Traditional Chinese medicine extract and application thereof in preparation of necrotic apoptosis inhibitor

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a traditional Chinese medicine extract and application thereof in preparation of a necrotic apoptosis inhibitor, the traditional Chinese medicine extract is prepared from the following traditional Chinese medicine composition: 10-14 parts of bupleurum chinense, 8-11 parts of angelica sinensis, 10-14 parts of parched white peony root, 8-12 parts of rhizoma atractylodis macrocephalae, 14-16 parts of poria cocos, 2-4 parts of liquorice, 10-14 parts of cortex moutan, 10-14 parts of raw fructus gardeniae and 10-14 parts of rhizoma acori graminei. 10-14 parts of polygala tenuifolia and 5-7 parts of mint; the preparation method comprises the following steps: mixing the traditional Chinese medicine composition, decocting, filtering, carrying out rotary evaporation concentration, and drying. The traditional Chinese medicine extract disclosed by the invention can be used for preparing a necroptosis inhibitor, and can be used for relieving damage to hippocampal neurons, so that the hippocampal neurons are normal in structure and form, round and full in edge and rich in nissl body.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a traditional Chinese medicine extract and an application thereof in preparing a cell necrosis apoptosis inhibitor. Background Art

[0002] The physiological functions of hippocampal neurons are closely related to human memory. If the hippocampal neurons are damaged, such as due to encephalitis, brain trauma or brain atrophy causing hippocampal nerve atrophy, memory impairment or dementia will occur.

[0003] HT-22 cells are a mouse hippocampal neuronal cell line derived from a subclone of the HT-4 cell line. The HT-22 cell line is an immortalized cell line obtained from the HT-4 cell line by subcloning technology.

[0004] Programmed necrosis has been a research hotspot in the field of death in recent years. More and more experimental data show that this highly regulated cell death mode is closely related to the body's innate immune response and the occurrence and development of inflammation-related diseases. When cells lack caspase-8, TNF-α binds to TNFR1, leading to the activation of RIPK1. Activated RIPK1 autophosphorylates on multiple residues, RIPK3 is phosphorylated on Ser227 in the necrosome, and RIPK3 homo-oligomers ultimately phosphorylate MLKL on Thr357 / Ser358 residues. MLKL then translocates from the cytoplasm to the cell membrane to destroy cell membrane integrity and induce programmed necrosis.

[0005] The programmed cell death of HT-22 cells causes damage to the hippocampal neurons of mice. Inhibiting the programmed cell death of HT-22 cells is an important means to avoid damage to the hippocampal neurons.

[0006] However, the existing programmed cell death inhibitors for HT-22 cells mainly include Ferrostatin-1, which has low bioavailability and short half-life, and requires frequent administration to maintain effective blood drug concentrations, thereby increasing the risk of adverse drug reactions. Traditional Chinese medicine is a type of drug with high safety and few side effects. Therefore, it is of great significance to use traditional Chinese medicine to prepare programmed cell death inhibitors for HT-22 cells. Summary of the invention

[0007] In order to develop a use of a traditional Chinese medicine in regulating cell necroptosis, the present invention provides a traditional Chinese medicine extract and its use in preparing a cell necroptosis inhibitor. The traditional Chinese medicine extract provided by the present invention can be used as a cell necroptosis inhibitor, thereby reducing the damage of hippocampal neurons, making the structure and morphology of hippocampal neurons normal, with rounded edges and abundant Nissl bodies, and further having a therapeutic effect on degenerative diseases, such as significantly improving depression.

[0008] The invention provides an application of a traditional Chinese medicine extract in preparing a cell necrosis inhibitor. The traditional Chinese medicine extract is prepared from the following components by weight: 10 to 14 parts of northern bupleurum, 8 to 11 parts of angelica, 10 to 14 parts of stir-fried white peony root, 8 to 12 parts of atractylodes, 14 to 16 parts of poria, 2 to 4 parts of liquorice, 10 to 14 parts of moutan bark, 10 to 14 parts of raw gardenia, 10 to 14 parts of calamus, 10 to 14 parts of polygala, and 5 to 7 parts of mint;

[0009] The Chinese medicine extract extraction step is: mixing the above Chinese medicine components with distilled water in a ratio of 1:10-11, decocting, filtering, rotary evaporation concentration, and drying to obtain the Chinese medicine extract;

[0010] The cell necroptosis inhibitor is used for inhibiting the necroptosis of HT-22 cells.

[0011] The Chinese medicinal extract provided by the invention can inhibit the necroptosis of mouse hippocampal neuron cells (HT-22) through its active ingredients as a cell necroptosis inhibitor, reduce the damage of hippocampal neurons, and make the structure and morphology of hippocampal neurons normal, with rounded edges and abundant Nissl bodies.

[0012] Furthermore, the cell necrosis apoptosis inhibitor is the Chinese herbal medicine extract, or a composition of the Chinese herbal medicine extract and an auxiliary material.

[0013] Furthermore, the auxiliary material is any one or a combination of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol and starch.

