CN114469946A

CN114469946A - Use of delamasil as a CXCL10 inhibitor

Info

Publication number: CN114469946A
Application number: CN202210171757.1A
Authority: CN
Inventors: 高孟秋; 乔敏; 逄宇; 李姗姗
Original assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Current assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Priority date: 2022-02-24
Filing date: 2022-02-24
Publication date: 2022-05-13
Anticipated expiration: 2042-02-24
Also published as: CN114469946B

Abstract

The invention relates to the technical field of medicines, in particular to application of delamanic to treatment of patients with multidrug-resistant tuberculosis. Use of delamasib in the preparation of a CXCL10 inhibitor. Use of delamanib and other CXCL10 inhibitors in the preparation of a medicament for the treatment of multi-drug resistant tuberculosis. The invention provides a new application of delaunay to the treatment of patients with multi-drug resistant tuberculosis, and delaunay can reduce the level of C-reactive protein of patients with multi-drug resistant tuberculosis and alleviate the inflammatory reaction of organisms. The delamanide can inhibit the expression of CXCL10 of multi-drug resistant tuberculosis patients, thereby reducing the accumulation of lymphocytes in focuses and relieving the inflammatory reaction of lungs.

Description

Use of delamasil as a CXCL10 inhibitor

Technical Field

The invention relates to the technical field of medicines, and particularly relates to application of delaminide as a CXCL10 inhibitor.

Background

Tuberculosis (TB) is one of the worldwide important causes of death and is still a major public health challenge today. The world health organization report in 2021 shows that about 1000 million new tuberculosis patients and about 149 million tuberculosis patients die in 2020. The treatment of multidrug-resistant tuberculosis (MDR-TB) patients is complicated and long in treatment period, and only about 59% of patients can obtain good treatment effect. The struggle against tuberculosis further goes into embarrassment since the end of 2019, with the widespread prevalence of new coronary pneumonia worldwide. Thus, there is a need for more potent antitubercular drugs.

Research has shown that antituberculosis drugs can affect the immune system by modulating the function of immune cells. Rifampicin impairs macrophage function by binding to aryl hydrocarbon receptors. However, bedaquiline enhances the body's ability to kill mycobacterium tuberculosis by increasing lysosomal activity and activating autophagy.

Delamanid (DLM) is a nitrodihydroimidazoloxazole drug that acts by inhibiting the synthesis of the cell wall of mycobacterium tuberculosis. DLM can effectively improve the negative conversion rate of sputum smears when patients with sputum smear positive MDR-TB are treated for 2 months, and is currently included in group C medicines for treating MDR-TB by WHO. Macrophages play an important role in resisting mycobacterium tuberculosis, and it is not clear whether DLM has influence on the immune system of the organism besides being capable of efficiently killing mycobacterium tuberculosis.

DLM has important practical significance on the action mechanism of macrophages and the exploration of new action effect on tuberculosis patients.

Disclosure of Invention

The invention provides a new application of delaunay to treatment of MDR-TB patients, which can relieve inflammatory reaction of organisms and local damage of lung tissues.

In order to achieve the purpose, the invention adopts the technical scheme that:

use of delamasib in the preparation of a CXCL10 inhibitor.

The invention also provides uses of delamanib and other CXCL10 inhibitors.

Use of delamanib and other CXCL10 inhibitors in the preparation of a medicament for the treatment of multi-drug resistant tuberculosis.

Further wherein the inhibitor of CXCL10 is an inhibitor of CXCL10 expression.

Further wherein the inhibitor of CXCL10 expression is ruxolitinib.

The invention provides a new application of delaunay to treatment of MDR-TB patients, and delaunay can reduce the level of C-reactive protein of multi-drug resistant tuberculosis patients and alleviate the inflammatory reaction of organisms. The delamanide can inhibit the expression of CXCL10 of multi-drug resistant tuberculosis patients, thereby reducing the accumulation of lymphocytes in focuses and relieving the inflammatory reaction of lungs.

According to the new application provided by the invention, Delamanib can reduce the inflammatory response of MDR-TB patients, and Delamanib brings greater benefit to patients with high CXCL10 expression.

Drawings

FIG. 1 is a comparison of C-reactive protein levels in two groups of humans.

FIG. 2 is a heat map of cytokine levels in two populations (a); (b) the level of expression.

FIG. 3 shows RT-qPCR and ELISA results.

FIG. 4 shows the results of migration experiments.

FIG. 5 shows the Western blot results.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.

Use of delamasib in the preparation of a CXCL10 inhibitor.

The invention also provides uses of delamanib and other CXCL10 inhibitors.

