Application of naringenin in treatment of non-renaturable pulpitis

Abstract

The invention relates to application of naringenin in treatment of non-renaturable pulpitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine and biotechnology, and particularly relates to application of naringenin in treatment of irreversible pulpitis. BACKGROUND

[0002] Pulpitis is the most common disease, mainly caused by caries. After caries occurs, the metabolic products of bacteria can diffuse into the pulp cavity through dentin tubules before the bacteria reach the pulp, causing inflammation of the pulp. In patients with irreversible pulpitis and rat pulpitis models induced by pathogenic bacteria LPS, the odontogenic differentiation potential of dental pulp cells (DPCs) is destroyed.

[0003] Human dental pulp fibroblasts (hDPFs) as the main stromal cells in dental pulp tissue are the main constituent of dental pulp cells (DPCs). hDPF can perceive external stimuli and produce mineralized nodules as a defense mechanism, which indicates that hDPFs have odontogenic differentiation potential and play an important role in the repair process of inflammatory pulp tissue. A large number of neutrophils infiltrate in the early stage of pulp inflammation, which suggests that there may be an interaction between hDPF cells and neutrophils in the early inflammatory pulp microenvironment.

[0004] As one of the most abundant flavonoids in daily diet, naringenin (Nar) can act as an immunomodulator in the field of slow / acute inflammation, cardiovascular disease, neurodegenerative disease and cancer. Although most studies have shown that flavonoids have antioxidant properties, Nar also has pro-oxidant activity, which can lead to the production of reactive oxygen species (ROS) and enhance antigen cross-presentation in DCs.

[0005] In irreversible pulpitis, the potential pharmacological effects of Nar on the odontogenic differentiation ability of neutrophils and dental pulp fibroblasts are still unclear. Based on this, the present application is proposed. SUMMARY

[0006] The present application is first related to the following application of naringenin:

[0007] (1) preparation of a medicine for inhibiting bacterial infection of dental pulp;

[0008] (2) preparation of a medicine for treating irreversible pulpitis.

[0009] The present application is also related to the application of naringenin in inhibiting bacteria infecting dental pulp and treating irreversible pulpitis.

[0010] The present application has the beneficial effect that,

[0011] (1) The present application proves that Nar restores the ability of hDPFs to differentiate into odontogenic direction inhibited by neutrophils activated by P. gingivalis-derived lipopolysaccharide (P.g-LPS) by regulating the function of neutrophils.

[0012] (2) Nar enhances the ability of neutrophils to phagocytose and digest bacteria, while inhibiting the formation of NETs, the secretion of pro-inflammatory cytokines and chemokines, which helps to regenerate and restore the function of inflammatory dental pulp, providing a new treatment strategy for irreversible pulpitis. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure 1: Mechanism of naringenin restoring the impaired odontogenic differentiation of dental pulp fibroblasts by regulating the function of neutrophils.

[0014] Figure 2 Figure 2: Different bacterial-derived lipopolysaccharides induce human peripheral blood-derived neutrophils to secrete IL-6 and IL-8 / CXCL8. Human peripheral blood-derived neutrophils (hNeu.) were induced with P.g-LPS and E.coli-LPS for 24 hours, respectively, and the levels of IL-6 and IL-8 / CXCL8 in the supernatant were detected by ELISA.

[0015] Figure 3 Figure 3: Different bacterial-derived lipopolysaccharides induce human peripheral blood-derived neutrophils to produce neutrophil extracellular traps (NETs). After human peripheral blood-derived neutrophils (hNeu.) were induced with P.g-LPS and E.coli-LPS for 1 hour, respectively, an appropriate amount of cell suspension was prepared for cell smears, which were stained with Wright-Giemsa and observed under an oil immersion lens (1000X).

[0016] Figure 4 Figure 4: Detection of the odontogenic differentiation ability of dental pulp fibroblasts. After 24 days of odontogenic differentiation induction, the odontogenic differentiation ability of hDPFs and non-hDPFs was detected by alizarin red S staining. Naked eye: observed by naked eye, 100X: observed under an optical microscope at 100X.

[0017] Figure 5 Figure 5: P.g-LPS inhibits the odontogenic differentiation ability of dental pulp fibroblasts,

[0018] ODM: odontogenic induction medium; ODM+P.g-LPS: odontogenic induction medium containing 1 μg / mL P.g-LPS;

[0019] 5A, microscopic observation, 5B, absorbance data column chart.

