CN110327322A - TLR4 inhibitor treats the application in healing of tooth extraction disorder remedies caused by BRONJ in preparation - Google Patents

Abstract

The invention discloses the new applications in the drug of TLR4 inhibitor healing of tooth extraction obstacle caused by preparation treatment diphosphonate jawbone necrosis, the present invention by the preventative use of TLR4 inhibitor in BRONJ mouse model after, mouse healing of tooth extraction speed and quality is obviously improved, the present invention is verified by experiments, the activation for inhibiting the macrophage TLR-4 signal path as caused by ZA can not only be tested in vitro using TLR4 inhibitor TAK-242, it can also promote the healing of tooth extraction of BRONJ, tooth extraction wound part sequestrum is reduced to be formed, therefore TLR-4 inhibitor can be used for preparing the preventive use drug or health care product of the healing of tooth extraction obstacle for BRONJ.

Description

TLR4 inhibitor in preparation of medicament for treating dental extraction wound healing disorder caused by BRONJ Applications

technical field

The invention belongs to the technical field of medicine, in particular to the application of a TLR4 inhibitor in the preparation of a medicament for the treatment of a tooth extraction wound healing disorder caused by bisphosphonate osteonecrosis of the jaw (BRONJ).

Background technique

Nitrogen-containing Bisphosphonates (NPBs) are widely used in patients with metastatic bone cancer, Paget's disease and osteoporosis. However, after receiving high-dose intravenous bisphosphonates such as Zoledronic Acid (ZA), patients are prone to develop BisphosphonateRelated Osteonecrosis of the Jaw (BRONJ). BRONJ refers to the phenomenon that maxillofacial wounds caused by tooth extraction, periodontitis or trauma after the use of bisphosphonates persist for more than eight weeks without the premise of radiotherapy. BRONJ is a rare but devastating disease in which 2/3 of cases occur in the mandible.

Previous studies have reported that inhibition of bone resorption, inhibition of angiogenesis, and vitamin D deficiency can lead to BRONJ. At present, the treatment of BRONJ is mainly based on anti-inflammatory conservative treatment. Surgical debridement treatment is mainly used in patients with poor conservative treatment effect and large area of bone necrosis, but in some cases, the disease will be aggravated.

Toll-like receptor 4 (Toll Like Receptors-4, TLR4), is a human gene. TLR 4 is a Toll-like receptor. It detects lipopolysaccharides on Gram-negative bacteria and therefore plays an important role in the activation of the innate immune system. As a member of the Toll-like receptor (Toll Like Receptors) family, TLR-4 is mainly involved in the regulation of systemic immune inflammatory response. The activation of its downstream pathway is closely related to the activation of the body's inflammatory response. The activation of the TLR4 signaling pathway can activate the activation of the downstream NF-κB signaling pathway to maintain a continuous inflammatory response in the body. As an important gene regulating natural systemic immune response, TLR-4 is mainly used to warn and control inflammation in the early stage of inflammation, and long-term and continuous activation of TLR-4 pathway can also cause damage to the body. It has been reported that this long-term TLR4-mediated inflammatory response will lead to poor bone healing.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of a TLR4 inhibitor in the preparation of a medicament for the treatment of dental extraction wound healing disorder caused by bisphosphonate osteonecrosis of the jaw (BRONJ) in view of the deficiencies in the treatment of BRONJ in the prior art.

The application of TLR4 inhibitor in the preparation of medicament for the treatment of dental extraction wound healing disorder caused by bisphosphonate osteonecrosis of the jaw (BRONJ). The inventor's experimental research found that TLR4 inhibitors can reduce the infiltration of M1 macrophages by inhibiting the activation of macrophage TLR4 signaling pathway, and ultimately improve the healing of BRONJ tooth extraction wounds.

Further, the TLR4 inhibitor is TAK-242. The molecular structural formula of TAK-242 is:

Further, the dosage form of the drug is a liquid preparation, which can be administered by local injection or tail vein.

Further, in the medicine, the concentration of the TLR4 inhibitor is 1 uM.

Further, the drug includes a TLR4 inhibitor and a composite sustained-release material.

