Use of aila in the treatment of anemia

Abstract

The application provides an application of aila in treating anemia. The present study finds that aila can inhibit the STAT3 signal pathway, inhibit the hepcidin level, increase the Fpn1 level, increase the iron excretion, increase the serum iron level, and achieve the effect of treating various anemias, and is a new type of drug for treating anemia.

Description

Technical Field

[0001] This invention relates to the field of medicine, and in particular to the application of Aila in the treatment of anemia. Background Technology

[0002] Iron is an essential trace element for all living organisms and the most abundant transition metal element in the human body. Iron homeostasis depends on the coordination of various stages of iron ion absorption, uptake, utilization, and storage. Problems in any of these stages will lead to changes in iron levels; both excessively high and low iron levels will affect the body's biological activity. Iron deficiency can cause metabolic disorders, with anemia as the main clinical symptom. Iron deficiency anemia is one of the most common nutrient deficiency diseases worldwide.

[0003] Hepcidin, primarily synthesized by the liver, plays a crucial role in maintaining iron homeostasis. It is a central molecule regulating iron homeostasis, binding to ferroportin (Fpn1), the only known iron export protein pump, leading to its degradation and thus reducing iron export from cells. Excessive hepcidin levels can result in decreased iron uptake, leading to anemia.

[0004] There are no existing reports on the use of ailanthone for the treatment of anemia. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an application of Aila in the treatment of anemia, thus providing a new technical means for the treatment of anemia.

[0006] One aspect of the present invention provides at least one of Aila or its hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer, or a precursor compound as an active ingredient for the preparation of a drug for treating anemia.

[0007] Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of this invention with acids. Suitable acids for salt formation include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; 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 benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0008] The term "tautomer" refers to functional group isomers that are produced by the rapid movement of an atom in two positions within a molecule, such as enols and their corresponding ketones.

[0009] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, such as cis-trans isomers, enantiomers, and conformational isomers.

[0010] The term "precursor compound" refers to a compound that is inactive in vitro but can be metabolized or chemically reacted in vivo to become the active ingredient of this invention, thereby exerting its pharmacological effects.

[0011] Furthermore, the anemia described is iron-deficiency anemia, such as cancer-related anemia, physiological anemia, or inflammatory anemia.

[0012] Cancer-related 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 leukemia.

[0013] Inflammatory anemia refers to anemia caused by inflammation or infection.

[0014] Physiological anemia refers to anemia that occurs in pregnant women or infants during pregnancy and growth and development, respectively.

[0015] Furthermore, the drug has at least one of the following effects:

[0016] a. Inhibit the STAT3 signaling pathway;

[0017] b. Inhibits hepcidin levels;

[0018] c. Increase serum iron levels.

[0019] One aspect of the present invention provides a medicament for treating anemia, wherein the medicament contains at least one of the following as an active ingredient: a therapeutically effective amount of Aila or its hydrate, a pharmaceutically acceptable salt, a tautomer, a stereoisomer, or a precursor compound.

[0020] Furthermore, the drug also contains a carrier that is acceptable to the human body.

[0021] Furthermore, 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.

[0022] Furthermore, the anemia is cancer-related anemia, physiological anemia, or inflammatory anemia.

[0023] Furthermore, the drug has at least one of the following effects:

[0024] a. Inhibit the STAT3 signaling pathway;

[0025] b. Inhibits hepcidin levels;

[0026] c. Increase serum iron levels.

[0027] One aspect of the present invention provides a pharmaceutical composition comprising the aforementioned drug for treating anemia. The drug of the present invention can be used alone or in the form of a pharmaceutical composition, wherein the pharmaceutical composition refers to a drug that, in addition to containing a major active ingredient, may also contain small amounts of minor components that do not affect the effective ingredient and / or pharmaceutically acceptable carriers and excipients necessary for various formulations.

