Application of aloperine in preparation of medicine for treating GPX4 related diseases

Abstract

The invention discloses application of aloperine in preparation of a medicine for treating GPX4 related diseases, and relates to the technical field of biological medicines. A molecular docking experiment shows that aloperine can be stably bound with GPX4 protein, and the binding energy is-8.2 kcal / mol. In-vitro GPX4 activation activity detection is carried out by using aloperine, and the result shows that aloperine has activation activity on GPX4 protein and is concentration-dependent, and the half effective concentration of aloperine is 55 mu M. Therefore, aloperine can be used as an activator of GPX4 protein and is used for preparing the medicine for treating GPX4 related diseases. The invention provides an important GPX4 activator resource for treating GPX4 related diseases, and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of matrine in the preparation of a medicament for treating GPX4-related diseases. Background Art

[0002] GSH can be oxidized by glutathione peroxidase 4 (GPX4) to oxidized glutathione (GSSH). GPX4 is involved in the metabolism of GSH and uses GSH as a substrate to reduce lipid peroxides and inhibit ferroptosis.

[0003] Recent research progress has shown that the promotion and inhibition of ferroptosis coexist in the development of pulmonary hypertension and are involved in the pathogenesis and progression of pulmonary hypertension through links such as PAECs dysfunction, PASMCs proliferation, and right ventricular hypertrophy. However, while the activity of PAECs weakens, it is accompanied by an increase in the levels of ferroptosis markers such as lipid peroxidation, mitochondrial damage, and ferroptosis-related proteins. Related ferroptosis inhibitors (such as glutathione peroxidase 4 (GPX4)) can down-regulate these markers while weakening the progression of pulmonary vascular remodeling. The above research suggests that inhibiting ferroptosis can reduce pulmonary vascular remodeling caused by endothelial cell dysfunction and can be a potential therapeutic target for pulmonary hypertension. Ferroptosis is a type of programmed cell death induced by lipid peroxidation and dependent on iron ions, which causes mitochondrial rupture, and peroxidation of the endoplasmic reticulum, Golgi apparatus, lysosomes, etc. under the condition of an intact cell nucleus. The occurrence of ferroptosis mainly involves processes such as polyunsaturated fatty acids (PUFA), generation of reactive oxygen species (ROS), lipid peroxidation, and iron metabolism. Its main biochemical characteristics include the accumulation of iron, lipid peroxides, and ROS, as well as depletion of glutathione (GSH). Ferroptosis can be triggered by extrinsic or intrinsic pathways, and the intrinsic pathway is mainly induced by blocking the activity of intracellular antioxidant enzymes (such as GPX4) or increasing the accumulation of intracellular free fatty acids. Nowadays, various defense mechanisms have evolved in cells to detoxify toxic lipid peroxides, and the most prominent of which is the GPX4 defense mechanism. As a structural protein and antioxidant enzyme, GPX4 can strongly inhibit lipid oxidation and play a major role in blocking ferroptosis by scavenging phospholipid hydroperoxides (such as PUFAs-OOH), and is a key regulatory factor for inhibiting ferroptosis. In recent years, it has been considered as a key regulator of ferroptosis, playing a role in lipid and amino acid metabolism and affecting cell senescence, tumorigenesis, and cell death.

[0004] Increasing evidence indicates that targeting ferroptosis regulated by GPX4 is a promising therapeutic strategy for diseases. GPX4 mainly inhibits ferroptosis by participating in the GPX4-GSH axis. The process is as follows: The subunit SLC7A11 of the cystine / glutamate antiporter (SystemXc-) transports cystine into the cell and glutamate out of the cell. The cystine entering the cell is synthesized into GSH under the catalytic action of glutamate-cysteine ligase (GCL) and glutathione synthetase (GSS). GPX4 uses GSH as a cofactor to detoxify toxic PL-OOH into lipid alcohol (PL-OH), participates in the antioxidant metabolism process, and inhibits ferroptosis.