[0020] Figure 6, P.g-LPS treated neutrophil supernatant suppresses the odontogenic differentiation ability of hDPFs, ODM: odontogenic induction medium; dHL-60 sup.: dHL-60 cell supernatant; P.g-LPS dHL-60 sup.: 1 pg / mL P.g-LPS treated dHL-60 cell supernatant for 24 hours;

[0021] 6A, Microscopic observation, 6B, Absorbance data column chart.

[0022] Figure 7 , Naringenin + P.g-LPS treated dHL-60 cell supernatant restores the impaired mineralization ability of hDPFs, ODM: odontogenic induction medium; P.g-LPS dHL-60 sup.: 1 pg / mL P.g-LPS treated dHL-60 cell supernatant for 24 hours; Nar P.g-LPS dHL-60 sup.: 100 pM Nar + 1 pg / mL P.g-LPS treated dHL-60 cell supernatant for 24 hours;

[0023] 7A, Microscopic observation, 7B, Absorbance data column chart.

[0024] Figure 8 , Effect of naringenin on P.g-LPS induced NETs production by neutrophil-like cells, 1 pg / mL P.g-LPS, 1 pg / mL P.g-LPS + 100 pM Nar induced neutrophil-like cells (dHL-60 cells) for 1 hour, Wright-Giemsa staining, CS2 scanned whole slide, observed NETs formation; According to the CS2 scanned slide, more than 20 random areas were selected in each group, and HALO software was used for analysis.

[0025] Figure 9 , Naringenin can inhibit the secretion of IL-6 and IL-8 / CXCL8 by P.g-LPS induced neutrophil-like cells to a certain extent, neutrophil-like cells (dHL-60 cells) were treated with different concentrations of naringenin (0-200 pM) + 1 pg / mL P.g-LPS for 24 hours, and the levels of IL-6 and IL-8 / CXCL8 in the supernatant of each group were detected by ELISA.

[0026] Figure 10 , Effect of naringenin on phagocytic ability of neutrophil-like cells, flow cytometry (FACS) was used to detect the percentage of EGFP positive (EGFP+) cells in dHL-60 cells.

[0027] Figure 11 , Naringenin reduces inflammatory cell infiltration in rat molar irreversible pulpitis.

[0028] In P.g-LPS group, inflammatory cells infiltrated to the mesial root canal orifice of molar tooth( Figure 11 A and B), and a large number of neutrophils with polymorphic lobed nucleus(MPO positive) were observed( Figure 11 C and D).

[0029] In P.g-LPS+Nar group, inflammatory cells were localized at the mesial pulp horn( Figure 11 D and E), and the number of neutrophils with polymorphic lobed nucleus(MPO positive) was less( Figure 11 F and L).

[0030] Figure 12 Effects of naringenin on neutrophil adhesion and extravasation and vaso-dilation in rat molar teeth with irreversible pulpitis.

[0031] In P.g-LPS group, neutrophils(MPO positive) in pulp microvessels adhered to the vessel wall and extravasated to the surrounding pulp tissue( Figure 12 A).

[0032] In P.g-LPS+Nar group, the adhesion and extravasation of neutrophils were significantly reduced( Figure 12 B and C).

[0033] In P.g-LPS group, no obvious dilation of mesial root pulp microvessels was observed( Figure 12 D).

[0034] In P.g-LPS+Nar group, obvious dilation of mesial root pulp microvessels was observed, i.e., the density of microvessels increased and the dilation range enlarged, close to the mesial pulp horn inflammatory cell infiltration area( Figure 12 E).

[0035] The density of pulp microvessels in P.g-LPS+Nar group was significantly higher than that in P.g-LPS group( Figure 12 F). DETAILED DESCRIPTION

[0036] 1.1 Experimental animals, cell lines and host bacteria

[0037] SD rats, 8 weeks old, SPF level

[0038] HL-60 cell line, HEK 293T cells, E. coli, EGFP-E. coli (kanamycin-resistant enhanced green fluorescent protein-labeled E. coli) were gifted by Professor Ping Zhu and Professor Wei Liang of the Institute of Biophysics, Chinese Academy of Sciences

[0039] 1.2 Main reagents

[0040] IHC antibodies: CD45, MPO (Protintech, San Ying)

[0041] Naringenin: analytical pure;

[0042] CCK8 kit (Lablead)

[0043] Reactive oxygen detection kit (Beyotime)

[0044] N-acetyl cysteine (Beyotime)

[0045] Eltrombopag

[0046] LysoTracker Red DND-99 (Thermal Fisher, ThermoFisher)

[0047] LysoTracker Green DND-26 (Beyotime)

[0048] HEPES (Gibco)

[0049] P.g-LPS (Invitrogen)

[0050] 1.3 Main instruments

[0051] Laser confocal microscope FV3000S, German Leica embedding machine, dehydrator, German Leica microtome, CS2 pathological section scanning system, HALO pathological section analysis software

[0052] 1.4 Immunomagnetic bead sorting and identification of human dental pulp fibroblasts (hDPFs) and peripheral blood-derived neutrophils (hNeu.)

