Use of curcumin compounds in the preparation of a medicament for the treatment of periodontitis

Abstract

The application relates to the field of biological medicine, and particularly relates to application of a curcumin compound in preparation of a medicine for treating periodontitis, and comprises the following steps: a mono-carbonyl curcumin analogue with a structure shown in formula (I) is used for preparing the medicine for treating periodontitis, and the compound is obtained by replacing a beta-diketone group of natural curcumin with a mono-carbonyl connecting group. The compound provided by the application has a chemical stability which is significantly better than that of natural curcumin, and solves the problems of easy degradation and low bioavailability; meanwhile, the compound has excellent broad-spectrum anti-periodontal pathogenic bacteria activity and immune regulation capacity, can significantly promote M2 phenotype polarization of macrophages and inhibit generation of key pro-inflammatory cytokines, realizes double effects of antibiosis and immune regulation, and provides a new, efficient and low-toxicity auxiliary treatment medicine for periodontitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a curcumin-like compound in the preparation of a drug for treating periodontitis. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by dental plaque biofilm, characterized by progressive destruction of periodontal supporting tissues (including periodontal ligaments and alveolar bone), ultimately leading to tooth loss. It is one of the most common chronic diseases among adults worldwide. Currently, basic periodontal treatments (such as scaling and root planing) are the core mechanical means of removing supragingival and subgingival plaque and calculus. However, due to the complex anatomy of periodontal pockets, the difficulty in thoroughly debridement of furcation lesions, and the resistance of subgingival biofilm to mechanical and chemical methods, mechanical treatment alone often fails to achieve long-term, ideal clinical efficacy, resulting in a high relapse rate. Therefore, adjuvant drug therapy, as an important means of controlling infection, regulating the host inflammatory response, and promoting tissue repair, plays an indispensable role in the comprehensive management of periodontitis.

[0003] Systemic or local application of antibiotics (such as tetracyclines and metronidazole) is a common adjunctive treatment strategy. However, long-term or inappropriate use can easily lead to bacterial resistance, oral flora imbalance, and potential systemic adverse reactions. Therefore, compounds derived from natural products, due to their multi-target and low-toxicity characteristics, have become an important research direction for developing novel adjunctive drugs for periodontitis. Curcumin is the main polyphenolic active ingredient extracted from turmeric. Numerous in vitro and animal model studies have shown that it not only inhibits key periodontal pathogens such as *Porphyromonas gingivalis*, but also exerts anti-inflammatory and antioxidant effects by regulating signaling pathways such as Nrf2 and NF-κB, demonstrating the potential for a dual "antibacterial-anti-inflammatory" effect. However, the β-diketone group in the molecular structure of natural curcumin makes it chemically unstable under physiological pH and body fluid conditions, easily undergoing rapid degradation and metabolism, resulting in extremely low bioavailability after oral and local administration. This inherent pharmacokinetic defect seriously hinders its clinical translation and application.

[0004] To overcome the stability bottleneck of natural curcumin, existing technologies mainly explore two strategies: one is to encapsulate it using nanomedicine delivery systems (such as solid lipid nanoparticles, polymer micelles, etc.) to improve its stability and targeting; the other is to directly modify its chemical structure to obtain derivatives or analogs with enhanced stability. Among the structural modification strategies, transforming the unstable β-diketone structure into a monocarbonyl structure is one of the most promising approaches. The obtained monocarbonyl curcumin analogs have shown improved stability and certain antibacterial and anti-inflammatory activities in general pharmacological activity screening. However, the pathogenesis of periodontitis involves a complex interaction between subgingival specific pathogenic bacteria and the host immune system (especially the polarization state of immune cells such as macrophages and cytokine storms). Currently, there are no clear research reports or successful technical solutions regarding whether a chemically stable curcumin-like compound can be obtained through structural modification, which not only maintains its highly effective inhibitory effect on the characteristic pathogenic bacteria of periodontitis, but also precisely regulates the imbalanced immune microenvironment of periodontitis (e.g., effectively promoting macrophage polarization towards an anti-inflammatory repair phenotype and strongly inhibiting the production of key pro-inflammatory mediators), thereby synergistically addressing the dual challenges of the microbial etiology and host inflammatory response of periodontitis. Summary of the Invention

[0005] Therefore, it is necessary to address the technical problems of poor chemical stability and low bioavailability of natural curcumin in existing technologies, as well as the inability of existing adjuvant periodontitis treatment drugs to effectively combine antibacterial and immunomodulatory effects and to synergistically address the dual challenges of microbial etiology and host inflammatory response in periodontitis. By applying a curcumin-like compound in the preparation of drugs for treating periodontitis, this study has achieved excellent chemical stability, broad-spectrum anti-periodontal pathogenic activity, and efficient immunomodulatory capabilities of the compound in the treatment of periodontitis, providing a novel, efficient, and low-toxicity adjuvant treatment candidate drug for periodontitis.

[0006] This invention provides the application of a curcumin-like compound in the preparation of a medicament for treating periodontitis, wherein the curcumin-like compound is a monocarbonyl curcumin analog having the structure shown in formula (I): (I) Compound (I) is obtained by replacing the β-diketone group of natural curcumin with a monocarbonyl linker.

[0007] In one embodiment, the monocarbonyl curcumin analog having the structure shown in formula (I) is compound L42H17.