Further wherein the inhibitor of CXCL10 is an inhibitor of CXCL10 expression.

Further, wherein the inhibitor of CXCL10 expression is an siRNA capable of inhibiting CXCL 10.

Yet another CXCL10 inhibitor is ruxolitinib.

Use of delamanic as an inhibitor of the JAK2/STAT1 signaling pathway.

Use of delamasib in the manufacture of a medicament for the treatment of an infectious disease.

MDR-TB patients were screened for optimal basal therapeutic regimens (OBR) alone and for MDR-TB patients with OBR in combination with Delamanib (ORR + DLM) according to inclusion and exclusion criteria. The plasma of these patients was retained on

days

1, 14, 28, and 56 of treatment, respectively, and the levels of C-reactive protein were measured by immunoturbidimetry and CXCL10 expression was measured by flow cytometry. The results show that the ORB + DLM group C reactive protein is obviously reduced compared with the OBR group, and the difference has statistical significance. The expression level of CXCL10 in the ORB + DLM group is obviously reduced compared with that in the OBR group, and the difference has statistical significance. Thus, DLM can inhibit the expression of CRP and CXCL10 of multi-drug resistant tuberculosis patients and can reduce inflammatory injury.

In cell experiments, after the DLM acts on macrophages for 24 hours, the level of CXCL10 is obviously reduced compared with that of a control group. Indicating that in vitro experiments, DLM can inhibit macrophage from expressing CXCL 10.

In the migration experiment, the chemotactic effect of the DLM on human Peripheral Blood Mononuclear Cells (PBMC) is weaker than that of a control group, which indicates that the DLM inhibits the migration of lymphocytes after inhibiting the CXCL10 expression and reduces the local inflammatory reaction of a focus.

In Western blot experiments, DLM inhibited expression of CXCL10 by inhibiting the JAK/STAT signal pathway.

Example 1

Collection of MDR-TB patients based on inclusion and exclusion criteria

(1) Inclusion criteria were: age 18-64 years old; positive mycobacterium tuberculosis in sputum culture or sputum smear; ③ TB patients caused by infection of mycobacterium tuberculosis resistant to isoniazid and rifampicin, or patients positive in sputum smear and positive in rifampicin resistance quick test; and fourthly, the breast CT examination result proves that the tuberculosis is the pulmonary tuberculosis.

(2) Exclusion criteria: firstly, the medicine allergy history of nitro-imidazoles and derivatives thereof is available; ② patients with serious complications or impaired liver and kidney functions; the clinically significant electrocardiogram change exists; fourthly, the medicine has a clinical significant history of cardiovascular diseases or suffers from the diseases, such as heart failure, coronary heart disease, hypertension, arrhythmia and the like; fifthly, the patients have diseases that the nitroimidazole and the derivatives thereof are contraindicated; sixthly, the patient has clinically significant metabolic, gastrointestinal, nervous, mental or endocrine diseases, malignant tumors or other abnormalities; the subject is judged by the researcher to have any disease that is not suitable for taking part in the trial or that may not allow the patient to reliably take part in the overall course of the trial.

Collecting 5ml of morning fasting venous blood of the two groups of patients in blood collecting tubes containing EDTA anticoagulation on

days

1, 14, 28 and 56 after treatment, sending the blood to a laboratory as soon as possible under the condition of low-temperature preservation, centrifuging at 3000rpm for 10min, subpackaging the blood plasma and storing in a refrigerator at-80 ℃ for later use.

Example 2

Immunoturbidimetry for plasma C-reactive protein levels

(1) Two groups of patient plasma samples were added to a Beckman Coulter full-automatic biochemical analyzer, respectively.

(2) CRP values were read for each plasma sample.

A line graph was drawn using GraphPad Prism8 software, and the results are shown in FIG. 1, in which the abscissa represents time and the ordinate represents the protein expression amount of CRP. The results showed that CRP was significantly lower in the DLM-treated group than in the control group. In FIG. 1, the horizontal line represents the mean. + -. standard error, and the symbol denotes P < 0.05. Symbol denotes P < 0.01.

Detection of cytokine levels in plasma by flow cytometry

(1) The lyophilized standard spheres of CXCL10 and CCL2 were placed in the same 15ml centrifuge tube, and 4ml of Assay Diluent was added thereto by pipette, and the mixture was pipetted and mixed well, equilibrated at room temperature for 15min, and labeled Top standard 1, at which the concentration was 2500 pg/ml. The lyophilized standard spheres of IL-6, IL-8, IL-1 β, TNF- α and IFN- γ were placed in another 15ml centrifuge tube, 4ml of Assay Diluent was added thereto by pipette, blown and mixed well, and equilibrated at room temperature for 15min at a concentration of 200,000 fg/ml.

