CN117462542A - Application of Aila in treating anemia - Google Patents

Abstract

The invention provides application of ailanthinone (Aila) in treating anemia. The study shows that Aila can inhibit the STAT3 signal path, inhibit the hepcidin level to further increase Fpn1 level so as to increase iron discharge and serum iron level, thereby achieving the effect of treating various anemias, and being a novel medicine for treating anemias.

Description

Application of Aila in treating anemia

Technical Field

The invention relates to the field of medicines, in particular to application of Aila in treating anemia.

Background

Iron is an essential trace element which is indispensable to all life bodies, and is also the most abundant transition metal element in human bodies. The iron stabilization is coordinated among the various links by means of absorption, ingestion, utilization and storage of iron ions, wherein any link has a problem, the iron content is changed, and the biological activity of the organism is influenced by the iron content which is too high or too low. Iron deficiency can cause metabolic disorders in the body with anemia as a major clinical symptom. Iron deficiency anemia is one of the most common nutritional deficiency diseases worldwide.

Hepcidin (hepcidin) is synthesized mainly by the liver, plays a leading role in maintaining iron homeostasis in the body, is a central molecule regulating iron homeostasis, and is degraded by binding to the only currently known iron exporter protein, ferritin (Fpn 1), thereby reducing iron export from the cell. Excessive body hepcidin results in reduced iron intake and thus in poor blood.

No report has been made in the prior art on ailanthinone (Ailanthone) for the treatment of anaemia.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an application of ala in treating anemia, and to provide a new technical means for treating anemia.

In one aspect of the invention, at least one of Aila or a hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer and precursor compound thereof is provided as an active ingredient for preparing a medicament for treating anemia.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid.

The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions, for example: enols and the corresponding ketones.

The term "stereoisomer" refers to an isomer produced by the spatial arrangement of atoms in a molecule, such as: cis-trans isomers, enantiomers, conformational isomers, and the like.

The term "precursor compound" refers to a compound that is inactive in vitro but is capable of undergoing metabolic or chemical reactions in vivo to be converted into the active ingredient of the present invention, thereby exerting its pharmacological effect.

Further, the anemia is iron deficiency anemia. Such as cancer anemia, physiological anemia or inflammatory anemia.

The cancer anemia refers to anemia caused by various cancers, such as liver cancer, kidney cancer, stomach cancer, lung cancer, uterine cancer, thyroid cancer, lymphoma, pancreatic cancer, and anemia caused by leukemia.

Inflammatory anemia refers to anemia caused by inflammation or infection.

Physiological anemia refers to anemia that results from pregnancy and growth, respectively, in a pregnant woman or infant.

Further, the medicament has at least any one of the following effects:

a. inhibiting STAT3 signaling pathway;

b. inhibiting hepcidin levels;

c. increasing serum iron levels.

In one aspect, the invention provides a medicament for treating anemia, which comprises at least one of Aila or hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer and precursor compound thereof in a therapeutically effective amount as an active ingredient for preparing the medicament for treating anemia.

Further, the medicine also contains a carrier acceptable to human bodies.

Further, the carrier includes at least one of a diluent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a cosolvent, a buffer, and a surfactant.

Further, the anemia is cancer anemia, physiological anemia or inflammatory anemia.

Further, the medicament has at least any one of the following effects:

a. inhibiting STAT3 signaling pathway;

b. inhibiting hepcidin levels;

c. increasing serum iron levels.

In one aspect, the invention provides a pharmaceutical composition comprising the above medicament for treating anemia. The medicine of the invention can be used alone or in the form of a pharmaceutical composition, wherein the pharmaceutical composition can contain minor ingredients which do not affect the active ingredients and/or pharmaceutically acceptable carriers, auxiliary materials necessary for various preparations and the like besides the major active ingredients.

The dosage form of the drug or the pharmaceutical composition of the present invention is not limited as long as it is a dosage form capable of allowing the active ingredient to reach the body effectively, and includes: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, electuaries, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches and the like; preferred are oral dosage forms, such as: capsules, tablets, oral liquid, granules, pills, powder, pills, paste and the like.