[0005] In previous work, the R & D team of the present invention used combined analysis of 16s rRNA sequencing and metabolomics to screen out the differential flora and differential metabolic pathways affected by pulmonary arterial hypertension, and found that there were significant differences in glutathione (GSH) metabolism in the cysteine metabolic pathway. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease, and its occurrence and development are closely related to pulmonary vascular structure and / or pulmonary vascular remodeling. Whether idiopathic or secondary to various diseases, patients with pulmonary arterial hypertension show similar pathophysiological changes, including abnormal pulmonary vascular contraction, pulmonary vascular inflammation, pulmonary vascular structural remodeling, and in-situ thrombosis, etc., ultimately leading to an increase in pulmonary vascular resistance, followed by right ventricular hypertrophy, heart failure, and even death. Epidemiology shows that in the population over 65 years old, the incidence of pulmonary arterial hypertension reaches 10%. It is defined as the mean pulmonary artery pressure (mPAP) ≥ 25 mmHg (1 mmHg = 0.133 kPa) at rest. Its prognosis is poor and the survival rate is low. Therefore, it is very important to find more effective drugs and clinical treatment methods for the treatment of pulmonary arterial hypertension.

[0006] Sophora alopecuroides has bitter and cold properties and has the effects of dispelling wind and drying dampness, relieving pain, killing insects, etc. Sophora alopecuroides mainly contains quinolizidine alkaloids, among which aloperine (Alo)( Figure 1 ) is one of its representative alkaloids and has various pharmacological activities such as anti-inflammatory, anti-cancer, anti-microbial, anti-viral, and anti-allergic effects. There is currently no report on using aloperine to target GPX4 for the treatment of GPX4-related diseases. Summary of the Invention

[0007] The object of the present invention is to provide the application of matrine in the preparation of a medicament for treating GPX4-related diseases, so as to solve the problems existing in the above-mentioned prior art. The present invention discovers through research that matrine can activate the activity of GPX4 protein, and thus can be used in the preparation of a medicament for treating GPX4-related diseases.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides the application of matrine in the preparation of a GPX4 activator.

[0010] The present invention also provides a GPX4 activator, and the active ingredient thereof includes matrine.

[0011] The present invention also provides the application of matrine in the preparation of a medicament for treating GPX4-related diseases.

[0012] Further, the GPX4-related disease is pulmonary hypertension.

[0013] Further, the medicament plays an active role in the treatment of pulmonary hypertension by regulating the GPX4-GSH axis and affecting the intestinal flora.

[0014] The present invention also provides a medicament for treating GPX4-related diseases, and the active ingredient thereof includes matrine.

[0015] Further, the medicament also includes pharmaceutically acceptable excipients.

[0016] Further, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives or colorants.

[0017] Further, the dosage form of the medicament is tablets, granules, capsules, pills or oral liquids.

[0018] The present invention discloses the following technical effects:

[0019] The present invention discovers through molecular docking experiments that matrine can stably bind to GPX4 protein, and the binding energy is -8.2 kcal / mol. The in vitro GPX4 activation activity detection is carried out by using matrine, and the results show that matrine has activation activity on GPX4 protein and shows concentration dependence, and its half maximal effective concentration is 55 μM. Thus, it can be seen that matrine can be used as an activator of GPX4 protein and is used in the preparation of a medicament for treating GPX4-related diseases. The present invention further confirms that matrine can play an active role in regulating the intestinal flora in the treatment of pulmonary hypertension through the GPX4-GSH axis.