[0028] The dosage form of the drug or drug composition described in this invention is not limited, as long as it enables the active ingredient to effectively reach the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc.; oral dosage forms are preferred, such as: capsules, tablets, oral liquids, granules, pills, powders, elixirs, ointments, etc.

[0029] Taking pharmaceutical compositions as an example, the compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. 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 aseptic conditions.

[0030] The drugs, formulations, or pharmaceutical compositions of the present invention can be administered to desired subjects (such as humans and non-human mammals) in a conventional manner. Representative administration methods include (but are not limited to): one or more routes of administration such as oral or injectable (including one or more of intravenous injection, intravenous drip, intramuscular injection, or subcutaneous injection). When using the pharmaceutical composition, a safe and effective amount of the drug is administered to the mammal. Of course, the specific dosage and method should also take into account factors such as the route of administration and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0031] As described above, the application of Aila of the present invention in the treatment of anemia has the following beneficial effects:

[0032] Aila treats anemia by inhibiting the STAT3 signaling pathway, suppressing hepcidin levels, and thus increasing Fpn1 levels, leading to increased iron excretion and serum iron levels. It is a novel drug for treating anemia. Attached Figure Description

[0033] Figure 1AILA inhibits the expression of HepG2 and MHCC97H hepcidin; among which, Figure 1A Aila inhibits the viability of HepG2 cells; Figure 1B Aila inhibits the viability of MHCC97H cells; Figure 1C Aila inhibits the transcriptional level of hepcidin in HepG2; Figure 1 shows that Aila inhibits the transcriptional level of hepcidin in MHCC97H. HepG2 and MHCC97H were treated with Aila (0, 0.3125, 0.625, 1.25, 2.5, 5 μmol) for 24 hours.

[0034] Figure 2A ILA inhibits the phosphorylation levels of HepG2 and MHCC97H STAT3 (Tyr705); Figure 2A Aila increases the transcriptional level of Fpn1 in HepG2; Figure 2B Aila (1.25 μmol) increased the transcriptional level of Fpn1 in MHCC97H; Figure 2 shows that Aila inhibited the phosphorylation levels of HepG2 and STAT3 (Tyr705) in MHCC97H.

[0035] Figure 3A ILA improves cancer-related anemia; among which, Figure 3A Tumor weight photos and tumor weight statistics; Figure 3B Serum iron; Figure 3C Unbound iron saturation force; Figure 3D Total iron bonding force; Figure 3E Iron saturation. Six-week-old C57BL / 6 mice were randomly divided into three groups according to body weight after one week of acclimatization: a CON group (n=6), an HCC group (n=5), and an HCC+Aila group (n=5 each). The CON group received saline; the HCC and HCC+Aila groups received hepatoprotective acid (HPA1-6) 3 x 10^6 mg / mL subcutaneously per mouse. 5 Mice were treated with 100 μl of Aila per 20 g mice. Starting on day 3, the CON and HCC groups were given saline every other day, while the HCC+Aila group was given 1 mg / kg Aila every other day. After 21 days, the mice were anesthetized, enucleated, and blood was collected. Subcutaneous tumors were removed, weighed, and photographed.

[0036] Figure 4A ILA inhibits LPS-induced hepcidin elevation in primary macrophages via the STAT3 pathway; among which... Figure 4A Aila inhibits the activity of peritoneally induced primary macrophages; Figure 4B Aila (1.25 μmol, 24 h) inhibited the increase of hepcidin in primary macrophages induced by LPS (1 μg / ml, 12 h) in the peritoneal cavity; Figure 4CAila (1.25 μmol, 24 h) inhibited LPS (1 μg / ml, 12 h)-induced phosphorylation of STAT3 (Tyr705) and the decrease in TfR1, Fpn1, FtH and FtL; Figure 4D Sttatic (10 μmol, 13 h) significantly reversed the increase in LPS-induced hepcidin induced by Aila inhibition.

