Prolyl hydroxylase inhibitors and their use

Abstract

The present invention discloses a prolyl hydroxylase inhibitor and its application in the prevention and treatment of iron deficiency and related diseases. Specifically, hydroxyproline di / tripeptides produced by collagen digestion and degradation are used as prolyl hydroxylase inhibitors, which upregulate the expression of iron absorption transporters such as divalent metal ion carrier 1, duodenal cytochrome b, ferroportin and ferrous oxidase in intestinal epithelial cells by activating the hypoxia-inducible factor 2α signaling pathway, thereby stimulating the body's iron absorption, and can be applied to foods, health foods and medicines for the prevention and treatment of iron deficiency and related diseases. The prolyl hydroxylase inhibitor used in the present invention is derived from food protein, so compared with conventional synthetic drugs, it has the advantage of no toxic side effects. The application of the present invention can be used as a supplement or alternative therapy for iron nutritional intervention, which can make up for the deficiencies of iron nutritional intervention in safety and effectiveness, and is particularly effective for functional iron deficiency caused by inflammation or renal injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a prolyl hydroxylase inhibitor and application thereof. Background Art

[0002] Iron deficiency is the most common type of malnutrition. According to the Global Burden of Disease Study, iron deficiency affected 1.1 billion people worldwide (61% of the total malnourished population) in 2017, causing 30 million disability-adjusted life years (DALYs). It is the number one factor causing DALYs in children aged 5-14 years and the fifth largest factor causing DALYs in women of childbearing age.

[0003] Currently, common oral iron supplements and food iron fortification are intervention measures to deal with iron deficiency. However, clinical studies have found that excessive iron intake has great health risks and can cause intestinal microecological dysfunction, thereby increasing the incidence of diarrhea and infectious diseases in children. According to the latest systematic review and meta-analysis, oral iron supplements have no preventive effect on approximately 30% of iron deficiency during pregnancy and 20% of iron deficiency in school-age children. Therefore, it is necessary to seek an effective and potentially safe intervention method to replace or supplement existing intervention measures.

[0004] The body's iron homeostasis is closely related to the oxygen supply of various tissues. The hydroxylation of proline residues in HIFα subunits by prolyl hydroxylases (PHDs) is the core part of the oxygen sensing pathway. In recent years, PHD inhibitors have become a hot target for regulating the body's iron homeostasis, but natural PHD inhibitors have not been fully recognized and utilized. Therefore, screening natural PHD inhibitors provides new ideas for nutritional intervention of iron deficiency by regulating iron homeostasis. No relevant reports on the inhibition of PHD by collagen peptides were found in the literature. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a natural prolyl hydroxylase inhibitor and its application in the prevention and treatment of iron deficiency and related diseases.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] The present invention firstly finds that hydroxyproline oligopeptides can be used as prolyl hydroxylase inhibitors, especially hydroxyproline di / tripeptides.

[0008] The hydroxyproline-containing di / tripeptide provided by the present invention comprises one or more of the following sequences: prolyl hydroxyproline

[0009] (Pro-Hyp), valine-hydroxyproline (Ala-Hyp), glutamyl-hydroxyproline (Glu-Hyp), leucyl-hydroxyproline (Leu-Hyp), seryl-hydroxyproline (Ser-Hyp), hydroxyprolylglycine (Hyp-Gly), valine-hydroxyproline-glycine (Ala-Hyp-Gly), glutamic acid-hydroxyproline-glycine (Glu-Hyp-Gly), proline-hydroxyproline-glycine (Pro-Hyp-Gly), serine-hydroxyproline-glycine (Ser-Hyp-Gly), glycine-proline-hydroxyproline (Gly-Pro-Hyp), and glycine-hydroxyproline-hydroxyproline (Gly-Hyp-Hyp).

[0010] The hydroxyproline-containing di / tripeptide of the present invention is produced by digestion and degradation of collagen, and the digestion and degradation of collagen includes any one or more of protease digestion, acid hydrolysis or alkaline hydrolysis.