Taking pharmaceutical compositions as an example, the compositions of the present invention may be formulated as injectable formulations, for example, using physiological saline or aqueous solutions containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.

The medicament, formulation or pharmaceutical composition of the invention may be administered to a subject in need thereof (e.g., human and non-human milk animals) by conventional means. Representative modes of administration include (but are not limited to): one or more routes of administration, such as oral or injectable (including one or more of intravenous, intravenous drip, intramuscular or subcutaneous injection, etc.). When used, the pharmaceutical composition is administered to a mammal in a safe and effective amount. Of course, the particular dosage and method will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

As described above, the application of the Aila in treating anemia has the following beneficial effects:

the Aila can inhibit STAT3 signal pathway, inhibit hepcidin level to increase Fpn1 level, increase iron discharge, increase serum iron level, and treat anemia, and is a novel medicine for treating anemia.

Drawings

FIG. 1Aila inhibits expression of HepG2 and MHCC97H hepcidin; wherein, FIG. 1A Aila inhibits HepG2 cell viability; FIG. 1B Aila inhibits MHCC97H cell viability; FIG. 1C Aila inhibits transcription levels of HepG2 hepcidin; FIG. 1D Aila inhibits the transcriptional level of MHCC97H hepcidin. Aila (0,0.3125,0.625,1.25,2.5,5. Mu. Mol) treated HepG2 and MHCC97H for 24 hours.

FIG. 2Aila inhibits phosphorylation levels of HepG2 and MHCC97H STAT3 (Tyr 705); FIG. 2A Aila increases the transcript level of Fpn1 of HepG 2; FIG. 2B Aila (1.25. Mu. Mol) increases the transcript level of Fpn1 of MHCC 97H; FIG. 2C Aila inhibits HepG2 and MHCC97H STAT3 (Tyr 705) phosphorylation levels.

FIG. 3Aila improves cancerous anemia; wherein, FIG. 3A is a photograph of tumor weight and statistics of tumor weight; FIG. 3B serum iron; FIG. 3C does not incorporate iron saturation forces; FIG. 3D total iron binding force; fig. 3E carrier saturation. After one week of adaptive feeding, 6 week-old C57BL/6 mice were randomly divided into 3 groups, 6 CON groups, 5 HCC groups and HCC+Aila groups according to body weight. The CON group is given with physiological saline; the HCC group and hcc+aila group were each given subcutaneously with hepa1-6 x 10 ⁵ Mu.l/100. Mu.l/20 g mice, starting on day 3, were given saline on alternate days for CON and HCC groups, and 1mg/kg of Aila on alternate days for HCC+Aila groups. After 21 days, the eyeballs are picked up for blood taking, subcutaneous tumors are taken out for weighing and photographing.

FIG. 4Aila inhibits LPS-induced primary macrophage hepcidin elevation by STAT3 pathway; wherein fig. 4A Aila inhibits the viability of peritoneal-induced primary macrophages; FIG. 4B Aila (1.25. Mu. Mol,24 hours) inhibits LPS (1. Mu.g/ml, 12 hours) induced peritoneal induced elevation of primary macrophage hepcidin; FIG. 4C Aila (1.25. Mu. Mol,24 hours) inhibits LPS (1. Mu.g/ml, 12 hours) induced STAT3 (Tyr 705) phosphorylation and decreases in TfR1, fpn1, ftH and FtL; FIG. 4D Sttatic (10. Mu. Mol,13 hours) significantly reversed Aila-inhibited LPS-induced elevation of hepcidin.