[0020] The present invention provides an important GPX4 activator resource for the treatment of GPX4-related diseases and has important clinical application value. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0022] Figure 1 is the structural formula of matrine;

[0023] Figure 2 is the molecular docking visualization diagram;

[0024] Figure 3 is the echocardiogram of rats in different groups; among them, A is the control group, B is matrine 100 mg / kg; C is the SuHx group; D is the SuHx + matrine 25 mg / kg group; E is the SuHx + matrine 50 mg / kg group; F is the SuHx + matrine 100 mg / kg group; G is the SuHx + sildenafil group;

[0025] Figure 4 is the statistical chart of echocardiogram index parameters of rats in different groups; among them, A is the statistical chart of the end-systolic internal diameter of the right ventricle; B is the statistical chart of the maximum pulmonary blood flow velocity (PV max); C is the statistical chart of the pulmonary acceleration time (PAAT); ## P < 0.01, compared with the control group; **P < 0.01, compared with SuHx; Alo: matrine; SuHx: SU5416 + hypoxia;

[0026] Figure 5 is the detection result diagram of the right ventricular systolic pressure of rats in different groups; among them, A is the control group, B is matrine 100 mg / kg; C is the SuHx group; D is the SuHx + matrine 25 mg / kg group; E is the SuHx + matrine 50 mg / kg group; F is the SuHx + matrine 100 mg / kg group; G is the SuHx + sildenafil group; H is the statistical chart of the right ventricular systolic pressure; ## P < 0.01, compared with the control group; **P < 0.01, compared with SuHx; RVSP: right ventricular systolic pressure; Alo: matrine, SuHx: SU5416 + hypoxia, Sildenafil: sildenafil;

[0027] Figure 6Graph showing the detection results of mean pulmonary artery pressure in rats of different groups; among them, A is the control group, B is the group treated with 100 mg / kg of aloperine, C is the SuHx group, D is the SuHx + 25 mg / kg aloperine group, E is the SuHx + 50 mg / kg aloperine group, F is the SuHx + 100 mg / kg aloperine group, G is the SuHx + sildenafil group, and H is the statistical graph of mean pulmonary artery pressure; ## P < 0.01, compared with the control group; **P < 0.01, compared with the SuHx group; mPAP: mean pulmonary artery pressure; Alo: aloperine, SuHx: SU5416 + hypoxia, Sildenafil: sildenafil;

[0028] Figure 7 Graph showing the detection results of right ventricular hypertrophy in rats of different groups; among them, A is the statistical graph of right heart hypertrophy index, and B is the statistical graph of right ventricular mass index; ## P < 0.01, compared with the control group; **P < 0.01 compared with the SuHx group; RVHI: right heart hypertrophy index; RVMI: right ventricular mass index; Alo: aloperine, SuHx: SU5416 + hypoxia, Sildenafil: sildenafil;

[0029] Figure 8 Graph showing the HE staining results of pulmonary arterioles in rats of different groups; among them, A - G are representative micrographs of small pulmonary arteries stained with HE (×400 magnification); A is the control group, B is the group treated with 100 mg / kg of aloperine, C is the SuHx group, D is the SuHx + 25 mg / kg aloperine group, E is the SuHx + 50 mg / kg aloperine group, F is the SuHx + 100 mg / kg aloperine group, G is the SuHx + sildenafil group, H is the statistical graph of the percentage of pulmonary vascular thickness (WT%), and I is the statistical graph of the percentage of pulmonary vascular area (WA%); ## P < 0.01, compared with the control group; **P < 0.01, compared with the SuHx group; SuHx: SU5416 + hypoxia; the arrow points to the media;

[0030] Figure 9 Species analysis and heatmap of biomarker - species composition;

[0031] Figure 10 Results of biomarker analysis of the correlation among groups by Heatmap;

[0032] Figure 11 Graph of important endogenous small molecule - related metabolic pathways constructed using the MetPA database;

[0033] Figure 12Figure showing the detection results of the expression level of GPX4 in rat lung tissue; among them, A is the figure of the detection results by Western blotting; B is the statistical chart of the protein expression level of GPX4 / GAPDH; GPX4: glutathione peroxidase 4; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Control: normal control group; Model: hypoxia + SU5416; ALO: hypoxia + SU5416 + ALO. Detailed implementation manners

[0034] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms used in the present invention are only for describing particular implementation manners and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0038] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] I. Molecular docking: GPX4 and aloperine

[0041] 1.1 Experimental method:

[0042] Dock the protein GPX4 and the ligand matrine using AutoDockTools software. Download the GPX4 protein structure from the PDB database and the matrine structure from the TCMSP database, and introduce them into the software to calculate the binding energy.

