Application of rhizoma acori graminei extract in preparation of medicine for inhibiting nerve cell iron deposition and ferroptosis

Abstract

The invention discloses application of a rhizoma acori graminei extract in preparation of a medicine for inhibiting nerve cell iron deposition and ferroptosis. In vitro and in vivo levels, the invention reveals that ATR reduces iron deposition and inhibits hippocampal neuron ferroptosis by activating Nrf2 / GPX4 axis, finally improves the pharmacological mechanism of AD cognitive expression, and preliminarily characterizes the main active components of ATR. And a new strategy direction and a material basis are provided for AD treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of an Acorus tatarinowii extract in preparing a medicine for inhibiting iron deposition and ferroptosis in nerve cells. Background Art

[0002] Alzheimer's disease (AD) is a severe neurodegenerative disorder with no known cure, and its incidence is increasing annually. Therefore, there is an urgent need to explore and expand treatment options. AD manifests itself in a variety of brain lesions, primarily characterized by neuronal loss leading to brain atrophy, hippocampal volume reduction, and ventricular enlargement. The etiology of these lesions remains incompletely elucidated, but various hypotheses have been proposed, including excessive deposition of β-amyloid protein, hyperphosphorylation of tau protein, neuroinflammation, oxidative stress, and blood-brain barrier dysfunction. Although numerous studies have demonstrated that modulating these pathological processes can alleviate the cognitive impairment and pathological manifestations of AD, the development of new drugs targeting these processes remains a bottleneck. In recent years, several new drugs and technologies have been proposed, including aducanumab, canerumab (lecanemab), mannitol (GV-971), and deep cervical lymphovenous anastomosis (LVA), which are believed to have some potential to alleviate AD, but their long-term efficacy remains to be determined.

[0003] The pathogenesis of AD remains unclear, significantly hindering the development of new drugs. Therefore, actively exploring the pathology and progression of AD, building on existing knowledge, will help deepen our understanding of AD pathology. Excessive iron deposition in the brains of AD patients was reported decades ago, but little attention was paid to this phenomenon in the following decades. It wasn't until the concept of ferroptosis was proposed in 2012 that people realized that neuronal ferroptosis, caused by excessive iron deposition, may be a key form of neuronal loss in AD.

[0004] Several studies have shown that there is significant neuronal ferroptosis in AD, and inhibiting neuronal ferroptosis has a significant effect on cognitive performance and AD-like pathology. Typical characteristics of ferroptosis include a significant increase in intracellular free iron levels and the production of a large amount of reactive oxygen species (ROS) through the Fenton reaction, leading to lipid peroxidation and ultimately triggering biomembrane destruction and cell death. In addition, this process may also produce a large amount of lipid peroxidation product malondialdehyde (MDA) and change the expression of some iron metabolism-related proteins, such as transferrin (TFR1) and membrane iron transporter (FPN1).

[0005] Glutathione peroxidase 4 (GPX4) is a glutathione-dependent antioxidant enzyme whose primary function is to inhibit cell membrane lipid peroxidation, thereby protecting cells from oxidative damage. GPX4 is the most important inhibitor of ferroptosis, promoting the clearance of lipid peroxides and protecting biomembranes. Upregulating or inhibiting GPX4 can inhibit or induce ferroptosis. Therefore, GPX4 is also considered a key target for regulating ferroptosis. As a multi-etiological and complex disease, treatment strategies based on a single target may not be applicable, requiring a multi-pathway, multi-target systemic intervention.

[0006] Nrf2 (nuclear factor E2-related factor 2) is a transcription factor that belongs to the CNC transcription factor family. Nrf2 is a transcription factor that is very important for oxidative stress response. It can translocate from the cytoplasm to the nucleus and bind to the antioxidant response element (ARE) to initiate the transcription of antioxidant and iron metabolism genes, including SLC7A11, GPX-4, ferritin heavy chain 1 (FTH1), FPN1, etc.