(2) To each of the 8 flow tubes, 500. mu.l of Assay Diluent was added by pipette, and the mixture was labeled 1:2, 1:4,1:8,1:16,1:32, 1:64,1:128 and 1: 256. After pipetting 500. mu.l of the well-balanced Top standard 1 and adding the well-balanced mixture into a flow tube labeled with 1:2, pipetting 500. mu.l of the well-balanced mixture into a flow tube labeled with 1:4, and performing concentration gradient dilution in the same manner until the flow tube labeled with 1:256 is reached, and preparing 1 flow tube with only 500. mu.l of Assay dilution as a negative control tube of 0 pg/ml.

(3) Another 7 flow tubes were labeled Top Standard 2,1:3,1:9,1:27,1:81,1:243 and 1:729, respectively. 460. mu.l of Assay Diluent were added to the flow tube labeled Top Standard 2, and 400. mu.l of Assay Diluent were added to the remaining 6 flow tubes, respectively. Pipette 40. mu.l of the Standard from the equilibrated 15ml centrifuge tube, add it to the flow tube labeled with Top Standard 2, and blow-mix it. And then sucking 200 mul from the flow tube marked with the Top Standard 2 by using a pipettor, adding the pipettor into the flow tube marked with the 1:3, blowing and mixing the pipettes uniformly, sucking 200 mul from the flow tube marked with the 1:3 by using the pipettor, adding the pipettes into the flow tube marked with the 1:9, and performing concentration gradient dilution in the same way until the flow tube marked with the 1:729 is obtained. Another 1 flow tube containing only 500. mu.l Assay dilution was prepared as a negative control tube of 0 fg/ml.

(4) And (3) fully and uniformly mixing the Capture beads of the CXCL10 and the CCL2 cytokines by using a micro vortex mixer for later use, and configuring the required Capture beads according to the total number of the samples. In consideration of the loss during the experiment, the amount of 2 to 3 samples may be used as appropriate. After the preparation according to the table 1.1, the mixture is fully mixed by a micro vortex mixer and is used after being incubated for 15min at room temperature.

TABLE 1.1 Capture Bead collocation method of CXCL10 and CCL2

Components	Amount of the composition used
		Capture Bead Diluent	48μl
CXCL10Capture Bead	1μl
		CCL2 Capture Bead	1μl

(5) The Capture beads of 5 cytokines IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma are fully and uniformly mixed by a micro vortex mixer, and the required Capture beads are configured according to the total number of samples according to the description in the table 1.2. In consideration of the loss during the experiment, the amount of 2 to 3 samples may be used as appropriate.

TABLE 1.2 Capture Bead collocation methods for IL-6, IL-8, IL-1 β, TNF- α and IFN- γ

Components	Amount of the composition used
		Capture Bead Diluent	15μl
IL-6Capture Bead	1μl
		IL-8Capture Bead	1μl
IL-1βCapture Bead	1μl
		TNF-αCapture Bead	1μl
IFN-γCapture Bead	1μl

(6) According to the table 1.3, PE Detection Reagent of two downward-shifting factors of CXCL10 and CCL2 is configured according to the total number of samples, and the prepared PE Detection Reagent is stored on ice in a dark place for later use.

Table 1.3 PE Detection Reagent configuration method of CXCL10 and CCL2

(7) According to the table 1.4, 5 cytokines, i.e., IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma, were prepared from the total number of samples by Human Detection Reagent (Part A), and then stored on ice in dark for further use.

TABLE 1.4 Human Detection Reagent (Part A) configuration method for IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma

(8) According to the total number of samples, Enhanced Sensitivity Detection Reagent (Part B) of 5 cytokines IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma was prepared, 550. mu.l Detection Reagent was used to resuspend the lyophilized Enhanced Sensitivity Detection Reagent (Part B), and the mixture was incubated at room temperature in the dark for 15 min. 4.5ml of Detection Reagent solution is added into a 15ml centrifuge tube, and 500. mu.l of incubated Part B is sucked into the 15ml centrifuge tube and mixed for standby.

(9) Preparing a flow tube for CXCL10 and CCL2 standard products and all samples, marking, and adding 50 mu l of standard products or samples with various concentrations which are diluted in a gradient way into each flow tube. After the prepared Capture Beads were vortexed for 5s, 50. mu.l of the mixture was added to each flow tube, gently mixed, and incubated at room temperature for 1 h.