[0014] Furthermore, the active ingredients in the cell necroptosis inhibitor include 33 alkaloids, 7 carbohydrates, 12 fatty acids, 5 polyketides, 38 shikimic acids and phenylpropanes, and 19 terpenes.

[0015] Furthermore, the active ingredients of the necroptosis inhibitor include: 2-Furoic acid, Marmesin, Procyanidin B2, 4-Piperidinemethanol, 4-fluoro-, Linamarin, Maltotetraose, Cycloleucin, Atractylenolide III, 180-181PE, Coniferyl alcohol, Parthenolide, Liquiritin, Agmatine, Pro-Arg, D-Proline, Aspartic acid, Aspartylphenylalanine, Asparagine, Methyl 3-aminobenzoate, Guanosine, Deoxyribose, Adenosine monophosphate, Luteolin, 1,5-Anhydro-D-sorbitol, Hypoxanthine, Glycerophosphocholine, Leu-Leu, Ganciclovir, Zearalenone, Leu-Phe, 4-Aminosalicylic acid, Deoxycorticosterone, Evodiamine, Luminol, Sulfadoxin, Caftaric acid, 2-Methoxycinnamic acid, Loxistatin acid, 1-.beta.-D-Arabinofuranosyluracil 5'-monophosphate, Anabasine, Licochalcone A, ST 638, Stanolone, Icariin II, Nicotinuric acid, Methyl 4-coumarate, Beclomethasone, Guanosine5'-diphospho-.beta.-L-fucose, Ethyl4-hydroxyquinoline-2-carboxylate, Tyr-Pro-Phe, Sucrose, Sulconazole, Lithocholylglycine, Ro 31-7549, D-(+)-Neopterin, R406, Isovitexin, Suramin, Eugenol gentiobioside, Folinic acid and Hypocrellin B.

[0016] Furthermore, the cell necroptosis inhibitor has the following effects:

[0017] Reduce damage to neurons in the hippocampus;

[0018] Treat depression, Alzheimer's disease or Parkinson's disease.

[0019] Furthermore, the Chinese medicine extract is made of the following components by weight: 12 parts of northern bupleurum, 10 parts of angelica, 12 parts of stir-fried white peony root, 10 parts of atractylodes, 15 parts of Poria, 3 parts of licorice, 12 parts of moutan bark, 12 parts of raw gardenia, 12 parts of calamus, 12 parts of polygala, and 6 parts of mint.

[0020] The present invention provides a method for preparing a Chinese medicine extract, comprising the following steps:

[0021] Weigh the following Chinese medicinal ingredients by weight: 10-14 parts of Bupleurum chinense, 8-11 parts of Angelica sinensis, 10-14 parts of stir-fried white peony root, 8-12 parts of Atractylodes macrocephala, 14-16 parts of Poria cocos, 2-4 parts of Licorice root, 10-14 parts of Cortex Moutan, 10-14 parts of Raw Gardenia jasminoides, 10-14 parts of Acorus calamus, 10-14 parts of Polygala tenuifolia, and 5-7 parts of Mentha arvense;

[0022] The above Chinese medicinal components and distilled water are mixed in a ratio of 1:10-11 and boiled for 40-50 minutes, and the residue and the first filtrate are collected after filtering, and the first filtrate is used as the first decoction;

[0023] The filter residue is added with 6-7 times distilled water and boiled for 40-50 minutes. After filtering, the second filtrate is collected to obtain the second decoction. The first decoction and the second decoction are then mixed evenly. After filtering, rotary evaporation concentration and freeze-drying, freeze-dried powder is obtained to obtain the Chinese medicine extract.

[0024] Furthermore, the parameters of the rotary evaporation concentration are: a drug volume of 300 ml, a condensation circulation pump temperature of -5.4°C, a vacuum pump of 0.095 MPa, a temperature of 50°C, a rotation speed of 100 RPM, and a concentrated drug volume of 30 ml is obtained;

[0025] The freeze-drying process includes: pre-freezing in a -80°C refrigerator for 48 hours, and freeze-drying at -60.3°C and 1.0 kPa for 24 hours.

[0026] The invention also provides a traditional Chinese medicine extract prepared by the preparation method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The potential targets of the active ingredients of the Chinese herbal extract of the present invention are related to programmed necrosis. The Chinese herbal extract can improve the morphology of HT-22 cells, inhibit cell swelling and cell membrane rupture, and simultaneously inhibit the expression and phosphorylation levels of the programmed necrosis signaling pathways RIPK1, RIPK3 and MLKL, and is dose- and time-dependent.