[0007] Traditional Chinese Medicine (TCM) theory, due to its holistic approach, is particularly well-suited for the treatment of complex diseases with multiple etiologies. Acorus tatarinowii Schott (ATR), one of the oldest Chinese medicinal herbs, is listed as a top-grade herb in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica) and is the most widely used herbal formula for the treatment of AD. In TCM clinical practice, the dried rhizome of Acorus tatarinowii is often used as a medicinal herb, with benefits such as invigorating the senses, clearing phlegm, invigorating the mind, and promoting intelligence, as well as removing dampness and stimulating the appetite. Modern pharmacological studies have demonstrated that ATR has significant neuroprotective activity, including improving AD by inhibiting neuroinflammation and reducing Aβ toxicity, improving depression by regulating the serotonin transporter, and improving epilepsy by regulating the intestinal microbiota. However, little has been reported on the effects of ATR on iron deposition and ferroptosis in neurons, or on how it acts through the Nrf2 / GPX4 axis. Summary of the Invention

[0008] The purpose of the present invention is to address the above problems and provide a use of an Acorus tatarinowii extract in the preparation of a drug for inhibiting iron deposition and ferroptosis in nerve cells.

[0009] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0010] A first aspect of the present invention provides use of an Acorus tatarinowii extract in the preparation of a medicament for inhibiting iron deposition and ferroptosis in nerve cells.

[0011] The Acorus calamus extract activates the Nrf2 / GPX4 axis, reduces iron deposition in nerve cells, and further reduces iron deposition in the brain and inhibits neuronal ferroptosis.

[0012] The Acorus calamus extract alleviates cognitive impairment and neuronal degeneration in a subject.

[0013] The Acorus calamus extract upregulates the expression of Nrf2 and GPX4.

[0014] The Acorus calamus extract protects the subject's nerve cells, alleviates cognitive impairment, improves cognitive function, and improves / treats neurodegenerative diseases characterized by iron overload.

[0015] The neurodegenerative disease characterized by iron overload is Alzheimer's disease.

[0016] A second aspect of the present invention provides the use of an Acorus tatarinowii extract in the preparation of a medicament for activating the Nrf2 / GPX4 axis and inhibiting iron deposition and ferroptosis in nerve cells.

[0017] In any of the above-mentioned application technical solutions, the Acorus calamus extract is prepared by using Acorus calamus as raw material through ethanol extraction.

[0018] Preferably, the ethanol extraction method comprises the following steps: mixing and soaking the Chinese medicine Acorus calamus slices with 50-90% ethanol in a mass-to-volume ratio of 1:(6-12), refluxing and extracting in a 75-90°C water bath, concentrating under reduced pressure, and freezing to prepare freeze-dried powder.

[0019] Further preferably, the ethanol extraction method comprises the following steps: mixing the Chinese medicine Acorus calamus slices with 50-90% ethanol in a mass-to-volume ratio of 1:(6-12), soaking for 0-4 hours, reflux extraction in a 75-90°C water bath for 1-4 hours, concentrating under reduced pressure and freezing at -70°C to -85°C for 8-16 hours, and then transferring to a freeze dryer and freeze-drying for 24-50 hours to prepare a freeze-dried powder.

[0020] The beneficial effects of the present invention are:

[0021] This study reveals, both in vitro and in vivo, the pharmacological mechanism by which ATR reduces iron accumulation and inhibits ferroptosis in hippocampal neurons, ultimately improving cognitive performance in AD. The study also preliminarily characterizes the main active ingredient in ATR, providing new strategic directions and a material basis for the treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 3 shows the effects of ATR on ferric citrate (FC)-induced mouse hippocampal neurons (HT22) cell viability, iron deposition, ROS levels, and lipid peroxidation: A. Effects of ATR on HT22 cell viability within 24 h; B. Effects of ATR on FC-induced HT22 cell viability; C. Representative images of FerroOrange, DCFH-DA, and C11-BODIPY staining; D. Fe2+ Relative content quantitative graph; E. Relative content quantitative graph of ROS in each group of cells; F. Quantitative graph of lipid peroxidation in each group of cells.

[0023] Figure 2 The transcriptomic analysis of ATR on FC-induced HT22 cells is shown: A. PCA plot of the three groups of samples; B. Volcano plot of differentially expressed genes between the FC group and the control group; C. Volcano plot of differentially expressed genes between the ATR group and the FC group; D. Venn diagram of differentially expressed genes between the two groups; E. Clustering heat map of common differentially expressed genes in the three groups of samples; F. GO enrichment analysis of common differentially expressed genes; G. KEGG enrichment analysis of common differentially expressed genes; H. Reactome enrichment analysis of common differentially expressed genes.