(10) Preparing a flow tube for the standard products of 5 cytokines IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma and all samples, marking, and adding 50 mu l of standard products with various concentrations or samples diluted in a gradient way into each flow tube. And (3) vortexing the prepared Capture Beads for 5s, adding 20 mu l of the vortexes into each flow tube, gently mixing the vortexes, and incubating the vortexes at room temperature for 2h in a dark place.

(11) To each flow tube for Detection of CXCL10 and CCL2 was added 50 μ l each of PE Detection Reagent, mixed gently and incubated at room temperature for 2 h.

(12) 20 mu l of Part A is added into each flow tube for detecting 5 cytokines of IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma, the mixture is mixed evenly and gently, and the mixture is incubated for 2 hours at room temperature in a dark place.

(13) To each flow tube for detection of CXCL10 and CCL2 was added 1ml of Wash Buffer, 200g at room temperature, centrifuged for 5min, and the supernatant was discarded.

(14) 1ml of Wash Buffer was added to each flow tube for detecting 5 cytokines IL-6, IL-8, IL-1. beta., TNF-. alpha., and IFN-. gamma., and 200g was centrifuged at room temperature for 5min to discard the supernatant.

(15) Add 100. mu.l of Wash Buffer to each flow tube for CXCL10 and CCL2 detection, vortex and mix well before detecting with flow cytometer.

(16) Add 100. mu.l of Part B into each flow tube for detecting 5 cytokines IL-6, IL-8, IL-1 beta, TNF-alpha and IFN-gamma, mix them evenly, incubate for 1h at room temperature in the dark. 1ml of Wash Buffer was added to each flow tube at room temperature, 200g was centrifuged for 5min, and the supernatant was discarded. And adding 100 mu l of Wash Buffer into each flow tube, vortex and mixing uniformly, and detecting by a flow cytometer.

The cytokine expression level was heat mapped using the R software and the results are shown in fig. 2(a), where each panel represents a patient and the ordinate represents the expression level of each cytokine at each time point. The cytokine expression levels were statistically analyzed and plotted using Graphpad Prism8, and the results are shown in FIG. 2(b), in which the abscissa represents time and the ordinate represents the protein expression levels of the respective cytokines. The results showed that the level of CXCL10 was significantly lower in MDR-TB patients in the DLM-treated group than in the control group. In fig. 2, the horizontal line represents the mean ± sem, and the symbol represents P <0.05 and P < 0.01.

Example 3

1. Cell culture

(1) Wiping the ultra-clean workbench surface irradiated by ultraviolet for 30min with 75% alcohol.

(2) Placing a sterile centrifuge tube, a suction tube and a cell culture bottle in the superclean bench in sequence.

(3) Taking out the cell culture bottle, opening the bottle cap, and pouring the cell culture solution into a centrifuge tube.

(4) Centrifuge at 1000rpm for 5 min.

(5) The upper medium was aspirated off, the cell pellet was resuspended in 1ml fresh medium, mixed well and transferred to a new cell culture flask in diluted proportion.

(6) Placing the cell culture flask into a container containing 5% CO at 37 deg.C₂The time for changing the culture solution is determined by the growth speed of the cells.

(7) At1 × 10⁶Density of U937 cells/ml in 12-well plates, 1ml of complete medium containing PMA at a concentration of 10ng/ml was added to each well and placed at 37 ℃ with 5% CO₂Cultured overnight in an incubator to induce adherent macrophages.

(8) Cells were washed 2 times with PBS and DLM (0.3. mu.g/ml) was added to the complete medium, blanketed with the same volume of DMSO.

(9) After DLM or DMSO acts for 0-24h, the supernatant is reserved for ELISA detection, 1ml PBS is added, cells are gently scraped by a cell scraper and placed in a 1.5ml EP tube, centrifugation is carried out at 3000rpm for 5min, the supernatant is discarded, and cell precipitates are frozen in a refrigerator at-80 ℃ for standby.

3. Method for detecting CXCL10 gene expression by RT-qPCR method

(1) Extraction of RNA

1.1 Add beta-mercaptoethanol to a final concentration of 1% in solution RD and absolute ethanol at a ratio of 1:4 in solution RW.

1.2 the cell pellets obtained in the above step are dissolved on ice, 540. mu.L of RD solution is added into each tube, vortex and shake to mix evenly, if insoluble pellets appear, centrifugation is carried out at 12000rpm for 2min, and the supernatant is transferred to another centrifuge tube.

