CN115414344B

CN115414344B - Application of L-citrulline in preparation of iron overload prevention and treatment drugs

Info

Publication number: CN115414344B
Application number: CN202211282808.4A
Authority: CN
Inventors: 戴汉川; 巴彤彤; 张骋; 赵黛; 牛赛; 陈怡沁; 李可可
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-10-19
Filing date: 2022-10-19
Publication date: 2024-01-05
Anticipated expiration: 2042-10-19
Also published as: CN115414344A

Abstract

The invention discloses an application of L-citrulline in preparing a medicament for preventing and treating iron overload. Belongs to the technical field of iron overload prevention and treatment medicines. The invention provides a new application of L-citrulline in preparing drugs for preventing and treating iron overload. Further, a novel application of L-citrulline in preparing medicaments for preventing and treating thymus injury caused by iron overload, a novel application of L-citrulline in preparing medicaments for preventing and treating iron autophagy caused by iron overload and resisting iron death, and an application of a composition of L-citrulline and siNCOA4 in preparing medicaments for preventing and treating iron overload are provided. Thereby solving the problem of lack of drugs for effectively preventing and treating iron overload at present.

Description

Application of L-citrulline in preparation of iron overload prevention and treatment drugs

Technical Field

The invention belongs to the technical field of iron overload prevention and treatment medicines, and particularly relates to application of L-citrulline in preparation of iron overload prevention and treatment medicines.

Background

Iron is an important trace element in the body, has important significance for maintaining physiological steady state, and participates in biochemical reactions such as oxygen transportation, oxidative phosphorylation, enzymatic reaction and the like, and is also a metal element which is critical for proliferation of all cells including immune cells. Iron is mainly from food and is absorbed through the small intestine as a component of hemoglobin, myoglobin and many key enzymes, and there is increasing evidence that iron metabolic disorders can lead to the development of many diseases. The iron metabolic process comprises the followingThe steps are as follows: in the first step of intake, iron is taken up into the blood by the intestinal cells and then acts, since ferric ions are not bioavailable, reduction to Fe by Dcytb (duodenal enzyme cytochrome b reductase) is required ²⁺ When intestinal cells ingest iron into the body, ionic iron is preferentially deposited in ferritin, iron in the cytoplasm exists mainly in ferrous form, or is bound to GSH (glutathione in reduced state), and can be transferred to a transporter through a cytoplasmic iron chaperone PCBP2 to release iron into blood; the second step of transportation and storage, wherein iron is combined with transferrin, endocytosed into endosomes containing proton pumps under the action of transferrin receptor, and then transported into cells at various parts of the body, and the iron is stored in cytoplasmic ferritin; in contrast, it has been converted to Fe by STEAP family reductase ²⁺ The extracellular iron in its state can be introduced directly into the cell by replacing surface transport proteins such as Zrt-Irt-like protein 14 (ZIP 14), and finally the iron is used by the body. And when iron homeostasis is deregulated, manifested as systemic iron deficiency or excess (iron overload), and maldistribution of iron in tissues, individual tissues or organs may be iron deficient or overloaded, which may be caused by genetic lesions that directly impair iron regulation or conditions that indirectly affect iron regulation.

Although the body can regulate the process of iron absorption by itself to prevent the adverse effect of iron overload, it is currently believed that the body can only control iron content by regulating iron metabolism upstream, i.e., iron absorption, so that transfusion-dependent patients who are suffering from iron overload can be potentially at risk of inducing iron overload when iron is largely supplemented, transfusions repeatedly, long-term slow hemolysis-induced anaemia, and genetic diseases such as aplastic anemia, thalassemia major, myelodysplastic syndrome, or sickle cell disease occur. Iron overload begins to produce a negative effect when the iron-bindable complex becomes saturated, and in particular, when the environment contains a lot of unbound iron, which accumulates as a result of being unable to be transported to the desired parts of the body, this physiological phenomenon, without being effectively inhibited, further enlarges the damaged parts, from cellular damage to tissue damage, especially for metabolically active organs (liver, heart), including immune organs. To prevent this, it is necessary to tightly control the storage and release of iron, and to regulate this in such a way that phagocytic ferritin has evolved in the cells. Iron autophagy is considered to be a selective autophagy modality, specifically for the degradation of intracellular ferritin, and is required to be mediated by the selective carrier nuclear receptor coactivator 4 (NCOA 4) in this process, and this physiological process is that NOCA4 is bound to LC3-PE after recognizing ferritin heavy chain 1 (FTH 1) on the autophagosome membrane and carrying FTH1, and both the ferritin-containing complex and NCOA4 retained in the autophagosome are degraded under the action of this physiological process, releasing bioavailable iron.