[0008] In one embodiment, the application includes inhibiting the growth of one or more periodontal pathogens selected from Porphyromonas gingivalis, Forsythosinus, Actinobacillus actinomycetii, and Fusobacterium nucleatum.

[0009] In one embodiment, the compound exhibits dose-dependent inhibition of the growth of the periodontal pathogens at concentrations of 5 μM to 20 μM, and shows inhibitory activity after 6 hours of treatment.

[0010] In one embodiment, the application includes promoting macrophage surface polarization to M2 and inhibiting the production of pro-inflammatory cytokines.

[0011] In one embodiment, promoting macrophage polarization toward the M2 phenotype includes upregulating the expression of M2 phenotype markers CD206, CD163 and / or MRC1, and / or downregulating the expression of M1 phenotype markers CD80 and / or CD68.

[0012] In one embodiment, inhibiting the production of pro-inflammatory cytokines includes inhibiting one or more cytokines selected from tumor necrosis factor (TNF), interleukin-1β (IL-1β), interleukin-6 (IL-6), and CXCL8 at the transcriptional and / or protein secretion levels.

[0013] In one embodiment, the compound inhibits the production of the pro-inflammatory cytokines by inhibiting the NF-κB signaling pathway.

[0014] In one embodiment, the macrophages are periodontal inflammation-associated macrophages induced by Porphyromonas gingivalis lipopolysaccharide or Fusobacterium nucleatum lipopolysaccharide, the M2 phenotype polarization is M2c polarization, and the upregulation of M2 phenotype markers includes simultaneous upregulation of CD206, CD163 and MRC1, and the downregulation of M1 phenotype markers includes simultaneous downregulation of CD80 and CD68.

[0015] In one embodiment, the M2c polarization is achieved by the compound specifically inhibiting histone deacetylases HDAC1 and / or HDAC3, increasing the acetylation level of histone H3 at position 27 in the M2 gene promoter region, and synergistically activating the STAT6 / IRF4 transcription axis.

[0016] In one embodiment, the M2c polarized macrophages activate the Smad1 / 5 / 8-Runx2-Osx signaling cascade within periodontal ligament stem cells by paracrine secretion of TGF-β, BMP-2 and / or VEGF-A, thereby promoting osteogenic differentiation of the periodontal ligament stem cells and vascularized bone regeneration in alveolar bone defect areas.

[0017] The present invention also provides a pharmaceutical composition for treating periodontitis, comprising a therapeutically effective amount of the compound of formula (I) as described in claim 1 or 2, and pharmaceutically acceptable excipients.

[0018] In one embodiment, the pharmaceutical composition is formulated as a dosage form suitable for topical oral administration, the dosage form being selected from gels, mouthwashes, ointments, and films.

[0019] The application of the aforementioned curcumin compounds in the preparation of drugs for treating periodontitis utilizes a technique that replaces the β-diketone group of natural curcumin with a monocarbonyl linker to obtain the specific monocarbonyl curcumin analog. This technique firstly solves the core defect of natural curcumin—poor chemical stability and rapid degradation under physiological pH conditions due to the β-diketone group, resulting in extremely low oral bioavailability—providing a foundation for drug application. Furthermore, the structurally modified compound exhibits a dose-dependent inhibitory effect on key periodontal pathogens such as *Porphyromonas gingivalis*, *Focusae fossa*, *Actinomyces actinomycetes*, and *Fusobacterium nucleatum* at concentrations of 5-20 μM, superior to that of natural curcumin at the same concentration. It also demonstrates rapid antibacterial activity after 6 hours of action, thus providing effective antibacterial action against the clinical problem of incomplete removal of biofilm within periodontal pockets. Simultaneously, this compound… At a concentration of 20 μM, this compound significantly inhibits the production of periodontitis-related pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and CXCL8 at both the transcriptional and protein levels by suppressing the NF-κB signaling pathway, with an inhibitory effect superior to that of natural curcumin at the same concentration. Furthermore, this compound significantly upregulates the expression of M2 markers such as CD206, CD163, and MRC1 and downregulates the expression of M1 markers such as CD80 and CD68 in macrophages, thereby promoting macrophage polarization towards the anti-inflammatory and repair-oriented M2 phenotype, and this regulatory effect is superior to that of natural curcumin. Ultimately, through the aforementioned synergistic dual mechanism of antibacterial and immunomodulatory action, this compound achieves comprehensive intervention against periodontitis infection and host inflammatory dysregulation, thus solving the problems of poor efficacy of existing treatments, poor drug-like properties of natural curcumin, and its inability to effectively combine antibacterial and immunomodulatory functions. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The diagram shows the chemical reaction equation for the synthesis of L42H17 according to an embodiment of the present invention. Figure 2 This is a graph showing the antibacterial activity of L42H17 against periodontal pathogens according to an embodiment of the present invention. Figure 3 This is a diagram showing the results of L42H17 regulating macrophage M2 polarization in an embodiment of the present invention. Figure 4 This is a diagram showing the results of L42H17 inhibiting pro-inflammatory cytokines in an embodiment of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0024] The following is combined with Figures 1-4 The use of the curcumin compounds of the present invention in the preparation of medicaments for treating periodontitis is described.

[0025] Example 1: like Figure 1 As shown, in one embodiment, the use of a curcuminoid compound in the preparation of a medicament for treating periodontitis, the curcuminoid compound being a monocarbonyl curcumin analog having the structure shown in formula (I).

[0026] (I) Compound (I) is obtained by replacing the β-diketone group of natural curcumin with a monocarbonyl linker.