[0037] Figure 5A ILA improves physiological anemia and LPS-induced inflammatory anemia; among which Figure 5A Serum iron; Figure 5B Unbound iron saturation force; Figure 5C Total iron bonding force; Figure 5D Iron saturation. Six-week-old C57BL / 6 mice were randomly divided into four groups according to body weight after one week of acclimatization: the LPS group (n=5 each) and the other three groups (n=6 each). The CON group received a single intraperitoneal injection of PBS; the Aila group received a single intraperitoneal injection of Aila 1 mg / kg Body Weight (24 h); the LPS group received a single intraperitoneal injection of LPS 1 mg / kg Body Weight (12 h); and the LPS+Aila group received a single intraperitoneal injection of Aila 1 mg / kg Body Weight (24 h) and LPS 1 mg / kg Body Weight (12 h). Blood was collected from the eyeballs after anesthesia.

[0038] Figure 6A ILA inhibited the LPS-induced increase in hepcidin in the spleen of mice; among which, Figure 6A -C Aila inhibited LPS-induced decreases in Fpn1 (6A, B) and FtL (6A, C) in mouse spleen; Figure 6D Aila inhibited the increase in hepcidin transcription levels in the spleen of mice induced by LPS. Six-week-old C57BL / 6 mice were randomly divided into four groups (n=6 per group) after one week of acclimatization. The CON group received a single intraperitoneal injection of PBS; the Aila group received a single intraperitoneal injection of Aila 1 mg / kg body weight (24 h); the LPS group received a single intraperitoneal injection of LPS 1 mg / kg body weight (12 h); and the LPS+Aila group received a single intraperitoneal injection of Aila 1 mg / kg body weight (24 h) and LPS 1 mg / kg body weight (12 h). Blood was collected from the eyeballs after anesthesia. Detailed Implementation

[0039] Studies have confirmed that the signal transduction and transcriptional activator protein (STAT3) is abnormally activated in cancer patients. In liver cancer patients, compensatory reduction in hepcidin increases iron absorption, leading to decreased serum iron and significant anemia. Furthermore, studies have demonstrated that inflammation induces abnormal increases in hepcidin through STAT3 (Y705) phosphorylation, resulting in inflammatory anemia. Our research team, through screening 2880 active substances from traditional Chinese medicine sources, discovered that the active component Aila from the traditional Chinese medicine Ailanthus altissima significantly inhibits the expression of hepcidin.

[0040] The applicant primarily studied the effects of Aila on cancer-related anemia, physiological anemia, and inflammatory anemia, while also investigating the mechanism by which Aila increases serum iron at the cellular and animal levels.

[0041] The effects of Aila on cancer-induced anemia were investigated: Human hepatocellular carcinoma cell lines HepG2 and MHCC97H were treated with different concentrations of Aila for 24 hours. Real-time PCR was used to detect the transcriptional level of hepcidin, and Western blot was used to detect the expression level of pump ferritin Fpn1 and the phosphorylation level of STAT3 (Tyr705). Six-week-old C57BL / 6 mice were acclimatized for one week and subcutaneously injected with hepatocellular carcinoma mouse cells hepa1-6 (3*10⁻⁶ cells). 5 On the third day, Aila (1 mg / kg) was administered intraperitoneally every other day. On the 21st day, the eyeballs were enucleated under anesthesia to collect blood and test serum iron, unsaturated iron binding capacity, total iron binding capacity, iron loading saturation, and to photograph and weigh subcutaneous tumors.

[0042] A study on the effects of Aila on inflammatory anemia: Primary macrophages induced by intraperitoneal injection were treated with LPS or Aila. The transcriptional level of hepcidin was detected by real-time PCR, and the expression levels of ferritin receptor TfR1 and pump ferritin Fpn1, as well as the phosphorylation level of STAT3 (Tyr705), were detected by Western blot. C57BL / 6 mice were anesthetized by intraperitoneal injection of LPS or Aila, and blood was collected by enucleation to detect serum iron, unsaturated iron binding capacity, total iron binding capacity, and iron saturation. The transcriptional level of hepcidin in the spleen was detected by real-time PCR, and the protein levels of Fpn1 and FtL in the spleen were detected by Western blot.