[0011] The protease used in the protease digestion includes any one or two or more of pepsin, trypsin, alkaline protease, papain, ginger protease, bromelain, ficin, and neutral protease.

[0012] The prolyl hydroxylase inhibitor of the present invention can be used to prepare food, health food and medicine for preventing and treating iron deficiency and related diseases.

[0013] Furthermore, the present invention also provides a drug for preventing and treating iron deficiency and related diseases, which uses the prolyl hydroxylase inhibitor as an active ingredient and is used in combination with an iron agent.

[0014] Furthermore, the iron agent is selected from one or more of ferrous sulfate, ferrous lactate, ferrous gluconate, ferrous fumarate, dextran iron, ferrous succinate, polysaccharide iron complex, ammonium ferric citrate, ferric citrate, heme chloride, ferric pyrophosphate, iron porphyrin, ferrous glycinate, reduced iron, sodium ferric ethylenediaminetetraacetate, carbonyl iron powder, ferrous carbonate, ferrous citrate, ferrous fumarate, heme iron, and electrolytic iron.

[0015] The iron deficiency and related diseases that can be prevented and treated by the medicine provided by the present invention include iron deficiency, iron deficiency anemia, renal anemia, sports anemia, chronic disease anemia, tumor-related anemia and hemorrhagic anemia.

[0016] The administration route of the drug is oral administration or parenteral administration. The parenteral administration method is one of intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and local injection.

[0017] The prolyl hydroxylase described in the present invention includes any one or more of prolyl hydroxylase domain protein 1 (PHD1), prolyl hydroxylase domain protein 2 (PHD2) and prolyl hydroxylase domain protein 3 (PHD3).

[0018] The experimental data of the present invention show that the hydroxyproline di / tripeptide produced by collagen digestion and degradation can produce competitive and non-competitive mixed inhibition on prolyl hydroxylase by chelating the coenzyme iron core and forming steric hindrance, and has activity equivalent to that of chemically synthesized PHD inhibitors. Secondly, the experiment further verified that the hydroxyproline di / tripeptide produced by collagen digestion and degradation activated the hypoxia-inducible factor 2α signaling pathway in colon cancer cells and rat duodenum, and by upregulating the expression of iron absorption transporters such as divalent metal ion carrier 1, duodenal cytochrome b, ferroportin and ferrous oxidase, the ability of intestinal cells to reduce, absorb and transport iron was improved, and at the same time, the functional iron deficiency caused by renal damage could be alleviated, thereby achieving the prevention and treatment of iron deficiency and related diseases.

[0019] Compared with the prior art, the technical advantages of the present invention are:

[0020] 1) The present invention is the first to use the hydroxyproline di / tripeptide produced by digestion and degradation of collagen as a prolyl hydroxylase inhibitor. The prolyl hydroxylase inhibitor is obtained by using collagen, a protein naturally present in food. Therefore, compared with conventional artificially synthesized prolyl hydroxylase inhibitors, the prolyl hydroxylase inhibitor of the present invention is expected to have no toxic side effects;

[0021] 2) Experimental data show that the hydroxyproline di / tripeptides produced by collagen digestion and degradation can produce competitive and non-competitive mixed inhibition of prolyl hydroxylases by chelating the coenzyme iron core and forming steric hindrance, and have activity comparable to that of chemically synthesized PHD inhibitors. For example, Pro-Hyp inhibits the EC of PHD3 50 Value and K i The values ​​can reach 10.62μM and 6.73μM respectively. Secondly, the experiment further verified that the hydroxyproline di / tripeptide produced by collagen digestion and degradation activated the hypoxia-inducible factor 2α signaling pathway in colon cancer cells and rat duodenum, and by upregulating the expression of iron absorption transporters such as divalent metal ion carrier 1, duodenal cytochrome b, membrane ferroportin and ferroxidase, the ability of intestinal cells to reduce, absorb and transport iron was improved, thereby achieving the prevention and treatment of iron deficiency and related diseases; therefore, the hydroxyproline di / tripeptide produced by collagen digestion and degradation can be used as a prolyl hydroxylase inhibitor to stimulate the body's own intestinal iron absorption. Compared with traditional iron nutritional intervention, it has significant application advantages in solving functional iron deficiency caused by inflammation or renal injury;