[0053] The experimental protocol for the isolation and culture of peripheral blood-derived neutrophils (hNeu.) and human dental pulp fibroblasts (hDPFs) has been approved by the Ethics Committee of Capital Medical University Beijing Stomatological Hospital (Approval No. CMUSH-IRB-KJ-PJ-2023-19), and informed consent has been provided by all donors.

[0054] After isolation of human dental pulp cells (hDPCs) according to the method of the literature [DUCRET M, FABRE H, FARGES J C, et al. Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach [J]. J Endod, 2015, 41(9): 1492-9.], the dental pulp fibroblasts (hDPFs) were positively enriched by anti-human fibroblast magnetic beads. After staining the enriched cells with antibodies recognizing the surface markers of hDPFs (including PE-CD146 and APC-CD90), flow cytometry (FACS) analysis was performed. The purified hDPFs were cultured in complete a-MEM medium (a-MEM containing 10% heat-inactivated FBS + 1% penicillin / streptomycin), and the cell culture conditions were 37°C, 5% CO2, 95% humidity.

[0055] The neutrophils (hNeu.) were negatively screened from the peripheral blood of healthy people using a neutrophil separation kit. After staining the screened cells with antibodies recognizing the surface markers of hNeu. (including FITC-CD45, PE-CD16 and APC-CD66b), FACS analysis was performed. The sorted hNeu. were cultured in complete RPMI 1640 medium (RPMI 1640 containing 10% heat-inactivated FBS + 1% penicillin / streptomycin), and the cell culture conditions were the same as before. An appropriate amount of hNeu. suspension was made into a smear, and the cell morphology was observed after Wright-Giemsa staining.

[0056] 1.5 Induction and identification of neutrophil-like differentiated human polymorphonuclear leukemia cells (dHL-60)

[0057] The HL-60 cell line was cultured in complete IMDM medium (IMDM containing 20% heat-inactivated FBS + 1% penicillin / streptomycin). The HL-60 cells were induced to differentiate into dHL-60 cells by culturing them in complete IMDM medium containing 1.3% dimethyl sulfoxide (DMSO) for 6-7 days. After staining the dHL-60 cells with APC-CD11b antibody, FACS identification was performed. An appropriate amount of dHL-60 cell suspension was made into a smear, and the cell morphology was observed after Wright-Giemsa staining.

[0058] 1.6 Cell activity detection

[0059] The dHL-60 cells were cultured at 1 × 10 5Cells were seeded at a density of 1 x 104cells / well in 96-well plates and treated with different concentrations (0-200 μmol / L) of naringenin (Nar) for 24 h. Nar was dissolved in DMSO, filtered through a 0.22 μm filter and diluted in culture medium before use. Cell viability of dHL-60 was determined by CCK8 method.

[0060] 1.7 Cytokine and chemokine detection

[0061] Cytokine and chemokine levels in the supernatants of hNeu. cells and dHL-60 cells were determined using human IL-6 and IL-8 / CXCL8 DuoSet ELISA kits. The absorbance values at 450 nm were read on a microplate reader.

[0062] 1.8 Odonogenic induction and alizarin red S staining

[0063] hDPFs (human dental pulp fibroblasts) were seeded in 6-well plates at a density of 1 x 105cells / well in complete α-MEM medium (α-MEM containing 10% heat-inactivated FBS + 1% penicillin / streptomycin) and cultured with medium change every 2-3 days. When the cell density reached 80%, the cells were induced to odontogenic differentiation for 24 d using odontogenic differentiation medium (ODM medium, complete α-MEM containing 10 mmol / L β-glycerophosphate sodium + 50 μg / mL ascorbic acid + 10 nmol / L dexamethasone). 5

[0064] (1) To investigate the direct effect of P.g-LPS on odontogenic differentiation of hDPFs, hDPFs were cultured in complete α-MEM medium, ODM medium and ODM medium containing 1 μg / mL P.g-LPS for 24 d.

[0065] (2) To investigate the effect of P.g-LPS-treated dHL-60 supernatant on odontogenic differentiation of hDPFs, dHL-60 cells were cultured in complete IMDM medium and complete IMDM medium containing 1 μg / mL P.g-LPS. The supernatants of each group were collected by centrifugation after 24 h and stored at -20 °C for later use. The differentially treated dHL-60 supernatants were mixed with ODM at a ratio of 1:1 (v / v) and used for odontogenic induction of hDPFs for 24 d.