[0043] Studies have found that Aila significantly inhibits hepatocellular carcinoma-induced anemia in hepatocellular carcinoma-bearing mice (HCC-B1-6). Further research revealed that Aila significantly inhibits the increase in Hepcidin induced by p-STAT3 activation in HCC cells. Aila also significantly inhibits LPS-induced decreases in serum iron and iron saturation in mice. Further research showed that Aila's significant inhibition of LPS-induced inflammatory anemia is achieved by inhibiting the increase in Hepcidin induced by p-STAT3.

[0044] Conclusion: Aila achieves its therapeutic effect on anemia by inhibiting the STAT3 signaling pathway, suppressing hepcidin levels, and thereby increasing Fpn1 levels, leading to increased iron excretion and serum iron levels. It is a novel drug for treating anemia.

[0045] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0046] Abbreviation Notes

[0047] CON (control) comparison

[0048] Aila (Ailanthone)

[0049] LPS (Lipopolysaccharide)

[0050] HCC (Hepatocellular carcinoma)

[0051] Fpn1 (Ferroportin) is a cell membrane iron transporter 1.

[0052] TfR1 (Transferrin Receptor)

[0053] Ferritin

[0054] FtL (Ferritin light chain)

[0055] FtH (Ferritin heavy chain)

[0056] Hepcidin

[0057] STAT3 (signal transducer and activator of transcription) is a protein that activates transcription and signal transduction.

[0058] Serum iron

[0059] UIBC (unsaturated iron-binding capacity)

[0060] TIBC (total iron-binding capacity)

[0061] TF (%) Iron Saturation

[0062] Western blot protein blotting (immunoblotting)

[0063] Materials and methods

[0064] 1. Six-week-old male C57BL / 6 mice were randomly divided into four groups according to body weight after one week of acclimatization: a blank control group (CON), an aila group (Aila), a lipopolysaccharide (LPS) group, and a LPS+Aila group (LPS + Aila). The LPS group consisted of five mice, and the other three groups each contained six mice. Mice were administered the drugs intraperitoneally. The blank control group received 100 μl / 20 g body weight PBS. The Aila group received 1 mg / kg body weight ailadronate (24 h), the LPS group received 1 mg / kg body weight LPS (12 h), and the LPS+Aila group received 1 mg / kg body weight ailadronate (24 h) and 1 mg / kg body weight LPS (12 h). After anesthesia, the mice were enucleated to collect blood, euthanized by cervical dislocation, and the spleen was harvested.

[0065] 2. Six-week-old male C57BL / 6 mice were randomly divided into four groups according to body weight after one week of acclimatization: blank control group (CON), hepatocellular carcinoma model group (HCC), and hepatocellular carcinoma and ailanthus ketone group (HCC+Aila). The CON group consisted of 6 mice, and the HCC and HCC+Aila groups each contained 5 mice. On day 1, the HCC and HCC+Aila groups were subcutaneously administered hepatocellular carcinoma 1-6 (3 x 10^6 ml) at a dose of 100 μl / 20 g body weight. 5 Mice were administered the drug via intraperitoneal injection every other day starting on day 3. The blank control group received 100 μl / 20g BodyWeight PBS, the HCC group received 100 μl / 20g BodyWeight PBS, and the HCC+Aila group received 1 mg / kg BodyWeight ostrichone. After 21 days of administration, the mice were anesthetized, enucleated to collect blood, euthanized by cervical dislocation, and the tumors were dissected, photographed, and weighed.

[0066] 3. Preparation of serum: Blood samples obtained by enucleation were left at room temperature for 1 hour, centrifuged at 7000 rpm for 10 minutes, and the supernatant was collected. The samples were then centrifuged again at 7000 rpm for 10 minutes and the supernatant was collected for use.