[0022] 3) Experiments have shown that the hydroxyproline-containing di / tripeptides produced by digestion and degradation of collagen are quite resistant to degradation by digestive tract and blood peptidases and can therefore be administered orally, which is a significant advantage of the present invention considering the fact that proteins are rarely administered by this route. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The specific implementation modes and beneficial effects of the present invention are further described in detail below with reference to the accompanying drawings.

[0024] Figure 1 The enzymatic reaction kinetics of recombinant human PHD3: (a) substrate concentration dependence; (b) effect of Pro-Hyp concentration (substrate concentration is Km); (c) double reciprocal plot at different Pro-Hyp concentrations.

[0025] Figure 2 The dominant binding conformations of PHD3 with (a) Pro-Hyp and (b) HIF19, respectively, and the conformations of the two after being merged in the (c) protein filling model and (d) ball-and-stick model.

[0026] Figure 3 Effects of hydroxyproline di / tripeptides (CH) and Pro-Hyp on the HIF-2α signaling pathway in intestinal epithelial cells: Western blotting of HIF-2α protein levels (a) image analysis; (b) density analysis; (c) dual luciferase reporter gene method to determine the activity of hypoxia response element (HRE); (d) HIF-2α transcription level.

[0027] Figure 4 Effects of collagen degradation-containing hydroxyproline di / tripeptides (CH) and Pro-Hyp on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) ferroportin (FPN); and (d) ferroxidase (HEPH).

[0028] Figure 5 Figure 3. The promoting effect of hydroxyproline di / tripeptides (CH) and Pro-Hyp on intestinal iron absorption by collagen degradation: (a) kinetics of iron absorption in Caco-2 cell monolayers; (b) concentration of divalent iron accumulated in the supernatant culture medium; (c) concentration of iron transported from the monolayer to the lower chamber in the Transwell iron transport device.

[0029] Figure 6Effects of oral administration of collagen-degrading hydroxyproline di / tripeptides (CH) or Pro-Hyp on the mRNA expression levels of duodenal ferroportin in rats: (a) divalent metal ion carrier 1 (DMT1) and duodenal cytochrome b (Dcytb); (b) ferroportin (FPN) and ferroxidase (HEPH); (c) Western blotting images of HIF-2α; (d) density analysis.

[0030] Figure 7 The improvement effect of hydroxyproline di / tripeptide (CH) from collagen degradation on renal anemia: (a) renal index; (b) urea nitrogen content in urine; (c) hemoglobin content in blood; (d) number of red blood cells in blood. DETAILED DESCRIPTION

[0031] Example 1

[0032] Evaluation of prolyl hydroxylase inhibitory activity and mechanism analysis of hydroxyproline di / tripeptides derived from collagen. The specific experimental process and experimental conclusions are as follows:

[0033] Figure 1 The enzymatic reaction kinetics of recombinant human PHD3: (a) substrate concentration dependence; (b) effect of Pro-Hyp concentration (substrate concentration is Km); (c) double reciprocal plot at different Pro-Hyp concentrations.

[0034] Figure 2 The dominant binding conformations of PHD3 with (a) Pro-Hyp and (b) HIF19, respectively, and the combined conformations of the two in (c) protein filling model and (d) ball-and-stick model.