Thymus is the primary cellular donor of the lymphatic system in the body; the primary lymphoid organs of the defense system monitor various pathogens, tumors, antigens, and tissue damaging mediators. Iron is required for the proliferation of thymocytes and T cells, and plays an important role in the expression of T cells, regulating expansion and function of subpopulations. When iron overload occurs due to exogenous iron supplement, iron can accumulate in organ parenchymal cells, after Fenton reaction between the overloaded iron and hydrogen peroxide occurs, reactive Oxygen Species (ROS) are used as a reaction product to cause oxidation injury and cell death in a large range, and when the iron is in a higher level, the probability of iron death is increased, and then inflammation is caused to cause injury to body immunity. The relationship between iron overload and thymus is still not defined at present.

L-citrulline (L-citrulline) is an amino acid produced during catabolism that does not participate in the synthesis of proteins required by the body, and was originally extracted from watermelon juice. L-citrulline plays a role in maintaining protein homeostasis in the body, has high specificity of metabolism, is produced with urea production, can bypass viscera extraction, is not absorbed by intestinal tracts and livers, and is a promising pharmaceutical nutrient in the problem of malnutrition. L-citrulline is converted from glutamine and proline by five mitochondrial enzymes, phosphate-dependent glutaminase, OAT, pyrroline-5-carboxylate synthase, OTCD (ornithine carbamoyltransferase) and carbamoylphosphate synthase-1. L-citrulline has good bioavailability due to being processable by a variety of amino acid transporters. L-citrulline, a non-essential amino acid, has not been known to regulate iron metabolism and immune response in iron overload thymus.

Disclosure of Invention

The embodiment of the invention provides application of L-citrulline in preparing a medicine for preventing and treating iron overload, and aims to solve the problem of lack of the medicine for effectively preventing and treating iron overload at present.

The technical scheme of the invention is realized by the following method:

the invention confirms the application of L-citrulline in preparing medicaments for preventing and treating iron overload.

The embodiment of the invention proves that the L-citrulline can be used for relieving thymus injury caused by iron overload, and further, the L-citrulline is applied to the preparation of medicaments for preventing and treating thymus injury caused by iron overload.

The embodiment of the invention proves that the L-citrulline can resist iron death by inhibiting iron autophagy, and further, the L-citrulline can be applied to the preparation of the medicine for inhibiting iron autophagy and resisting iron death caused by iron overload.

As a preferred embodiment, the use of L-citrulline in the manufacture of a medicament for the prevention and treatment of iron overload, preferably the use of a combination of L-citrulline and sinccoa 4 in the manufacture of a medicament for the prevention and treatment of iron overload.

Compared with the prior art, the invention has the following beneficial effects:

the invention analyzes the influence of iron overload on thymus morphological structure, iron metabolism and immunoregulation from aspects such as in vivo and in vitro by preparing thymus and mTEC1 cell models of mice with iron overload, and clarifies the effect of L-citrulline on iron overload, iron autophagy caused by the iron overload and thymus immune injury caused by the iron overload. The invention discovers that the L-citrulline has remarkable effect on preventing and treating iron overload, and can inhibit oxidative stress damage and iron death caused by the iron overload; the L-citrulline can inhibit iron autophagy to relieve thymus immune injury induced by iron overload; and further finds application in preparing medicaments for preventing and treating iron overload by using the combination of L-citrulline and sinCOA 4. The invention proves that the L-citrulline is applied to the preparation of the medicine for preventing and treating iron overload and iron autophagy and thymus injury caused by the iron overload. And provides a regulating target for relieving immune injury caused by iron overload.