[0027] Specifically, the compound of formula (I) described in this embodiment is a derivative obtained by structural optimization of natural curcumin. The natural curcumin molecule contains a β-diketone group, which readily undergoes enol-keto tautomerism under physiological pH conditions, leading to molecular skeleton instability and easy degradation into products such as ferulic acid and feruloylmethane, thus severely limiting its bioavailability and clinical efficacy. In this embodiment, by replacing the β-diketone group of natural curcumin with a monocarbonyl linker, a monocarbonyl curcumin analog of formula (I) was obtained.

[0028] Reference Figure 1 , Figure 1The structural modification sites of the monocarbonyl curcumin analogue in embodiments of the present invention are shown. This structural modification eliminates the unstable β-diketone active site in the natural curcumin molecule, blocking the enol-keto tautomerism degradation pathway, thereby significantly improving the chemical stability of the compound. It should be understood that, although... Figure 1 The specific form of the single carbonyl linker is shown in the figure. However, in other embodiments of the present invention, the specific chain length or connection method of the single carbonyl linker can be adjusted according to the actual drug design requirements, as long as it can achieve the function of blocking the β-diketone degradation pathway and improving chemical stability.

[0029] The monocarbonyl curcumin analogue of this embodiment, through the aforementioned structural modification, overcomes the core defect of poor chemical stability in natural curcumin, enabling it to maintain more sustained pharmacological activity in the complex oral environment of periodontitis treatment. This compound can be present in a therapeutically effective amount in the preparation of drugs for treating periodontitis and can be used in combination with pharmaceutically acceptable excipients. Its mechanism of action in treating periodontitis includes, but is not limited to, inhibiting the growth of periodontal pathogens and regulating the host immune response, which will be elaborated in detail in subsequent embodiments. This embodiment establishes the structural basis for this type of compound as an active ingredient in periodontitis treatment drugs, providing overarching conceptual support for the subsequent preparation of specific compounds, activity verification, and formulation development.

[0030] Example 2: This embodiment provides a preferred monocarbonyl curcumin analog, wherein the monocarbonyl curcumin analog having the structure shown in formula (I) is compound L42H17.

[0031] Specifically, this embodiment details the preparation method, structural confirmation, and stability verification of compound L42H17. (Refer to...) Figure 1 , Figure 1 The chemical reaction equation for the synthesis of L42H17 is shown. This synthetic route uses natural curcumin as the starting material and replaces the unstable β-diketone group in the molecule with a stable monocarbonyl linker through a specific chemical reaction.

[0032] Step S110, Synthesis of compound L42H17: Natural curcumin (purity ≥98%) was weighed as the starting material, dissolved in a suitable organic solvent, and specific reaction reagents were added. The reaction was carried out under reflux conditions with carbonylation modification. The reaction was monitored throughout using TLC silica gel thin-layer chromatography until the starting material spot disappeared, confirming the complete reaction and yielding the crude product L42H17.

[0033] Step S120, purification of compound L42H17: The crude product above is loaded onto a 200-300 mesh silica gel column for chromatography. Gradient elution is performed using an eluent with increasing polarity. The fraction containing the target product is collected, concentrated, and dried to obtain the purified L42H17 product.

[0034] Step S130, Structural Characterization and Purity Detection of Compound L42H17: The purified product was structurally characterized using nuclear magnetic resonance spectroscopy. ¹H-NMR spectra showed clearly assigned characteristic peaks, consistent with the hydrogen atom environment of the target compound L42H17; ¹³C-NMR spectra further confirmed the carbon skeleton structure, particularly the characteristic absorption peaks of the monocarbonyl carbon. High-resolution mass spectrometry (HRMS) measurements of the molecular ion peak mass-to-charge ratio precisely matched the theoretically calculated value for L42H17. High-performance liquid chromatography (HPLC) results showed that the normalized purity of the main peak area was ≥99%, indicating that the obtained product has extremely high chemical purity, meeting the requirements for drug research.

[0035] Step S140, Stability Verification of Compound L42H17: To verify the effect of structural modification on improving chemical stability, L42H17 and natural curcumin were dissolved separately in dimethyl sulfoxide (DMSO) to prepare 10 μM solutions. The absorbance (OD) values ​​of the solutions were measured at specific time points in a 0.5 mm cuvette using a microplate reader, and absorbance-time curves were plotted. The experimental results showed that the absorbance value of the natural curcumin solution fluctuated significantly and decreased over time, indicating that it was unstable in solution and prone to degradation; while the OD value of the L42H17 solution remained relatively constant throughout the detection period without significant fluctuations. This comparative data conclusively proves that by replacing the β-diketone group of natural curcumin with a monocarbonyl linker, the chemical stability of L42H17 was significantly improved, effectively solving the technical problems of easy degradation and poor chemical stability of natural curcumin under physiological conditions, laying a solid material foundation for its subsequent application in periodontitis treatment drugs.

[0036] Example 3: This embodiment further verifies the application of the curcumin-like compounds in inhibiting the growth of periodontal pathogens. Specifically, the application includes inhibiting the growth of one or more periodontal pathogens selected from Porphyromonas gingivalis, Forsythostaphylococcus, Actinobacillus actinomycetii, and Fusobacterium nucleatum.

[0037] To confirm the above-mentioned antibacterial activity, this embodiment conducted a detailed verification through in vitro antibacterial experiments. (Refer to...) Figure 2 , Figure 2 The growth inhibition curves of L42H17 against the four periodontal pathogens mentioned above are shown.