[0035] Using in vitro recombinant PHD3, the inhibitory activity of prolylhydroxyproline (Pro-Hyp) on it was determined. The PHD3 gene was inserted between the EcoRV and SalI sites of the pET-32a vector to construct the plasmid pET-32a-PHD3. The expression host of the plasmid is Escherichia coli. After heat shock (42°C, 30s), the plasmid was transformed. A single colony of pET-32a-PHD3 was selected on a standard agar plate containing ampicillin and inoculated into LB broth. It was cultured overnight in a 37°C shaker until the optical density at 600nm reached between 0.6-0.8. Then 200μL IPTG was used for induction and cultured in a 25°C shaker for 4h to overexpress PHD3 in Escherichia coli. Purification was performed by nickel (Ni) affinity chromatography. E. coli was centrifuged at 11000×g for 9 min, resuspended in 8 mL of pre-cooled binding buffer (20 mM Tris-HCl buffer at pH 7.9, containing 0.5 M NaCl and 5 mM imidazole), and centrifuged at 14000×g for 20 min after ultrasonic treatment on ice. The supernatant after centrifugation was loaded onto a 2.5 mL Ni-NTA agarose column at a flow rate of 20 mL / h. After adsorption of recombinant PHD3, the column was washed with 10 column volumes of binding buffer. The recombinant PHD3 was then eluted with 6 column volumes of elution buffer (20 mM Tris-HCl buffer at pH 7.9, containing 0.5 M NaCl and 1 M imidazole). The obtained PHD3 was dialyzed twice in sodium phosphate buffer (20 mM, pH 7.0) containing 2 M NaCl and 5 mM DTT to remove imidazole. PHD belongs to the 2-ketoglutarate (2-OG)-dependent oxygenase superfamily. 2+ Combines with enzyme to form Fe 2+ -enzyme complex, which binds 2-OG and HIFα subunits in sequence, so the progress of the enzyme reaction can be judged according to the consumption of 2-OG. In the entire reaction system, the substrate is HIF 19 peptide (sequence DLDLEMLAPYIPMDDDFQL). When performing inhibition experiments, different concentrations of Pro-Hyp were added to the entire system, and the reaction was initiated by adding 4 μL 2-OG (160 mM) to the reaction mixture. The concentration of 2-OG remaining after the catalytic reaction was quantified by detecting the fluorescence intensity of the derivatized product of 2-OG on a Synergy H4 fluorescence microplate reader (Bio-Tek) (340 nm excitation; 420 nm emission).

[0036] Figure 1 a It can be seen that the Michaelis constant of the synthesized recombinant human PHD3 protein is 2.15±0.22μM, which is close to the 2.9±0.4μM reported in the literature; Pro-Hyp can inhibit the activity of recombinant human PHD3 protein, and its half-effective concentration EC50 is 10.62μM ( Figure 1 b), inhibition constant K i6.73μM( Figure 1 c), which is comparable to the activity of chemically synthesized PHD inhibitors in the literature. Therefore, Pro-Hyp can effectively inhibit the activity of PHD and stabilize the level of HIF.