FIG. 5Aila improves the physiological anemia and LPS-induced inflammatory anemia; wherein fig. 5A serum iron; FIG. 5B does not incorporate iron saturation forces; FIG. 5C total iron binding force; fig. 5D carrier saturation. After 6-week-old C57BL/6 mice were fed adaptively for one week, they were randomly divided into 4 groups according to body weight, 5 LPS groups, and 6 groups of the other three groups. Disposable intraperitoneal injection PBS of CON group; the Aila group is injected into the abdominal cavity once only with Aila1mg/kg Body Weight (24 h); LPS group is injected with 1mg/kg Body Weight (12 h) of LPS at a time; the LPS+Aila group was injected intraperitoneally with Aila1mg/kg Body Weight (24 h) and LPS 1mg/kg Body Weight (12 h), and the eyeballs were harvested after anesthesia.

FIG. 6Aila inhibits LPS-induced elevation of mouse spleen hepcidin; wherein FIGS. 6A-C Aila inhibited LPS-induced reduction in mouse spleens Fpn (6A, B) and FtL (6A, C); FIG. 6D Aila inhibits LPS-induced elevation of spleen hepcidin transcription in mice. After 6-week-old C57BL/6 mice were fed adaptively for one week, they were randomly divided into 4 groups of 6 animals each according to body weight. Disposable intraperitoneal injection PBS of CON group; the Aila group is injected into the abdominal cavity once only with Aila1mg/kg Body Weight (24 h); LPS group is injected with 1mg/kg Body Weight (12 h) of LPS at a time; the LPS+Aila group was injected intraperitoneally with Aila1mg/kg Body Weight (24 h) and LPS 1mg/kg Body Weight (12 h), and the eyeballs were harvested after anesthesia.

Detailed Description

Studies have shown that cancer patients 'signaling and transcriptional activator protein (STAT 3) are abnormally activated, liver cancer patients' hepcidin compensatory decreases increase iron absorption, serum iron decreases, and significant anemia occurs. In addition, studies have shown that inflammation leads to inflammatory anaemia by inducing abnormal elevation of hepcidin by STAT3 (Y705) phosphorylation. The research team discovers that the Chinese medicine Aila active ingredient remarkably inhibits the expression of Hepcidin by screening 2880 Chinese medicine-derived active substances.

The applicant has studied mainly the effect of alas on cancer, physiological and inflammatory anaemia, while studying the mechanism of increasing serum iron in alas at cellular and animal level:

among them, the effect of Aila on cancer anemia: different concentrations of Aila treated human liver cancer cell lines HepG2 and MHCC97H24 hours, real-time PCR detected the transcript level of hepcidin and Western blot detected the expression level of Pump ferritin Fpn1 and the STAT3 (Tyr 705) phosphorylation level. Adaptive feeding for 1 week using 6 week old C57BL/6 mice, subcutaneous injection of liver cancer mice cells hepa1-6 (3 x 10) ⁵ One/one), the administration of Aila (1 mg/kg) by intraperitoneal injection was started on the third day, and blood was collected from the eyes after anesthesia and the 21 st day, and serum iron was detected withoutSaturated iron binding capacity, total iron binding capacity, iron carrying saturation, and taking subcutaneous tumors for photographing and weighing;

research on the effect of Aila on inflammatory anemia: abdominal induced primary macrophages were treated with LPS or Aila, and hepcidin transcript levels and Western blot were detected by Real-time PCR for expression levels of the transferrin receptor TfR1 and transferrin Fpn1 and STAT3 (Tyr 705) phosphorylation. C57BL/6 mice are utilized, LPS or Aila is injected into the abdominal cavity, eyeballs are taken out for blood after anesthesia, serum iron is detected, the binding capacity of unsaturated iron, the total iron binding capacity and the iron carrying saturation are detected. Transcription levels of spleen hepcidins were detected by Real-time PCR and protein levels of spleen Fpn and FtL were detected by Western blot.

The research finds that: further research shows that the Aila significantly inhibits liver cancer cell p-STAT3 activation induced Hepcidin elevation. Further studies have found that significant inhibition of LPS-induced inflammatory anaemia by Aila is achieved by inhibiting p-STAT 3-induced elevation of Hepcidin.