[0043] 1.2 Experimental results:

[0044] If the binding energy is less than 0, it indicates that the ligand and the receptor can bind. The smaller the binding energy value, the higher the binding activity. The binding energy of molecular docking is -8.2 kcal / mol( Figure 2 ), indicating that the binding activity between GPX4 and matrine is stable.

[0045] II. Detection of the activation activity of matrine on GPX4

[0046] GPX4 can catalyze GSH to produce oxidized glutathione (GSSH), while glutathione reductase can use nicotinamide adenine dinucleotide phosphate (NADPH) to catalyze GSSG to produce GSH. The activity level of GPX4 can be calculated by detecting the decrease in the amount of NADPH. Since NADPH has the maximum absorption at 340 nm, and the absorbance value at 340 nm will gradually decrease with the gradual consumption of NADPH, the activity of GPX4 enzyme can be detected by detecting the consumption of NADPH.

[0047] The in vitro activation activity of matrine on GPX4 was detected. The results showed that matrine had an activation activity on GPX4 and was concentration-dependent, and its half-maximal effective concentration (EC 50 ) was 55 μM.

[0048] Example 2

[0049] I. Experimental materials

[0050] 1.1 Main experimental drugs and instruments

[0051] Matrine (purity > 98%) was purchased from Ningxia Dushun Pharmaceutical Factory; Sildenafil (H20020528) was purchased from Pfizer Inc., USA; SU5416 (205994) was purchased from medkoo biosciences; BCA Protein Quantification Kit (KGPBCA) was purchased from Jiangsu KeyGen Biotech Co., Ltd.; Western Blot Primary and Secondary Antibody Diluent (15127C17) was purchased from Boster Biological Technology; Mouse Anti-GPX4 Monoclonal Antibody and Mouse Anti-GAPDH Monoclonal Antibody were purchased from Wuhan Sanying Co., Ltd.; Hypoxic Chamber (XBS-03B) was purchased from Hangzhou AIPU Instrument and Equipment Co., Ltd.; Functional Experiment System (BL-420S) was purchased from Chengdu Taimeng Co., Ltd.; Freezing Grinder, JXFSTPRP-CL, was purchased from Shanghai Jingxin Co., Ltd.; Microplate Reader (Multiskan G0) was purchased from Thermo Fisher Scientific, USA; High-Speed Low-Temperature Centrifuge (5430R) was purchased from Eppendorf AG, Germany.

[0052] 1.2 Animal treatment

[0053] Male Sprague-Dawley rats with an average body weight of 270 ± 20 g, 8 weeks old, were provided by the Animal Experiment Center of Ningxia Medical University, with the certificate SYXK 2020-0001, and were allowed to eat and drink freely.

[0054] 1.3 Grouping and administration of experimental animals

[0055] Male Sprague-Dawley rats were randomly divided into 7 groups: normal control group, matrine control group, SuHx group, SuHx + matrine (25 mg / kg / d, 50 mg / kg / d, 100 mg / kg / d) groups, and SuHx + sildenafil (30 mg / kg / d) positive drug group. A 12-hour light / dark cycle was maintained, with constant temperature and humidity, and standard laboratory drinking water and food.

[0056] Rats in the control group were only injected with the solvent and maintained in room air. Rats in each experimental group were given matrine and sildenafil for 3 weeks. Rats in the matrine control group were maintained in room air and were gavaged with matrine and sildenafil at the beginning of gavage administration for 3 weeks.