Beneficial effects of the present invention: The present invention discloses a new use of TLR4 inhibitors in the preparation of medicines for the treatment of dental extraction wound healing disorders caused by bisphosphonate osteonecrosis of the jaw. The present invention uses TLR4 inhibitors prophylactically in BRONJ mice After entering the model, the healing speed and quality of tooth extraction wounds in mice are significantly improved. The use of the medicine of the present invention can not only inhibit the activation of macrophage TLR-4 signaling pathway caused by ZA in vitro experiments, but also promote the healing of tooth extraction wounds of BRONJ. To reduce the formation of local sequestrum in tooth extraction wounds, TLR-4 inhibitor (TAK-242) can be used to prepare preventive medicines or health care products for BRONJ's tooth extraction wound healing disorders.

Description of drawings

Figure 1 shows the western blot detection results of the expression of TLR4 signaling pathway specific markers CD86 and CD206 in macrophages treated with ZA;

Figure 2 shows the results of western blot detection of the activation of TLR-4 signaling pathway and its downstream key targets in macrophages treated with ZA;

Figure 3 shows the results of western blot detection of intracellular NF-κB nuclear translocation in macrophages treated with different concentrations of ZA;

Figure 4 shows the test situation of the healing of tooth extraction wounds in mice after receiving zoledronic acid injection;

Figure 5 is an immunofluorescence image of frozen tissue sections of tooth extraction wounds of mice after receiving zoledronic acid injection;

Figure 6 shows the healing of tooth extraction wounds in mice treated with TLR4 inhibitor TAK-242 after injection of zoledronic acid;

Fig. 7 is an immunofluorescence image of a frozen tissue section of a tooth extraction wound in a mouse to which TAK-242 was prophylactically administered after zoledronic acid injection.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The TLR-4 inhibitor (TAK-242) in the following examples was purchased from APExBIO Company, product number A3850, and the quantitative experiments in the following examples were set up to repeat the experiments three times, and the results were averaged.

In the following experiments, the involved culture medium and formula are as follows:

Macrophage conditioned medium (BMDM): DMEM high glucose (25mM, GIBCO company) + 10% fetal bovine serum + penicillin (100U/ml) + 100ng/ml recombinant mouse macrophage colony stimulating factor (M-CSF) (Peprotech Corporation).

Example 1 Experimental test of zoledronic acid promoting M1 polarization of macrophages.

In order to simulate the effect of ZA on macrophages in vivo, the inventors treated primary macrophages derived from mouse bone marrow with ZA (zoledronic acid, 10 μM, Zometa, Novartis, USA) for 24 hours, and then extracted the total cell protein and used Western blot detection of the polarity transition specific markers CD86 and CD206 expression.

Methods as below:

①Extraction of total cell protein

A. Take out the regular culture flask of cells, pour out the culture medium, add 3ml of 4 ℃ pre-cooled PBS solution (0.01mol/L, pH 7.4) to each bottle of cells, wash the cells by gently shaking for 1min, then discard the washing solution, Repeat the above operation three times, and invert the culture flask on absorbent paper to blot dry.

B. Add 10ul PMSF (100mM) to 1ml of lysis solution, shake well and place on ice, add 300ul of PMSF-containing lysis solution to each bottle of cells, ensure that the lysis solution is spread to the bottom of the entire culture flask, and place it on ice for lysis for 20min. The cells were fully lysed, and the ice box was shaken on a shaker.

C. After the lysis, use a clean scraper to scrape the cells on one side of the culture flask, and then use a pipette to transfer the cell debris and lysate to a pre-cooled 1.5ml centrifuge tube.

D. The collected lysate containing cell debris was sonicated for 1 min using an ultrasonic cell disrupter.

E. Centrifuge the centrifuge tube at 12000rpm for 5min at 4°C.

F. Transfer the supernatant after centrifugation into 0.5ml centrifuge tubes to obtain total protein.

② The total protein extracted above was subjected to protein quantification (Braford method), and 5×Loading Buffer (protein loading buffer, Biyuntian) was added in a volume ratio of 4:1, mixed well, boiled for 5 minutes, aliquoted and stored at -70°C , avoid repeated freezing and thawing.

Determination of protein content:

A. Make standard curve: Take out 1mg/ml BSA from -20℃, melt at room temperature, and set aside. Add various reagents to each tube according to the table below, and after mixing, place at room temperature for 2 minutes, perform colorimetric analysis on a biospectrophotometer, and calculate and make a standard curve equation.

B. Absorb 10ul sample protein respectively, add 10ul 0.15mol/L NaCl to 1ml G250 Coomassie brilliant blue solution and shake well, perform colorimetric analysis on a biospectrophotometer to obtain the absorbance value, and substitute it into the standard curve to calculate the sample protein content .

③Sampling and electrophoresis: prepare polyacrylamide separating gel and polyacrylamide stacking gel.