Conclusion: the Aila can inhibit STAT3 signal pathway, inhibit hepcidin level to increase Fpn1 level, increase iron discharge, increase serum iron level, and treat anemia, and is a novel medicine for treating anemia.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the spirit of the invention, and various modifications and adaptations of the invention may be made to the details of the present description based on various points of view and applications. It should be understood that the process equipment or devices not specifically identified in the examples below are all conventional in the art. Furthermore, it is to be understood that the reference to one or more method steps in this disclosure does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise indicated; it should also be understood that the combined connection between one or more devices/means mentioned in the present invention does not exclude that other devices/means may also be present before and after the combined device/means or that other devices/means may also be interposed between these two explicitly mentioned devices/means, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention which may be practiced, as such changes or modifications in the relative relationships are deemed to be within the scope of the invention which may be practiced without substantial modification.

Abbreviation remarks

CON (control) control

Aila (Ailanthone) ailanthinone

LPS (Lipopolysaccharide) lipopolysaccharide

HCC (Hepatocellular carcinoma) liver cancer

Fpn1 (Ferroportin) cell membrane iron transporter 1

TfR1 (Transferrin Receptor) transferrin receptor 1

Ferritin Ferritin

FtL (Ferritin light chain) ferritin light chain

FtH (Ferritin heavy chain) ferritin heavy chain

Hepcidin

STAT3 ((signal transducer and activator of transcription) signaling and transcriptional activator proteins

Serum iron of Serum iron

UIBC (unsatured ironbinding capability) unsaturated iron binding force

TIBC (total iron binding capacity)

TF (%) iron saturation

Western blot protein printing method (immunoblotting)

Materials and methods

1. C57BL/6 male mice, 6 weeks old, after 1 week of adaptive feeding, were randomly divided into four groups according to body weight, respectively a blank control group (CON), an ailanthinone group (Aila), a lipopolysaccharide group (LPS), a lipopolysaccharide and ailanthinone group (LPS+Aila), 5 LPS groups, and 6 of the other three groups. Mice were given intraperitoneal injections, with 100 μl/20g Body Weight PBS given to the blank, aila group given to ailanthinone (24 h) at 1mg/kg Body Weight, LPS group given to lipopolysaccharide (12 h) at 1mg/kg Body Weight, LPS+aila group given to 1mg/kg Body Weight ailanthinone (24 h) and 1mg/kg Body Weight lipopolysaccharide (12). After anesthesia, the eyeballs are picked up for blood taking, the neck is broken for sacrifice, and spleen is dissected and taken.

2. C57BL/6 male mice, 6 weeks old, after 1 week of adaptive feeding, were randomly divided into four groups according to body weight, respectively, a blank control group (CON), a liver cancer model group (HCC), liver cancer and ailanthinone group (HCC+Aila), CON group 6, HCC group and HCC+Aila group 5 each. On day 1, HCC group and HCC+Aila group were given subcutaneously with hepa1-6 (3X 10) at 100 μl/20g Body Weight ⁵ 100 μl), mice were started on day 3 with intraperitoneal injections every other day, and the blank group was given 100 μl/20g Body Weight PBS,HCC group and 1mg/kg Body Weight ailanthone was given according to 100 μl/20g Body Weight PBS,HCC+Aila group. 21 days after administration, the eyeballs are picked up for blood taking after anesthesia, the neck is broken for sacrifice, the tumor is dissected and taken, and the tumor is photographed and weighed.

3. Preparation of serum: blood samples obtained by taking the eyeballs are placed at room temperature for 1 hour, centrifuged at 7000 rpm for 10 minutes, the supernatant is taken, and centrifuged at 7000 rpm for 10 minutes again, and the supernatant is taken for later use.

Serum iron and unsaturated iron binding capacity detection

The Iron related index of serum was measured using the POINTE's ion/TIBC Reagent Set kit.