[0024] Figure 3 The effects of ATR on the Nrf2 / GPX4 axis in HT22 cells are shown: A. Effect of ATR on the Nrf2 mRNA level in HT22 cells; B. Effect of ATR on the GPX4 mRNA level in HT22 cells; C. Representative images of Nrf2 immunofluorescence staining; D. Quantitative graph of Nrf2 immunofluorescence; E. Effect of ATR on GPX4 protein expression in HT22 cells; F. Representative images of immunofluorescence staining of Nrf2 protein expression induced by FC in HT22 cells by ATR; G. Quantitative graph of Nrf2 immunofluorescence in HT22 cells by ATR in FC; H. Effect of ATR on GPX4 protein expression induced by FC in HT22 cells; I. Representative images of DCFH-DA fluorescence in HT22 cells induced by FC by ATR; J. Quantitative graph of ROS; K. Representative images of C11-BODIPY staining; L. Quantitative graph of lipid peroxidation.

[0025] Figure 4 Shown are the effects of ATR on the Nrf2 / GPX4 axis in the hippocampus of APP / PS1 mice: A. Effect of ATR on the Nrf2 mRNA level in the hippocampus of APP / PS1 mice; B. Effect of ATR on the GPX4 mRNA level in the hippocampus of APP / PS1 mice; C. Representative immunofluorescence images of ATR on Nrf2 protein expression in the hippocampus of APP / PS1 mice; D. Quantitative immunofluorescence images of ATR on Nrf2 protein expression in the hippocampus of APP / PS1 mice; E. Representative immunofluorescence images of ATR on GPX4 protein expression in the hippocampus of APP / PS1 mice; F. Quantitative immunofluorescence images of ATR on GPX4 protein expression in the hippocampus of APP / PS1 mice.

[0026] Figure 5The effects of ATR on iron deposition and expression of iron metabolism-related proteins in the hippocampus of APP / PS1 mice are shown: A. Effect of ATR on iron content in the hippocampus of APP / PS1 mice; B. Effect of ATR on MDA content in the hippocampus of APP / PS1 mice; C. Correlation analysis between iron content in the hippocampus of mice in each group and the ratio of time spent exploring new / old objects; D. Correlation analysis between MDA content in the hippocampus of mice in each group and the ratio of time spent exploring new / old objects; E. Representative images of Perls staining in the brains of mice in each group and quantitative graphs of Perls staining-positive cells; F. Representative images of TFR1 immunohistochemistry in the brains of mice in each group and quantitative graphs of its expression; G. Representative images of FPN1 immunohistochemistry in the brains of mice in each group and quantitative graphs of its expression.

[0027] Figure 6 Figure 3 shows the effects of ATR on cognitive function and neuronal degeneration in APP / PS1 mice: A. Representative images of heat maps of movement trajectories of mice in each group in the novel object recognition experiment; B. Quantitative graph of the ratio of the number of explorations of new / old objects by mice in the novel object recognition experiment; C. Quantitative graph of the ratio of the exploration time of new / old objects by mice in the novel object recognition experiment; D. Nesting latency of mice in the nesting experiment; E. Nesting score of mice in the nesting experiment; F. Representative images of HE staining of mice in each group. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0030] Example 1

[0031] 1 Preparation and identification of Acorus calamus extract

[0032] Dried slices of the traditional Chinese medicine Acori Tatarinowii Rhizoma (ATR) were soaked in 70% ethanol at a ratio of 1:10 (w / v) for 2 hours. Extraction was then performed in an 80°C water bath under reflux for 2 hours. After vacuum concentration, the extract was frozen in a -80°C freezer for 12 hours and then freeze-dried in a freeze dryer for 48 hours to obtain a powdered extract, which served as the ATR used in this study. The extract was weighed and the extraction yield (i.e., the percentage of extract powder in the A. tatarinowii slices) was calculated to be 13.82%. For cell-based experiments, the ATR extract was dissolved in dimethyl sulfoxide (DMSO) to an 80 mg / mL stock solution, stored at -20°C, and diluted in culture medium to the appropriate working concentration for cell intervention. For animal experiments, an appropriate amount of ATR extract powder was weighed, ground into a fine powder, dissolved in water, and administered orally. The solution was freshly prepared before administration to the animals. The oral dose was 180 mg / kg of extract powder, equivalent to 1.3 g / kg of the original drug. The main chemical components of the ATR extract powder were identified by UPLC-QTOF-MS. The results are shown in Table 1.