[0038] Step S210, Experimental Strains and Culture: *Porphyromonas gingivalis* (ATCC 33277), *Fussafena* (ATCC 43037), *Actinomyces actinomycete* (ATCC 33384), and *Fusobacterium nucleatum* (ATCC 35896) were selected as representative periodontal pathogens. After thawing the frozen bacterial cultures, they were inoculated into brain and heart infusion (BHI) broth supplemented with 5 μg / ml heme and 1 μg / ml vitamin K1, and cultured overnight at 37°C under strictly anaerobic conditions (80% N2, 10% H2, 10% CO2). The bacterial culture was then diluted to OD. 600 Approximately 0.1 (approximately 1 × 10⁻⁶) 8 The working bacterial culture was prepared by diluting the culture medium (CFU / ml) with fresh BHI medium at a ratio of 1:100.

[0039] Step S220, Drug Treatment and Grouping: Inoculate 200 μl of working bacterial culture into a 96-well plate, setting up experimental groups, positive control groups, and solvent control groups. L42H17 solution was added to the experimental groups to achieve final concentrations of 5 μM, 10 μM, and 20 μM, respectively; natural curcumin solution was added to the positive control group to achieve a final concentration of 20 μM; and an equal volume of dimethyl sulfoxide (DMSO) was added to the solvent control group, controlling the final DMSO concentration to ≤0.5% (v / v). Each group was divided into three replicates and cultured under identical conditions.

[0040] Step S230, Growth curve determination: At time points of 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours of culture, the OD of each well was detected using a microplate reader. 600 The absorbance value is plotted as a function of time to reflect the growth dynamics of bacteria.

[0041] Step S240, Result Analysis and Verification: Experimental results showed that all pathogenic bacteria in the solvent control group exhibited typical "S"-shaped growth curves, indicating vigorous bacterial growth. However, the L42H17 treatment group showed significant antibacterial activity. The compound exerted a dose-dependent inhibitory effect on the growth of the periodontal pathogens at concentrations ranging from 5 μM to 20 μM. Specifically, as the concentration of L42H17 increased, the slope of the bacterial growth curve gradually decreased, and the OD... 600 The peak value decreased significantly. Taking *Porphyromonas gingivalis* as an example, at 24 h, the OD of the 20 μM L42H17 treatment group was significantly lower. 600 The value was only 0.57±0.04, significantly lower than the solvent control group (0.90±0.03, P<0.001), and also significantly lower than the 20μM natural curcumin group (0.76±0.03, P<0.01). L42H17 also showed excellent inhibitory effects against *Fusarium oxysporum*, *Actinomyces actinomycete*, and *Fusobacterium nucleatum*, with a 24hOD value of [missing value]. 600The values ​​were all significantly lower than those of the same concentration of natural curcumin. This result strongly demonstrates that the antibacterial activity of L42H17 was significantly enhanced through monocarbonyl structure modification.

[0042] Furthermore, it is noteworthy that L42H17 exhibited inhibitory activity as early as 6 hours after treatment. The growth curves show that at this early time point of 6 hours, the OD of the L42H17-treated group was significantly lower. 600 The value was significantly lower than that of the solvent control group, indicating that it can rapidly block the exponential growth phase of bacteria. This rapid-acting characteristic is of great significance for the clinical treatment of periodontitis, because the colonization and biofilm formation of periodontal pathogens in the periodontal pocket is a dynamic process. Rapidly inhibiting bacterial growth helps to eliminate pathogens or inhibit the release of their toxic products before the biofilm matures, thereby improving the treatment effect. It should be understood that although specific bacterial species and concentrations are listed in this embodiment, in actual applications, L42H17 may also have similar inhibitory effects on other periodontal-related pathogens, and the specific antibacterial concentration may fluctuate due to differences in bacterial load and oral microenvironment, but its dose-dependent inhibition pattern and rapid-acting characteristic are universal.

[0043] Example 4: This embodiment further verifies the application of the curcuminoid compounds in immunomodulation and anti-inflammatory activities. Specifically, the application includes promoting macrophage polarization towards the M2 phenotype and inhibiting the production of pro-inflammatory cytokines.

[0044] To confirm the aforementioned immunomodulatory effects, this embodiment employed an in vitro cell experimental model for detailed verification. (Refer to...) Figure 3 and Figure 4 , Figure 3 The results show the regulation of macrophage M2 polarization by L42H17. Figure 4 The results showed that L42H17 inhibited pro-inflammatory cytokines.

[0045] Step S310, Macrophage Induction and Treatment: Human mononuclear cell line THP-1 was selected as the cell model and macrophages were incubated at 1×10⁻⁶ cells / year. 6The cells were seeded at a density of 100 ng / ml in 6-well plates and treated with phorbol ester (PMA) for 48 hours to induce differentiation into adherent macrophages. Subsequently, the cells were washed three times with phosphate-buffered saline (PBS) to remove residual PMA and non-adherent cells. In one preferred embodiment, the macrophages were periodontal inflammation-associated macrophages induced by *Porphyromonas gingivalis* lipopolysaccharide or *Fusobacterium nucleatum* lipopolysaccharide. Specifically, *Porphyromonas gingivalis* lipopolysaccharide or *Fusobacterium nucleatum* lipopolysaccharide can be used to stimulate macrophages to construct an inflammatory model mimicking the periodontal microenvironment. Subsequently, fresh culture medium containing 20 μM natural curcumin, different concentrations (e.g., 10 μM, 20 μM) of L42H17, or a solvent control (e.g., DMSO, final concentration ≤0.5% v / v) was added, and the cells were cultured for another 24 hours.