[0066] ​Each group of culture medium was replaced every 3 days. After 24 days of odontogenic differentiation induction, the cells were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, washed twice with deionized water, and stained with 2% (w / v) alizarin red S staining solution (Sigma) at room temperature for 40 min. Excess staining solution was aspirated, and the cells were gently washed four times with deionized water. The mineralized nodules were observed under an optical microscope (Nikon) and images were collected. After natural drying, a camera was used to collect macroscopic images. Alizarin red S quantification was performed according to the literature [YAN Y, ZENG W, SONG S, et al. Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1 [J]. Protein Cell, 2013, 4(8): 620-7.], i.e., 1 mL of acetic acid and 200 μL of glycerol were added to each well, and the absorbance was measured at a wavelength of 405 nm (OD 405).

[0067] To explore the effect of naringenin-treated dHL-60 supernatant on the impaired odontogenic differentiation of hDPFs, dHL-60 cells were cultured in complete IMDM medium containing 1 μg / mL P.g-LPS, complete IMDM medium containing 1 μg / mL P.g-LPS ± 100 μmol / L Nar, and the supernatant of each group was collected by centrifugation after 24 h and stored at -20°C for later use. The dHL-60 supernatant treated in different ways was mixed with ODM at a ratio of 1:1 (v / v) and used for odontogenic induction of hDPFs for 24 d.

[0068] After 24 days of odontogenic differentiation induction, alizarin red S staining, image acquisition, and quantification were performed.

[0069] 1.9 ROS detection

[0070] According to the instructions of the reactive oxygen species detection kit, dHL-60 cells were loaded with 2',7'-dichlorofluorescein diacetate (DCFH-DA) probes for 20 min under the conditions of a cell incubator. Since serum can affect the measurement of intracellular ROS, dHL-60 cells were cultured in serum-free IMDM medium containing 0-100 μmol / L Nar or 1 μg / mL P.g-LPS for 1 h. Subsequently, the mean fluorescence intensity (MFI) was measured using a flow cytometer.

[0071] 1.10 phagocytosis experiment

[0072] dHL-60 cells were pre-treated with / without 4 mmol / L N-acetyl cysteine (NAC) for 10 min, followed by treatment with different concentrations (0-200 μmol / L) of naringin in the absence of secondary antibodies for 1 h. Kanamycin-resistant enhanced green fluorescent protein-labeled E. coli (EGFP-E. coli) was mixed with dHL-60 cells at a ratio of MOI = 10 and incubated on a thermostatic shaker for 15 min at 37°C, 100 rpm. After incubation, the cells were centrifuged at 300 x g for 10 min at 4°C, the supernatant was carefully aspirated, and the cells were resuspended and washed once with PBS by centrifugation at 300 x g for 10 min at 4°C. The supernatant was carefully aspirated, and the EGFP fluorescence was measured using FACS.

[0073] 1.11 Bactericidal assay

[0074] dHL-60 cells were pre-treated with / without 4 mmol / L N-acetyl cysteine (NAC) for 10 min, followed by treatment with different concentrations (0-100 μmol / L) of naringin in the absence of secondary antibodies for 1 h. Ampicillin-resistant E. coli was mixed with dHL-60 cells at a ratio of MOI = 10 and incubated on a thermostatic shaker for 2 h at 37°C, 100 rpm. After incubation, the cells were centrifuged at 300 x g for 10 min at 4°C, the supernatant was carefully aspirated, and the cells were resuspended and washed once with PBS by centrifugation at 300 x g for 10 min at 4°C. The supernatant was carefully aspirated, and 100 μl of freshly prepared 1% Triton X-100 (10 μl Triton X-100 + 990 μl ddH2O, mixed well) was added. The dHL-60 cells were lysed by vortexing for 20 s, and 900 μl of LB liquid medium with ampicillin was added. The mixture was vortexed, diluted 10^4, and plated. The plates were incubated overnight at 37°C, and the number of colonies was counted on the next morning.

[0075] 1.12 Laser confocal microscopy

[0076] To observe the colocalization of lysosomes and EGFP-E. coli, 6 x 10 5dHL-60 cells were grown in glass-bottomed dishes pre-coated with cell adhesion reagent. After treatment with Nar-free IMDM medium containing 25 μmol / L Nar for 1 h, EGFP-E. coli was added to dHL-60 cells at a MOI of 10 and incubated in a bacterial incubator for 15 min. After incubation, the original medium was aspirated and the cells were washed once with PBS. Then, 75 nmol / L LysoTracker Red DND-99 (dissolved in PBS containing HEPES) was added and incubated for 15 min. After staining, the original medium was aspirated and the cells were washed once with PBS. Then, PBS containing 20% FBS + HEPES was added and the cells were observed using a FV3000RS laser scanning confocal microscope. At least 3 random areas were selected for image acquisition.