[0033] Table 1 List of main components of ATR

[0034]

[0035] 2 Cell experiments

[0036] 2.1ATR improves iron accumulation and ferroptosis in mouse hippocampal neurons (HT22)

[0037] To investigate the protective effect of ATR on HT22 cell damage induced by ferric citrate (FC), this study first evaluated the safety of ATR on HT22 cells through a cell viability experiment: HT22 cells were seeded in 96-well plates at 8000 cells / well, and different ATR treatment groups were set up. The final concentrations of ATR in the wells were 0, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 μg / mL. After incubation for 24 h, the absorbance of each group of cells at 570 nm (OD 570 ), and then the relative cell viability was calculated. The protective effect of ATR on FC-induced HT22 cell damage was then observed within the safe concentration range of ATR: experimental groups without / with FC (100 μmol / L) were set up, and 8 ATR concentrations (0, 1.2, 2.5, 5, 10, 20, 40, 80 μg / mL) were set up in the experimental group with FC added. A positive group was also set up, namely the ferroptosis inhibitor Ferrostatin-1 (Fer1, concentration of 10 μmol / L) group, in which FC and ATR or Fer1 were added at the same time for intervention. After 24 hours of intervention in all groups, the OD values ​​of each group were determined using the MTT method. 570 , and then the relative cell viability was calculated.

[0038] The results showed that the maximum tolerance concentration of HT22 cells to ATR was 80 μg / mL ( Figure 1 A). 100 μmol / L FC can significantly reduce the viability of HT22 cells. However, under the intervention of ATR, cell viability can be restored in a dose-dependent manner. At a concentration of 80 μg / mL, cell viability is restored to about 90% of the normal group, which is similar to the effect of the ferroptosis-positive drug Fer1 ( Figure 1 B). Since the typical characteristics of ferroptosis are excessive deposition of intracellular iron ions, increased reactive oxygen species (ROS), and lipid peroxidation, in order to further evaluate the effect of ATR on ferroptosis-related indicators, this study detected the effect of ATR on the above indicators using a variety of fluorescence staining techniques. In the fluorescence staining experiment, 200,000 HT22 cells were seeded in a 6-well plate. After the cells adhered to the wall, they were divided into a control group (no intervention conditions were imposed), an FC group (100μmol / LFC intervention), three ATR concentration groups (100μmol / LFC+20 / 40 / 80μg / mLATR) and a Fer1 group (100μmol / LFC+10μmol / LFer1). FC and ATR or Fer1 were added to the wells at the same time for intervention. All groups were subjected to corresponding fluorescence staining after 24 hours of intervention, and the fluorescence intensity was quantified using ImageJ software. The results showed that ATR significantly reduced the intracellular iron content, ROS levels and lipid peroxidation ( Figure 1 CD). These results indicate that ATR can significantly improve FC-induced iron overload and inhibit ferroptosis in HT22 cells.

[0039] 2.2 Transcriptomics results showed that ATR-regulated differentially expressed genes were significantly associated with iron metabolism and ferroptosis

[0040] In order to understand the regulatory effect of ATR on gene expression in HT22 cells as a whole, the three treatment groups mentioned above were incubated for 24 hours and then the cell RNA was extracted for transcriptome sequencing: control group (no intervention conditions), FC group (100 μmol / LFC intervention), and ATR group (100 μmol / LFC + 80 μg / mATR intervention at the same time).