[0067] Serum iron and unsaturated iron binding capacity detection

[0068] Serum iron levels were measured using POINTE's Iron / TIBC Reagent Set kit.

[0069] A. Serum iron

[0070] 1. Prepare a 96-well plate and label it blank, standard, and sample.

[0071] 2. Add 100 μl of iron buffer to each well.

[0072] 3. Add 20 μl of deionized water to blank, 20 μl of Iron standard (500 μg / dl) to standard, and 20 μl of sample to sample.

[0073] 4. Using a microplate reader with blank as 0, measure the absorbance reading (A1) of each well at 560 nm.

[0074] 5. Add 2 μl of iron color reagent to each well, mix well, and incubate at 37 degrees Celsius for 10 minutes.

[0075] 6. Using a microplate reader with blank as 0, measure the absorbance reading at 560 nm for each well (A2).

[0076] Serum iron (μg / dl) = (A2sample - A1sample) / (A2std - A1std) * concentration of St

[0077] B. Serum UIBC

[0078] 1. Prepare a 96-well plate and label it blank, standard, and sample.

[0079] 2. Add 80 μl of UIBC buffer to each well.

[0080] 3. Add 40 μl of deionized water to blank, and add 20 μl of deionized water and 20 μl of Ironstandard to standard.

[0081] Add 20 μl of sample and 20 μl of Iron standard to the sample.

[0082] 4. Using a microplate reader with blank as 0, measure the absorbance reading (A1) of each well at 560 nm.

[0083] 5. Add 2 μl of iron color reagent to each well, mix well, and incubate at 37 degrees Celsius for 10 minutes.

[0084] 6. Using a microplate reader with blank as 0, measure the absorbance reading (A2) at 560 nm for each well.

[0085] UIBC(μg / dl)=Conc of std-(A2sample-A1sample) / (A2std-A1std)*Concentration of std

[0086] TIBC (μg / dl) = serum iron + UIBC

[0087] TF (%) = Serum Iron / TIBC

[0088] 4. Preparation of tissue proteins:

[0089] Take 0.01g of spleen or tumor tissue, add 100μl of RIPA lysis buffer, shake on a shaker at 60 Hz for 3 minutes, centrifuge at 13200 rpm for 15 minutes, collect the supernatant, and use Western blot to detect the expression levels of ferritin receptor TfR1 and ferroexclusion protein Fpn1, as well as the phosphorylation level of STAT3 (Tyr705).

[0090] 5. Peritoneal-induced primary macrophages and RAW264.7

[0091] Six-week-old male C57B6 mice were acclimatized for one week and then intraperitoneally injected with 3% mercaptoacetic acid (MGA) medium. Three days later, the mice were euthanized by cervical dislocation. The cells were soaked in 75% ethanol solution for 10 seconds, fixed in a supine position on a dissecting board, and the abdominal skin was thoroughly cut open with surgical scissors to expose the abdominal muscle layer. The abdominal muscle layer was then disinfected with an alcohol swab. 10 ml of PBS was injected into the peritoneal cavity using a 10 ml syringe, and the abdomen was gently massaged for 2 minutes. Cell suspension was then aspirated using a 5 ml syringe, centrifuged at 300 g for 10 minutes, and diluted to 1 × 10⁶ cells / mL. The cells were seeded into 6-well plates, with 2 mL of cell suspension added to each well. After incubation in a 5% CO₂ incubator for 3 hours, the medium was changed, and the cells were washed twice with PBS to remove non-adhering cells. DMEM high-glucose complete medium (10% fetal bovine serum, 1% penicillin, and streptomycin) was added. Cells were treated with the drugs 24 hours later. The control group received PBS, the Aila group received 1.25 μmol of ailanthus ketone (for 24 hours), the LPS group received 1 μg / ml of lipopolysaccharide (for 12 hours), and the ailanthus ketone and lipopolysaccharide groups received 1.25 μmol of ailanthus ketone (for 24 hours) and 1 μg / ml of lipopolysaccharide (for 12 hours). Cells were then collected for RNA and protein extraction. RAW26.7 cells were cultured at a density of 5*102 5 Inoculate per milliliter into a 6-well plate, using the same administration method as above.