Drawings

FIG. 1 shows the morphology of L-citrulline against thymus tissue, CD8 ⁺ A graph of the results of the effect of T cell number; a is HE staining slice result diagram of thymus tissue; b is analysis CD8 ⁺ Immunofluorescent staining results for T lymphocyte numbers.

FIG. 2 is a graph showing the results of detection of the effect of L-citrulline on thymic ferric ion deposition and ferric ion concentration; a is a Prussian blue staining result diagram, and B is an iron ion concentration detection result diagram.

FIG. 3 is a graph showing the effect of L-citrulline on LDH, MDA, SOD, GSH-Px in thymus iron overload; wherein A is a serum LDH activity detection result graph; b is an MDA content detection result diagram; c is a graph of SOD enzyme activity detection results; d is a GSH-Px enzyme activity detection result graph.

FIG. 4 is a graph showing the results of detection of the effect of L-citrulline on iron autophagy and the expression of inflammation-associated gene proteins. Wherein A is a protein expression detection result diagram of L-citrulline on iron autophagy related genes TfR1, NCOA4, FTH, GPX4 and LC 3; b is a graph of the detection result of the L-citrulline pair to verify the protein expression of related genes TNF-alpha, IL-6, IL-1 beta, p-p65 and p-p 65.

FIG. 5 is a graph showing the results of detection of CCK8 screening drug concentration and time, LDH activity and iron ion concentration, wherein A is a graph showing the results of detection of cell activity of FAC treated for 24 hours at different concentrations; b is a graph of cell activity detection results of FAC treatment for 48 hours at different concentrations; c is a cell activity detection result graph of L-cit treatment for 16h and 200 mu M FAC treatment for 24h at different concentrations; d was 2mM L-cit treated for 16h, 200. Mu.M FAC treated for 24h, and the cell status results were visualized; e is an LDH activity detection result graph; f is a graph of the detection result of the concentration of the iron ions in the cells.

FIG. 6 is a graph showing the effect of L-cit on iron overload mTEC1 cells MDA, SOD, GSH-Px, ROS, and the results of screening for the concentration of siNCOA 4; wherein A is an MDA content detection result diagram, B is an SOD activity detection result diagram, C is a GSH-Px activity detection result diagram, D is a siNCOA4 concentration screening result diagram, E is an ROS average fluorescence intensity detection result diagram, and F is a cytoplasmic ROS detection result diagram.

FIG. 7 is a graph showing the results of regulation of the level of the mitochondrial membrane potential in combination with L-cit and sincOA4 treatment groups for the expression of the Nrf2 protein; wherein A is an expression result graph of Nrf2 protein in L-cit treatment, B is an expression result graph of Nrf2 protein in combined treatment of L-cit and sincOA4, and C is a result graph of mitochondrial membrane potential level.

FIG. 8 is a graph of the results of the effect of L-cit and combination treatment with siNCOA4 on TfR1, NCOA4, FTH, GPX4, LC3 and lipid ROS; wherein A is a graph of the effect of the L-cit protection group on TfR1, NCOA4, FTH, GPX4 and LC3 protein expression, B is a graph of the effect of L-cit synergistic siNCOA4 on TfR1, NCOA4, FTH, GPX4 and LC3, and C is a graph of the effect of L-cit synergistic siNCOA4 on lipid ROS.

FIG. 9 is a graph showing the results of immunofluorescent staining of TNF-. Alpha., IL-6, IL-1β, p65, p-p65 protein expression and p65 in a combination treatment group with siNCOA 4. Wherein A is a graph of the result of L-cit treatment group affecting the NF- κB pathway protein expression of iron overload mTECl cells, and B is a graph of the result of combined treatment group affecting the NF- κB pathway protein expression of iron overload mTEC1 cells; c is a graph of the effect of the L-cit protective group and the combined treatment group on the nuclear penetration of the iron overload mTEC1 cells p 65.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Currently, effective prevention and treatment medicines are lacking in aspects of iron autophagy, thymus injury and the like caused by iron overload. In order to solve the technical problems, the invention provides application of L-citrulline in preparing drugs for preventing and treating iron overload.