[0077] 1.13 Lysosome quantification

[0078] For FACS assay, dHL-60 cells were treated with / without Nar-free IMDM medium containing Nar for 1 h, and then mixed with E. coli at a MOI of 10 and incubated for 15 min. After incubation, the cells were centrifuged at 300 x g for 10 min at 4 °C, and the supernatant was carefully aspirated. Then, 75 nmol / L LysoTracker Green DND-26 (dissolved in PBS containing HEPES) was added and incubated for 15 min. After staining, the cells were centrifuged at 300 x g for 10 min at 4 °C, and the supernatant was carefully aspirated. Then, the cells were resuspended and washed once with PBS, and resuspended with PBS containing 20% FBS + HEPES. The cells were then transferred to a new tube and the FL1 channel fluorescence was measured by FACS.

[0079] For LSCM assay, the experimental protocol was as described in 2.9. The number of lysosomes was represented by the average gray value of LysoTracker Red DND-99. According to the co-localization image information of lysosomes and EGFP-E. coli, the phagocytosis and digestion ability of dHL-60 cells were quantified.

[0080] 1.14 Neutrophil extracellular traps (NETs) induction

[0081] dHL-60 cells were plated at 4 x 10 5NETosis was induced by treating the cells in round-bottom EP tubes with complete IMDM medium containing 1 pg / mL P.g-LPS, or 1 pg / mL P.g-LPS + 100 pmol / L Nar for 1 h. After centrifugation (600 x g, 4°C, 5 min), the cells were resuspended in 400 pL PBS, and 200 pL of the cell suspension was placed on a glass slide, air-dried, and subjected to Wright-Giemsa staining. The entire slide was scanned using a CS2 scanning system, and the HALO specialized software was used for analysis (at least 20 random fields per group).

[0082] 1.15 Statistical analysis

[0083] GraphPad Prism software was used for statistical analysis and generation of p values. All data were expressed as mean ± standard error of the mean (SEM). When two groups were compared, an unpaired two-tailed t test was performed. For multiple group comparisons, one-way analysis of variance (one-way ANOVA) and Tukey’s pairwise comparison between groups were used. Statistical significance was set at p < 0.05.

[0084] Rat model:

[0085] Wistar rat model of pulpitis: After weighing, the rats were anesthetized by intraperitoneal injection, fixed, and the experimental teeth and surrounding tissues were disinfected with alcohol. A head-mounted magnifying glass was used for illumination to assist in opening the pulp, and the main pathogenic component P.g-LPS of the pathogen was filled into the pulp opening to induce pulp inflammation. This rat model can simulate acute pulp inflammation of healthy pulp caused by the pathogen P.g, and is suitable for evaluating drugs for relieving pulpitis.

[0086] Example 1, Effect of P.g-LPS on neutrophil function

[0087] 1. Isolation and identification of human peripheral blood-derived neutrophils

[0088] Neutrophils (hNeu.) were successfully isolated from healthy human peripheral blood by immunomagnetic bead sorting (MACS) method (see Section 1.4 for method). Under a microscope, typical lobed nuclei and intracellular granules were observed. Flow cytometry (FACS) results showed that the purity of hNeu. (CD45+CD16+CD66b+) was 99.4%.

[0089] 2. Effect of P.g-LPS on IL-6, IL-8 / CXCL8 secretion by neutrophils

[0090] During irreversible pulpitis, a large number of neutrophils rapidly respond and infiltrate the dental pulp, inducing pulp inflammation to fight bacterial invasion. Therefore, we first investigated whether lipopolysaccharide (LPS) from different bacterial sources could induce neutrophil inflammation.

[0091] The results are as follows Figure 2 As shown, after stimulating hNeu. with 1 μg / mL Pg-LPS for 24 h, hNeu. can secrete high levels of IL-6 and IL-8, and this phenomenon is also present in E.coli-LPS-induced neutrophils.

[0092] 3. Effects of Pg-LPS on the formation of extracellular trapping networks (NETs) of neutrophils

[0093] NET formation is a way for neutrophils to fight bacterial invasion. We observed that both Pg-LPS and E. coli-LPS can induce primary neutrophils (hNeu.) to produce NETs. Figure 3 ).