[0037] The possible mechanism of PHD inhibition by collagen-derived hydroxyproline di / tripeptides was further studied using molecular simulation technology. The crystal structure of PHD3 (EGLN1, protein Data Bank ID: 2G19) was used as a template, free water and nonpolar hydrogen were removed, Gasteiger charges and partial atomic charges were added, and desolvation parameters were assigned using AutoDock Tools in MGL Tools 1.5.6 software (The Scripps Research Institute, CA, USA). The three-dimensional protein structure of PHD3 (EGLN3) was simulated by homology modeling. The three-dimensional structures of typical collagen-derived di / tripeptides and Roxadustat were constructed using ChemDraw 19.0, and energy minimization was performed using Chem3D 19.0 (PerkinElmer, MA, USA). The collagen-derived di / tripeptides and Roxadustat were molecularly docked with PHD3 using Autodock 4.2.6 software. The semi-flexible docking method was used to allow the ligand to be flexible and the receptor backbone atoms to remain rigid during the calculation. A grid with a spacing of 10000 points was constructed with the Fe(II) atom at the active site of PHD3 as the center. A high-resolution rectangular grid frame The optimal docking configuration was selected from the lowest binding energy cluster of 100 Lamarckian genetic algorithm docking solutions. The initial position of the ligand was set randomly, and the rest of the docking parameters were set by default. The docking results were analyzed using the PyMOL program ( NY, USA). Pro-Hyp has a good fit to the hydrophobic pocket in the active site of PHD3, where the imino N atom and amide O atom of proline form coordination bonds with the iron core in the active pocket of PHD3 ( Figure 2 a), hydrogen bonds between its hydroxyproline residue and Y125, R205 and T209 residues of the enzyme ( Figure 2 b), the hydrophobic group in Pro-Hyp forms good hydrophobic contacts with the hydrophobic amino acid residues Y132, V198, W211, and M121 in the active pocket of PHD3 ( Figure 2 c). Apparently, Pro-Hyp can act as an iron chelator to block the active site of PHD3 to some extent, which explains its non-competitive inhibition ( Figure 2c). The amide O atom of P9 in the HIF19 peptide forms a hydrogen bond with R144 in the active pocket of PHD3, guiding P9 to approach the iron ion for hydroxylation. The docking models of PHD3 with Pro-Hyp and HIF19 were placed in the protein filling model ( Figure 2 c) and the ball-and-stick model ( Figure 2 d), it was found that Pro-Hyp would cause steric hindrance to the proximity of P9 in the HIF19 peptide to the iron ion, indicating that there is a competitive antagonistic relationship between Pro-Hyp and the HIF19 peptide. Pro-Hyp can competitively and sterically hinder the binding of the substrate to the enzyme active center, which explains its competitive inhibition ( Figure 2 c).

[0038] As shown in Table 1 , it is predicted that the binding energy value of Pro-Hyp is close to that of the oral PHD inhibitor Roxadustat (Table 1 ), indicating that Pro-Hyp has great potential as a dietary PHD inhibitor. At the same time, the binding energy values ​​of prolylhydroxyproline (Pro-Hyp), valinehydroxyproline (Ala-Hyp), glutamylhydroxyproline (Glu-Hyp), leucylhydroxyproline (Leu-Hyp), serylhydroxyproline (Ser-Hyp), hydroxyprolylglycine (Hyp-Gly), valine-hydroxyproline-glycine (Ala-Hyp-Gly), glutamic acid-hydroxyproline-glycine (Glu-Hyp-Gly), proline-hydroxyproline-glycine (Pro-Hyp-Gly), serine-hydroxyproline-glycine (Ser-Hyp-Gly), glycine-proline-hydroxyproline (Gly-Pro-Hyp) and glycine-hydroxyproline-hydroxyproline (Gly-Hyp-Hyp) with PHD were calculated, and it was found that the presence of hydroxyproline residues can greatly enhance the binding of di / tripeptides with PHD3. The above in vitro experiments show that, therefore, the hydroxyproline-containing di / tripeptides produced by collagen digestion and degradation can produce competitive and non-competitive mixed inhibition of prolyl hydroxylase by chelating the coenzyme iron core and forming steric hindrance, and have activity comparable to that of chemically synthesized PHD inhibitors.

[0039] Table 1. Predicted binding energy values ​​of collagen-derived hydroxyproline di / tripeptides and Roxadustat to human PHD3 protein calculated by molecular docking

[0040] Ligand Binding energy value (kcal / mol) Ala-Hyp -7.48 Glu-Hyp -7.05 Leu-Hyp -7.69 Ser-Hyp -7.39 Pro-Hyp -7.91 Hyp-Gly -7.06 Gly-Pro-Hyp -8.33 Ala-Hyp-Gly -8.80 Glu-Hyp-Gly -9.00 Pro-Hyp-Gly -9.42 Ser-Hyp-Gly -7.85 Gly-Hyp-Hyp -8.58 Roxadustat -8.11

[0041] Example 2

[0042] The specific experimental process and experimental conclusions of the inhibitory effect of hydroxyproline di / tripeptide (CH) and Pro-Hyp on PHD and the regulatory effect on iron absorption and transport in Caco-2 intestinal epithelial cells in vitro are as follows:

[0043] Figure 3 Effects of collagen degradation-containing hydroxyproline di / tripeptides (CH) and Pro-Hyp on the HIF-2α signaling pathway in intestinal epithelial cells: Western blotting of HIF-2α protein levels (a) image analysis; (b) density analysis; (c) dual luciferase reporter gene method to determine the activity of hypoxia response element (HRE); (d) HIF-2α transcription level.