[0057] 1.4 Preparation of a rat model of pulmonary hypertension induced by SuHx

[0058] Rats in the SuHx group were subcutaneously injected with SU5416 (20 mg / kg). Starting from the first day after injection of SU5416, the nitrogen flow was adjusted so that the O2 concentration in the hypoxic chamber was 10% and hypoxia was induced for 3 weeks.

[0059] II. Experimental procedure

[0060] (I) Detection of echocardiogram parameters

[0061] 1.1 Experimental method:

[0062] Before the examination, all rats were anesthetized with sodium pentobarbital. After shaving the chest, a GE VIVID7 color Doppler ultrasound diagnostic instrument (General Electric, CO, USA) was used to detect the echocardiogram parameters of the rats. The pulsed wave Doppler signal of the pulmonary artery valve blood flow was obtained from the left ventricular short-axis parasternal section, and the pulmonary artery acceleration time (PAAT) and the maximum pulmonary velocity (PVmax) were measured. All echocardiogram examinations were performed under anesthesia at a heart rate of 350 - 400 beats / min.

[0063] 1.2 Experimental results:

[0064] The echocardiogram results showed that the shape of the velocity profile of the rats in the SuHx group changed from the "round" ( Figure 3 in A) of the control group rats to the typical "dome and spike" ( Figure 3 in B) of PAH. After administration of sophoridine (200 mg / kg and 400 mg / kg) and sildenafil (30 mg / kg) ( Figure 3 in D - F), the above abnormal changes were significantly improved. Compared with the control group, the echocardiogram index parameters PATT and PVmax of the rats in the SuHx group were significantly shortened, and PA was significantly increased (P < 0.01, Figure 4 ), while after treatment with sophoridine (200 mg / kg and 400 mg / kg) and sildenafil (30 mg / kg), the echocardiogram index parameters PATT and PVmax of the rats were significantly increased and PA was significantly decreased (P < 0.01, Figure 4 ). All the above results indicated that sophoridine improved SuHx - induced PAH in rats.

[0065] (II) Hemodynamic detection

[0066] 2.1 Experimental method:

[0067] The rats were weighed and anesthetized by intraperitoneal injection of 2% sodium pentobarbital. A micro - catheter was inserted into the right ventricular cavity of the rats through the right jugular vein, and the intubation position was adjusted until the typical curves of the right ventricular systolic pressure and the pulmonary artery pressure appeared. The mean pulmonary artery pressure (mPAP) and the right ventricular systolic pressure (RVSP) were measured and recorded through the waveform curve, and the pressure values were displayed on the panel of the physiological recorder.

[0068] 2.2 Experimental results:

[0069] Forty-two days after a single subcutaneous injection of SuHx into the nape of the neck of rats, compared with the control group, the mPAP and RVSP of rats in the SuHx group were significantly increased (P<0.01, Figure 5 and Figure 6 ), indicating that the PAH rat model induced by SuHx has been successfully established. Twenty-one days after SuHx treatment, rats were given matrine (100 mg / kg, 200 mg / kg, and 400 mg / kg) and sildenafil (30 mg / kg) for continuous treatment for 21 days. Compared with the SuHx group, after treatment with matrine (200 mg / kg and 400 mg / kg) and sildenafil (30 mg / kg), the hemodynamic parameters mPAP and RVSP of rats were significantly decreased (P<0.01, Figure 5 and Figure 6 ). The above research results all indicate that matrine can improve SuHx-induced PAH in rats.

[0070] (III) Detection of right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI)

[0071] 3.1 Experimental method:

[0072] After hemodynamic detection, rats in each group were immediately sacrificed, the chest was opened to dissect the heart, the atria were removed, and the right ventricle RV was separated from the left ventricle LV and the interventricular septum S. The calculation formulas are RVHI = RV / (LV + S); RVMI = RV / BW (body weight).