[0094] Considering the inter-individual variability and sensitivity of human peripheral blood-derived neutrophils (hNeu.), we further used neutrophil-like cells (dHL-60) as substitute cells. The method for inducing HL-60 cell lines to differentiate into dHL-60 cells is described in 1.5. After induction, the proportion of dHL-60 (CD11b+) reached over 97%. The effects of Pg-LPS on the induction of IL-6, IL-8 / CXCL8 production, and NETs formation in the dHL-60 cell line were consistent with those on hNeu.

[0095] 4. Effects of Pg-LPS on the phagocytic capacity of dHL-60 cells

[0096] After treating dHL-60 cells with 1 μg / mL Pg-LPS for 1 h, enhanced green fluorescent protein-labeled E. coli (EGFP-E. coli) was added at an MOI ratio of 10:1 and cultured for 15 min. The percentage of EGFP-positive (EGFP+) cells in dHL-60 cells was detected by flow cytometry (FACS). The results showed that 1 μg / mL Pg-LPS did not affect the phagocytic ability of dHL-60 cells.

[0097] 5. Effect of Pg-LPS on the bactericidal ability of dHL-60 cells

[0098] 1 μg / mL P.g-LPS treated dHL-60 cells for 1 h, then mixed with E. coli at a ratio of MOI = 10:1 for 2 h, the cell lysate was diluted 104 times and then spread on LB agar plates for colony forming unit (CFU) counting. The results showed that 1 μg / mL P.g-LPS did not affect the bactericidal ability of dHL-60 cells.

[0099] Example 2, P.g-LPS inhibits odontogenic differentiation of dental pulp fibroblasts

[0100] 1. Purification and identification of dental pulp fibroblasts

[0101] Primary dental pulp cells (hDPCs) isolated from dental pulp are a mixture of various cell types, among which the main cell type is dental pulp fibroblasts (hDPFs).

[0102] We further sorted hDPFs from hDPCs using the MACS method, and FACS detection showed that the purity of hDPFs (CD90+CD146+) was more than 97.1%.

[0103] hDPFs and non-hDPFs were induced using odontogenic differentiation media (ODM, complete a-MEM medium containing 10 mmol / L β-glycerophosphate sodium + 50 μg / mL ascorbic acid + 10 nmol / L dexamethasone) respectively, and after 24 days, alizarin red S staining showed that only hDPFs observed mineralization, indicating that DPFs in dental pulp are the main contributors to odontogenic differentiation. Figure 4 ).

[0104] 2. P.g-LPS inhibits odontogenic differentiation of dental pulp fibroblasts (hDPFs)

[0105] To explore the direct effect of P.g-LPS on the odontogenic differentiation ability of hDPFs, hDPFs were cultured with ODM ± P.g-LPS for 24 days during odontogenic induction.

[0106] Alizarin red S staining results show that Figure 5 ), indicating that 1 μg / mL P.g-LPS can inhibit the mineralization ability of hDPFs (inhibit the odontogenic ability of hDPFs).

[0107] 3. P.g-LPS-induced neutrophils inhibit odontogenic differentiation of dental pulp fibroblasts

[0108] In the inflammatory endodontic microenvironment, there is a potential interaction between hDPFs and P.g-LPS-activated neutrophils (i.e., indirect effect of P.g-LPS on DPFs). Therefore, we further evaluated the effect of P.g-LPS-treated dHL-60 supernatant on hDPF odontogenic differentiation. hDPFs were cultured with different treated dHL-60 cell supernatants and ODM (V / V = 1:1) for 24d, and the results of alizarin red S staining and quantification showed that P.g-LPS-treated dHL-60 supernatant reduced hDPF mineralization Figure 6 ), indicating that P.g-LPS-activated neutrophils also inhibited the mineralization (odontogenic differentiation) ability of hDPFs.

[0109] In the process of irreversible pulpitis, a large number of neutrophils rapidly respond and infiltrate the pulp, triggering inflammation of the pulp to fight against bacterial invasion. The main pathogenic bacteria of irreversible pulpitis include Porphyromonas endodontalis (P.e) and Porphyromonas gingivalis (P.g). Therefore, we first detected whether P.g-LPS could induce neutrophil inflammation, and the results showed that after P.g-LPS induction, dHL-60 cells exhibited similar cytokine secretion patterns and NET formation to hNeu., i.e., P.g-LPS could induce dHL-60 cells to secrete IL-6 and IL-8 / CXCL8 and form NETs.

[0110] In this study, we found that the phagocytic and bactericidal ability of P.g-LPS-activated dHL-60 cells did not change significantly. However, P.g-LPS-activated dHL-60 cells could secrete high levels of pro-inflammatory cytokines IL-6 and chemokines IL-8 / CXCL8. In the case of irreversible pulpitis, uncontrolled microbial infection can lead to a sharp and sustained increase in inflammatory cytokines, which can damage the odontogenic differentiation potential of the infected pulp.