The primers and amino acid sequences used in the following examples are as follows:

according to the sequence information of NCOA4 and GAPDH genes recorded in NCBI, primers are designed for fluorescent quantitative detection through Primer software Primer Premier 5. Primers were synthesized by Wohan engine biotechnology Co. The sincOA4 and NC (negative control) were synthesized from Shanghai Ji Ma gene and specific sequence information is shown in Table 1.

TABLE 1 qPCR primer sequence listing

The L-citrulline used in the examples of the present invention was purchased from Solarbio company; iron dextran was purchased from Aladin.

Example one treatment of iron overload and L-citrulline animal and cell tests the treatment was as follows:

animal experiment: the environmental temperature for raising the C57BL/6 mice is 25 ℃, the light and dark environment is circulated for 12 hours, the mice eat and drink water freely, and the mice are adaptively raised for one week. Subsequently, experiments were performed, and 42 four-week-old SPF-class C57BL/6 mice were randomly divided into 6 groups of 8 mice each. Normal control group, L-citrulline group (1 g/Kg), IO (iron overload) model group, IO+L-citrulline low dose group (0.5 g/Kg), IO+L-citrulline medium dose group (1 g/Kg), IO+L-citrulline high dose group (2 g/Kg). The test groups were perfused with L-citrulline daily at different doses in the morning and were intraperitoneally injected with iron dextran (50 mg/Kg) in the afternoon, and after 14 days of treatment, the eyes were collected blood and thymus tissue was collected. One part for tissue embedding, one part in liquid nitrogen and one part fixed in 4% paraformaldehyde solution.

Cell assay: the survival conditions required for mTEC1 cells (mouse thymus epithelial cells) are 1640 medium containing 10% FBS, placed at 37deg.C, 5% CO ₂ Culturing in an incubator. Changing the liquid of the cells every 24 hours, discarding the old culture medium when the cells grow to about 80%, rinsing 2-3 times by using sterile PBS, adding a proper amount of 0.25% pancreatin to digest the cells, placing the cells in an incubator for 2-3min, and observing under an inverted microscope. When the cells become round and small and the cell gap is enlarged, the pancreatin is discarded, the medium is added to stop digestion, and the cells are repeatedly blown until the cells are completely fallen off and dispersed into single cellsThe subsequent experiments were performed in separate flasks or in corresponding well plates. Cells were mixed at 1 x 10 ⁵ The density of/mL is inoculated in a 6-hole plate, each hole is 2mL of culture solution, and after the cells are cultured to the logarithmic phase, the cells are treated by adding medicines. And screening the drug treatment concentration and treatment time by CCK8 test.

The following were analyzed by detection of iron overload and L-citrulline treated animal experiments and cell experiments:

1. the iron overload and thymus tissue damage following L-citrulline treatment were observed by histopathology. As shown in FIG. 1, in the HE stained sections (FIG. 1A), compared with the control group, the intratympanic cells in the IO group were abnormal in morphology, bleeding was obvious, the medullary cortex was not clearly limited, the IO+L-cit0.5 group was slightly bleeding, and no bleeding was seen in IO+L-cit1 and IO+L-cit 2. Thymus is responsible for the selection and recruitment of CD8 in the innate immune system ⁺ Critical organs of T lymphocytes, thymus injury affects immune cell number and function, thus analyzing CD8 ⁺ T lymphocyte number. Immunofluorescence staining results show that IO group CD8 ⁺ T cell number was significantly reduced and CD8 in the L-cit treatment group ⁺ The T cell numbers were dose-dependent elevated compared to the IO group (fig. 1B). In addition, in Prussian blue staining results (FIG. 2A), iron deposition in the IO group was significant, iron deposition in the L-cit treated group was reduced, and the high dose of L-cit (IO+L-cit2) had the most significant effect of inhibiting iron deposition. Based on the qualitative result of Prussian blue staining, the iron ion concentration was detected (FIG. 2B), and the result shows that the iron ion concentration of the iron overload group is about 2 times that of the control group, and the serum iron ion concentration of the L-cit treatment group is reduced in a dose-dependent manner. The results show that the L-citrulline has the effect of inhibiting iron overload, can inhibit thymus tissue injury and iron deposition caused by the iron overload, and plays a role in protecting in a dose-dependent manner.