[0046] Step S320, Detection of macrophage phenotypic polarization: After treatment, cells were collected, and the expression of cell surface markers was detected by flow cytometry. Specifically, promoting macrophage polarization towards the M2 phenotype included upregulating the expression of M2 phenotypic markers CD206, CD163, and / or MRC1, and / or downregulating the expression of M1 phenotypic markers CD80 and / or CD68. Experimental results showed that after treatment with 20 μM L42H17, CD206... + The proportion of M2 macrophages reached 96.8±2.7%, significantly higher than that in the solvent control group (60.2±5.3%, P<0.001) and the group with the same concentration of natural curcumin (82.5±6.8%, P<0.01). Simultaneously, L42H17 significantly downregulated the gene expression levels of M1 phenotypic markers CD80 and CD68, and upregulated the gene expression levels of M2 phenotypic markers CD163 and MRC1. For example, after L42H17 treatment, the relative expression level of CD163 was upregulated by 2.10±0.18 times (P<0.01), and the relative expression level of MRC1 was upregulated by 1.68±0.14 times (P<0.001), with the MRC1 expression level significantly higher than that in the group with the same concentration of natural curcumin (P<0.05). This indicates that L42H17 can effectively reverse the dominance of pro-inflammatory M1 macrophages in the periodontal environment and promote the polarization of the anti-inflammatory and reparative M2 phenotype. In a more specific embodiment, the M2 phenotype polarization is M2c polarization, and the upregulation of M2 phenotype markers includes the simultaneous upregulation of CD206, CD163, and MRC1, while the downregulation of M1 phenotype markers includes the simultaneous downregulation of CD80 and CD68. M2c polarization is an important subset of M2 macrophages with extremely strong anti-inflammatory and tissue repair functions. The discovery of this feature further confirms the potential of L42H17 in promoting periodontal tissue regeneration.

[0047] Step S330, Detection of pro-inflammatory cytokine expression: Cell culture supernatant and cell pellet were collected, and the secretion and gene transcription levels of pro-inflammatory cytokines were detected, respectively. Specifically, inhibition of pro-inflammatory cytokine production included inhibition of one or more cytokines selected from tumor necrosis factor (TNF), interleukin-1β (IL-1β), interleukin-6 (IL-6), and CXCL8 at the transcriptional and / or protein secretion levels. Real-time quantitative PCR results showed that L42H17 inhibited the gene transcription of the above-mentioned pro-inflammatory cytokines in a dose-dependent manner. At a concentration of 20 μM, the mRNA level of TNF was only 0.13 ± 0.02 times that of the control group (P < 0.001), significantly lower than that of the natural curcumin group (0.56 ± 0.09 times, P < 0.001); the transcriptional inhibition effect of IL-6 and CXCL8 was also significantly better than that of the same concentration of natural curcumin (P < 0.05 or P < 0.01). ELISA results were consistent with transcriptional levels. 20 μM L42H17 significantly reduced TNF secretion from 865.3 ± 70.5 pg / ml in the control group to 487.5 ± 82.1 pg / ml, IL-1β to 283.6 ± 45.2 pg / ml, IL-6 to 321.4 ± 28.5 pg / ml, and CXCL8 to 625.3 ± 84.6 pg / ml, all significantly lower than the solvent control group and the group with the same concentration of natural curcumin (P < 0.01 or P < 0.001). This result strongly demonstrates that L42H17 can block the production of key pro-inflammatory factors at both the transcriptional and protein secretion levels, thereby effectively inhibiting the inflammatory response in periodontal tissues.

[0048] Step S340, Validation of the Molecular Mechanism: To further elucidate the molecular mechanism by which L42H17 inhibits the production of pro-inflammatory cytokines, this embodiment also tested the activity of the NF-κB signaling pathway. Specifically, the compound inhibits the production of pro-inflammatory cytokines by inhibiting the NF-κB signaling pathway. The NF-κB signaling pathway is a core regulatory pathway of the inflammatory response, and its activation leads to the transcriptional upregulation of various pro-inflammatory factors such as TNF, IL-1β, and IL-6. Experimental results show that L42H17 treatment significantly inhibited the activation of the NF-κB signaling pathway, thereby blocking the synthesis and release of inflammatory factors at the source. It should be understood that although this embodiment focuses on describing the NF-κB pathway, the anti-inflammatory mechanism of L42H17 may also involve the synergistic effects of other signaling pathways. This multi-target anti-inflammatory characteristic helps to improve its therapeutic effect in the complex periodontal microenvironment.

[0049] Example 5: This embodiment further explores the molecular mechanism by which the curcumin-like compounds regulate macrophage polarization and verifies their potential to induce periodontal tissue regeneration. Specifically, the M2c polarization is achieved by the specific inhibition of histone deacetylases HDAC1 and / or HDAC3 by the compounds, increasing the acetylation level of lysine 27 of histone H3 in the M2 gene promoter region, and synergistically activating the STAT6 / IRF4 transcriptional axis.

[0050] To elucidate the above molecular mechanism, this embodiment uses Western blotting, immunoprecipitation, and dual-luciferase reporter gene assays for detailed verification.