[0029] The Chinese medicine extract of the present invention can improve the morphology of HT-22 cells, inhibit cell swelling and cell membrane rupture, reduce the damage of hippocampal neurons, and treat depression, Alzheimer's disease or Parkinson's disease. By studying the effect of the Chinese medicine extract on depressed mice, it was found that the Chinese medicine extract can significantly improve the sugar water preference rate and increase the number of autonomous activities of rats, indicating that it has a significant improvement on depression. Through the analysis of mouse hippocampal neurons, the Chinese medicine extract prepared by the present invention can reduce the damage of hippocampal neurons, make the hippocampal neurons structure and morphology normal, the edges are rounded, and the Nissl bodies are abundant. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The brain-penetrating components and chemical composition distribution of the Chinese medicine extract prepared in Example 1 of the present invention;

[0032] In the figure, A is the base peak chromatogram (BPC) of the positive mode;

[0033] B is the base peak chromatogram (BPC) in negative ion mode;

[0034] C is the standard peak of Chinese medicine in positive ion mode;

[0035] D is the standard peak in the negative ion mode of traditional Chinese medicine;

[0036] E is the distribution ratio of Chinese medicine in each chemical classification;

[0037] F is the distribution ratio of each chemical classification of Chinese medicine-containing tissues.

[0038] Figure 2 Correlation analysis between potential targets of active ingredients of the Chinese herbal extract prepared in Example 1 of the present invention and programmed necrosis;

[0039] In the figure, A is KEGG analysis;

[0040] B is the biological process analysis in GO analysis;

[0041] C is the component analysis in GO analysis;

[0042] D is the molecular function analysis in GO analysis.

[0043] Figure 3 Effects of the Chinese herbal extract prepared in Example 1 of the present invention on TSZ-induced HT-22 cell damage;

[0044] In the figure, A shows HT-22 cells exposed to TSZ for different time periods, and the cell viability was assessed using the CellTiter-Glo chemiluminescence method.

[0045] B is a representative image of HT-22 cells.

[0046] C: HT-22 cells were pretreated with different concentrations of TCM extracts for 30 min and then treated with TSZ for the indicated time. Subsequently, all cells were lysed and immunoblotted with the indicated antibodies.

[0047] Figure 4 Effects of the Chinese herbal extract prepared in Example 1 of the present invention on depressive-like behavior and neural damage in mice induced by CUMS;

[0048] In the figure, A is the result of the sucrose preference test;

[0049] B is the immobility time in the tail suspension test;

[0050] C is the time spent in the central area during the field test;

[0051] D is the total distance traveled in OFT;

[0052] E is a representative motion trajectory.

[0053] F is a representative micrograph of Nissl-stained neurons. Hippocampus represents the entire hippocampus, CA1, CA3, and DG. CA1 region of the hippocampus, CA3 region of the hippocampus, and dentate gyrus.

[0054] Figure 5 Effect of the Chinese herbal extract prepared in Example 1 of the present invention on programmed necrosis of hippocampal neurons;

[0055] In the figure, A is a representative immunoblot image of RIPK1 / 3 and MLKL.

[0056] B shows the distribution and expression levels of NEUN and MLKL in brain tissue. DETAILED DESCRIPTION

[0057] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0058] Example 1: Use of a traditional Chinese medicine extract in the preparation of a cell necrosis inhibitor.

[0059] 1. Chinese herbal medicine extracts and preparation methods

[0060] Chinese medicine ingredients: 12g of northern bupleurum, 10g of angelica, 12g of stir-fried white peony root, 10g of atractylodes, 15g of Poria, 3g of licorice, 12g of moutan bark, 12g of raw gardenia, 12g of calamus, 12g of polygala, and 6g of mint.

[0061] Preparation process: 11 kinds of Chinese medicine components are soaked in distilled water for 2 hours (116g of Chinese medicine, 1160ml of water) according to the material-liquid ratio of 1:10, then boiled for 40 minutes, filtered and collected the filtrate to obtain the first decoction, and the filtered drug residue is collected for use. 6 times the amount of distilled water is added to the drug residue, boiled for 40 minutes, filtered and collected the filtrate to obtain the second decoction. Then the first decoction and the second decoction are mixed evenly, filtered through gauze, the filtrate is collected, and then rotary evaporation is performed. The parameters of rotary evaporation are: 300ml of drug volume at a time, condensation circulation pump temperature: -5.4℃, vacuum pump: 0.095MPa, temperature: 50℃, speed: 100RPM, and the concentrated drug volume is 30ml; the concentrated drug solution is pre-frozen in a -80℃ refrigerator for 48h, and freeze-dried at -60.3℃ and 1.0kPa for 24h to obtain freeze-dried Chinese medicine extract freeze-dried powder. The final yield is 21.7%.

[0062] Dosage concentration and dosage:

[0063] (1) Cell experiment: The freeze-dried powder of the Chinese herbal medicine extract was resuspended in the cell growth medium to a final concentration of 1.0 g / mL, filtered through a 0.22 μm membrane, and stored at -20°C until use.

[0064] (2) Mouse experiment: The lyophilized powder of Chinese herbal medicine extract was dissolved in pure water at a concentration of 35.2 g / kg (twice the clinical dose) for mouse experiment.

[0065] (3) Rat experiment: The lyophilized powder of Chinese herbal extract was dissolved in pure water at a concentration of 24.4 g / kg (twice the clinical dose) for rat experiment.