Pour the separating glue evenly between the glass plates, add 2/3 of the height, and level the liquid level with double-distilled water; after the separating glue solidifies, pour out the double-distilled water, dry it with absorbent paper, add the concentrated glue, and insert it immediately. Comb; after the gel is completely coagulated, put the glass plate into the electrophoresis tank, add electrophoresis buffer, add 20ug of total sample protein to each lane, calculate the corresponding sample volume, add 10ul to the protein marker, and keep the stacking gel at a constant pressure of 60V, and separate gel electrophoresis at 80V constant voltage.

④ Transfer film

Before taking out the electrophoresis gel, soak the sponge and filter paper in the transfer buffer for 10 minutes; cut the gel and spread the gel on the filter paper, cover the front of the infiltrated PVDF membrane on the gel, remove the air bubbles between them, and put the sponge into the gel. Clamp it with filter paper, put it into an electrophoresis tank, add electrophoresis buffer, and transfer the membrane under a constant current of 300 mA in an ice bath for 75 min.

⑤ Closed

Take out the PVDF membrane, face up, rinse with PBST 3 times for 5 min at room temperature to remove SDS on the PVDF membrane, put the PVDF membrane in 2.5g/50ml PBST prepared skim milk, shake on a shaker, and block at room temperature for 2h.

⑥ Antibody hybridization

A. Pack PVDF membrane with parafilm, add TβR1 (1:1000 skim milk dilution), TβR2 (1:1000 skim milk dilution), mouse-derived β-actin monoclonal antibody (1:1000 skim milk dilution) 1ml to block, Incubate overnight at 4°C.

B. Rinse the PVDF membrane 3 times with PBST for 10 min, bag the PVDF membrane with parafilm, add 1 ml of goat anti-rabbit IgG (1:10000) and 1 ml of goat anti-mouse IgG (1:10000) at 37°C and incubate for 1 h at 37°C. The PVDF membrane was rinsed 10 times for 10 min.

⑦Detection

Add chemiluminescence substrate (Millipore company) A:B=1:1, dark room exposure, dark box closed development, fixing, washing, drying, scanning the film.

Figure 1 shows the western blot detection results of the expression of TLR4 signaling pathway specific markers CD86 and CD206 in macrophages treated with ZA, wherein, Figure 1A is the western blot picture, Figure 1B is the quantitative statistical result of Figure A, * indicates p<0.05. It can be seen that after adding ZA to treat macrophages, the total protein of macrophages showed high expression of CD86 and low expression of CD206. This indicated that ZA induced M1 polarization in macrophages.

Example 2 Experimental test of activation of TLR4 signaling pathway in macrophages treated with ZA.

The inventors detected the activation of TLR-4 signaling pathway and its downstream key targets in macrophages treated with ZA (10 μM, Zometa) for 24 hours, and the method was the same as that of Example 1.

Figure 2 shows the western blot detection results of the activation of TLR-4 signaling pathway and its downstream key targets in macrophages treated with ZA. In Figure 2, Figure 2A is the western blot chart, and Figure 2B is the quantitative statistical results of Figure 2A. * means p<0.05, ** means p<0.01. It can be seen that ZA-treated macrophages promoted the expression of TLR-4, MYD88, TRIF, and IRAK-4 at the protein level. It indicated that the treatment of ZA could activate the TLR-4 signaling pathway.

Example 3 Experimental test that zoledronic acid promotes nuclear translocation of NF-κB in macrophages.

Macrophages were treated with different concentrations of ZA (5μM, 10μM, 15μM) for 30min, and then the nuclear protein was extracted. Western blot was used to detect the nuclear translocation of NF-κB in macrophages after being stimulated by zoledronic acid.

The method of extracting nuclear protein is as follows:

a. Take the treated cells, centrifuge at 4°C, 500×g for 3 min to collect the cells, discard the culture medium, and wash twice with pre-cooled PBS;

b. Aspirate the supernatant as much as possible without leaving any residual liquid. Add 200 μL of pre-cooled Buffer A to each flask of cells (add 1 μL of DTT, 5 μL of 100mM PMSF, 5 μL of protease inhibitor to each mL of Buffer A, prepared before use), and vortex at maximum speed. Shake vigorously for 15s and place on ice for 10-15min;

c. Add 11 μL of cold Bμffer B, vortex vigorously for 5 s at maximum speed, and place on ice for 1 min;

d. After vortexing vigorously for 5s at maximum speed again, centrifuge at 4°C, 16000g, 5min;