1.3 Add 700. mu.l of RB solution and mix until precipitation may occur. The resulting solution was transferred to a purification column (purification column + collection tube) together with the precipitate, centrifuged at 12000rpm for 60s, the waste liquid was decanted, and the purification column was returned to the collection tube.

1.4 Add 350. mu.l of solution RP to the purification column, centrifuge at 12000rpm for 1min, discard the waste and put the purification column back into the collection tube.

1.5 to the purification column is added 500 u l has added absolute ethanol solution RW, room temperature static 2min, 12000rpm centrifugation for 30s, discarded waste liquid, purification column placed back to the collection tube.

1.6 repeat the above steps.

1.7 the purification column is placed back into the collection tube, and the collection tube is continuously emptied at 12000rpm for 2min to remove the residual liquid, the purification column is placed at room temperature for a plurality of minutes, and the residual rinsing liquid in the adsorption material is completely dried.

1.8 transferring the purification column into a new RNA enzyme-free 1.5ml centrifuge tube, adding 30. mu.L of RNA enzyme-free water, standing at room temperature for 2min, centrifuging at 12000rpm for 2min to obtain RNA solution, and immediately performing reverse transcription to obtain cDNA.

(2) Reverse transcription

Reverse transcription was performed using Hifair II 1st strand cDNA synthesis supermix from Yeasen Biotech, Inc., 20. mu.L in total. The reaction system was prepared on ice as follows:

components	Amount of the composition used
		2*Hifair ^R II SuperMix	10μl
Template RNA	2ng
		RNase free dd H₂O	Adding to 20. mu.l

The prepared reaction system carries out the following reactions on a PCR instrument: 5min at 25 ℃; 45 deg.C, 30min, 85 deg.C, 5 min. After the reaction was complete, the reaction was stored at-20 ℃.

(3) Real-time fluorescent quantitative PCR

3.1 primer sequences

3.2 real-time fluorescent quantitative PCR detection of CXCL10

Using the cDNA obtained in the step (2) as a template, a kit of Yeasen Biotech was used

qPCR SYBR green master mix (Low Rox) detects the expression level of CXCL10 gene, and selects beta-actin as internal reference gene. The detection was carried out using ABI 7500 real-time fluorescent quantitative PCR instrument from Applied Biosystems. The reaction system is as follows:

the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 35s, 40 cycles.

3.3 data processing and statistical analysis

According to the result of real-time fluorescent quantitative PCR, the result is analyzed by ABI 7500v2.3 software, beta-actin gene is taken as an internal reference gene, and 2^-△△ctThe method of (4) calculates the relative expression amounts of CXCL10 genes in the DLM group and the DMSO group. The difference is statistically significant by adopting a two-factor analysis of variance method and taking P less than 0.05 as the difference. Histogram was generated using GraphPad Prism8 software, and the results are shown in fig. 3(a), in which the abscissa represents time and the ordinate represents relative expression. The results showed that the CXCL10 gene was expressed at a significantly lower level in the DLM-treated group than in the control group. In FIG. 3(a), the horizontal line represents the mean. + -. standard deviation, and the symbol represents P < 0.05. Symbol represents P<0.01。

4. Detection of changes in expression of CXCL10 by ELISA

(1) Taking out the batten required by the experiment from the sealed bag which is balanced to the room temperature;

(2) and (4) sucking 1ml of standard substance and specimen universal diluent into the freeze-dried standard substance, standing for 15min, and fully dissolving. The concentration in this case was 500 pg/ml.

(3) 8 1.5ml EP tubes were prepared and labeled. Adding 500 mul of standard substance and specimen universal diluent into each tube;

(4) by ddH₂O diluting 20 × concentrated wash to 1 ×;

(5) diluting the dissolved standard substance by times. Pipette 500. mu.l from the standard into the 1st EP tube, and pipette 500. mu.l into the 2 nd EP tube after mixing well. Up to the 7 th EP tube. The concentration gradients were 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.62pg/ml, 7.8pg/ml, 3.9pg/ml, 0pg/ml, respectively.

(6) Adding standard substance and universal diluent for the sample into the blank hole, adding the sample or standard substances with different concentrations (100 μ l/hole) into the other corresponding holes, sealing the reaction hole with sealing plate adhesive paper, and incubating in a dark place at 37 ℃ for 90 min;

(7) washing the plate for 5 times;

(8) adding biotinylated antibody diluent into the blank hole, adding 100 μ l of biotinylated antibody working solution into the rest holes, sealing the reaction holes with new sealing plate adhesive paper, and incubating in a 37 deg.C incubator for 60min in dark;

(9) washing the plate for 5 times;

(10) adding enzyme conjugate diluent into blank holes, adding 100 μ l enzyme conjugate working solution into the rest holes, sealing the reaction holes with new sealing plate adhesive paper, and incubating at 37 deg.C in dark for 30 min;

(11) washing the plate for 5 times;

(12) adding 100 μ l chromogenic substrate (TMB) into each well, and incubating in a 37 deg.C incubator for 15min in the dark;

(13) adding 100 μ l reaction stop solution into each well, mixing well, and measuring OD within 3min₄₅₀The value is obtained.