2. The contribution of L-citrulline to oxidative stress caused by iron overload thymus is analyzed through MDA content, SOD enzyme activity and GSH-Px enzyme activity detection. As shown in fig. 3, the degree of cell damage under different treatment conditions of iron overload and L-citrulline was analyzed by detecting LDH activity in serum. Compared with a control group, the LDH of the IO group is obviously up-regulated, and the high-dose L-cit treatment group (IO+L-cit2) has obvious effect of inhibiting the LDH up-regulation. Malondialdehyde (MDA) is the end product of oxidative stress injury, and the IO group is increased about 3 times than the control group by MDA detection, and the L-citrulline treatment group MDA is dose-dependent reduced. SOD and GSH-Px are antioxidant substances, the organism resists oxidative stress injury of the organism by increasing the activity of the antioxidant substances, and the detection result shows that after the excessive iron agent is applied, the activity of the antioxidant enzymes SOD and GSH-Px of the organism is obviously reduced, and after the high-dose L-citrulline (IO+L-cit2) is treated, the activity of the SOD and GSH-Px is up-regulated. The results prove that the L-citrulline relieves thymus injury caused by iron overload through antioxidation.

L-citrulline inhibits iron autophagy and regulates thymic inflammatory factor in iron-overloaded mice via NF- κB signaling pathway

Ferritin upregulation is an important indicator of iron overload by analysis of ferritin content of FTH (ferritin heavy chain) to verify whether the model was successfully established, as shown in fig. 4, the results were consistent with expectations, IO groups were significantly upregulated compared to control groups, and the high dose L-citrulline (io+l-cit2) treatment group ferritin upregulation trend was reversed (fig. 4A). Iron overload and iron transport, iron autophagy are indistinguishable, and Transferrin (Trf) and Fe under physiological pH conditions ³⁺ Transferrin receptor (TfR 1) is a widely expressed membrane protein that mediates cell uptake of Trf with high affinity binding. The ferritin degradation pathway iron autophagy relies on the selective autophagy cargo receptor (NCOA 4) while playing a central role in regulating iron homeostasis, a process generally understood as a buffer system, i.e. whether activation of iron autophagy is inversely related to the concentration of iron ions, and as a result it was found that the iron overload group had an up-regulated transferrin receptor 1 (TfR 1) content, i.e. a high amount of iron ions entered the cell, compared to the control group, while NCOA4 and LC3 were up-regulated, i.e. iron autophagy was activated. Iron ions are taken up by TfR1, thereby greatly increasing the probability of cell-induced iron death by promoting Fenton-like reactions of preformed lipid hydrogen peroxide. GPX4 is a key factor involved in the occurrence or absence of iron death, and thus its expression level was analyzed. Compared with the control group, GPX4 is obviously down-regulated, namely iron death occurs in the iron overload model, the expression trend of IO histone is partially reversed after L-citrulline treatment, and the L-citrulline treatment group is in concentration dependenceTfR1, NCOA4, LC3, FTH were down-regulated, especially iron autophagy-related proteins were inhibited, and GPX4 was up-regulated for protection. Previous studies have demonstrated that accumulation of iron results in inflammation, while L-citrulline has anti-inflammatory effects, and thus the present test was conducted to investigate whether L-citrulline can resist iron overload-induced damage, including TNF- α, interleukin 6, β (IL-6, IL- β), up-regulated inflammatory factor expression in the IO group, and that the L-cit treated group resists such up-regulation and exerts a concentration-dependent down-regulation effect. The classical pathway of inflammatory factor expression is NF- κB, protein analysis shows that the pathway is activated in IO group, the proinflammatory medium is down regulated after L-citrulline is used, the pathway is NF- κB dependent, and the effect of high-dose L-cit (IO+L-cit2) treatment group is obvious. The above shows that L-citrulline inhibits iron autophagy, and by inhibiting NF- κB signaling pathway, anti-inflammatory effects can be exerted to avoid thymus damage during iron overload.