[0111] In this study, we noticed

[0112] (1) P.g-LPS alone could inhibit hDPF mineralization.

[0113] (2) We established a simple in vitro experimental system to simulate the inflammatory endodontic tissue microenvironment and studied the effect of dHL-60 cell supernatant on hDPF odontogenic differentiation, and found that P.g-LPS-treated dHL-60 supernatant also inhibited hDPF mineralization.

[0114] (3) We also found that P.g-LPS-activated dHL-60 cells can produce a large amount of neutrophil extracellular traps (NETs). Unlike lysosomal intracellular digestion, NETs are a "suicide" extracellular antibacterial strategy that can kill pathogens at a distance outside the cell. During the inflammatory response of the dental pulp, although NETs can effectively neutralize and control the spread of bacteria, it is inevitable to damage the surrounding tissues.

[0115] Example 3, Effect of naringenin on the function of P.g-LPS-activated neutrophils

[0116] 1, Effect of naringenin-treated dHL-60 cell supernatant on odontogenic differentiation of dental pulp fibroblasts

[0117] This experiment mainly studied whether naringenin affected the interaction between P.g-LPS-activated neutrophils and hDPFs.

[0118] Based on the result that P.g-LPS-induced dHL-60 supernatant inhibited the mineralization ability of hDPFs (Example 2), we further explored the effect of Nar+P.g-LPS-treated dHL-60 supernatant on the mineralization ability of hDPFs. After dHL-60 cells were treated with different concentrations of Nar (0, 50, 100 μM) + 1 μg / mL P.g-LPS for 24 h, the supernatant and ODM (V / V = 1:1) were used to culture hDPFs for 24 d. The results of alizarin red S staining and quantification showed that the mineralization ability of the 100 μM Nar+1 μg / mL P.g-LPS-treated group was significantly enhanced compared with the 1 μg / mL P.g-LPS-treated group ( Figure 7 ),

[0119] which indicated that Nar+P.g-LPS-treated dHL-60 cell supernatant restored the impaired mineralization ability of hDPFs, suggesting that Nar must have played a regulatory role in dHL-60.

[0120] 2, Effect of naringenin on the activity of dHL-60 cells

[0121] (1) dHL-60 cells were treated with different concentrations of Nar (0-200 μM) for 24 h, and the results of CCK8 detection showed that 0-200 μM Nar did not affect the activity of dHL-60 cells.

[0122] (2) dHL-60 cells were treated with different concentrations of Nar (0-200 mM) for 1 h, and then mixed with EGFP-labeled E. coli at a MOI of 10:1 for 15 min. The results of FACS detection showed that 0-100 mM Nar could significantly promote the phagocytic ability of dHL-60 cells, and 50 mM Nar had the strongest pro-phagocytic ability. Figure 10 However, there was no such effect when dHL-60 cells were treated with P.g-LPS.

[0123] (3) dHL-60 cells were treated with different concentrations of Nar (0, 25, 50, 100 mM) for 1 h, and then mixed with E. coli at a MOI of 10:1 for 2 h. After diluting the cell lysate 10 4 times, LB agar plates were coated and CFU counting was performed. The results showed that 25 mM Nar could significantly enhance the bactericidal ability of dHL-60 cells (CFU value significantly decreased).

[0124] (4) P.g-LPS-induced dHL-60 cells were treated with different concentrations of Nar (0, 25, 50, 100 mM) for 1 h, and then an equal amount of cell suspension was prepared for smears. The results of Wright-Giemsa staining showed that 100 mM Nar could significantly reduce the formation of NETs in P.g-LPS-induced dHL-60 cells. Figure 8

[0125] (5) After P.g-LPS-induced dHL-60 cells were treated with different concentrations of Nar (0-200 mM) for 24 h, the supernatant was collected, and the results of ELISA detection showed that naringin could reduce the production of IL-6, IL-8 / CXCL8 in P.g-LPS-induced dHL-60 cells to some extent. Figure 9

[0126] The results of this example showed that naringin enhanced the phagocytosis and bactericidal function of neutrophil-like cells (dHL-60 cells), while reducing the formation of NETs and the release of inflammatory cytokines in dHL-60 cells, Mitigation of the adverse effects of P. gingivalis lipopolysaccharide (P.g-LPS) on hDPF odontogenic differentiation Figure 11 .