e. Transfer the supernatant to another pre-cooled clean microcentrifuge tube as soon as possible and place it on ice to obtain cytoplasmic protein;

f. Add 100 μL of pre-chilled Buffer C to the centrifuge pellet (add 1 μL DTT, 5 μL 100 mM PMSF, 5 μL protease inhibitor for each mL of Buffer C, prepared before use), vortex vigorously for 15 s at maximum speed, and place on ice for 40 min at intervals. Vortex vigorously for 15s for 10min;

g. Centrifuge at 4°C, 16000g, 10min, transfer the supernatant to a pre-cooled clean microcentrifuge tube as soon as possible to obtain nucleoprotein;

h. Perform protein quantification (Braford method) on the cytoplasmic and nuclear proteins extracted above, add 5× protein loading buffer by volume, mix well, boil for 5 minutes, aliquot and store at -70°C to avoid repeated freezing and thawing.

Figure 3 shows the results of western blot detection of the nuclear translocation of NF-κB in macrophages treated with different concentrations of ZA, Figure 3A shows the results of P65 expression in the nucleoprotein of macrophages, and Figure 3B shows the cytoplasm of macrophages protein results. * indicates p<0.05. ** indicates p<0.01. It can be seen that the stimulation of zoledronic acid increases the expression of P65 in the nucleus of macrophages, while the phosphorylation level of P65 in the cytoplasm increases, and IκB is degraded. This indicates that zoledronic acid can activate the NF-κB pathway in macrophages and promote the nuclear translocation of NF-κB in macrophages.

Example 4 Experimental test of zoledronic acid causing delayed healing of dental extraction wounds in mice.

The inventors used a mouse model to simulate the healing of a patient's tooth extraction wound after receiving zoledronic acid injection. The mice were given tail vein injection of zoledronic acid (125 μg/kg, Zometa) twice a week for a total of four weeks. The left maxillary first molars of the mice were extracted after the first week of injection. The specific mouse BRONJ model was induced and constructed as follows:

(1) C57/B6J mice aged 4-6 weeks were divided into two groups, ten mice in each group. ZA was injected into the tail vein at a dose of 300 mg/kg twice a week.

(2) After the ZA injection in the first week, the maxillary first molars of the mice were extracted.

(3) Then inject the drug for three consecutive weeks.

(4) Micro-CT was taken at the third and fifth week of modeling to observe the healing of tooth extraction wounds in mice.

(5) The mice were sacrificed in the fifth week, and their maxillary bones were obtained and fixed with 4% paraformaldehyde for the study of subsequent experiments.

Figure 4 is the test of the healing of tooth extraction wounds in mice after receiving zoledronic acid injection; Figure 4A is the micro-CT image of the maxillary tooth extraction wound in mice, 3W represents 3 weeks, 5W represents 5 weeks, and Figure 4B is a mouse tooth extraction Statistical results of wound bone density, Fig. 4C shows the statistical results of the volume fraction of the tooth extraction wound. # means p>0.05, * means p<0.05, ** means p<0.01. It can be seen that the left upper first molar of the mice was extracted one week after receiving zoledronic acid injection, and the mice in the zoledronic acid injection group appeared to be more severe in the 3rd and 5th weeks after the extraction wound was healed compared with the mice in the PBS injection group. Delayed healing of extraction wounds.

Example 5 Zoledronic acid induces increased M1 polarization in mouse tooth extraction wound macrophages.

To further clarify the role of zoledronic acid in macrophages in mouse tooth extraction wounds. The inventors detected the polarity transformation of macrophages around the tooth extraction wound of the mice in Example 4 by using the technique of frozen section immunofluorescence. The specific steps for immunofluorescence of frozen tissue sections are as follows:

(1) Obtain fresh mouse maxilla and the soft tissue around the extraction wound and fix with 4% paraformaldehyde;

(2) Dehydrate the soft tissue around the tooth extraction wound in mice with 30% sucrose solution at room temperature overnight;

(3) The dehydrated soft tissue around the tooth extraction wound of the mouse was embedded with OCT, and sliced in a low temperature environment, and the slices were stored at -80°C;

(4) Take the section and place it in PBS at room temperature to dissolve OCT;

(5) Wash the slices three times by shaking slowly on a PBS shaker, 10 min each time;

(6) Wash the sections with PBST (containing 0.2% Triton) for 10 min;

(7) Goat serum was blocked for 30min;

(8) Repeat step 5;

(9) Add primary antibody and incubate at 37°C for 2 hours in the dark, or overnight at 4°C in the dark;

(10) Repeat step 5;

(11) Add the diluted fluorescent secondary antibody and incubate for 1 h;

(12) repeat steps;

(13) DAPI staining for 90s;

(14) Repeat step 5;

(15) Mount the slide with anti-fluorescence quenching mounting medium.