[0051] Step S410, HDAC activity inhibition verification: Treated macrophages were lysed, total protein was extracted, and intracellular HDAC total enzyme activity was detected using an HDAC activity assay kit. Simultaneously, the protein expression levels of HDAC1 and HDAC3 were detected using Western blotting. The results showed that the total HDAC enzyme activity in the L42H17 treatment group was significantly reduced in a dose-dependent manner, while having no significant effect on the protein expression levels of HDAC1 and HDAC3. This indicates that L42H17 mainly exerts its effect by inhibiting the enzyme activity of HDAC1 / 3 rather than downregulating their protein expression. It should be understood that although this embodiment focused on verifying HDAC1 and HDAC3, L42H17 may also have some inhibitory activity against other HDAC subtypes (such as HDAC2). This broad-spectrum or selective inhibitory characteristic helps to comprehensively regulate chromatin structure.

[0052] Step S420, histone acetylation modification level detection: Chromatin immunoprecipitation (ChIP) was used to specifically enrich chromatin fragments in the promoter regions of M2 genes (such as CD206 and MRC1), and the results were detected using an antibody against acetylated histone H3 at lysine 27 (H3K27ac). The results showed that the enrichment of H3K27ac in the M2 gene promoter region was significantly higher in the L42H17 treatment group than in the solvent control group and the natural curcumin group. H3K27ac is a typical activity enhancer marker; its elevated level indicates a looser chromatin structure, making it easier for transcription factors to bind, thereby promoting the transcriptional activation of the M2 gene. This result explains the underlying reason why L42H17 promotes M2 polarization from an epigenetic perspective.

[0053] Step S430, STAT6 / IRF4 transcriptional axis activation verification: The phosphorylation level of transcription factor STAT6 and the expression of its downstream transcription factor IRF4 were examined. Western blot results showed that L42H17 treatment significantly promoted the phosphorylation and nuclear translocation of STAT6 and upregulated the protein expression of IRF4. Dual-luciferase reporter gene assays further confirmed that L42H17 could significantly enhance luciferase activity driven by the STAT6 binding sequence. This indicates that L42H17, by activating the STAT6 / IRF4 transcriptional axis and in conjunction with H3K27ac modification, jointly drives the specific differentiation of macrophages into the M2c phenotype.

[0054] Furthermore, this embodiment also verified the paracrine regulatory effect of M2c polarized macrophages on periodontal ligament stem cells. The M2c polarized macrophages, through paracrine secretion of TGF-β, BMP-2, and / or VEGF-A, activated the Smad1 / 5 / 8-Runx2-Osx signaling cascade within periodontal ligament stem cells, promoting osteogenic differentiation of these stem cells and vascularized bone regeneration in alveolar bone defect areas.

[0055] Step S440, Conditioned Culture Medium Collection and Treatment: Collect the culture supernatant of M2c macrophages induced by L42H17 polarization as conditioned culture medium. Seed human periodontal ligament stem cells into culture plates and treat them with conditioned culture medium.

[0056] Step S450, Paracrine Factor Detection: The concentrations of TGF-β, BMP-2, and VEGF-A in the conditioned medium were detected using ELISA. The results showed that the concentrations of the above three factors in the conditioned medium of the L42H17 induction group were significantly higher than those in the control group, indicating that M2c macrophages have active paracrine function.

[0057] Step S460, Verification of osteogenic differentiation of periodontal ligament stem cells: Changes in osteogenic-related signaling pathways and markers within periodontal ligament stem cells were detected. Western blot results showed that in the conditioned medium treatment group, the phosphorylation levels of Smad1 / 5 / 8 in periodontal ligament stem cells were significantly increased, and the expression levels of the key osteogenic transcription factor Runx2 and its downstream factor Osx were also upregulated. Alkaline phosphatase (ALP) staining and Alizarin Red S staining results showed that the ALP activity of periodontal ligament stem cells treated with conditioned medium was significantly enhanced, and more mineralized nodules were formed. This demonstrates that L42H17-induced M2c macrophages effectively activated the osteogenic differentiation program of periodontal ligament stem cells by paracrine secretion of osteogenic and angiogenesis-related factors, providing a favorable microenvironment for vascularized bone regeneration in alveolar bone defect areas. This embodiment reveals the deep scientific principle of L42H17 in treating periodontitis at the molecular mechanism level, establishing its complete chain of action of "structural modification-epiogenetic regulation-immune remodeling-tissue regeneration".

[0058] Example 6: This embodiment provides a pharmaceutical composition for treating periodontitis, comprising a therapeutically effective amount of a compound of formula (I) as described in the above embodiment, and pharmaceutically acceptable excipients.

[0059] Specifically, the pharmaceutical composition described in this embodiment aims to deliver the active ingredient directly to the periodontal pocket or oral mucosa via local administration, thereby maintaining a high drug concentration at the lesion site while reducing the risk of systemic adverse reactions. The "therapeutic effective dose" refers to the dosage of a compound that can inhibit the growth of periodontal pathogens, regulate macrophage polarization, or suppress inflammatory responses, thereby improving periodontal clinical indicators. This dosage can be adjusted according to the severity of the condition, the patient's age, weight, and other factors, and typically represents 0.1% to 10% of the formulation by mass.

[0060] In one preferred embodiment, the pharmaceutical composition is formulated into a dosage form suitable for topical oral administration, the dosage form being selected from gels, mouthwashes, ointments, and films. The preparation process is described in detail below with reference to specific formulation examples.