[0041] The results are as follows: The principal component analysis (PCA) graph shows that the three groups of samples are completely separated, suggesting that the gene expression patterns of the three groups of cells are significantly different ( Figure 2 A). The volcano plot of differentially expressed genes in the pairwise comparisons showed that a large number of genes were up-regulated or down-regulated in the FC group compared with the control group, and in the ATR group compared with the FC group, suggesting that the gene expression pattern of HT22 cells changed significantly when exposed to FC, and ATR intervention could significantly change the gene expression pattern of HT22 cells when exposed to FC ( Figure 2 BC). The VENN diagram of the two groups of differentially expressed genes showed that there were 2378 common genes ( Figure 2 D), cluster heat map shows that the expression patterns of these genes in the three groups of samples are significantly different ( Figure 2 E). Enrichment analysis of these 2378 common genes showed that these genes were significantly enriched in intracellular iron homeostasis, iron ion transport, and reactive oxygen metabolism ( Figure 2 F), KEGG enrichment results showed that ferroptosis, reactive oxygen species and other processes were significantly enriched ( Figure 2 G), Reactome enrichment results showed that lipid metabolism, iron uptake and transport were significantly enriched ( Figure 2 H). These results clearly indicate that the differentially expressed genes in HT22 cells mediated by ATR-regulated FC are significantly correlated with iron metabolism and ferroptosis.

[0042] The mechanism by which ATR inhibits iron overload and neuronal ferroptosis is related to activation of the Nrf2 / GPX4 axis

[0043] Nrf2, as the most important redox signal, regulates the expression of numerous genes related to iron metabolism and ferroptosis, such as the iron transporter FPN1 and glutathione peroxidase 4 (GPX4). GPX4 is a key protein in regulating ferroptosis. To understand the regulatory effects of ATR on Nrf2 and GPX4 expression, two treatments were designed: in the first, HT22 cells were treated with three ATR concentrations (20, 40, and 80 μg / mL) without the addition of FC for 24 hours, and then the mRNA and protein expression of Nrf2 and GPX4 were measured. In the second, HT22 cells were treated with ATR (80 μg / mL) alone or in combination with the Nrf2 selective inhibitor ML385 (5 μmol / L) in the presence of FC (100 μmol / L), and the mRNA and protein expression of Nrf2 and GPX4 were measured after 24 hours. The results showed that without the addition of FC, ATR increased the mRNA levels of Nrf2 and GPX4 in a dose-dependent manner ( Figure 3 AB) and protein expression ( Figure 3 CE); When FC was added, FC intervention alone led to increased Nrf2 expression and decreased GPX4 expression in HT22 cells. Compared with FC intervention alone, FC+ATR combined intervention further increased Nrf2 expression and significantly increased GPX4 expression. The Nrf2 specific inhibitor ML385 significantly weakened the upregulation of Nrf2 and GPX4 by ATR ( Figure 3FH). Under the second treatment method mentioned above, the ROS and lipid peroxidation in the cells were further detected using DCFH-DA and BODIPY-C11 fluorescent probes. The results showed that the inhibitory ability of ATR on ROS and lipid peroxidation was offset or weakened by the Nrf2 inhibitor ML385 ( Figure 3 These results suggest that ATR inhibits FC-mediated ferroptosis in HT22 cells and is associated with the activation of the Nrf2 / GPX4 axis.

[0044] 3 Animal experiments

[0045] 3.1 Experimental treatment methods

[0046] To confirm the in vivo effects of ATR on activating the Nrf2 / GPX4 axis and improving iron accumulation in neurons, we conducted animal experiments using APP / PS1 double transgenic mice. APP / PS1 double transgenic mice are a model of Alzheimer's disease (AD). They begin to exhibit cognitive impairment at 6 months of age, accompanied by AD-related pathology. Their brains exhibit significant iron accumulation and abnormal iron metabolism, making them a common tool for studying the effects of drugs on cognitive function.