Figure 5 is the immunofluorescence image of the frozen tissue section of the tooth extraction wound of the mice after receiving zoledronic acid injection. In the figure, Veh represents the PBS injection group, ZA represents the zoledronic acid injection group, and Veh represents the tooth extraction wound of the control group. ZA is the tooth extraction wound of the mice in the zoledronic acid injection group, M1Macs are M1 macrophages, and M2Macs are M2 macrophages. It can be seen that after the injection of zoledronic acid, the soft tissue macrophages around the tooth extraction wound in the mice M1 polarization increased and M2 macrophage polarization decreased.

Example 6 TLR4 inhibitor (TAK-242) treatment of delayed healing of dental extraction wounds caused by zoledronic acid test.

To explore the clinical effect of TLR-4 as a target for the prevention and treatment of delayed healing of dental extraction wounds caused by zoledronic acid. The inventors administered the TLR4 inhibitor TAK-242 (300 mg/kg) to the mice one week before the injection of zoledronic acid, and then administered the tail vein injection of zoledronic acid (125 μg/kg, Zometa) twice a week. times, for a total of four weeks. The left maxillary first molars of the mice were extracted after the first week of injection. The healing of tooth extraction wounds in mice was assessed by micro-CT.

Figure 6 shows the healing of tooth extraction wounds in mice treated with TLR4 inhibitor TAK-242 after zoledronic acid injection. 242 mice, Figure 6A is the micro-CT image of the maxillary tooth extraction wound in mice, 3W means 3 weeks, 5W means 5 weeks; Figure 6B is the statistical results of the bone density of the tooth extraction wounds in mice, and Figure 6C is the statistical results of the volume fraction of the tooth extraction wounds ;* means p<0.05, ** means p<0.01, *** means p<0.001. It can be seen that prophylactic use of TAK-242 can significantly alleviate the delayed healing of dental extraction wounds caused by zoledronic acid.

Example 7 Test of TLR4 inhibitor (TAK-242) reducing M1 macrophage polarization by zoledronic acid.

To verify the effect of a small molecule inhibitor of TLR4 (TAK-242) on the polarization of macrophages in mouse tooth extraction wounds. The inventors used the method of frozen section immunofluorescence to detect the polarity transformation of macrophages in tooth extraction wounds of mice using TLR-4 small molecule inhibitor (TAK-242) prophylactically. The specific operation of immunofluorescence of frozen tissue sections is described in Example 5.

Figure 7 is the immunofluorescence image of the frozen tissue section of the tooth extraction wound of the mice that received zoledronic acid injection after prophylactic use of TAK-242, Veh represents the PBS injection group, ZA represents the zoledronic acid injection group, and Veh represents the control The tooth extraction wound of the mice in the group, ZA is the tooth extraction wound of the zoledronic acid injection group, M1Macs are M1 macrophages, M2Macs are M2 macrophages, # means p>0.05, * means p<0.05. ZA+PBS indicated that the mice were normally induced with ZA for the BRONJ model, and ZA+TAK-242 indicated that the mice were given prophylactic TAK-242 one week before the induction of the model. Figure 7A represents the frozen section immunofluorescence photograph; 7B represents the statistics for the proportion of M1 type macrophages; 7C represents the statistics for the proportion of M2 type macrophages.

It can be seen that the infiltration of M1-type macrophages around the jawbone extraction wound of the TAk-242-treated mice was significantly reduced compared with the mice injected with ZA alone, while the M2-type macrophages showed no obvious difference in infiltration. This indicates that excessive M1 macrophage infiltration by ZA around tooth extraction wounds in mice was alleviated after prophylactic administration of TAK-242.

Claims (5)

1. Application of a TLR4 inhibitor in the preparation of a medicament for the treatment of dental extraction wound healing disorder caused by BRONJ. 2. The use according to claim 1, wherein the TLR4 inhibitor is TAK-242. 3. The application according to claim 1, wherein the dosage form of the medicine is a liquid preparation. 4. The application of claim 1, wherein in the medicine, the concentration of the TLR4 inhibitor is 1 uM. 5. The use according to any one of claims 1 to 4, wherein the drug comprises a TLR4 inhibitor and a composite sustained-release material.