CN113440513B - Application of small molecular compound honeysuckle biflavone D - Google Patents

Abstract

The invention relates to an application of NRF2 protein inhibitor in preparing a medicine for treating diseases induced by infection of mycobacterium tuberculosis, which takes NRF2 protein as a target to inhibit the growth of mycobacterium tuberculosis in macrophages. The invention discloses an important role of an NRF2-SOD2 signal channel in the regulation of mycobacterium tuberculosis survival by macrophages, and also provides a small molecular compound Japoflavanone D capable of regulating the signal channel in a targeted manner.

Description

Application of small molecular compound honeysuckle biflavone D

Technical Field

The invention relates to the technical field of medicines, and in particular relates to application of a small molecular compound honeysuckle biflavone D.

Background

Tuberculosis is a chronic infectious disease caused by infection with mycobacterium tuberculosis. The number of tuberculosis attacks is over 1000 thousands of cases and the number of tuberculosis deaths is over 160 thousands of cases every year in the world, and the tuberculosis attacks are one of ten causes of death in the world for many years. Although the success rate of the existing first-line tuberculosis treatment drugs such as isoniazid, rifampicin, pyrazinamide and the like on tuberculosis caused by sensitive tubercle bacillus reaches 90%, the tuberculosis still recurs in about 3-9% of cases at present due to long treatment course (6-9 months), patient compliance, drug toxicity and side effects and the like. More importantly, the success rate of the existing drugs for treating the multi-drug resistant tuberculosis (MDR-TB) is less than 50 percent, and the patients face the serious challenge of 'no drug cure'. And the increase in multidrug-resistant tuberculosis cases from 250,000 cases per year in 2009 to 490,000 cases per year in 2016, is approximately 95%. Therefore, the search of an effective new scheme for treating the multi-drug resistant tuberculosis is a difficult problem to be solved urgently in clinic.

Currently, the anti-tuberculosis treatment scheme for MDR-TB mainly comprises the following aspects: firstly, the existing antituberculosis drugs are recombined for use. However, the method greatly prolongs the treatment time of the patient and greatly increases the treatment cost. Secondly, research and development of novel antituberculosis drugs, such as Delamanid, Bedaquiline and the like. But because of high biological toxicity, the compound can not be used as an anti-tuberculosis medicament alone, and the use condition is harsh. Moreover, these new drugs, although acting on different mechanisms, act essentially directly on the tubercle bacillus itself, and therefore there is always a risk of cross-resistance. And thirdly, cell immunotherapy. Although the application of immune preparations for adjuvant therapy of drug-resistant tuberculosis has been reported to achieve good curative effects in recent years, the overall immune therapy has the disadvantages of inaccurate curative effect, high cost and large difficulty in large-scale clinical application.

Because the existing multi-drug resistant tuberculosis treatment scheme still has a plurality of defects, tuberculosis Host-directed therapy (HDT) is receiving more and more attention. At present, all antituberculosis drugs directly act on tubercle bacillus, so that the risk of cross drug resistance exists, and the effect of host immunity on antituberculosis is ignored. In all tubercle bacillus infected people, more than 90% of people do not develop tuberculosis and become latent infected, which indicates that host immunity can effectively eliminate tubercle bacillus infection. HDT is of great value not only in drug-sensitive tubercle bacillus, but also in the treatment of multidrug-resistant tubercle bacillus. Therefore, improving the host anti-tuberculosis immunity is an important idea for developing a multi-drug resistant tuberculosis treatment scheme.

Disclosure of Invention

The invention aims to provide application of a small molecular compound honeysuckle xanthone Japoflavanone D aiming at the defects in the prior art.

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

provides an application of NRF2 protein inhibitor in preparing medicine for treating diseases induced by infection of mycobacterium tuberculosis, which uses NRF2 protein as a target to inhibit the growth of mycobacterium tuberculosis in macrophages.

Preferably, the NRF2 protein inhibitor comprises one or more of honeysuckle biflavone Japoflavone D or pharmaceutically acceptable salts, solvates, hydrates and prodrugs thereof.

Preferably, the diseases induced by infection with mycobacterium tuberculosis include tuberculosis, intestinal tuberculosis, tuberculous peritonitis, tuberculous meningitis.