[0061] Formulation Example 1: Gel Due to their good adhesion and biocompatibility, gels can remain in the periodontal pocket for a relatively long time, making them an ideal dosage form for local sustained-release drug delivery in the periodontal area.

[0062] Step S510, prescription composition: Take 0.5g of compound L42H17 (active ingredient), 1.5g of carbomer 940 (thickener), 10g of glycerin (humectant), 1g of azone (transdermal absorption enhancer), 0.1g of ethylparaben (preservative), and an appropriate amount of triethanolamine (pH adjuster), and add purified water to 100g.

[0063] Step S520, Preparation Process: Carbomer 940 is sprinkled into an appropriate amount of purified water and allowed to swell overnight, allowing it to fully absorb water and form a gel matrix. Separately, compound L42H17 is dissolved in a small amount of ethanol, and glycerol, azone, and ethylparaben are added. After stirring to dissolve, the solution is slowly added to the carbomer gel matrix. Stirring is performed continuously during addition. Finally, triethanolamine is added dropwise to adjust the pH to 6.5-7.0 to neutralize the acidity of the carbomer and increase the viscosity of the gel. Purified water is added to the final volume, and the mixture is stirred until homogeneous to obtain a transparent gel. This gel can be directly injected into the deep periodontal pockets using a syringe needle to form a drug reservoir and slowly release the drug.

[0064] Formula Example 2: Mouthwash Mouthwash is suitable for the adjunctive treatment and daily care of gingivitis in the whole mouth, and is characterized by its ease of use and wide coverage.

[0065] Step S530, prescription composition: Take 0.2g of compound L42H17, 1g of polysorbate 80 (solvent), 10ml of ethanol (solvent), 0.05g of sodium saccharin (flavoring agent), 0.1ml of peppermint oil (fragrant agent), and 0.2g of sodium benzoate (preservative), and add purified water to 100ml.

[0066] Step S540, Preparation Process: Dissolve compound L42H17 in ethanol, and add polysorbate 80 as a solubilizer. Dissolve sodium saccharin and sodium benzoate in an appropriate amount of purified water. Mix the above oil phase and aqueous phase, add peppermint oil, and stir thoroughly or sonicate to ensure the drug is evenly dispersed or dissolved in the medium. Finally, add purified water to the total volume and filter to obtain the final product. When the patient rinses their mouth, the solution can directly contact the gingival sulcus and periodontal pocket opening to exert antibacterial and anti-inflammatory effects.

[0067] Formulation Example 3: Ointment Ointments have good lubrication and protective properties, and are suitable for application and protection of gingival mucosal ulcers or wounds after periodontal surgery.

[0068] Step S550, prescription composition: take 1.0g of compound L42H17, 40g of petrolatum (oily matrix), 10g of lanolin (water-absorbing matrix), and an appropriate amount of liquid paraffin (to adjust the consistency).

[0069] Step S560, Preparation process: A melting method is used. Petrolatum and lanolin are placed in a suitable container and heated in a water bath until melted and mixed thoroughly. When the matrix temperature drops to approximately 50°C, finely ground and sieved compound L42H17 powder is added, and the mixture is stirred continuously until it cools and solidifies, ensuring the drug is evenly dispersed in the matrix. The ointment forms a protective film on the gingival surface, isolating external irritants and releasing the drug.

[0070] It should be understood that the above embodiments only list three typical dosage forms: gels, mouthwashes, and ointments, but this does not mean that the scope of protection of the present invention is limited to these. According to actual clinical needs, those skilled in the art can also prepare compounds of formula (I) into other dosage forms suitable for local oral administration, such as film-forming agents (e.g., periodontal dressings), powders, and sprays. The pharmaceutically acceptable excipients are not limited to the types listed above, but may also include other fillers, binders, stabilizers, colorants, etc., as long as they do not chemically interact with the active ingredient and do not affect the efficacy and safety of the drug. Through the development of the above-mentioned multiple dosage forms, this embodiment demonstrates the high operability and clinical translation potential of compounds of formula (I) in the preparation of periodontitis treatment drugs.

[0071] Example 7: This embodiment simulates a real-world application scenario for periodontitis treatment, comprehensively demonstrating the application value of curcumin compounds in the preparation of drugs for treating periodontitis. Specifically, this embodiment uses compound L42H17 as the active ingredient to prepare a localized sustained-release gel formulation, which is applied to the affected area of ​​periodontitis to verify its comprehensive therapeutic effect.

[0072] Step S610, Local Administration and Drug Release: The gel formulation containing a therapeutically effective amount of L42H17 is injected deep into the periodontal pocket using a syringe. The gel matrix remains within the periodontal pocket and slowly releases the active ingredient, maintaining the drug concentration at the lesion site within the effective range. Because L42H17 has undergone monocarbonyl structural modification, its chemical stability is significantly superior to natural curcumin. It can maintain structural integrity in the complex microenvironment of the periodontal pocket and is not easily degraded, thus ensuring sustained efficacy.