[0047] Four experimental groups were used in this study: (1) normal control group: 7-month-old wild-type (WT) mice; (2) APP / PS1 model group: 7-month-old APP / PS1 mice were gavaged with 10 mL / kg of drinking water daily; (3) ATR group: 7-month-old APP / PS1 mice were gavaged with 180 mg / (kg·d) of ATR extract powder; (4) ATR+ML385 treatment group: 7-month-old APP / PS1 mice were gavaged with 180 mg / (kg·d) of ATR extract powder and, in addition, received a daily intraperitoneal injection of ML385 (30 mg / kg) for the last two weeks. All groups of mice were treated according to the established method for 6 consecutive weeks. Behavioral experiments, including novel object recognition and nesting tests, were performed on the last 3 days of treatment. After the behavioral experiment, the brains were removed and Perls staining was used to detect iron deposition. The expression of Nrf2, GPX4, TFR1, and FPN1 proteins in the hippocampus of mice in each group was detected by immunofluorescence and immunohistochemistry, respectively. The hippocampal tissues were isolated and the iron content, MDA content, and Nrf2 and GPX4 mRNA levels were detected.

[0048] 3.2ATR activates the Nrf2 / GPX4 axis

[0049] RT-PCR and immunofluorescence results showed that there was no significant difference in the mRNA levels of Nrf2 and GPX4 between APP / PS1 mice and WT mice, but the mRNA levels of Nrf2 and GPX4 were significantly increased in APP / PS1 mice after ATR treatment. However, the effect of ATR was offset when the Nrf2 inhibitor ML385 was applied. Figure 4AB). Immunofluorescence results showed that the expression of Nrf2 in the CA3 and DG regions of the hippocampus of APP / PS1 mice was significantly lower than that of WT mice, and there was no significant difference in the CA1 region. Nrf2 expression in the CA1, CA3, and DG regions of the hippocampus of APP / PS1 mice treated with ATR was significantly increased, and the translocation of Nrf2 from the cytoplasm to the nucleus was promoted, but this effect could be offset by the Nrf2 inhibitor ML385 ( Figure 4 CD). APP / PS1 mice showed significantly lower GPX4 expression in the CA1, CA3, and DG regions of the hippocampus compared to WT mice. ATR treatment significantly increased GPX4 protein expression in the above regions of APP / PS1 mice, and this effect was also offset by the Nrf2 inhibitor ML385 ( Figure 4 EF). These results indicate that ATR can activate the Nrf2 / GPX4 axis in vivo.

[0050] 3.3ATR significantly reduces brain iron deposition in APP / PS1 mice

[0051] Due to the excessive iron deposition in the brain of APP / PS1 mice, neurons suffer from ferroptosis, and studies have found that these factors are important causes of cognitive impairment. A significant increase in iron content and malondialdehyde (MDA) content is an important sign of ferroptosis. To observe the effect of ATR on iron deposition and MDA in the brain of APP / PS1 mice, this study took mouse hippocampal tissue to detect iron content and MDA content. The results showed that the iron content and MDA content in the hippocampus of APP / PS1 mice were significantly increased compared with WT mice, while ATR significantly reduced the iron content and MDA content ( Figure 5 AB). Correlation analysis found that iron content and MDA content were negatively correlated with the ratio of new / old object exploration time of mice, and the P value was less than 0.05, indicating that cognitive impairment of APP / PS1 mice was significantly correlated with iron content and MDA content in the hippocampus ( Figure 5 CD).

[0052] Perls staining can mark cells with iron overload. TFR1 and FPN1 are key proteins that mediate cellular iron absorption and excretion, respectively. This study used Perls staining and immunohistochemistry to detect iron deposition and iron metabolism-related protein expression in the hippocampus. The results showed that compared with WT mice, APP / PS1 mice had significantly increased numbers of Perls-positive cells, significantly increased TFR1 protein expression, and significantly decreased FPN1 protein expression. After ATR treatment, the above indicators were significantly improved, but the Nrf2 inhibitor ML385 could offset or weaken some of the effects of ATR ( Figure 5 These results suggest that ATR can alleviate brain iron accumulation in APP / PS1 mice, and its mechanism is partly dependent on Nrf2.