Preferably, the medicament is a solid formulation or a liquid formulation.

Preferably, the solid preparation comprises tablets, capsules, granules, pills, dripping pills, mixture and powder; the liquid preparation comprises injection, oral liquid, soft capsule and aerosol.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention discloses an important role of an NRF2-SOD2 signal channel in the regulation of mycobacterium tuberculosis survival by macrophages, and also provides a small molecular compound Japoflavone D capable of regulating the signal channel in a targeted manner.

Drawings

In FIG. 1 (a) is the chemical structure of Japoflavanone D; (b) effect on macrophage viability for 72 hours of treatment with different concentrations of Japoflavone D; (c) influence on the extracellular growth of tubercle rods after treatment of Japoflavone D with different concentrations for 72 hours; (d) effect of treatment with different concentrations of Japoflavone D for 72 hours on the growth of mycobacterium tuberculosis within macrophages; (e) the effect of treatment with different concentrations of Japoflavone D on phagocytosis of mycobacterium macrophagus by macrophages;

FIG. 2 (a-b) is a graph showing the effect of Japoflavone D treatment for 24 hours on apoptosis of tubercle bacillus-infected macrophages; (c) effect of Japoflavone D treatment for 24 hours on caspase3/7/8 activity in tubercle bacillus infected macrophages; (d) detecting the influence of 24-hour treatment of the Japoflavone D on the shearing of caspase3 in the mycobacterium tuberculosis infected macrophage for Western blot; (e) detecting the influence of the 24-hour treatment of the Japoflavone D on mitochondrial membrane potential in the macrophage infected by the mycobacterium tuberculosis for Western blot; (f) effects of zVAD treatment for 24 hours on Japoflavone D in promoting macrophage clearance of Mycobacterium tuberculosis;

FIG. 3 (a) is a Western blot to detect the effect of Japoflavone D treatment for 24 hours on AKT/mTOR signaling pathway in tubercle bacillus infected macrophages; (b) detecting the effect of Japoflavone D treatment for 24 hours on MAPK signaling pathway in tubercle bacillus infected macrophages for Western blot; (c) effect of 24 hour treatment of Dor on japoflavanone D-induced caspase3 shear in mycobacterium tuberculosis infected macrophages; (d) effect of Dor treatment for 24 hours on Japoflavone D-induced apoptosis of macrophage cells infected with mycobacterium tuberculosis; (e) the 24 hour treatment of Dor on Japoflavone D promoted macrophage clearance of mycobacterium tuberculosis;

FIG. 4 (a-b) is a graph of the effect of Japoflavone D treatment for 6 hours on ROS levels in tubercle bacillus infected macrophages; (c-D) effects of Japoflavone D treatment for 24 hours on ROS levels in tubercle bacillus infected macrophages; (e) effect of NAC treatment for 24 hours on Japoflavone D-induced cleavage of caspase3 in mycobacterium tuberculosis-infected macrophages; (f) effect of NAC treatment for 24 hours on Japoflavone D-induced apoptosis of tubercle bacillus infected macrophages; (g) effect of 24 hours of NAC treatment on Japoflavone D promoting macrophage clearance of mycobacterium tuberculosis;

FIG. 5 (a) is a qPCR assay to examine the effect of Japoflavone D treatment for 24 hours on the increase in expression of SOD2 induced by Mycobacterium tuberculosis; (b) detecting the influence of the 24-hour treatment of the Japoflavone D on the expression increase of the SOD2 induced by the mycobacterium tuberculosis for Western blot; (c) detecting the influence of the Japoflavone D treatment for 24 hours on the expression increase of the SOD2 induced by the tubercle bacillus; (d) the influence of Japoflavanone D on the stability of SOD2 protein; (e) is the effect of Japoflavanone D on the degradation of SOD2 protein; (f) the effect of 24 hours of MTO treatment on japoflavanoned in promoting macrophage clearance of mycobacterium tuberculosis;