Histograms were generated using GraphPad Prism8 software, and the results are shown in fig. 3(b), in which the abscissa represents time and the ordinate represents the protein expression amount of CXCL 10. The results showed that CXCL10 protein was expressed at significantly lower levels in the DLM-treated group than in the control group. In FIG. 3(b), the horizontal line represents the mean. + -. standard deviation, and the symbol represents P < 0.05. Symbol denotes P < 0.01.

5. The influence of DLM on PBMC migration after the inhibition of CXCL10 expression is explored through a Transwell experiment

(1) Starving PBMCs with serum free medium 24 hours in advance to the upper chamber;

(2) placing macrophages in the lower layer of the chamber, treating the macrophages with DLM, DMSO, CXCL10 and AMG487 (antagonist acting on CXCL10 and CXCR 3) for 24h respectively, and then incubating with the upper chamber for 12 h;

(3) the number of cells penetrating the membrane of the chamber was observed using a cell imaging microplate detection system and the mobility was calculated.

The photograph was taken using a cell imaging microplate detection system, as shown in fig. 4 (a). Histograms were plotted using GraphPad Prism8 software, with results see fig. 4(b), where the abscissa represents different treatment regimes and the ordinate represents mobility. The results showed that the expression level in the DLM treated group was significantly lower than that in the control group. In FIG. 4(b), the horizontal line represents the mean. + -. standard deviation, and the symbol represents P < 0.05. Symbol denotes P < 0.01. Symbol denotes P < 0.0001.

6. Western blot experiment is used for exploring a mechanism for inhibiting CXCL10 of macrophage by DLM

And (3) stimulating the macrophages for 0h, 4h, 8h, 12h and 24h by using DLM and DMSO respectively, collecting cell precipitates, extracting protein, quantifying, and carrying out Western blot detection.

Extraction of macrophage total protein and determination of protein concentration by BCA method

(1) The cell lysate required for each sample was prepared as shown in the following table, and in consideration of the loss, it was prepared as much as necessary, and was thoroughly mixed and then placed on ice for use.

Components	Amount of the composition used
		NP-40	100μl
PMSF	1μl
		Protein phosphatase inhibitors	1μl
Cocktail	1μl

(2) To each 1.5ml EP tube containing the sample, 100. mu.l of the prepared cell lysate was added, mixed well by a pipette, and placed on ice to lyse the protein for 10 min.

(3) After sufficient lysis, at 4 ℃, 12000rpm, centrifugation was carried out for 10min, and the supernatant was transferred to a fresh sterile 1.5ml EP tube, and 5. mu.l each was diluted 20-fold with PBS.

(4) Preparing a working solution: based on the standard and the number of samples, 50 volumes of BCA reagent plus 1 volume of Cu reagent (50:1) were prepared as BCA working solution, which was mixed well.

(5) Diluting the standard substance: BSA standard (5mg/ml) was diluted 10-fold to 0.5 mg/ml. The standards were added to protein standard wells of a 96-well plate in 0. mu.l, 2. mu.l, 4. mu.l, 6. mu.l, 8. mu.l, 12. mu.l, 16. mu.l, 20. mu.l, and PBS was added to make up to 20. mu.l, with 2 replicates at each concentration setting.

(6) Samples were taken 20. mu.l each into sample wells of a 96-well plate, with 2 replicates for each sample.

(7) Add 200. mu.l BCA working solution into each well, and react at 37 ℃ for 15-30 min.

(8) A562nm of each well is measured by a multifunctional microplate reader, the average value of the A562 concentration of the standard group is used as the ordinate, the corresponding standard substance concentration is used as the abscissa, and a standard curve is drawn.

(9) And (4) calculating the concentration of each diluted sample according to a standard curve formula, and then calculating the protein concentration of the original sample.

(10) The lowest concentration sample was found and the concentration of the remaining samples was adjusted to match the lowest concentration sample with PBS.

(11) To each sample was added 5X protein loading buffer to give a final concentration of 1X.

(12) All samples were heated with a metal bath at 95 ℃ for 10 min.

(13) After heating, centrifuging at 2000rpm for 2min, and freezing at-80 deg.C for use.