4. In the iron overload model of mTEC1 cells caused by ferric citrate amine (FAC), the activity of mTEC1 cells is reduced, and the influence of L-citrulline on the iron overload cell model is detected. Wherein the cell count uses CCK8 cell count kit. As shown in FIG. 5, mice thymus epithelial cells were stimulated for 24h by 200. Mu.M FAC (FIGS. 5A and 5B). Based on the FAC time concentration, the L-citrulline treatment concentration was screened, and finally 2mM was selected as the L-citrulline treatment concentration (fig. 5C). The subsequent cell test is carried out according to the concentration and time of the medicine, the cell state is obviously changed, as shown in fig. 5D, the number of adherent cells of the iron overload group is obviously reduced, the original form of the cells is damaged, and the number and the state of the cells are obviously improved after the L-citrulline is treated. And from the LDH activity test result (figure 5E), the activity of the FAC group is obviously enhanced compared with that of the control group, and the LDH activity of the L-cit treated group is reduced compared with that of the FAC treated group, so that the L-citrulline is effectively prevented from damaging cells. In addition, by analyzing the concentration of iron ions in the cells (fig. 5F), it was found that iron overload was significantly increased compared to the concentration of iron ions in the control group, and the concentration of iron ions was decreased after L-citrulline treatment, preventing the occurrence of iron overload. The above results indicate that L-citrulline is able to reverse the damage to the cellular state caused by iron overload and reduce the iron ion concentration.

5. The established cell model is adopted to treat cells, in vitro verification test is carried out, the kit detects the oxidation stress product and the antioxidant enzyme activity, and in vivo and in vitro results are consistent. As shown in fig. 6, MDA showed an increasing trend after FAC treatment, and the L-citrulline protective group resisted this abnormal increase (fig. 6A), as well as the FAC group SOD (fig. 6B) and GSH-Px (fig. 6C) significantly decreased, and the L-citrulline treated group resisted this decrease. By detecting cytoplasmic ROS (fig. 6E, 6F), FAC-treated groups showed a significant increase in ROS, whereas ROS levels decreased after pretreatment with L-citrulline. This suggests that L-citrulline reduces oxidative damage during iron overload by increasing the antioxidant capacity of the cells.

ROS production is closely related to iron metabolism during iron overload and animal protein expression results suggest that L-citrulline may be involved in iron metabolism and iron autophagy is overactivated during iron overload, thus further confirming the mechanism of L-citrulline action by sinccoa 4. The concentration of sinccoa 4 was first screened and by measuring the mrna levels and protein expression (fig. 6D), the concentration of sinccoa 4 was determined to be 50nM, with an interference efficiency of about 50%. After interference, the cellular oxidative stress levels were detected by detecting cytoplasmic ROS (fig. 6E, and fig. 6F), and it was found that both L-cit and sinccoa 4 treatments down-regulated the iron overload-induced oxidative stress levels, and the combination treatment group greatly enhanced the ability of the cells to avoid iron overload-induced oxidative stress. The above experiments demonstrate that the combined treatment of L-citrulline and sinSOA 4 can exert a synergistic effect, and that inhibition of iron autophagy is involved in alleviating oxidative stress induced by iron overload.

6. The expression of Nrf2 protein in the nuclei was examined in the different treatment groups. Nrf2 is a factor that plays a key role in regulating or resisting cellular oxidative reactions, nrf2 controlling gene transcription for both antioxidant and iron metabolism. As shown in fig. 7, by examining the expression of Nrf2 protein in the nuclei, it was found that Nrf2 was down-regulated compared to the control group when iron overload occurred, whereas after L-citrulline treatment, nrf2 was up-regulated compared to the iron overload group (fig. 7A, fig. 7B), indicating that L-citrulline enhanced nuclear translocation of Nrf2 and that the combination treatment group was better. When the free iron content was too high, a large amount of ROS was generated by the Fenton reaction, and most of these ROS were generated in mitochondria, so that mitochondria were extremely damaged, and thus mitochondrial membrane potential levels were examined (FIG. 7C), and as a result, it was found that the mitochondrial membrane potential was drastically decreased in the iron overload group compared with the control group and the pure L-citrulline treatment group, and the decrease in mitochondrial membrane potential was alleviated in the L-citrulline treatment group, the interference group and the combination treatment group, where the combination treatment group of L-citrulline and siNCOA4 had the best effect of alleviation. The test results show that L-citrulline plays an antioxidant role by increasing nuclear translocation of Nrf2, and plays the best antioxidant role by combining with siNCOA 4.

