Western blot.The results demonstrated a significant increase in the expression ofαTAT-1,Lsy 40α-tubulin,and NLRP3 in macrophages stimulated with LPS.Moreover,knocking downαTAT-1 significantly inhibited the expression of Lsy 40α-tubulin and NLRP3.Therefore,there is a strong correlation between the level of microtubule acetylation mediated byαTAT-1 and the inflammatory response in macrophages upon LPS stimulation.In chapter 4,we evaluated the inhibitory effect of Nb₂C nanosheets on macrophage inflammation.Firstly,Nb₂C nanosheets were synthesized using an acid etching method,and their physicochemical characterization and biocompatibility were assessed.The regulatory effects of Nb₂C nanosheets on macrophage polarization and migration were evaluated using flow cytometry,immunofluorescence staining,and the Transwell assay.The regulatory effects of Nb₂C nanosheets on the expression of NLRP3,caspase-1,and IL-1βwere assessed using RT-q PCR,Western blot,and immunofluorescence staining.The results demonstrated the successful synthesis of Nb₂C nanosheets,and at concentrations of 10μm/ml and below,no significant cytotoxicity or toxicity to major organs was observed.Flow cytometry and immunofluorescence staining results revealed that Nb₂C nanosheets promoted M2 polarization of macrophages.The Transwell assay results indicated that Nb₂C nanosheets significantly inhibited macrophage migration.RT-q PCR,Western blot,and immunofluorescence staining results showed that Nb₂C nanosheets significantly downregulated the expression of NLRP3,caspase-1,and IL-1β.Therefore,Nb₂C nanosheets possess an inhibitory effect on macrophage inflammation stimulated by LPS.In chapter 5,we investigated the mechanism underlying the inhibitory effect of Nb₂C nanosheets on macrophage inflammation.The regulatory effects of Nb₂C nanosheets on the expression ofαTAT-1 and Lsy 40α-tubulin were assessed using RT-q PCR,Western blot,and immunofluorescence staining.By employing si RNA technology to knock downαTAT-1,the protein expression ofαTAT-1 and Lsy 40α-tubulin was detected using RT-q PCR and Western blot,aiming to evaluate the relationship betweenαTAT-1 and the regulatory effects of Nb₂C nanosheets on the expression ofαTAT-1 and Lsy 40α-tubulin.The molecular biology results indicated that Nb₂C nanosheets significantly inhibited the protein expression ofαTAT-1 and Lsy40α-tubulin.Immunofluorescence staining revealed a significant downregulation of microtubule acetylation levels in macrophages treated with Nb₂C nanosheets.Molecular biology results showed that knocking downαTAT-1 significantly inhibited the regulatory effects of Nb₂C nanosheets on the expression ofαTAT-1 and Lsy 40α-tubulin.Therefore,Nb₂C nanosheets exert their inhibitory effect on macrophage inflammation by downregulating microtubule acetylation levels throughαTAT-1.In chapter 6,we evaluated the therapeutic effect of Nb₂C nanosheets on rat periodontitis.The rat periodontitis model was established using the silk ligature method.Immunohistochemistry,RT-q PCR,and Western blot methods were employed to detect the expression changes of NLRP3,caspase-1,and IL-1β.RT-q PCR,Western blot,immunohistochemistry,and immunofluorescence staining methods were used to assess the expression changes ofαTAT-1 and Lsy 40α-tubulin proteins.The results revealed that Nb₂C nanosheets significantly inhibited the m RNA and protein expression of NLRP3,caspase-1,and IL-1βin the gingival tissue.RT-q PCR,Western blot,and immunohistochemistry results showed that Nb₂C nanosheets significantly suppressed the expression ofαTAT-1 and Lsy 40α-tubulin in the gingival tissue.Immunofluorescence staining results demonstrated a significant downregulation of microtubule acetylation levels in the gingival tissue upon treatment with Nb₂C nanosheets.Immunofluorescence staining results demonstrated a significant reduction in gingival microtubule acetylation levels upon treatment with Nb₂C nanosheets.These findings highlight the remarkable therapeutic effect of Nb₂C nanosheets on rat periodontitis.In summary,Nb₂C nanosheets can effectively treat experimental periodontitis in rats by downregulating the microtubule acetylation levels throughαTAT-1,thereby inhibiting macrophage inflammation.