FIG. 6 (a) is a qPCR assay to examine the effect of Japoflavone D treatment for 24 hours on transcription factor expression levels; (b) detecting the influence of 24-hour treatment of the Japoflavone D on the expression level of the transcription factor for Western blot; (c) detecting the influence on the nuclear entry of the transcription factor for Western blot; (d) detecting the effect of Japoflavone D treatment for 24 hours on NRF2 nuclear entry for immunofluorescence; (e) effect of Japoflavone D treatment for 24 hours on binding of NRF2 to SOD2 promoter region; (f) the effect of Lut treatment for 24 hours on the increase in the expression of SOD2 induced by mycobacterium tuberculosis was examined for qPCR; (g) detecting the influence of 24-hour Lut treatment on the increase of the expression of SOD2 induced by the tubercle bacillus for Western blot; (h) effect of treatment with different concentrations of Japoflavone D for 72 hours on the growth of mycobacterium tuberculosis within macrophages;

FIG. 7 is a graph showing molecular docking predictions for binding of Japoflavone D to NRF2/KEAP1 protein;

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without inventive exercise, are within the scope of the present invention.

It should be noted that the experimental methods in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available from the public unless otherwise specified; the embodiments and features of the embodiments of the invention may be combined with each other without conflict.

The invention is further described with reference to the following figures and specific examples, which are not intended to be limiting.

Example (b): study of Japoflavone The in vitro and in vivo antitubercular activity and the action mechanism are clarified.

(I) an experimental method

1) Bacterial strains and culture conditions

Mycobacterium tuberculosis strains H37Ra, H37Rv and GFP-labeled H37Ra used in this study were cultured in Middlebrook 7H9 broth (BBL Microbiology Systems) supplemented with 10% oleic acid-albumin-glucose-catalase (OADC; Becton, Dickinson), 0.05% Tween80 and 0.2% glycerol. After 5 to 7 days at 37 ℃ with shaking, the bacteria were resuspended in serum-free RPMI medium and sonicated to obtain single cell suspensions which were then reused.

2) Cell culture and cell viability assay

The human monocyte line THP-1 was purchased from the institute of cell research, academy of sciences, China, Shanghai. RPMI 1640(Corning, New York, USA) medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, USA) at 37 ℃ and 5% CO ₂ Culturing THP-1 cells. THP-1 cells were plated at 2X 10 ⁵ cells/mL were plated in 24-well plates and differentiated with 40ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 24h, after which fresh medium was replaced and incubation continued for 12 h. The effect of Japoflavone D on cell viability was determined using WST-1 reagent. The cells were cultured at 1X 10 ⁴ The density of each well was inoculated into a 96-well plate, Japoflavone D at different concentrations was added after differentiation, and an equal amount of DMSO solvent was added to control wells as a control, and incubation was continued for 24 hours. After the treatment, the medium was removed, a medium containing 10% WST-1 solution was added, and after further incubation for 2 to 4 hours, absorbance at 450nm was measured.

3) Western blot detection of protein level

The drug-treated cells were lysed with a lysis solution containing protease and phosphatase inhibitors. The isolation of the nucleoplasm of the cells was carried out using a commercial kit according to the instructions. Proteins of different molecular weights were separated on 8-15% SDS-PAGE gels and then transferred to PVDF membranes by wet transfer. After blocking the membrane with 5% skim milk for 1 hour, incubation with the specific primary antibody solution for 1 hour at room temperature or overnight at 4 ℃, followed by 3 washes to remove unbound primary antibody. The membrane was then incubated with HRP conjugated secondary antibody for 1 hour and washed three more times to remove unbound secondary antibody. The target protein content was detected using immunoblot chemiluminescence reagents.

4) qPCR detection of Gene expression levels

RNA from cells was extracted using a commercial kit and an equivalent amount of RNA was inverted to cDNA using a reverse transcription kit. Specific primers are designed aiming at different genes, and the commercial qPCR premix is adopted for carrying out fluorescent quantitative PCR analysis. Calculation of Gene expression level normalization with reference Gene Gapdh (2) ^-ΔΔCT Analytical method).