[0044] Figure 4 Effects of collagen degradation by hydroxyproline di / tripeptides (CH) and Pro-Hyp on the expression of intestinal iron homeostasis-related proteins: (a) divalent metal ion transporter (DMT1); (b) duodenal cytochrome b (Dcytb); (c) ferroportin (FPN); and (d) ferroxidase (HEPH).

[0045] Figure 5 Promoting effects of collagen-degraded hydroxyproline di / tripeptides (CH) and Pro-Hyp on intestinal iron absorption: (a) Kinetics of iron absorption in Caco-2 cell monolayers; (b) Concentration of ferrous iron accumulated in the supernatant culture medium; (c) Concentration of iron transported from the monolayer to the lower chamber in the Transwell iron transport device.

[0046] This experiment selected colon cancer cells Caco-2, which have similar structures and functions to small intestinal epithelial cells. The cell lines used in the experiment were purchased from the cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee. The cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B mixed solution on a 25 cm 2 The cells were cultured in cell culture flasks, which were placed in a cell culture incubator at 37°C and 5% CO2 with constant humidity. The culture medium was changed every 2 days. When the cell confluence reached 80%, the cells were plated at a certain ratio according to the experimental needs. Caco-2 cells were diluted to a certain concentration with high-glucose DMEM complete medium, with 2×10 cells per well. 5The Caco-2 suspension was inoculated into a 6-well cell culture plate at a density of 10 cells. The culture medium was replaced every 2 days. After the cells were fully confluent (about 3-4 days), the culture medium was replaced every 1 day. After about 10 days, the cells were completely differentiated into a monolayer, and the in vitro intestinal cell model was obtained. Subsequently, hydroxyproline di / tripeptide (CH), Pro-Hyp and characteristic amino acids in the three collagen peptides were added to the collagen degradation at various concentrations for incubation, and a medium without drugs was set as a control group. After the cells were cultured in the incubator for 24 hours, the culture medium was removed and the above solutions of various concentrations were continued to be added for incubation. The total time for the cells to act on the cells was 48 hours. Western blotting was used to study the effects of different components on cellular HIF-2α expression and intestinal iron homeostasis. Figure 3 The Western blotting results of a and 3b show that after 48 h of incubation, CH and Pro-Hyp can significantly increase the expression level of HIF-2α in intestinal cells. The main mechanism is to inhibit PHD activity rather than to increase the transcription level of HIF-2α ( Figure 3 c). Afterwards, the plasmid containing the HIF-2α receptor response element (HRE) fragment was introduced into Caco-2 cells using Lipofectamine 3000, and the transcriptional activity of HIF-2α downstream genes was determined using the dual luciferase reporter gene method. It was found that the transcriptional activity of downstream genes increased in a dose-dependent manner with the increase in Pro-Hyp concentration ( Figure 3 d). We further detected the expression of intestinal iron homeostasis-related proteins downstream of HIF-2α. Figure 4 It can be seen that CH and Pro-Hyp played a significant role in promoting the expression of divalent metal ion carrier 1 (DMT-1) for transporting iron in the intestinal lumen and duodenal cytochrome b (Dcytb) responsible for iron reduction, as well as the expression of iron transporter (FPN) and ferroxidase (HEPH) on the intestinal basal side (P<0.05).