Western blot detection of protein level changes

(1) Carefully cleaning the glass plate, the 15-hole comb and the bracket by using a detergent and ultrapure water, and naturally drying for later use.

(2) A sterile 50ml centrifuge tube is taken, separation gel is prepared firstly, and the preparation method of the 10% SDS-PAGE separation gel is as follows:

H₂O	5.9ml
		30% glue solution (29:1)	5.0ml
1.5M Tris-HCL(pH8.8)	3.8ml
		10％SDS	0.15ml
10% ammonium persulfate	0.15ml
		TEMED	0.006ml
Total volume	15ml

(3) Mixing the above materials, sucking the separation gel with a pipette, slowly adding into the glass plate along the gap to a proper height, adding 1ml isopropanol, flattening the gel surface, and standing at room temperature for 30 min. After the gel was solidified, the isopropanol was poured off, and the gel surface was blotted dry with filter paper.

(4) SDS-PAGE concentrated gel (5% Acrylamide) was prepared by the following method:

H₂O	4.1ml
		30% glue solution (29:1)	1.0ml
1.0M Tris-HCL(pH6.8)	0.75ml
		10％SDS	0.06ml
10% ammonium persulfate	0.06ml
		TEMED	0.006ml
Total volume	6ml

(5) Adding the mixed concentrated gel along the glass gap by using a pipettor until the gel overflows, vertically inserting a comb into the concentrated gel, standing at room temperature for 30min, and slightly pulling out the comb vertically upwards after the gel is solidified.

(6) And (3) mounting the glass plate filled with the glue on an electrophoresis device, placing the glass plate into an electrophoresis tank, adding 1 xSDS electrophoresis buffer solution, and enabling the electrophoresis solution to at least flow over a guide wire at the bottom of the electrophoresis tank.

(7) Loading: and (3) sucking 18 mu l of rainbow 180 broad-spectrum protein Marker and each sample by using a pipette, adding the rainbow 180 broad-spectrum protein Marker and each sample into a corresponding lane, and complementing the vacant lanes with 1 x protein loading buffer solution.

(8) Electrophoresis: after electrophoresis at 80V for 30min, the voltage was adjusted to 120V and electrophoresis was carried out for about 1 h. And (5) stopping electrophoresis in due time according to the Marker separation condition.

(9) After electrophoresis, the glass plate is taken down from the bracket, the two glass plates are pried, the concentrated glue on the two glass plates is cut off, and then the glass plates are soaked in a 1 multiplied membrane buffer solution.

(10) The PVDF membrane was activated in methanol for 1 min.

(11) The clamp, the spongy cushion and the filter paper for film transfer are fully soaked in a 1 multiplied film transfer buffer solution, and then the spongy cushion, the filter paper, the gel, the PVDF film, the filter paper and the spongy cushion are sequentially placed in the direction from the negative electrode to the positive electrode. Avoiding bubble formation during the placing process.

(12) After confirming that the placement direction was correct, the clips were closed and fastened, the clips were placed in an electric rotating bath, and 1 Xrotary membrane buffer was added thereto until the PVDF membrane was completely submerged. Connecting the anode and the cathode, placing the whole electric rotating device in an ice-water bath, and rotating for 1.5h at 80V.

(13) After the electrotransformation is finished, the PVDF membrane is cut according to the size of the target protein, marked, and sealed for 2 hours by using a sealing liquid at a constant temperature.

(14) The primary antibody was diluted to an appropriate concentration with a blocking solution (p-JAK 21: 500, JAK 21: 1000, p-STAT11:2000, STAT11: 1000, p-651: 1000, p-p 651: 1000, p-381: 1000, p-p 381: 500, beta-actin 1:5000), and the PVDF membrane was placed in a heat-sealable hybridization bag, and an appropriate amount of the diluted primary antibody solution was added, sealed after air bubbles were removed, and incubated overnight at 4 ℃.

(15) The PVDF membrane was removed with forceps and washed 3 times with 1 XTSST buffer for 10min each.

(16) Diluting the secondary antibody (1:10000) with a sealing solution, putting the PVDF membrane into a heat-sealable hybridization bag, adding a proper amount of diluted secondary antibody solution, discharging bubbles, sealing, and incubating at room temperature for 2 h.

(17) The PVDF membrane was removed with forceps and washed 3 times with 1 XTSST buffer for 10min each.

(18) Placing the PVDF membrane in an AX-II X-ray radiography dark box, mixing the ECL hypersensitive luminescent liquid A and the ECL hypersensitive luminescent liquid B according to the quantity of the PVDF membrane according to the ratio of 1:1, and uniformly dripping the mixture on the surface of the PVDF membrane by using a pipette.