5) CFU assay and phagocytosis assessment of H37Ra

PMA differentiated THP-1 macrophages (2X 10) ⁵ Individual cells) were incubated with H37Ra or H37Rv at 37 deg.C, 5% CO ₂ Under conditions of multiplicity of infection (MOI) of 10 for 6 hours. Then, the cells were washed 3 times with pre-warmed PBS to remove extracellular bacteria. Thereafter, infected THP-1 cells were treated with the indicated drugs for an additional 72 hours. Cells were washed 3 times with PBS and then lysed with 500 μ L PBS containing 0.1% SDS. Three experimental groups for each treatment were plated on 7H10 agar supplemented with 10% OADC. After incubation at 37 ℃ for 2 weeks, colonies were counted. To assess phagocytosis of H37Ra, THP-1 cells were infected with GFP-labeled H37Ra at a multiplicity of infection (MOI) of 10 for 6 hours. Then, the cells were washed 3 times with pre-warmed PBS, trypsinized, and then quantified using a BD FACSAria II flow cytometer and analyzed using FlowJo _ V10 software.

6) Apoptosis assay

Detection of apoptotic cells was performed using the FITC Annexin V apoptosis detection kit (C1062L, Beyotime) according to the manufacturer's protocol. Briefly, cells were harvested after treatment, washed twice with ice-cold Phosphate Buffered Saline (PBS), and then incubated with Propidium Iodide (PI) and FITC-conjugated Annexin V in the dark for 15 minutes. The number of stained cells was then determined by BD FACSAria II flow cytometry and analyzed using FlowJo _ V10 software.

7) Measurement of Caspase-3, -7 and-8 Activity

Caspase-3, -7 and-8 activity was determined using Caspase-Glo 3/7 or the 8 detection kit (Promega) according to the manufacturer's instructions. Briefly, 1.0X 10 ⁴ Individual cells were seeded in 96-well plates. After treatment, an equal volume of Caspase-Glo 3/7 or 8 reagent was added to the cell culture medium that had equilibrated to room temperature for 1 hour, the cells were shaken for 5 minutes, and incubated for 30 minutes at room temperature. Luminescence was recorded using a Synergy H1 microplate reader (Bioteck).

8) RNA interference

PMA differentiated THP-1 macrophage cells (2X 10) were transfected with SOD2 siRNA (RiboBio, Guangzhou, China) using Lipofectamine RNAIMAAX (ThermoFisher, Volterm, USA) according to the manufacturer's protocol ⁵ Cells/well). 48 hours after transfection, cells were washed once with PBS before lysis and silencing efficiency was determined by real-time quantitative PCR and Western blotting. For CFU counting, THP-1 cells were infected with H37Ra (MOI 10:1) for 6H after transfection for 48H. The remaining procedure was the same as described above.

9) Measurement of ROS and mitochondrial Membrane potential

PMA-differentiated THP-1 macrophages (2X 10) with H37Ra (with or without JFD) ⁵ Cells/well) for 24 hours (MOI 10:1) for 6 or 24 hours. Extracellular bacteria were removed by washing 3 times with pre-warmed PBS. The fluorescence intensity of the cells was measured with CM-H2DCF-DA (Invitrogen, Carlsbad, USA), MitoSOX red mitochondrial superoxide indicator (Invitrogen, Carlsbad, USA) and Mitotrack red (Invitrogen, Calsbad USA, following the manufacturer's instructions, respectively, BD FACSAria II flow cytometer and analyzed with FlowJo _ V10 software.

10) Immunofluorescent staining

After treatment as indicated, cells cultured on the coverslips were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Cells were then incubated with PBS containing 0.25% Triton X-100 for membrane permeabilization, followed by blocking with 5% BSA for 1 hour. Thereafter, the cells were incubated with the specific primary antibody overnight at 4 ℃. The coverslips were then washed 3 times with PBS and then incubated with fluorescent secondary antibody for 1 hour in the dark. Then, staining was performed with Hoechst for 10 minutes, and photographing was performed with a fluorescence microscope.

11) Chromatin immunoprecipitation analysis

ChIP analysis was performed by following the Upstate Biotechnology chromatin immunoprecipitation (ChIP) assay kit protocol (17-10085). Briefly, disrupted chromatin was immunoprecipitated using anti-NRF 2 antibody (16396-1-AP, Proteintech) as recommended by the manufacturer. Rabbit IgG was used as a negative control. DNA purified from both immunoprecipitates and from pre-immunoprecipitate samples was diluted 1:100 and PCR amplified using the following SOD2 promoter region primers: a forward direction 5'-tgctccccgcgctttcttaag-3'; and reverse direction 5'-gctgccgaagccaccacag-3'.