[0053] 3.4ATR alleviates cognitive impairment and neuronal degeneration in APP / PS1 mice

[0054] Since iron overload in the brains of APP / PS1 mice leads to neuronal ferroptosis, a major cause of neuronal loss in AD, inhibiting neuronal ferroptosis is effective in improving cognitive function. The above results from this study have demonstrated that ATR can reduce brain iron accumulation and inhibit neuronal ferroptosis. To further investigate the protective effects of ATR on cognitive function, this study evaluated cognitive function in ATR-treated APP / PS1 mice using novel object recognition and nesting tests, and observed neuronal lesions using hematoxylin and eosin staining. The novel object recognition test is a behavioral experiment designed to exploit mice's curiosity and tendency to explore novel objects. In this study, mice were placed in a 40 × 40 cm square box. Two objects of identical shape, color, and material were placed in the northeast and southwest corners of the box. The mice were placed facing the wall in the northwest direction and allowed to explore freely for 5 minutes. After 24 hours, the northeast object was replaced with a novel object of completely different shape, color, and material. The mice were also allowed to explore freely for another 5 minutes. The mice's movement patterns were recorded, as well as the time and frequency of exploration of the two objects. The results showed that the ratio of the number of times WT mice entered the new object and old object areas was greater than 1, and the ratio of the time they explored the new / old objects was also greater than 1, indicating that they had a significant tendency towards new objects, reflecting their obvious ability to distinguish between new and old objects, and reflecting their good memory ability. The above indicators of APP / PS1 mice were close to 1, indicating that they did not show a clear preference for new and old objects, suggesting that their memory ability was reduced. The above indicators of APP / PS1 mice treated with ATR were significantly improved, suggesting that ATR can significantly improve the learning and memory ability of APP / PS1 mice ( Figure 6 AC).

[0055] Nesting experiments can be used to evaluate the nesting ability of mice, reflecting their ability to take care of themselves and cognitive function. The results of this study showed that the nesting latency of APP / PS1 mice was significantly prolonged and the nesting score was significantly reduced, indicating that their cognitive ability declined. After ATR treatment, the above indicators were significantly improved, suggesting that ATR can improve the cognitive function of APP / PS1 mice ( Figure 6 DE). Hematoxylin-eosin (HE) staining was used to further evaluate the protective effect of ATR on neurons. The results showed that compared with WT mice, the neurons in the CA1, CA3, and DG regions of the hippocampus of APP / PS1 mice were disordered, and some cell bodies were enlarged or atrophied. These pathological phenomena were significantly improved after ATR treatment ( Figure 6F) These results suggest that ATR can improve cognitive impairment and neuronal degeneration in APP / PS1 mice. However, in all of the above experimental results, the protective effects of ATR on cognitive function and neuronal degeneration in APP / PS1 mice were offset or attenuated by the Nrf2 inhibitor ML385, indicating that the protective effects of ATR on cognitive function and neuronal degeneration in APP / PS1 mice are dependent on Nrf2.

[0056] 4 Summary and Analysis

[0057] Studies have shown that the iron content in the brain increases with age, and excessive iron deposition in the brain has been found to be associated with a variety of diseases, among which AD is a widely reported brain iron overload disease. Currently, there is no cure for AD, and only a few drugs can relieve symptoms but cannot prevent disease progression. Due to the unclear pathogenesis of AD, drug development often ends in failure. Neuronal ferroptosis caused by excessive iron deposition is considered to be an important cause of AD. Many studies have confirmed that AD can be improved by regulating iron metabolism and inhibiting neuronal ferroptosis.

[0058] To investigate the inhibitory effect of ATR on neuronal ferroptosis, we treated HT22 cells in vivo with iron-rich medium to simulate the iron-overload microenvironment in the AD brain. The results showed that under these conditions, HT22 cell viability decreased, intracellular free iron content, ROS levels, and lipid peroxidation increased, and ATR significantly reversed these phenomena. Transcriptomics results also confirmed that ATR-regulated differentials were significantly enriched in ferroptosis, lipid metabolism, and iron uptake and transport pathways. These results confirm the inhibitory effect of ATR on neuronal ferroptosis. In addition, NFE2L2 (also known as Nrf2)-related pathways were also significantly enriched. Studies have reported that Nrf2 can regulate the expression of multiple ferroptosis genes, such as GPX4, FPN1, and SLC7A11, and is considered an important regulator of ferroptosis. In fact, multiple components in ATR have been reported to activate Nrf2. For example, betaine can protect Leydig cells from the effects of hyperglycemia by activating the Nrf2 / HO-1 pathway through antioxidants. Schaftoside activates the Nrf2 / GPX4 axis to inhibit ferroptosis and reduce liver damage. Cyathuloidin inhibits ferroptosis and reduces bronchial epithelial damage by activating the Nrf2 / SLC / 7A11GPX4 axis. The activation of Nrf2 by these active components of ATR may be the basis of its inhibitory effect on neuronal ferroptosis.