A. Serum iron

1. A96-well plate was prepared and labeled with blank, standard and sample, respectively.

2. Mu.l of iron buffer was added to each well.

3. Mu.l of deionized water was added to the blank, 20. Mu.l of Iron standard (500. Mu.g/dl) was added to the standard, and 20. Mu.l of sample was added to the sample.

4. The absorbance reading (A1) was measured at 560nm for each well using a microplate reader with a blank of 0.

5. Mu.l of iron color reagent was added to each well, mixed well and left at 37℃for 10 minutes.

6. The absorbance reading (A2) of each well at 560nm was measured with an enzyme-labeled instrument using a blank as 0

Serum iron (. Mu.g/dl) = (A2 sample-A1 sample)/(A2 std-A1 std) & concentration of St

B. Serum UIBC

1. 96-well plates were prepared and labeled with blank, standard and sample, respectively.

2. Mu.l of UIBC buffer was added to each well.

3. To the blank, 40. Mu.l deionized water was added, and to the standard, 20. Mu.l deionized water and 20. Mu.l Ironstandard were added.

To sample, 20. Mu.l of sample and 20. Mu.l of Iron standard were added.

4. The absorbance reading (A1) was measured at 560nm for each well using a microplate reader with a blank of 0.

5. Mu.l of iron color reagent was added to each well, mixed well and left at 37℃for 10 minutes.

6. The absorbance reading (A2) was measured at 560nm for each well using a microplate reader with a blank of 0.

UIBC(μg/dl)＝Conc of std-(A2sample-A1sample)/(A2std-A1std)*Concentration of std

TIBC (μg/dl) =serum iron+UIBC

TF (%) =serum iron/TIBC

4. Preparation of tissue protein:

0.01g of spleen or tumor tissue is taken, 100 mu l of RIPA lysate is added, the oscillator is vibrated, 60 Hz is performed, the centrifugation is performed for 3 minutes at 13200 rpm for 15 minutes, and the supernatant is taken and used for Western blot detection of the expression level of a transferrin receptor TfR1 and transferrin Fpn1 and the phosphorylation level of STAT3 (Tyr 705).

5. Abdominal induced primary macrophages and RAW264.7

C57B6 male mice, 6 weeks old, after adaptive feeding for one week, 3% thioglycollic acid culture medium was injected intraperitoneally, after three days, the mice were sacrificed by cervical scission, immersed in 75% ethanol solution for 10 seconds, fixed on dissecting plates in supine position, and the abdomen was fully cut off by direct surgical scissorsThe skin was partially exposed to the abdominal layer, and the abdominal layer was sterilized with alcohol cotton ball. 10ml of PBS was injected into the abdominal cavity using a 10ml syringe, the abdomen was gently warmed for 2 minutes, the cell suspension was aspirated using a 5ml syringe, centrifuged for 10 minutes at 300g, the cells were diluted to 1X 106 cells/ml, inoculated into 6-well plates, 2 ml of cell suspension was added to each well, incubated for 3 hours in a 5% CO2 incubator, the solution was changed, washed 2 times with PBS, the non-adherent cells were removed, and DMEM high-sugar complete medium (10% fetal bovine serum, 1% penicillin and streptomycin) was added. Cells were treated 24 hours later with drug, PBS was added to the control group, aila group was added to 1.25. Mu. Mol of ailanthinone (24 hours), LPS group was added to 1. Mu.g/ml of lipopolysaccharide (12 hours), ailanthinone and lipopolysaccharide group were added to 1.25. Mu. Mol of ailanthinone (24 hours) and lipopolysaccharide (12 hours) at 1. Mu.g/ml of final concentration, and cell extract RNA and protein were collected. RAW26.7 according to density 5 x 10 ⁵ Each milliliter was inoculated into a 6-well plate and the administration method was the same.

6. Liver cancer cell

Resuscitates liver cancer cells MHCC97H and HepG2 cells, transmits for 2 generations, and then reaches 5×10 ⁵ At a concentration of 1.25. Mu. Mol of ailanthinone (24 h), lipopolysaccharide (LPS) at a final concentration of 1. Mu.g/ml (12 h), lipopolysaccharide and ailanthinone (LPS+Aila) at a final concentration of 1.25. Mu. Mol of ailanthinone (24 h) and 1. Mu.g/ml of lipopolysaccharide (12 h), and cell extract RNA and protein were collected.