[0066] Preparation of brain tissue containing Chinese herbal extracts: 12 male SD rats were randomly divided into two groups and gavaged with Chinese herbal extracts and purified water once a day. After 14 days, the rats were anesthetized and the brains were removed by transcardial perfusion of physiological saline. The whole brain tissue was immediately removed and placed on ice to obtain blank brain tissue and brain tissue samples treated with Chinese herbal extracts.

[0067] Example 2: Use of a traditional Chinese medicine extract in the preparation of a cell necrosis inhibitor.

[0068] The steps are the same as those in Example 1, except that:

[0069] The Chinese medicine components are: 12g of northern bupleurum, 10g of angelica, 12g of stir-fried white peony root, 10g of atractylodes, 15g of Poria, 3g of licorice, 12g of moutan bark, 12g of raw gardenia, 12g of calamus, 12g of polygala, and 6g of mint.

[0070] Example 3: Use of a traditional Chinese medicine extract in the preparation of a cell necrosis inhibitor.

[0071] The steps are the same as those in Example 1, except that:

[0072] The Chinese medicine components are: 10g of northern bupleurum, 8g of angelica, 10g of stir-fried white peony root, 8g of atractylodes, 14g of Poria, 2g of licorice, 10g of moutan bark, 10g of raw gardenia, 10g of calamus, 10g of polygala, and 5g of mint.

[0073] Example 4: Use of a traditional Chinese medicine extract in the preparation of a cell necrosis inhibitor.

[0074] The steps are the same as those in Example 1, except that:

[0075] The Chinese medicine components are: 14g of northern bupleurum, 11g of angelica, 14g of stir-fried white peony root, 12g of atractylodes, 16g of Poria, 4g of licorice, 14g of moutan bark, 14g of raw gardenia, 14g of calamus, 14g of polygala, and 7g of mint.

[0076] Taking Example 1 as an example, the application of the Chinese herbal medicine extract obtained by the present invention is studied.

[0077] 1. Association between Chinese herbal medicine extracts and programmed necrosis

[0078] 1. Analysis of components of Chinese herbal medicine extracts that enter the brain

[0079] 1.1 Sample preparation

[0080] 1.1.1 Extraction of Chinese herbal medicine samples

[0081] Take an appropriate amount of sample into a 1.5mL EP tube; add 400μL pure methanol and vortex mix for 10s; take 200μL of the above solution, add 200μL 40% methanol aqueous solution; vortex mix for 10s; centrifuge for 15min (16000g, 4℃), and take the supernatant.

[0082] 1.1.2 Extraction of blank tissue samples

[0083] Take 100 mg of tissue sample, place it in a homogenizer tube, add 300 μL of water, grind it on a freezing grinder, then add 700 μL of methanol, ultrasonicate in a cold water bath for 20 min, centrifuge at 16000g at 4℃ for 15 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex thoroughly, centrifuge at 16000g at 4℃ for 15 min, and take the supernatant.

[0084] 1.1.3 Blank tissue + Chinese medicine extracts Extraction of Chinese medicine samples

[0085] Take 100 mg of blank tissue, place it in a homogenate tube, add 300 μL of water, and grind it on a freezing grinder. After the grinding, add 700 μL of methanol and 100 μL of the Chinese medicine test solution, ultrasonicate in a cold water bath for 20 min, centrifuge at 16000g and 4℃ for 15 min, take the supernatant and vacuum dry, add 80 μL of 40% methanol aqueous solution to the residue, vortex thoroughly, centrifuge at 16000g and 4℃ for 15 min, and take the supernatant.

[0086] 1.1.4 Extraction of tissue samples for administration of Chinese herbal medicine extracts

[0087] Take 100 mg of blank tissue, place it in a homogenate tube, add 300 μL of water, and grind it on a freezing grinder. After the grinding, add 700 μL of methanol and 100 μL of the Chinese medicine test solution, ultrasonicate in a cold water bath for 20 min, centrifuge at 16000g and 4℃ for 15 min, take the supernatant and vacuum dry, add 80 μL of 40% methanol aqueous solution to the residue, vortex thoroughly, centrifuge at 16000g and 4℃ for 15 min, and take the supernatant.

[0088] 1.2 Chromatographic-mass spectrometry conditions

[0089] 1.2.1 Chromatographic conditions

[0090] The samples were separated using a Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column; the column temperature was 35°C; the flow rate was 0.3 mL / min; the mobile phase composition was A 0.1% formic acid in water, B 0.1% formic acid in acetonitrile; and the gradient elution was performed according to the table below.

[0091] Table 1 Gradient elution parameters

[0092] Time (minutes) Mobile phase A (%) Mobile phase B (%) Initial 95 5 17.0 2 98 17.2 95 5 20.0 95 5

[0093] 1.2.2 Mass spectrometry conditions

[0094] The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system, and the electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃; the detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, the primary and secondary resolutions were 60000 and 15000, respectively, the top 10MS1 ions were used to obtain MS / MS spectra, and the collision energies (CEs) used step-normalized energy levels of 20, 40, and 60; the primary mass-to-charge ratio scanning range was 104-1300.