[0047] In order to further verify its promoting effect on iron absorption, the calcein fluorescence method was used to measure the changes in cellular iron absorption dynamics. Caco-2 cell model was used to simulate the pH value of 5.5 in the proximal small intestine. 4 The cells were seeded at a density of 100 cells / well in a collagen-coated 24-well plate. After the cells were completely differentiated, the serum-free MEM medium was replaced for starvation treatment for 24 h. Calcein-AM was then added and incubated at 37°C for 30 min. Then, 970 μL of HBSS solution containing MES (adjusted to pH 5.5) and 1 mM nitrilotriacetic acid-Fe were added. 3+The sample solution was incubated at 37°C for 30 minutes in a Synergy H4 fluorescence microplate reader (Bio-Tek), and the calcein fluorescence (excitation at 485 nm, emission at 530 nm) was recorded every 3 minutes. Calcein-AM is a membrane-permeable non-fluorescent molecule that emits fluorescence after being cleaved by cytoplasmic esterases in cells, which can mark the content of divalent metal ions in the weakly bound iron pool of cells. In the Calcein fluorescence iron absorption experiment ( Figure 5 a), it can be seen that the iron absorption capacity of cells incubated with CH was significantly enhanced (P<0.01), and the iron absorption capacity was also improved to a certain extent after incubation with Pro-Hyp (P<0.05). In order to study the cell's ability to reduce divalent iron, sodium bathophenanthroline disulfonate (BPDS) was used, which can quickly bind Fe 2+ A pink complex is formed, and the amount of Fe 3+ The restoration capability. Figure 5 b As can be seen, the content of complexes formed by cells incubated with CH and Pro-Hyp increased significantly (P<0.05). Subsequently, the iron content transported from the basal side of the cells was further verified, and a Transwell iron transport model was constructed to determine the cell iron absorption and transport capacity by measuring the iron content in the lower chamber. Figure 5 c shows the results of the Transwell iron transport experiment. It can be seen that the iron content transported to the lower chamber by the monolayer cells is consistent with the results of the calcein iron absorption experiment and the ferrous iron accumulation experiment. The ability of cells to transport iron after incubation with CH and Pro-Hyp is significantly enhanced (P<0.05). Therefore, it is shown that the hydroxyproline di / tripeptide containing collagen degradation can inhibit PHD activity and stabilize HIF-2α, thereby increasing the expression of intestinal iron homeostasis-related proteins and promoting intestinal iron transport and absorption.

[0048] Example 3

[0049] The specific experimental process and conclusions of the inhibitory effect of hydroxyproline di / tripeptide (CH) and Pro-Hyp on rat duodenal PHD and the regulation of iron absorption and transport by collagen degradation are as follows:

[0050] Figure 6 Effects of oral administration of collagen-degrading hydroxyproline-containing di / tripeptides (CH) or Pro-Hyp on the mRNA expression levels of duodenal ferroportins in rats: (a) divalent metal transporter 1 (DMT1) and duodenal cytochrome b (Dcytb); (b) ferroportin (FPN) and ferroxidase (HEPH); Western blotting of HIF-2α (c) image analysis and (d) density analysis.

[0051] In order to verify whether the hydroxyproline di / tripeptide containing collagen degradation can stimulate the absorption of intestinal non-heme iron in vivo, female Sprague-Dawley rats aged 21±3 days were selected. The temperature of the animal room was 24±2℃, the humidity was 55±15%, the lights were turned on at 6 am, and the light and dark were alternating 12 / 12h. During the whole study, the animals were free to eat and drink ultrapure water. After 1 week of adaptation, they were randomly divided into 5 groups. The mice were first fasted for 12h (ultrapure water was normally supplied) to eliminate dietary interference, and then gavage CH at a high dose of 1.2g / kg and a low dose of 0.6g / kg, and Pro-Hyp at a high dose of 0.2g / kg and a low dose of 0.1g / kg. 2h after gavage, anesthesia was performed, and the duodenum and jejunum were obtained for mRNA and protein extraction. The transcription levels of duodenal genes Dcytb, DMT1, FPN and HEPH were measured, such as Figure 6 As shown in a and 6b, CH and Pro-Hyp induced a dose-dependent increase in the gene transcription levels of Dcytb, DMT1, FPN, and HEPH in the duodenum of rats, confirming the direct stimulatory effect of CH and Pro-Hyp on intestinal non-heme iron absorption in vivo. According to the results of Western blotting ( Figure 6 c and 6d), CH and Pro-Hyp dose-dependently increased the protein level of HIF-2α in the duodenum of rats, verifying the activation of HIF-2α signaling pathway by CH in vivo.