[0092] 6. Liver cancer cells

[0093] Resuscitate liver cancer cells MHCC97H and HepG2, passage them for 2 generations, and then increase the cell count to 5×10⁻⁶. 5Cells were seeded at a concentration of 1 / mL into 6-well plates. The blank control group (CON) was given PBS (24h), the ailanthophyll group (Aila) was given ailanthophyll at a final concentration of 1.25 μmol (24h), the lipopolysaccharide group (LPS) was given lipopolysaccharide at a final concentration of 1 μg / mL (12h), and the lipopolysaccharide and ailanthophyll group (LPS+Aila) was given ailanthophyll at a final concentration of 1.25 μmol (24h) and lipopolysaccharide at a final concentration of 1 μg / mL (12h). RNA and protein were extracted from the cells.

[0094] 7. Preparation of cell protein samples

[0095] Immediately place the cells on ice after removal, wash them twice with PBS, add 80 μl of cell lysis buffer with protease inhibitor to each well, scrape the cells off, collect them in 1.5 ml centrifuge tubes, incubate on ice for 30 minutes, centrifuge at 13200 rpm for 15 minutes, and aspirate the supernatant.

[0096] Example 1: Aila significantly inhibited the expression of HepG2 and MHCC97Hhepcidin.

[0097] To investigate the effect of Aila on hepcidin expression in human hepatocellular carcinoma cells, we treated HepG2 and MHCC97H2 cells with different concentrations of Aila for 4 hours. The study found that Aila significantly inhibited the cell viability of both cell types. Figure 1A B). Simultaneously, it significantly inhibited the expression of hepcidin, a central molecule regulating iron in two cells. Figure 1C (D). Considering the effects of Aila on cell viability and hepcidin expression, we selected an Aila concentration of 1.25 μmol for our subsequent experiments.

[0098] Example 2: Aila inhibits the phosphorylation levels of HepG2 and MHCC97H STAT3 (Tyr705).

[0099] The above studies suggest that Aila inhibits hepcidin expression. Previous research has shown that hepcidin is degraded by binding to Fpn1, the only known iron export protein pump, while STAT3 phosphorylation into the nucleus and binding to the hepcidin promoter increases its expression. Studies have found that 1.25 μmol of Aila significantly increases the transcriptional level of Fpn1. Figure 2A Further mechanistic studies revealed that treatment of both cell lines with different concentrations of Aila significantly inhibited the phosphorylation level of STAT3 (Tyr705), and this inhibition was concentration-dependent. Figure 2CStudies have shown that STAT3 is abnormally activated in cancer. The above research suggests that Aila inhibits the expression of hepcidin by suppressing the abnormally activated STAT3 (Tyr705) in liver cancer cells. Hepcidin can internalize and degrade Fpn1. Aila inhibits the expression of hepcidin through STAT3, thereby increasing the expression of Fpn1.

[0100] Example 3: Aila significantly inhibited the decrease in serum iron and carrier saturation in liver cancer-bearing mice.