② experimental results

1) JFD promotes the elimination of mycobacterium tuberculosis in THP-1 cells

We found that Japoflavone D (JFD, fig. 1a) can significantly reduce the survival of H37Ra in THP-1 cells in a dose-dependent manner without significant effect on cell viability and growth of H37Ra itself (fig. 1 b-c). Similar effects were observed in THP-1 cells infected with H37Rv, a virulent strain of M.tuberculosis (FIG. 1 c). At the same time, JFD did not affect the growth of H37Ra in 7H9 medium (fig. 1d) nor the phagocytosis of H37Ra by macrophages (fig. 1 e).

2) JFD induces apoptosis in H37Ra infected THP-1 cells

To investigate the underlying mechanism of anti-tubercular activity of JFD, we examined the effect of JFD treatment on H37 Ra-infected THP-1 cells. The results show that JFD treatment induced more apoptosis in THP-1 cells infected with H37Ra at 24 hours (FIGS. 2 a-b). We observed an increase in pro-apoptotic caspase3/7/8 activity following JFD treatment of H37Ra infected THP-1 cells for 24 hours (fig. 2 c). Furthermore, the induction of cleavage by caspase3 and PARP1 was demonstrated by THP-1 cells infected with immunoblot H37Ra (FIG. 2 d). Flow cytometry analysis showed that after JFD treatment, the mitochondrial membrane potential of H37 Ra-infected THP-1 cells decreased (fig. 2 e). These data indicate that JFD treatment can induce apoptosis in H37 Ra-infected THP-1 cells. To verify whether JFD-induced apoptosis is required for its anti-tubercular activity, we used a caspase inhibitor fmk-zvad to inhibit JFD-induced apoptosis, and then measured the amount of H37Ra in the JFD-treated and control THP-1 cells. As shown in fig. 2f, blocking apoptosis with fmk-zvad can partially abrogate the anti-tubercular effect of JFD, suggesting that induction of apoptosis contributes to the anti-tubercular activity of JFD.

3) JFD induces apoptosis through p38 signal activation

Apoptosis is regulated by a complex signaling network. Among them, the cascade connection of AKT-mTOR signaling pathway and MAPK signaling plays a crucial role. Therefore, we examined whether both pathways of JFD are activated. As shown in fig. 3a and b, JFD treatment specifically activated p38 MAPK signaling, whereas AKT-mTOR signaling, JNK and ERK signaling were unaffected. To verify whether JFD-induced apoptosis in H37 Ra-infected THP-1 cells requires p38 MAPK activation, we examined caspase-3 cleavage in H37 Ra-infected THP-1 cells after JFD treatment in the presence of the potent p38 MAPK inhibitor, dorapilimod. As we expected, dorapremimod treatment significantly eliminated JFD-induced cleavage of caspase3 (fig. 3 c). Flow cytometry analysis also demonstrated that doratimod treatment inhibited JFD-induced apoptosis of H37 Ra-infected THP-1 cells (fig. 3 d). Furthermore, doratimod treatment partially reversed H37Ra clearance within macrophages induced by JFD (fig. 3 e).

4) JFD promotes accumulation of ROS in H37 Ra-infected THP-1 cells

Since the apoptosis inhibitor fmk-zVAD is unable to completely block anti-tubercular activity, and ROS is another important mediator of anti-tubercular immunity, we also examined the production of Reactive Oxygen Species (ROS) in H37 Ra-infected THP-1 cells after JFD treatment. As shown in fig. 4a-d, accumulation of ROS was significantly promoted in H37 Ra-infected THP-1 cells after JFD treatment for 6 hours or 24 hours. These data indicate that JFD treatment significantly increases ROS accumulation in H37Ra infected THP-1 cells, which may contribute to their anti-tubercular activity. To further confirm this hypothesis, we used the ROS scavenger NAC to scavenge JFD-induced ROS. As expected, NAC can dose-dependently reverse the anti-tubercular effect of JFD (fig. 4 e). Many research groups reported that ROS can activate p38 MAPK signaling, so we further validated whether NAC can block JFD-induced activation and apoptosis of p38 in H37 Ra-infected THP-1 cells. The results were consistent with other reports (fig. 4 f-g).