[0095] 1.3 Sample testing and analysis

[0096] 6 μL of each of the Chinese medicine extract samples, blank group samples, Chinese medicine extract-administered tissue samples, and blank tissue + Chinese medicine extract samples were accurately aspirated and analyzed by LC-MS. Each batch of blank group samples and drug-administered group samples was injected once, the blank group + Chinese medicine extract samples were injected three times, and the Chinese medicine extract samples were injected five times.

[0097] 1.4 Data Analysis Process

[0098] The original data file in .raw format was imported into proteoWizard and converted into .mzXML format. XCMS software was used for peak alignment, retention time correction and peak extraction. Compound identification was performed by searching the local high-resolution mass spectrometry database of traditional Chinese medicine of China Science and New Life Science. The primary mass error was less than 25ppm, and the secondary fragmentation spectrum matching score was greater than 0.7. The higher the score, the higher the spectrum similarity. It is generally believed that the identification result is reliable if it is above 0.7.

[0099] Ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to collect data from four different sample groups: blank group samples, drug group samples, blank + Chinese medicine samples, and Chinese medicine samples. Figure 1 A) and negative ( Figure 1 The bottom peak chromatogram (BPC) images of B) were compared and analyzed. Compound identification was performed by searching the local high-resolution mass spectrometry database. The first-order mass error was kept below 25ppm, and the second-order fragmentation spectrum matching score was greater than 0.7. The chemical components of traditional Chinese medicine and the corresponding tissue samples were analyzed and identified. A total of 2941 traditional Chinese medicine ingredients were identified, of which 143 ingredients were detected in brain tissue after administration. In the BPC diagram describing the positive and negative ions of traditional Chinese medicine, the chromatographic peaks with higher abundance were determined based on the shape of the peaks and verified by secondary spectral analysis. These chromatographic peaks were then numbered in the positive ion diagram and the negative ion diagram in turn ( Figure 1 C and D). The compounds were annotated using the NPClassifier classification method. The classification distribution of Chinese herbal medicine compounds in Chinese herbal medicine extracts is as follows: Figure 1 As shown in E, there are 560 alkaloids, 107 carbohydrates, 292 fatty acids, 172 polyketides, 707 shikimic acids and phenylpropanoids, and 499 terpenes. The distribution of Chinese herbal medicine extracts in the administration tissues is shown in Figure 1 As shown in F, there are 33 alkaloids, 7 carbohydrates, 12 fatty acids, 5 polyketides, 38 shikimic acids and phenylpropanoids, and 19 terpenes.

[0100] 2. Analysis of the association between the potential targets of the active ingredients of traditional Chinese medicine extract (MXYS) and programmed necrosis

[0101] We detected the targets of active ingredients of Chinese herbal medicine extracts that enter brain tissue on Gene Cards. Among the 143 components that enter brain tissue, we found 3164 targets of 61 components (see Table 2). These 3164 targets were analyzed using KEGG and GO analysis.

[0102] Table 2 Active ingredients of Chinese herbal medicine extracts entering brain tissue

[0103]

[0104] The results are as follows Figure 2 As shown in Figure 2, KEGG analysis showed that the Chinese herbal extract (MXYS) may exert its effects through multiple signaling pathways, including PI3K-AKT, MAPK, JAK-STAT pathways, and necroptosis ( Figure 2A). The enrichment analysis results showed that the potential drug targets of MXYS were mainly related to the programmed necrosis pathway, thus indicating that the potential targets of the active ingredients of the Chinese medicine extract of the present invention were related to programmed necrosis.

[0105] 2. Effect of Chinese herbal extract (MXYS) on TSZ-induced HT-22 cell injury

[0106] 1. Cell culture: HT-22 cells were obtained from Procell Life Science Technology Co., Ltd. (CL-0697) and cultured in a culture medium (HyClone, SH30022.01) supplemented with 10% fetal bovine serum (BI, C04001-500) and 100 U / mL penicillin / streptomycin (Gibco, 15140-122). The cells were stored at 37°C and 5% CO2.

[0107] 2. Anti-programmed necrosis activity of Chinese herbal medicine extract (MXYS)

[0108] HT-22 cells were seeded in 96-well plates at a density of 8000 cells per well, with 3 wells per group. The herbal extracts were diluted in growth medium to a final concentration of 6, 4 mg / mL and 2 mg / mL. After 12 h of incubation, the cells were treated with SM-164 hydrochloride (10 nM) and caspase inhibitor z-VAD-fmk (20 μM) for 30 min. They were then incubated with h-TNF-α (20 ng / mL) for another 12 h. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay Kit from Beyotime (C0065L). Luminescence was recorded using a Molecular Devices Spectrometer M5. Subsequently, comprehensive analysis was performed using a NIKON ECLIPSE TI-SR microscope manufactured in Japan, and the images obtained were then carefully examined and processed.