[0101] In vitro studies have shown that Aila can inhibit the expression of hepcidin in human liver cancer cells. Previous research has indicated that decreased hepcidin levels in the body increase iron absorption. Studies have shown that liver cancer patients have high hepcidin expression, decreased serum iron, and significant anemia. To investigate whether Aila can increase iron absorption in vivo, we used subcutaneous hepa1-6 tumor-bearing C57BL / 6 mice. The study found that Aila significantly inhibited tumor growth. Figure 3A Compared to control mice, hepa1-6 tumor-bearing mice showed significantly reduced serum iron, total iron-binding capacity, and iron saturation. Figure 3B (D, E), the saturation force of unbound iron remains unchanged. Figure 3C This indicates that serum iron levels in hepatocellular carcinoma-bearing mice are significantly decreased, leading to cancer-related anemia. Aila treatment significantly inhibited the decrease in serum iron, total iron-binding capacity, and iron saturation induced by hepatocellular carcinoma (HCC) 1-6. Figure 3B (D, E). In vivo studies have shown that Aila can improve serum iron deficiency caused by liver cancer and treat cancer-related anemia.

[0102] Example 4: Aila inhibits LPS-induced hepcidin elevation in primary macrophages via the STAT3 (Tyr705) pathway.

[0103] We further investigated whether Aila also had an effect on inflammatory anemia. Treatment of peritoneal-induced primary macrophages with Aila amide significantly inhibited cell viability, while treatment with Aila (1.25 μmol) had no significant effect on cell viability. Figure 4A In subsequent experiments, Aiila was used at a concentration of 1.25 μmol. Aiila significantly inhibited the increase in hepcidin expression induced by LPS treatment (Figure 4B), significantly inhibited LPS-induced phosphorylation of STAT3 (Y705), and significantly inhibited the decrease in ferritin TfR1, pump ferrin Fpn1, ferritin heavy chain (FtH), and ferritin light chain (FtL) induced by LPS. Figure 4CThe STAT3 inhibitor Sttatic significantly reversed the LPS-induced increase in hepcidin induced by Aila inhibition. Figure 4D This confirms that the LPS-induced increase in hepcidin inhibited by Aila is mediated by STAT3 (Tyr 705) phosphorylation.

[0104] Example 5: Under physiological conditions, Aila significantly increased serum iron and iron saturation. Under inflammatory conditions, Aila significantly inhibited LPS-induced decreases in serum iron and iron saturation.

[0105] To investigate the effects of Aila on serum iron levels in mice under physiological and LPS-induced inflammatory conditions, we used SPF-grade C57BL / 6 mice, 6 weeks old, which were randomly divided into four groups of 6 mice each after a week of acclimatization. All mice received a single dose of Aila for 24 hours and LPS for 12 hours. The study found that under physiological conditions, Aila administration significantly increased serum iron, unbound iron saturation, and total iron-binding capacity (LPS) 24 hours after administration. Figure 5A -C), has no effect on iron saturation. Figure 5D In inflammatory conditions, Aila significantly inhibited LPS-induced decreases in serum iron, unbound iron saturation, total iron-binding capacity, and iron loading saturation. Figure 5A Further analysis revealed that the increase in serum iron was more significant under inflammatory conditions compared to physiological conditions (148% vs 215%). In conclusion, injection of 1 mg / kg Aila in animals can significantly increase serum iron levels, thus improving both physiological and inflammatory anemia.

[0106] Example 6: Aila inhibits LPS-induced decrease in Fpn1 and increase in hepcidin in mouse spleen.

[0107] To verify the effects of Aila in vitro in peritoneally induced primary macrophages and to elucidate the mechanism by which Aila increases serum iron in mice, we treated C57BL / 6 mice with LPS or Aila. The study found that Aila significantly inhibited LPS-induced decrease in Fpn1 levels in the mouse spleen. Figure 6A B) and the decrease of FtL ( Figure 6A C) and hepcidin increase ( Figure 6D ).

[0108] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in methods and compositions, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The use of ailanthus ketone or its pharmaceutically acceptable salt as the sole active ingredient in the preparation of drugs for treating anemia.

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

3. Use according to claim 1, characterized in that: The drug has at least one of the following effects: a. Inhibit the STAT3 signaling pathway; b. Inhibit hepcidin levels; c. Increase serum iron levels.

4. Use according to claim 1, characterized in that: The drug also contains a carrier that is acceptable to the human body.

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