7. The results of the related protein expression verification test at the cellular level are shown in FIG. 8, and the results in vitro and in vivo are consistent. After iron overload, the expression level of TfR1, NCOA4, FTH, LC3II/I proteins was up-regulated in the iron overload group, the L-cit and FAC co-treated group was reduced compared to the FAC group, and GPX4 was significantly down-regulated in the FAC group, i.e. iron death occurred, and the L-cit up-regulated the expression of GPX4 (FIG. 8A). After successful inhibition of protein expression levels of NCOA4 as determined by the mechanism of the interference treatment, the results of detection of protein expression of TfR1, NCOA4 and LC3II/LC3I showed that the combined treatment groups were lower than those of the single treatment groups of siNCOA4 and L-citrulline, and GPX4 expression was significantly increased after the combined treatment (FIG. 8B). Also, in the staining results of lipid ROS, FAC group oxidative expression was found to be up-regulated compared to control group, and treatment group reduced lipid peroxidation caused by iron overload (fig. 8C). The above results indicate that L-citrulline resists iron death of mTEC1 cells by inhibiting iron autophagy and that the synergy of the combination treatment group of L-citrulline and sinccoa 4 is significant.

8. And detecting inflammatory factors in the iron overload or iron death state. Inflammation is accompanied by iron overload or iron death, and thus proinflammatory factors in cells are studied and detected. The results are shown in FIG. 9, in cells, the FAC group inflammatory factor protein and p-p65 were significantly up-regulated compared to the control group, indicating that after iron overload, the body up-regulated inflammatory factors through NF- κB signaling pathways and L-citrulline resistance was up-regulated (FIG. 9A). Inflammatory factor upregulation was also inhibited following sinccoa 4 treatment, and inhibition was optimal in the combination treatment group (fig. 9B). Furthermore, it was found visually from the p65 fluorescent staining results that, consistent with the above proteins, the FAC group induced p65 nuclear invasion, and the combination treatment group effectively inhibited p65 nuclear invasion (fig. 9C). The above demonstrates that L-citrulline inhibits the iron autophagy process and thereby down regulates NF- κB mediated cell inflammation during iron overload.

As a result of the above study, the application of L-citrulline in preparing a medicament for preventing and treating iron overload is embodied in the following four aspects:

1. l-citrulline relieves thymus and mTEC1 cell oxidative damage caused by iron overload

Compared with the control group, the IO group has obvious bleeding and obvious iron deposition, the IO group increases oxidation stress products MDA and cell membrane damage markers LDH, but SOD and GSH-Px activities show a trend of decreasing, and L-citrulline treatment obviously improves thymus structure, relieves iron deposition, improves the antioxidant capacity of thymus and shows a certain dose dependence. Cell model is built, CCK8 is screened for drug treatment concentration and time, and the scheme is that L-citrulline is treated for 16 hours by 2mM and FAC is treated for 24 hours by 200 mu M. Spectrophotometry is used for analyzing the iron content and the antioxidant enzyme activity, and cell chromatin ROS and Western blot are used for analyzing the expression content of Nrf2 protein. The results show that: compared with a control group, the FAC group has the advantages that the contents of oxidative stress products MDA, cell membrane injury markers LDH and cytoplasmic ROS are increased, SOD and GSH-Px activities show a trend of reduction, the expression of nuclear Nrf2 protein is down-regulated, the concentration of iron ions in cells and oxidative stress are obviously reduced after L-citrulline treatment, and the antioxidation capability is improved. The above shows that L-citrulline can alleviate thymus and cell damage caused by iron overload through antioxidation.