[0109] The results are as follows Figure 3 As shown in A, TSZ can induce a decrease in the activity of Ht-22 cells in a time-dependent manner. Figure 3 As shown in B, the Chinese herbal extract prepared in Example 1 of the present invention can improve the morphology of HT-22 cells and inhibit cell swelling and cell membrane rupture. Figure 3 As shown in C, the Chinese herbal extract prepared in Example 1 of the present invention can inhibit the expression and phosphorylation levels of programmed necrosis signaling pathways RIPK1, RIPK3 and MLKL, and is dose- and time-dependent.

[0110] 3. Effects of Chinese herbal extracts on depression

[0111] 1. Effect of the Chinese herbal extract prepared in Example 1 on the depressive behavior of mice induced by CUMS

[0112] (1) CUMS-induced depression model: The animals were randomly divided into a normal group (Con) and a treatment group. After one week of adaptive feeding, the rest of the animals except the normal group were subjected to CUMS modeling. The CUMS animal model was established by subjecting mice to discontinuous random mild stress. The stimulation factors included 15 types of stimuli, including tilted cages, rocking cages, cage changes, wet cages, water and food deprivation, strobes, noise, continuous light, continuous darkness, intermittent lighting, ice water swimming, warm water swimming, restraint, and foreign objects. Two types of stimuli were randomly received every day, which was in line with multi-factor modeling. The stimulation order was randomly adjusted every week to ensure unpredictability.

[0113] (2) Experimental grouping: Six weeks after CUMS modeling, the animals were randomly divided into a model group (CUMS), a Chinese herbal extract group (MXYS), a positive control group (Paroxetine), and a necroptosis inhibitor control group (Nec-1) (10 mg / kg); among them, 1.8 mg / kg of Chinese herbal extract was equivalent to the clinical dose, and the dose of the positive control group (Aladdin, P129711-1g) was 20 mg / kg. The drugs were administered orally for 4 consecutive weeks, and the normal group and the model group were administered with an equal volume of saline. After the last administration, behavioral experiments were observed, blood was collected from the eyeballs, serum was separated, and hippocampal tissue was immediately separated on ice and quickly frozen in liquid nitrogen for index detection.

[0114] (3) Sucrose preference test (SPT):

[0115] Before the experiment, mice were pre-adapted to 1% sugar water. Two bottles of sugar water were given for 12 hours, and then one of the bottles was replaced with pure water for 12 hours. The animals were fasted for 12 hours before the formal experiment, and two bottles of water were given to the animals, one of which was 1% sucrose water and the other was pure water. After 12 hours, the water bottles were collected to record the liquid consumption and calculate the sugar water preference ratio. Sugar water preference ratio (%) = [sugar water intake / (sugar water intake + pure water intake)] × 100%.

[0116] (4) Tail suspension test (TST):

[0117] The tail of the mouse was fixed to the top of the test box with tape, and the behavior of the mouse was recorded for 6 minutes using mouse behavioral video recording and analysis software, and the immobility time of the mouse in the last 4 minutes was calculated.

[0118] (5) Open field test (OFT):

[0119] The mice were placed one by one in the center of the open field box, and the mouse behavior video recording and analysis software was used to record the autonomous activities of the mice within 5 minutes, and the number of activities, activity distance, etc. After each session, the bottom of the device was wiped with alcohol.

[0120] The sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT) were used to evaluate the effect of MXYS on the depressive-like behavior of mice.

[0121] The results are as follows Figure 4 As shown, there was no significant difference in the baseline sucrose preference rate among the control group, CUMS group, paroxetine group, Nec-1 group, and MXYS group. However, after CUMS modeling, the sucrose preference rate in the CUMS group was significantly reduced compared with the control group (p<0.05) ( Figure 4 In the tail suspension test, the tail suspension time of the MXYS group was significantly shorter than that of the model group (p<0.01) ( Figure 4 In the open field test, the central walking distance of the MXYS group mice was significantly increased (p<0.05) ( Figure 4 C), the total walking distance also increased significantly (p<0.01) ( Figure 4 D).

[0122] 3. Effects of Chinese herbal extracts on mouse hippocampal neurons

[0123] Nissl staining was used to detect the effect of the Chinese medicine extract prepared in Example 1 on mouse hippocampal neurons. The specific method is as follows:

[0124] After the last administration, behavioral experiments were observed, blood was collected from the eyeballs, serum was separated, and brain tissue was immediately separated on ice for Nissl staining. First, paraffin sections were dewaxed to water: the sections were placed in environmentally friendly dewaxing solution for 20 minutes, environmentally friendly dewaxing solution for 20 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, 75% alcohol for 5 minutes, and rinsed with tap water. Frozen sections were warmed and fixed: the frozen sections were taken out of the -20°C refrigerator, returned to room temperature, fixed with tissue fixative for 15 minutes, and then rinsed with running water. Second, Nissl staining: the tissue sections were placed in the staining solution for 2-5 minutes, rinsed with water, differentiated with 0.1% glacial acetic acid, and terminated by rinsing with tap water. The degree of differentiation was controlled under a microscope, and then rinsed with tap water. The sections were placed in an oven for baking and drying. Transparent sealing: the sections were transparent in clean xylene for 10 minutes and sealed with neutral glue.