[0059] The present invention further confirmed the role of ATR in upregulating Nrf2 in vitro and found that ATR upregulated GPX4 expression, changes that were attenuated or reversed by the Nrf2-selective inhibitor ML385. Application of ML385 also counteracted ATR's inhibitory effects on ROS and lipid peroxidation. These results strongly suggest that ATR's inhibition of neuronal ferroptosis is related to activation of the Nrf2 / GPX4 axis. Although in vitro results suggest that ATR inhibits neuronal ferroptosis and activates the Nrf2 / GPX4 axis, its in vivo effects remain to be explored. Therefore, we used APP / PS1 mice for in vivo validation. The results showed that compared with WT mice, Nrf2 and GPX4 mRNA levels did not change significantly in APP / PS1 mice, but protein expression was significantly decreased. This is contrary to the results observed for Nrf2 in vitro but consistent with previous reports. ATR also significantly modulated iron accumulation and the expression of iron metabolism-related proteins TFR1 and FPN1 in the APP / PS1 mouse brain. These results suggest that ATR has an ameliorative effect on iron accumulation and iron metabolism in the APP / PS1 mouse brain. Finally, behavioral experiments and HE staining results confirmed that ATR can improve cognitive function and neuronal degeneration in APP / PS1 mice. These experimental results fully demonstrate that ATR can improve neuronal iron deposition and iron metabolism by activating the Nrf2 / GPX4 axis, thereby alleviating cognitive impairment in AD.

[0060] In addition, since ATR is an extract with rich chemical components, in order to preliminarily characterize its main chemical components, 36 compounds were identified by UPLC-QTOF-MS, which provided a basis for further research and key active substances of ATR or its quality control.

Claims

1. Application of Acorus tatarinowii extract in the preparation of drugs for inhibiting iron deposition and ferroptosis in nerve cells.

2. The use according to claim 1, characterized in that: The Acorus calamus extract activates the Nrf2 / GPX4 axis, reduces iron deposition in nerve cells, and further reduces iron deposition in the brain and inhibits neuronal ferroptosis.

3. The use according to claim 2, characterized in that: The Acorus calamus extract alleviates cognitive impairment and neuronal degeneration in a subject.

4. The use according to claim 2, characterized in that: The Acorus calamus extract upregulates the expression of Nrf2 and GPX4.

5. The use according to claim 2, characterized in that: The Acorus calamus extract protects the subject's nerve cells, alleviates cognitive impairment, improves cognitive function, and improves / treats neurodegenerative diseases characterized by iron overload.

6. The use according to claim 5, characterized in that: The neurodegenerative disease characterized by iron overload is Alzheimer's disease.

7. Application of Acorus tatarinowii extract in the preparation of drugs for activating the Nrf2 / GPX4 axis and inhibiting iron deposition and ferroptosis in neurons.

8. The use according to any one of claims 1 to 7, characterized in that: The Acorus calamus extract is prepared by using Acorus calamus as a raw material and adopting an ethanol extraction method.

9. The use according to claim 8, characterized in that: The ethanol extraction method comprises the following steps: mixing and soaking the Chinese medicinal Acorus calamus slices with 50-90% ethanol in a mass-to-volume ratio of 1:(6-12), refluxing and extracting in a 75-90°C water bath, concentrating under reduced pressure, and freezing to prepare freeze-dried powder.

10. The use according to claim 9, characterized in that: The ethanol extraction method comprises the following steps: mixing the Chinese medicinal Acorus calamus slices with 50-90% ethanol in a mass-to-volume ratio of 1:(6-12), soaking for 0-4 hours, reflux extraction in a 75-90°C water bath for 1-4 hours, concentrating under reduced pressure and freezing at -70-85°C for 8-16 hours, and then transferring to a freeze dryer for freeze drying for 24-50 hours to prepare a freeze-dried powder.

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