2. L-citrulline inhibits iron autophagy and iron death of thymus and mTECl cells caused by iron overload

In vitro test is carried out for 14 days, blood is collected from the eyeball to separate tissues, and thymus protein is extracted; the cell test treatment method is L-citrulline 2mM treatment for 16h and FAC 200 mu M treatment for 24h, and the test groups are as follows: control, L-citrulline, FAC, FAC+L-cit. Analysis of expression of TfR1, NCOA4, FTH, LC3, GPX4, cell staining analysis of MMP (mitochondrial membrane potential), lipid ROS levels. Wherein, the iron overload group GPX4 in tissues and cells is down-regulated, the rest proteins are up-regulated, the cell membrane potential level is reduced, the oxidized lipid ROS is up-regulated, and the L-citrulline is treated to reverse the above changes, which indicates that the L-citrulline can relieve thymus and cell injury caused by iron overload by participating in iron regulation, inhibiting iron autophagy and iron death.

3. L-citrulline relieves thymus and mTEC1 cell inflammation caused by iron overload

In vitro test is carried out for 14 days, blood is collected from the eyeball to separate tissues, and thymus protein is extracted; the cell test treatment method is L-citrulline 2mM treatment for 16h and FAC 200 mu M treatment for 24h, and the test groups are as follows: control, L-citrulline, FAC, FAC+L-cit. Analysis of TNF- α, IL-6, IL-1β, p65, p-p65 protein results, cell staining analysis of p65 nuclear entry. Analysis of the results shows that: compared with an untreated group, IL-6, TNF-alpha, IL-1 beta and p-p65 in the iron overload group show up-regulation trend, p65 is obvious to enter a nucleus, and L-citrulline is down-regulated after treatment, which shows that the L-citrulline can play an anti-inflammatory role through NF-KB signal channels and relieve thymus and cell injury caused by iron overload.

Combination of L-citrulline and siNCOA4 to exert anti-inflammatory and antioxidant effects by inhibiting autophagy of iron against iron death

The concentration of sincOA4 was screened by qPCR and Western blot and finally 50nM was selected for the assay. Cell assay treatment was 50nM siNCOA4 treatment for 48h, L-citrulline 2mM treatment for 16h, FAC 200. Mu.M treatment for 24h, assay groups: control, L-citrulline, FAC, FAC+siNCOA4, FAC+L-cit, FAC+siNCOA4+L-cit. Western bolt analysis of iron metabolic protein, inflammatory protein, nrf2 protein expression, and cell staining analysis of cytoplasmic ROS, mitochondrial membrane potential levels, lipid ROS levels. The result shows that: compared with a control group, the GPX4 protein in the FAC group and the Nrf2 expression in the nucleus are down-regulated, the other proteins are up-regulated, and the FAC+siNCOA4 group, the FAC+L-cit group and the FAC+siNCOA4+L-cit group reverse the changes, wherein the L-citrulline and the siNCOA4 combined treatment group resist the damage induced by iron overload optimally, which indicates that the L-citrulline resists iron death by inhibiting iron autophagy, exerts anti-inflammatory and antioxidant effects and relieves the damage of iron overload to cells.

In conclusion, the invention confirms the application of L-citrulline in preparing medicaments for preventing and treating iron overload.

In particular, the embodiment of the invention proves that the L-citrulline can be used for relieving thymus injury caused by iron overload, and further, the L-citrulline is applied to the preparation of medicaments for preventing and treating thymus injury caused by iron overload.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

Application of L-citrulline in preparing medicines for preventing and treating thymus injury caused by iron overload.
2. The use of L-citrulline according to claim 1 for the manufacture of a medicament for the prevention and treatment of thymus injury caused by iron overload, wherein the use of a combination of L-citrulline and sinccoa 4 for the manufacture of a medicament for the prevention and treatment of thymus injury caused by iron overload;

the upstream primer of the siNCOA4 is as follows: CCAUCAGGACACAUGUAAATT;

the downstream primer of the siNCOA4 is as follows: UUUACAUGUGUCCUGAUGGTT.