[0125] like Figure 4 As shown in F, the CUMS group showed obvious neuronal damage, as evidenced by decreased cell number, irregular cell morphology, and increased staining intensity. In contrast, the MXYS and paroxetine-treated groups showed significant improvement in neuronal integrity, with significantly less neuronal damage and preservation of cell number.

[0126] 4. Effects of Chinese herbal extracts on programmed necrosis of hippocampal neurons

[0127] Immunoblotting and immunofluorescence experiments were used to detect the effect of the Chinese herbal extract prepared in Example 1 on programmed necrosis of mouse hippocampal neurons. The specific methods are as follows:

[0128] After the last administration, behavioral experiments were observed, blood was collected from the eyeballs, serum was separated, and brain tissue was immediately separated on ice for immunofluorescence detection; hippocampal tissue was separated and quickly frozen in liquid nitrogen for immunoblotting detection.

[0129] (1) Immunofluorescence: The immunofluorescence procedure was performed according to the standardized protocol. Brain tissues were fixed with 4% paraformaldehyde and embedded in paraffin for IF staining. Primary antibodies (NeuN, 1:20 00; MLKL, 1:300”) were used at 4°C, rinsed 4 times with PBS, and then incubated with secondary antibodies for 1 hour in the dark. DAPI (G1012, Servicebio, China) was stained for 10 minutes at room temperature, rinsed with PBS, and treated with anti-fluorescence quencher. After the primary antibody, goat anti-rat IgG (1:300) (GB21303, Servicebio, China), Alexa Fluor488 goat anti-mouse IgG (GB25301, Servicebio, China) (1:400) were used as secondary antibodies. Fluorescence images of the samples were captured using a confocal microscope.

[0130] (2) Immunoblotting: HT-22 cells or brain tissue were collected with RIPA lysis buffer. The protein concentration was determined, separated on a 10% SDS-PAGE gel, and transferred to a PVDF membrane. After blocking, the membrane was incubated with the primary antibody (1:1000 dilution) at 4°C overnight, followed by incubation with an enzyme-labeled secondary antibody for 1 hour. The membrane was then scanned using a Bio-Rad imaging system. The grayscale values ​​of the bands were then analyzed using Image-J software.

[0131] The results are as follows Figure 5 As shown in A, the Chinese herbal extract prepared in Example 1 of the present invention can inhibit the phosphorylation levels of RIPK1, RIPK3 and MLKL, and reduce the effect on programmed necrosis of mouse neurons. In addition, we used the neuronal marker NeuN and the marker MLKL associated with necrosis to double stain the neuronal cells. Figure 5 As shown in B, the CUMS group showed a decrease in neuronal number accompanied by upregulation and increased distribution of MLKL. In contrast, treatment with MXYS, Nec-1, and paroxetine attenuated neuronal loss and inhibited the progression of necroptosis.

[0132] In summary, the potential targets of the active ingredients of the Chinese herbal extract prepared by the present invention are related to programmed necrosis. The Chinese herbal extract can improve the morphology of HT-22 cells, inhibit cell swelling and cell membrane rupture, and inhibit the expression and phosphorylation levels of the programmed necrosis signaling pathways RIPK1, RIPK3 and MLKL, and show dose and time dependence. By studying the effects of Chinese herbal extracts on depressed mice, it was found that the Chinese herbal extract can significantly increase the sugar water preference rate and increase the number of autonomous activities of rats, indicating that it has a significant improvement on depression. Through the analysis of mouse hippocampal neurons, the Chinese herbal extract prepared by the present invention can reduce the damage of hippocampal neurons, so that the structure and morphology of hippocampal neurons are normal, the edges are rounded, and the Nissl bodies are abundant.

[0133] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0134] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a Chinese herbal medicine extract in the preparation of a cell necrosis inhibitor, characterized in that: The Chinese medicine extract is prepared from the following Chinese medicine composition by weight: 10 to 14 parts of northern bupleurum, 8 to 11 parts of angelica, 10 to 14 parts of stir-fried white peony root, 8 to 12 parts of atractylodes, 14 to 16 parts of poria, 2 to 4 parts of liquorice, 10 to 14 parts of moutan bark, 10 to 14 parts of raw gardenia, 10 to 14 parts of calamus, 10 to 14 parts of polygala, and 5 to 7 parts of mint; The Chinese medicine extract extraction step is: mixing the above Chinese medicine components with distilled water in a ratio of 1:10-11, decocting, filtering, rotary evaporation concentration, and drying to obtain the Chinese medicine extract; The cell necroptosis inhibitor is used for inhibiting the necroptosis of HT-22 cells.