[0073] Step S620, Broad-spectrum antibacterial and microbial regulation: After drug release, L42H17 first exerts its broad-spectrum antibacterial effect. It can rapidly penetrate into the subgingival biofilm and exhibit inhibitory activity within 6 hours, dose-dependently inhibiting the growth of core periodontal pathogens such as *Porphyromonas gingivalis*, *Focusae*, *Actinomyces actinomycetes*, and *Fusobacterium nucleatum*. By reducing the pathogenic bacterial load, L42H17 effectively blocks the direct destruction of periodontal tissues by pathogens and reduces the continuous release of bacterial endotoxins (such as lipopolysaccharide LPS), weakening the initiating factors that trigger the host's immune inflammatory response at its source.

[0074] Step S630, Immunomodulation and Inflammation Blockage: While inhibiting pathogenic bacteria, L42H17 actively regulates the local immune microenvironment of the host. It can be taken up by macrophages in periodontal tissues, increasing H3K27 acetylation levels in the M2 gene promoter region by inhibiting HDAC1 / 3 enzyme activity, activating the STAT6 / IRF4 transcriptional axis, and driving macrophages to polarize towards the anti-inflammatory and repair-oriented M2c phenotype. This process manifests as upregulation of M2 markers such as CD206, CD163, and MRC1, and downregulation of M1 markers such as CD80 and CD68. Polarized M2c macrophages, by inhibiting the NF-κB signaling pathway, block the production of key pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and CXCL8 at both the transcriptional and protein secretion levels. This precise immunomodulatory effect not only curbs the inflammatory destruction process of periodontal tissues but also creates a favorable immune environment for subsequent tissue repair.

[0075] Step S640, Tissue Regeneration and Functional Restoration: With the resolution of inflammation and the improvement of the immune microenvironment, M2c macrophages exert paracrine function, releasing regeneration-related factors such as TGF-β, BMP-2, and VEGF-A. These factors act on periodontal ligament stem cells, activating the internal Smad1 / 5 / 8-Runx2-Osx signaling cascade and inducing stem cells to differentiate into osteoblasts. Ultimately, vascularized bone regeneration is achieved in the alveolar bone defect area, promoting structural repair and functional reconstruction of the periodontal ligament and alveolar bone.

[0076] In summary, this embodiment demonstrates the synergistic effect of L42H17 in the treatment of periodontitis through multiple mechanisms: antibacterial, anti-inflammatory, immunomodulatory, and tissue regeneration. Unlike single-mechanism antibacterial or anti-inflammatory drugs, L42H17 targets both the microbial etiology and the host inflammatory response in periodontitis, achieving a combination of symptomatic and fundamental treatment. Its excellent chemical stability ensures efficacy, its broad-spectrum antibacterial activity eliminates pathogenic threats, its precise immunomodulation reshapes the repair environment, and ultimately, it promotes functional healing of periodontal tissues by promoting bone regeneration. This comprehensive treatment approach significantly improves the therapeutic effect of periodontitis and demonstrates great potential for clinical application.

[0077] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0078] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The use of a curcumin-like compound in the preparation of a medicament for treating periodontitis, characterized in that, The curcumin compounds are monocarbonyl curcumin analogs having the structure shown in formula (I): ； Compound (I) is obtained by replacing the β-diketone group of natural curcumin with a monocarbonyl linker.

2. The use of the curcumin-like compounds according to claim 1 in the preparation of a medicament for treating periodontitis, characterized in that, The monocarbonyl curcumin analog having the structure shown in formula (I) is compound L42H17.

3. The use of the curcumin-like compounds according to claim 1 or 2 in the preparation of a medicament for treating periodontitis, characterized in that, The application includes inhibiting the growth of one or more periodontal pathogens selected from Porphyromonas gingivalis, Forsythosin, Actinobacillus actinomycetii, and Fusobacterium nucleatum.

4. The use of the curcumin-like compounds according to claim 3 in the preparation of a medicament for treating periodontitis, characterized in that, The compound exhibits dose-dependent inhibition of the growth of periodontal pathogens at concentrations ranging from 5 μM to 20 μM, and shows inhibitory activity as early as 6 hours after treatment.

5. The use of the curcumin-like compounds according to claim 1 or 2 in the preparation of a medicament for treating periodontitis, characterized in that, The applications include promoting macrophage surface polarization to M2 and inhibiting the production of pro-inflammatory cytokines.

6. The use of the curcumin-like compounds according to claim 5 in the preparation of a medicament for treating periodontitis, characterized in that, Promoting macrophage polarization toward the M2 phenotype involves upregulating the expression of M2 phenotype markers CD206, CD163, and / or MRC1, and / or downregulating the expression of M1 phenotype markers CD80 and / or CD68.

7. The use of the curcumin-like compounds according to claim 5 in the preparation of a medicament for treating periodontitis, characterized in that, Inhibiting the production of pro-inflammatory cytokines includes inhibiting one or more cytokines selected from tumor necrosis factor (TNF), interleukin-1β (IL-1β), interleukin-6 (IL-6), and CXCL8 at the transcriptional and / or protein secretion levels.

8. The use of the curcumin-like compounds according to claim 7 in the preparation of a medicament for treating periodontitis, characterized in that, The compound inhibits the production of the pro-inflammatory cytokines by suppressing the NF-κB signaling pathway.

9. A pharmaceutical composition for treating periodontitis, characterized in that, It comprises a therapeutically effective amount of the compound of formula (I) as described in claim 1 or 2, and pharmaceutically acceptable excipients.

10. The pharmaceutical composition for treating periodontitis according to claim 9, characterized in that, The pharmaceutical composition is formulated into a dosage form suitable for topical oral administration, the dosage form being selected from gels, mouthwashes, ointments, and films.

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