7. Preparation of cellular protein samples

Immediately taking out the cells, placing the cells on ice, washing the cells with PBS for 2 times, adding 80 mu l of cell lysis solution added with protease inhibitor into each well, scraping the cells, collecting a 1.5ml centrifuge tube, standing on ice for thirty minutes, centrifuging at 13200 rpm for 15 minutes, and sucking the supernatant.

Example 1Aila significantly inhibited HepG2 and MHCC97Hhepcidin expression

To study the effect of Aila on human hepatoma cell hepcidin expression, we treated HepG2 and MHCC97H for 24 hours with varying concentrations of Aila, and found that Aila significantly inhibited the cell viability of both cells (FIGS. 1A, B). Simultaneously significantly inhibited both cellular iron-regulated central molecule hepcidin expression (FIGS. 1C, D). In combination with the effect of Aila on cell viability and hepcidin expression, the concentration of Aila was chosen to be 1.25. Mu. Mol in our subsequent experiments.

Example 2Aila inhibits phosphorylation levels of HepG2 and MHCC97H STAT3 (Tyr 705)

The above studies suggest that Aila inhibits the expression of hepcidin, which has been shown to be degraded by binding to the only known iron exporter protein, pump ferritin Fpn1, whereas phosphorylation of STAT3 increases its expression by binding to the hepcidin promoter. 1.25. Mu. Mol of Aila was found to significantly increase the transcript level of Fpn1 (FIGS. 2A, B). Further mechanical studies have found that treatment of these two cell lines with different concentrations of sala significantly inhibited the phosphorylation levels of STAT3 (Tyr 705) and were concentration dependent (fig. 2C). Studies have shown that STAT3 is abnormally activated in cancer, and the above studies suggest that Aila inhibits hepcidin expression by inhibiting STAT3 (Tyr 705) which is abnormally activated by hepatoma cells, while hepcidin can inhibit hepcidin expression by internalization degradation Fpn1 and Aila by STAT3 thereby increases Fpn expression.

Example 3Aila significantly inhibits the decrease in serum iron and carrier saturation in tumor-bearing mice of hepatoma cells

In vitro studies indicate that Aila can inhibit the expression of human hepatoma cell hepcidin, and studies have shown that lowering of human hepcidin increases iron uptake. Studies show that hepcidin of liver cancer patients is highly expressed, serum iron is reduced, and anemia is remarkable. To investigate whether Aila can increase iron uptake in vivo, we used C57BL/6 mice to subcutaneously hepa1-6 tumor-bearing, and found that Aila significantly inhibited tumor growth (FIG. 3A). Compared with the control mice, the serum iron, the total iron binding capacity and the iron-carrying saturation of the hepa1-6 tumor-bearing mice are obviously reduced (figures 3B, D and E), and the non-binding iron saturation capacity is unchanged (figure 3C), which shows that the serum iron of the liver cancer tumor-bearing mice is obviously reduced and cancerogenic anemia is caused. While the Aila treatment can significantly inhibit the decrease of serum iron, total iron binding force and carrier saturation induced by hepa1-6 tumor-bearing (FIGS. 3B, D, E). The in vivo research result shows that Aila can improve the reduction of serum iron caused by liver cancer and treat cancerous anemia.