2. The use of the Chinese herbal extract according to claim 1 in the preparation of a cell necrosis inhibitor, characterized in that: The cell necrosis apoptosis inhibitor is the traditional Chinese medicine extract, or a composition obtained by compounding the traditional Chinese medicine extract with auxiliary materials.

3. The use of the Chinese herbal extract according to claim 2 in the preparation of a cell necrosis inhibitor, characterized in that: The auxiliary material is any one or a combination of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol and starch.

4. The use of the Chinese herbal extract according to claim 1 in the preparation of a cell necrosis inhibitor, characterized in that: The active ingredients in the cell necroptosis inhibitor include 33 alkaloids, 7 carbohydrates, 12 fatty acids, 5 polyketides, 38 shikimic acids and phenylpropanes, and 19 terpenes.

5. Use of the Chinese herbal extract according to claim 4 in the preparation of a cell necrosis inhibitor, characterized in that: The active ingredients of the cell necroptosis inhibitor include: 2-Furoic acid, Marmesin, Procyanidin B2, 4-Piperidinemethanol, 4-fluoro-, Linamarin, Maltotetraose, Cycloleucin, Atractylenolide III, 180-181PE, Coniferyl alcohol, Parthenolide, Liquiritin, Agmatine, Pro-Arg, D-Proline, Aspartic acid, Aspartylphenylalanine, Asparagine, Methyl 3-aminobenzoate, Guanosine, Deoxyribose, Adenosinemonophosphate, Luteolin, 1,5-Anhydro-D-sorbitol, Hypoxanthine, Glycerophosphocholine, Leu-Leu, Ganciclovir, Zearalenone, Leu-Phe, 4-Aminosalicylic acid, Deoxycorticosterone, Evodiamine, Luminol, Sulfadoxin, Caftaric acid, 2-Methoxycinnamic acid, Loxistatin acid, 1-.beta.-D-Arabinofuranosyluracil 5'-monophosphate, Anabasine, Licochalcone A, ST 638, Stanolone, Icariin II, Nicotinuric acid, Methyl 4-coumarate, Beclomethasone, Guanosine5'-diphospho-.beta.-L-fucose, Ethyl4-hydroxyquinoline-2-carboxylate, Tyr-Pro-Phe, Sucrose, Sulconazole, Lithocholylglycine, Ro 31-7549, D-(+)-Neopterin, R406, Isovitexin, Suramin, Eugenol gentiobioside, folinic acid and hypocrellin B.

6. Use of the Chinese herbal extract according to claim 1 in the preparation of a cell necrosis inhibitor, characterized in that: The cell necrosis inhibitor has the following effects: Reduce damage to neurons in the hippocampus; Treat depression, Alzheimer's disease or Parkinson's disease.

7. Use of the Chinese herbal extract according to claim 1 in the preparation of a cell necrosis inhibitor, characterized in that: The Chinese medicine extract is made of the following components by weight: 12 parts of northern bupleurum, 10 parts of angelica, 12 parts of stir-fried white peony root, 10 parts of atractylodes, 15 parts of Poria, 3 parts of licorice, 12 parts of moutan bark, 12 parts of raw gardenia, 12 parts of calamus, 12 parts of polygala, and 6 parts of mint.

8. A method for preparing a Chinese medicine extract, characterized in that: The Chinese medicine extract is prepared from the Chinese medicine composition described in claim 1; comprising the following steps: Weigh the following Chinese medicinal ingredients by weight: 10-14 parts of Bupleurum chinense, 8-11 parts of Angelica sinensis, 10-14 parts of stir-fried white peony root, 8-12 parts of Atractylodes macrocephala, 14-16 parts of Poria cocos, 2-4 parts of Licorice root, 10-14 parts of Cortex Moutan, 10-14 parts of Raw Gardenia jasminoides, 10-14 parts of Acorus calamus, 10-14 parts of Polygala tenuifolia, and 5-7 parts of Mentha arvense; The above Chinese medicinal components and distilled water are mixed in a ratio of 1:10-11 and boiled for 40-50 minutes, and the residue and the first filtrate are collected after filtering, and the first filtrate is used as the first decoction; The filter residue is added with 6-7 times distilled water and boiled for 40-50 minutes. After filtering, the second filtrate is collected to obtain the second decoction. The first decoction and the second decoction are then mixed evenly. After filtering, rotary evaporation concentration and freeze-drying, freeze-dried powder is obtained to obtain the Chinese medicine extract.

9. The method for preparing the Chinese medicine extract according to claim 8, characterized in that: The parameters of the rotary evaporation concentration are: a drug volume of 300 ml, a condensation circulation pump temperature of -5.4°C, a vacuum pump of 0.095 MPa, a temperature of 50°C, a rotation speed of 100 RPM, and a concentrated drug volume of 30 ml is obtained; The freeze-drying process includes: pre-freezing in a -80°C refrigerator for 48 hours, and freeze-drying at -60.3°C and 1.0 kPa for 24 hours.

10. A Chinese medicine extract obtained by the preparation method according to any one of claims 8 to 9.

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