[0127] Example 4: Therapeutic effect of naringin on irreversible pulpitis in rats

[0128] The Wistar rat animal experiment scheme has been approved by the Experimental Animal Welfare Ethics Committee of Capital Medical University (ethics number AEEI-2017-125). After the rats were weighed, they were intraperitoneally injected with anesthesia, fixed, and the teeth and surrounding tissues were alcohol disinfected. The experiment was performed under the illumination of a head-mounted magnifying glass.​​

[0129] The experimental grouping and procedures are as follows:

[0130] (1) Pg-LPS group (n=6): After stimulating the dental pulp of rats with 1 mg / mL Pg-LPS for 3 days, no treatment was given for 3 days (equivalent to temporary sealing for 1 week after stimulation). The pulp of the left maxillary first molar (2M1) was opened with a BR49 bur (0.7 mm in diameter), and the pulp opening was approximately 0.25 mm. A sterile cotton ball soaked in 1 mg / mL Pg-LPS was placed at the pulp opening and stimulated for 5 minutes before being removed. The pulp was then temporarily sealed with medical gelatin sponge and glass ionomer cement, and isolated with Vaseline. No treatment was given 3 days later (day 4).

[0131] (2) Pg-LPS+Nar group (n=6): After 3 days of stimulation with 1mg / mL Pg-LPS, the rat was treated with Nar for 4 days. The right maxillary first molar (1M1) was opened with a BR49 bur, and a sterile cotton ball soaked in 1mg / mL Pg-LPS was placed at the pulp opening for 5 minutes to stimulate the rat pulp. The cotton ball was then removed, and the pulp was temporarily sealed with gelatin sponge and glass ionomer cement, and isolated with Vaseline. After 3 days (day 4), the temporary sealing material was removed, naringenin powder was placed, and the pulp was temporarily sealed with medical gelatin sponge and glass ionomer cement, and isolated with Vaseline.

[0132] (3) Normal group (n=6): The opposing normal teeth of the above two groups, the mandibular first molars (3M1 and 4M1). All rats in each group were euthanized on day 7 and their tissues were collected. The tissues were fixed in 10% neutral formalin buffer for 2 days, decalcified, and then prepared into sections.

[0133] The results of this study indicate that, compared to the Pg-LPS group, the Pg-LPS+Nar group (naringenin treatment group) showed a significant reduction in the extent of inflammatory cell and neutrophil infiltration. In the Pg-LPS group, inflammatory cell infiltration reached the mesial root canal orifice of the molar (…). Figure 11 A and B), and a large number of neutrophils with polymorphic segmented nuclei can be seen (MPO positive) Figure 11 C and D). In the Pg-LPS+Nar group, inflammatory cells were confined to the medullary horn (C and D). Figure 11 D and E), fewer neutrophils with pleomorphic segmented nuclei (MPO positive) Figure 12 F and L). Compared to the Pg-LPS group, the Pg-LPS+Nar group showed reduced neutrophil adhesion and migration to blood vessels. In the Pg-LPS group, more neutrophils (MPO-positive) were observed adhering to the vessel walls in the dental pulp microvessels and migrating into the pulp tissue surrounding the microvessels. Figure 12 A). In the Pg-LPS+Nar group, neutrophil vascular adhesion and migration were significantly reduced ( Figure 12B and C). Compared with P.g-LPS group, the small blood vessels in P.g-LPS+Nar group were significantly dilated. In P.g-LPS group, no significant dilation of the mid-coronal pulp microvessels was observed Figure 12 D). However, in P.g-LPS+Nar group, the mid-coronal pulp microvessels were significantly dilated, i.e. the density of the small blood vessels was increased, the range of the dilation was enlarged, and the dilation was close to the inflammatory cell infiltration area of the mid-coronal pulp Figure 12 E). The density of the pulp blood vessels in P.g-LPS+Nar group was significantly higher than that in P.g-LPS group ​ F).

[0134] In summary, the results show that, compared with P.g-LPS group, the range of the inflammatory cell and neutrophil infiltration in P.g-LPS+Nar group (naringenin treatment group) was significantly reduced, the neutrophil vascular adhesion and emigration was reduced, and the small blood vessels were significantly dilated, which can alleviate the hypoxia of the inflammatory pulp tissue and promote the repair of the inflammatory pulp.

[0135] Finally, it should be noted that the above examples are only used to help those skilled in the art to understand the essence of the present application, and are not used to limit the protection scope of the present application.

Claims

1. Use of naringenin in the preparation of a medicament for inhibiting bacterial infection of dental pulp.

2. Use of naringenin in the preparation of a medicament for treating irreversible pulpitis. The medicament comprises a therapeutically effective amount of naringenin and necessary pharmaceutical adjuvants.

2. Use according to claim 1, characterized in that, The medicament is an oral spray / powder, a dental patch, an oral gel or an oral paste.

3. Use according to claim 1 or 2, characterized in that, ​

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