Example 4Aila inhibits LPS-induced primary macrophage hepcidin elevation by STAT3 (Tyr 705) pathway

We continue to study whether alas also have an effect on inflammatory anaemia. The primary giant phagocytes induced by the abdominal cavity are treated by the Aila amide, so that the cell viability of the primary giant phagocytes can be remarkably inhibited, the treatment of the Aila (1.25 mu mol) has no remarkable influence on the cell viability (figure 4A), and the concentration of the Aila in the subsequent experiment is 1.25 mu mol. The ala significantly inhibited the LPS-treatment-induced increase in hepcidin expression (fig. 4B), the LPS-induced phosphorylation of STAT3 (Y705), while the LPS-induced decrease in transferrin TfR1, transferrin Fpn1, ferritin heavy chain (FtH) and ferritin light chain (FtL) was significantly inhibited (fig. 4C), and the STAT3 inhibitor Sttatic was able to significantly reverse the ala-inhibited LPS-induced increase in hepcidin (fig. 4D). The LPS-induced hepcidin elevation by Aila inhibition was demonstrated to be mediated by STAT3 (Tyr 705) phosphorylation.

Example 5Aila significantly increases serum iron and carrier saturation in physiological conditions and Aila can significantly inhibit LPS-induced decrease in serum iron and carrier saturation in inflammatory conditions

To study the effect of sala on serum iron content in mice under physiological and LPS-induced inflammatory conditions, we utilized SPF grade C57BL/6 mice, 6 weeks old, after 1 week of adaptive feeding, randomly divided into four groups of 6 animals per group, one dose, sala treatment for 24 hours, LPS treatment for 12 hours according to body weight. It was found that in physiological conditions, the administration of ala for 24 hours significantly increased serum iron, unbound iron saturation forces and total iron binding forces (fig. 5A-C), with no effect on iron-carrying saturation (fig. 5D). In the case of inflammation, aila significantly inhibited LPS-induced serum iron, unbound iron saturation force, total iron binding force and iron-loaded saturation reduction (fig. 5A-D). Further analysis found that the serum iron increasing effect was more pronounced in inflammatory conditions than in physiological conditions (148% vs 215%). In summary, it is known that injection of 1mg/kg Aila into animals can significantly increase serum iron content, i.e. improve physiological and inflammatory anaemia.

Example 6Aila inhibits LPS-induced decrease in mouse spleen Fpn1 and increase in hepcidin

To verify the effect of alas in vitro peritoneal induced primary macrophages and elucidate the mechanism by which alas increase serum iron in mice in vivo, we treated C57BL/6 mice with LPS or alas, and studied to find that alas significantly inhibited LPS-induced reduction in mouse spleen Fpn1 (fig. 6a, b) and FtL (fig. 6a, C) and hepcidin increase (fig. 6D).

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims (10)

1. At least one of ailanthinone or hydrate, pharmaceutically acceptable salt, tautomer, stereoisomer and precursor compound thereof is used as an active ingredient for preparing the medicine for treating anemia.

2. Use according to claim 1, characterized in that: the anemia is iron deficiency anemia, preferably cancer anemia, physiological anemia or inflammatory anemia.

3. Use according to claim 1, characterized in that: the medicament has at least any one of the following effects:

a. inhibiting STAT3 signaling pathway;

b. inhibiting hepcidin levels;

c. increasing serum iron levels.

4. Use according to claim 1, characterized in that: the medicine also contains a carrier acceptable to human bodies.

5. A medicament for treating anemia, characterized in that the medicament contains a therapeutically effective amount of at least one of Aila or a hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer and a precursor compound thereof as an active ingredient for preparing the medicament for treating anemia.

6. A medicament according to claim 5, characterized in that: the medicine also contains a carrier acceptable to human bodies.

7. A medicament according to claim 6, characterized in that: the carrier includes at least one of a diluent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a co-solvent, a buffer, or a surfactant.

8. A medicament according to claim 5, characterized in that: the anemia is iron deficiency anemia, preferably cancer anemia, physiological anemia or inflammatory anemia.

9. A medicament according to claim 5, characterized in that: the medicament has at least any one of the following effects:

a. inhibiting STAT3 signaling pathway;

b. inhibiting hepcidin levels;

c. increasing serum iron levels.

10. A pharmaceutical composition characterized by: the pharmaceutical composition contains the medicament as claimed in any one of claims 5 to 9.