5) JFD promotes mROS accumulation by inhibiting SOD2 transcription

Considering the important role of SOD2 in regulating oxidative stress during H37Ra infection, we examined the effect of JFD treatment on SOD2 expression levels. The results showed that JFD treatment inhibited SOD2 expression induced by H37Ra infection (FIG. 5a-b), which was further confirmed by immunofluorescence assay (FIG. 5 c). To demonstrate the role of SOD 2-mediated ROS regulation in the promotion of JFD-induced H37Ra clearance, we confirmed whether treatment with MTO can reverse the anti-tubercular effect of JFD. As shown in fig. 5d, MTO can dose-dependently reverse the anti-tubercular effect of JFD. To verify the effect of JFD treatment on SOD2 stability, we used cycloheximide to block protein synthesis. As shown in fig. 5e, JFD treatment did not affect the stability of SOD 2. In addition, proteasome inhibitor mg132 and lysosomal inhibitors CHQ and BAF were unable to reverse JFD-induced inhibition of SOD2 expression (fig. 5 f). In conclusion, JFD treatment can inhibit SOD2 transcription induced by H37Ra infection.

6) JFD inhibits SOD2 transcription by inhibiting nuclear localization of NRF2

To reveal the potential mechanism of JFD inhibition of SOD2 transcription, we examined the expression levels of NRF2, Foxo3a, SP1 and SP3, which are reported transcription factors that regulate SOD2 transcription. Surprisingly, JFD treatment did not affect the expression of these transcription factors (fig. 6 a-b). Further, we detected the effect of JFD treatment on nuclear localization of these transcription factors. As shown in fig. 6c, JFD inhibited NRF2 nuclear localization caused by H37Ra infection, whereas JFD did not affect the localization of Foxo3a, SP1 and SP3 at all. Immunofluorescence further confirmed these results (fig. 6 d). Furthermore, chromatin immunoprecipitation analysis indicated that JFD treatment blocked the interaction of NRF2 with the SOD2 transcription start site (fig. 6 e). More importantly, luteolin, an NRF2 inhibitor, inhibited SOD2 transcription induced by H37Ra infection dose-dependently (fig. 6f-g) and promoted H37Ra clearance within macrophages (fig. 6H). Taken together, these observations indicate that JFD treatment reduces SOD2 transcription induced by H37Ra infection by inhibiting nuclear localization of NRF 2.

③ Effect summary

In this patent, we disclose a novel flavonoid, Japoflavanone D (JFD), which promotes the elimination of Mycobacterium tuberculosis in THP-1 cells. JFD inhibits NRF2 from migrating into the nucleus, thereby reducing SOD2 transcription resulting from H37Ra infection. SOD2 induced attenuation leading to Reactive Oxygen Species (ROS) accumulation in H37Ra infected THP-1 cells. Excessive ROS can directly lead to more m. tuboculosis killing, while apoptosis can be induced by activating p38 MAPK signaling. In summary, Japoflavone D can promote clearance of mycobacterium tuberculosis by inhibiting NRF 2-mediated SOD2 transcription and can be used for host-directed therapy against mycobacterium tuberculosis infection.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (4)

1. An application of an NRF2 protein inhibitor in preparing a medicament for treating diseases induced by infection of mycobacterium tuberculosis is characterized in that the NRF2 protein inhibitor is honeysuckle biflavone D or a medicinal salt thereof, and NRF2 protein is used as a target point to inhibit the growth of mycobacterium tuberculosis in macrophages.

2. The use according to claim 1, wherein the diseases induced by infection with mycobacterium tuberculosis include pulmonary tuberculosis, intestinal tuberculosis, tuberculous peritonitis, tuberculous meningitis.

3. The use according to claim 1, wherein the medicament is a solid formulation or a liquid formulation.

4. The use according to claim 3, wherein the solid formulation comprises a tablet, capsule, granule, pill, powder; the liquid preparation comprises injection, oral liquid, mixture and aerosol.

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