[0052] Example 4

[0053] Evaluation of the improvement effect of collagen degradation-containing hydroxyproline di / tripeptide on functional iron deficiency caused by renal injury. The specific experimental process and experimental conclusions are as follows:

[0054] Figure 7 Improvement of functional iron deficiency caused by renal injury by hydroxyproline di / tripeptide (CH) from collagen degradation: (a) renal index; (b) urea nitrogen content in urine; (c) hemoglobin content in blood; (d) number of red blood cells in blood

[0055] Abbreviations: high-dose group of hydroxyproline di / tripeptide for collagen degradation (CH-H), low-dose group of hydroxyproline di / tripeptide for collagen degradation (CH-L).

[0056] Six-week-old C57BL / 6 mice were used in each group. The animal room temperature was 24±2℃, the humidity was 55±15%, and the light cycle was 12 / 12h (lighting from 8 am to 8 pm). The animals were free to eat and drink ultrapure water during the entire study. The animal experiment ethics committee of Ocean University of China was approved (approval number: SPXY20220702).

[0057] After the mice adapted for 1 week, each group was gavaged with 100 μL of 50 mg / kg body weight of adenine (in 0.5% sodium carboxymethyl cellulose solution) daily for modeling. At the same time, according to the grouping, 100 μL of collagen peptide (in 0.5% sodium carboxymethyl cellulose solution) was gavaged daily, with the high dose being 600 mg / kg and the low dose being 150 mg / kg. The Roxadustat group was gavaged with 100 μL of 25 mg / kg (in 0.5% sodium carboxymethyl cellulose solution), and the control group was gavaged with 100 μL of PBS (in 0.5% sodium carboxymethyl cellulose solution). Modeling and treatment lasted for a total of four weeks.

[0058] like Figure 7 As shown in a, after a long period of adenine modeling damage, the kidneys of the PBS group were significantly enlarged and the renal index was high, while CH could significantly alleviate the enlargement of the kidneys (P<0.05). The presence of adenine caused renal damage, and a large amount of urea nitrogen could not be excreted from the urine. Therefore, the urea nitrogen in the urine of the PBS group was significantly reduced (P<0.05) ( Figure 7 b), while CH alleviates this trend. At the same time, CH can increase the number of hemoglobin and red blood cells in the blood ( Figure 7 c, 7d), improving the problem of iron deficiency caused by renal damage, which is consistent with the effect of the positive drug Roxadustat. Therefore, CH can effectively alleviate renal damage and is particularly effective in improving functional iron deficiency caused by renal damage.

[0059] The above embodiments show and describe the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for illustrating the principles of the present invention, and are not intended to limit the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the protection claimed.

Claims

1. The use of collagen digestion and degradation oligopeptides in the preparation of a drug for treating iron deficiency renal anemia, characterized in that: The collagen digestion and degradation oligopeptides are valine hydroxyproline (Ala-Hyp), glutamyl hydroxyproline (Glu-Hyp), leucyl hydroxyproline (Leu-Hyp), seryl hydroxyproline (Ser-Hyp), valine-hydroxyproline-glycine (Ala-Hyp-Gly), glutamic acid-hydroxyproline-glycine (Glu-Hyp-Gly), proline-hydroxyproline-glycine (Pro-Hyp-Gly), serine-hydroxyproline-glycine (Ser-Hyp-Gly), glycine-proline-hydroxyproline (Gly-Pro-Hyp) and glycine-hydroxyproline-hydroxyproline (Gly-Hyp-Hyp).

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