(19) Pouring the developing solution and the fixing solution into two enamel plates respectively in a darkroom for standby, putting the film into an AX-II X-ray photographic cassette, fastening, photographing for different times according to different protein expression quantities, putting the film into the developing solution, and putting the film into the fixing solution after the film presents a clear strip. And finally, placing the film in a shady and ventilated place for natural drying.

The results, see figure 5(a), indicate that DLM inhibits the expression of macrophage CXCL10 by inhibiting the JAK2/STAT1 signaling pathway.

JAK/STAT pathway inhibition assay

Macrophages were treated with DLM, DMSO and JAK/STAT pathway inhibitor Ruxolitinib phosphate (Ruxolitinib phosphate) for 4h, 8h, and 24h, respectively, and cell supernatants were collected and assayed for CXCL10 expression levels by ELISA.

Histograms were generated using GraphPad Prism8 software and the results are shown in fig. 5(b), where the abscissa represents time and the ordinate represents CXCL10 protein expression level. The result shows that the ruxolitinib phosphate can also inhibit the expression of CXCL10 through a JAK/STAT signal channel, and the inhibition effect of the ruxolitinib phosphate is stronger than that of DLM. In FIG. 5(b), the horizontal line represents the mean. + -. standard deviation, and the symbol represents P < 0.05. Symbol denotes P < 0.001. Symbol denotes P < 0.0001.

Example 4

1. General condition analysis of two groups of people

A total of 23 MDR-TB patients were enrolled, with an OBR group of 10 and an OBR + DLM group of 13, and the general condition of both groups was found by analysis (table 1): the DLM + OBR group and the DLM group have no statistical difference in age, sex and body mass index.

TABLE 1 demographic and baseline clinical characteristics of OBR and OBR + DLM groups

2. C-reactive protein expression level of two groups of people

CRP expression levels in both groups of patients are shown in FIG. 1, and DLM + OBR group C reactive protein levels were lower in the OBR group at

weeks

4, 5, 6, and 8 (week 4: 6.15. + -. 6.07mg/dl, week 5: 3.72. + -. 2.70mg/dl, week 6: 2.81. + -. 2.16mg/dl, and week 8: 2.22. + -. 2.30 mg/dl). It is shown that DLM can reduce the expression level of CRP in MDR-TB patients, thereby reducing local inflammatory response.

3. Cytokine expression levels in both populations

On

day

14, 28 and 56 of treatment, patients with CXCL10 in the OBR + DLM group expressed significantly lower levels than in the OBR group. There was no statistical difference in the plasma levels of CCL2, IL-6, IL-8 and IL-1 β between the two groups of patients, with TNF- α and IFN- γ below the detection limits. Indicating that the DLM can inhibit the expression level of CXCL10 of MDR-TB patients. DLM may be more effective in treating MDR-TB patients with high CXCL10 expression.

4. Effect of DLM on CXCL10 secretion from macrophages in vitro experiments

When macrophages are treated by DLM and DMSO respectively for 24h, the gene expression level and the protein expression level of CXCL10 in the DLM group are lower than those in the control group. The DLM has an immunoregulation effect on macrophages, and is particularly characterized in that the expression level of CXCL10 can be reduced.

5. Functional verification of DLM after CXCL10 expression inhibition

After macrophages are treated by the DLM group, the migration capacity of the DLM group to PBMCs is obviously weaker than that of the control group, and the DLM can inhibit the accumulation of the PBMCs in local focuses and reduce local inflammatory response after the expression of CXCL10 is inhibited.

6. Mechanism for inhibiting CXCL10 expression by DLM

DLM inhibits p-JAK2 and p-STAT1 in a time-dependent manner, does not affect the expression of p-65 and p-38, and shows that the DLM inhibits CXCL10 expression through a JAK2/STAT1 signal channel.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are included in the protective scope of the present invention.

It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. Use of delamasib in the preparation of a CXCL10 inhibitor.

2. Use of delamanib and other CXCL10 inhibitors in the preparation of a medicament for the treatment of multi-drug resistant tuberculosis.

3. Use according to claim 2, characterized in that: wherein the inhibitor of CXCL10 is an inhibitor of CXCL10 expression.

4. Use according to claim 3, characterized in that: wherein the CXCL10 inhibitor is ruxolitinib.

5. Use of delamanic as an inhibitor of the JAK2/STAT1 signaling pathway.

6. Use of delamasib in the manufacture of a medicament for the treatment of an infectious disease.