[0073] 3.2 Experimental results:

[0074] Compared with the control group, the right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) of the SuHx group were significantly increased (P<0.01, Figure 7 ), suggesting that SuHx can induce right ventricular hypertrophy in rats. Compared with the SuHx group, the RVHI and RVMI of rats in the sildenafil (30 mg / kg) and matrine (50 mg / kg and 100 mg / kg) groups were significantly decreased (P<0.01, Figure 7 ). The above results show that matrine can significantly improve SuHx-induced right ventricular hypertrophy in rats.

[0075] (IV) HE staining to observe pathological changes of tissue structure

[0076] 4.1 Experimental method:

[0077] After hemodynamic detection, the rats in each group were immediately sacrificed. The chest cavity was opened to remove the lung tissue. The upper right lobe of the right lung was cut, and the lung tissue blocks were placed in an embedding cassette and immersed in 10% formaldehyde for fixation for 48 hours. Subsequently, dehydration, clearing, paraffin infiltration, embedding, wax trimming, sectioning, spreading, and picking were carried out, and dried in an incubator at 60°C for 2 hours. The paraffin sections were baked at 70°C for 2 hours, rinsed with xylene for 10 minutes, twice; rinsed with alcohol for 5 minutes, and rinsed with tap water for 5 minutes, three times. Stained with hematoxylin for 5 minutes, rinsed with tap water for 1 minute, differentiated with 50% acetic acid for 2 minutes, rinsed with tap water for 15 minutes, and stained with eosin staining solution for 1 minute. Dehydrated and sealed, and its histological images were collected with an optical microscope to measure the outer diameter (ED), inner diameter (ID), total vascular area (TA), lumen area (LA), wall thickness (WT), and wall area (WA) of the small pulmonary arteries. Calculate WT% = ((ED - ID) / ED × 100%) and WA% = ((TA - LA) / TA × 100%) according to the following formula.

[0078] 4.2 Experimental results:

[0079] In the SuHx group, the walls of the small pulmonary arteries in the lungs of rats were thicker, the lumens were narrower, the intima proliferated, and there were more inflammatory cell infiltrations compared with the control group and the matrine control group ( Figure 8 in A - C in the figure). After treatment with different doses of matrine and sildenafil, the above pathological changes were significantly improved, the degree of vascular wall thickening was also improved, and the lumen became larger ( Figure 8 in D - G in the figure). Compared with the control group, the WA% and WT% of the rats in the SuHx group were significantly increased (P < 0.01, Figure 8 in H in the figure). Compared with SuHx, after treatment with matrine (200 mg / kg and 400 mg / kg) and sildenafil (30 mg / kg), the WA% and WT% of the rats were significantly decreased (P < 0.01, Figure 8 in I in the figure). The above results indicate that matrine significantly improves the vascular remodeling of small pulmonary arteries in rats induced by SuHx.

[0080] (V) Combined analysis of 16s rRNA sequencing and metabolomics

[0081] 5.1 16s rRNA sequencing

[0082] 5.1.1 Experimental method:

[0083] Using the 16S rRNA high-throughput sequencing method, the effects of matrine administration on the intestinal flora of SuHx-induced pulmonary hypertension were studied, the differences in α-diversity and β-diversity among groups were analyzed, and the differential microorganisms between groups were found through variance analysis. Correlation analysis was used to analyze the correlation between species and metabolites, and the species significantly related to metabolites were screened to explain the effects of the pulmonary hypertension model on the body's intestinal flora and whether matrine intervention has a regulatory effect on the differential flora of pulmonary hypertension.

[0084] 5.2.1 Experimental results:

[0085] From the heat map of species composition ( Figure 9 ), it was found that the abundances of the genera SMB53 and Turicibacter were significantly decreased, and the abundances of genera such as Allobaculum were significantly increased. Compared with the model group, the abundance of the genus SMB53 in the administration group was significantly decreased, and the abundance of the genus Lactobacillus was significantly increased. Through the comparison of the three groups, the abundances of the genera in Firmicutes and Bacteroidetes were significantly increased, and Firmicutes and Bacteroidetes had the highest abundances in the metabolic category, including metabolic pathways such as amino acids and lipids, suggesting that matrine may resist pulmonary hypertension through these genera and their metabolites.

[0086] 5.2 Metabolomics analysis

[0087] 5.2.1 Experimental method:

[0088] The LC-MS / MS analysis technology was used to conduct metabolomics research on the sera of rats in the normal control group, model control group, and matrine administration group. Combining chemometric methods, differential metabolites and metabolic pathways were found to study the effects of matrine on the endogenous metabolites of rats with pulmonary hypertension.

[0089] 5.2.2 Experimental results:

[0090] From the heat map of hierarchical clustering analysis ( Figure 10 ), the longitudinal clustering showed the clustering of the expression patterns of metabolite contents among samples. Using differential metabolites as molecular characteristics, each group was not under the same branch and there was no overlap, and the blank group and the model group could be clearly distinguished, indicating that the endogenous metabolic pattern in the lung tissue changed after pulmonary hypertension occurred. The MetaboAnalyst website was used to conduct enrichment analysis of the metabolic pathways of metabolites ( Figure 11) The metabolic pathway impact value (-lgP) represents the significance level of metabolic pathway enrichment analysis. The larger the -lgP, the higher the correlation of metabolic differences between different groups. MetPA was used to analyze differential metabolic pathways, and the impact value criterion was set at 0.10. Metabolic pathways with an impact value higher than 0.10 are expected to become potential target pathways. As shown in Table 1, the GSH metabolism in the cysteine metabolic pathway has significant differences, and its impact value is equal to 1.

[0091] Table 1 Results of MetPA analysis of differential metabolic pathways (Impact>0.10)

[0092]

[0093]

[0094] (VI) Detection of GPX4 protein expression in rat lung tissue by Western blotting

[0095] 6.1 Experimental method:

[0096] Take out the lung tissue of the required groups from the refrigerator. Weigh 100 mg of rat lung tissue for each group and add 1 mL of protein lysate into a pre-cooled centrifuge tube. Turn on the homogenizer to grind and lyse the lung tissue to obtain tissue homogenate. Place the tissue homogenate in a centrifuge, take the supernatant and transfer it to a centrifuge tube, and then perform protein quantification using a BCA kit.

[0097] SDS-PAGE gel electrophoresis: (1) Prepare separating gel and stacking gel; (2) Perform electrophoresis, and after electrophoresis, perform wet transfer. After the transfer is completed, use the milk powder blocking method and incubate on a shaker at room temperature for 2 hours. After blocking, incubate with primary antibody and secondary antibody. After each antibody incubation, wash the membrane three times with PBST for 10 minutes each time. Finally, use a gel image analysis and imaging system to scan and analyze the PVDF membrane, record and analyze the gray value of the target band.

[0098] 6.2 Experimental results:

[0099] To detect the relationship between GPX4 and pulmonary hypertension, the expression of GPX4 in the lung tissue of PAH rats was further detected by Western blotting. The Western blotting results showed that the expression level of GPX4 in the lung tissue of rats in the SuHx group was significantly higher than that in the model group (P<0.05, Figure 12 ).

[0100] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of alopecurine in the preparation of GPX4 activators.

2. A GPX4 activator, characterized in that The active ingredients include alopecurine.

3. Application of sophora flavescens in the preparation of drugs for treating GPX4-related diseases.

4. The use according to claim 3, characterized in that: The GPX4-related disease is pulmonary arterial hypertension.

5. The use according to claim 4, characterized in that: The drug affects the intestinal flora by regulating the GPX4-GSH axis, thereby playing a positive role in the treatment of pulmonary hypertension.

6. A drug for treating GPX4-related diseases, characterized in that: The active ingredients include alopecurine.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The auxiliary materials include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives or colorants.

9. The drug according to claim 7, characterized in that The dosage form of the medicine is tablet, granule, capsule, pill or oral solution.

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