Traditional Chinese medicine compound for treating bortezomib-induced peripheral neuropathy and application thereof

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a traditional Chinese medicine compound for treating bortezomib-induced peripheral neuropathy and application of the traditional Chinese medicine compound. The traditional Chinese medicine preparation comprises the following active pharmaceutical ingredients in parts by weight: 8-14 parts of honey-fried licorice root, 5-12 parts of licorice root, 10-20 parts of cassia twig, 8-16 parts of Chinese angelica, 5-13 parts of white peony root, 1-5 parts of asarum, 3-9 parts of tetrapanax papyriferus, 6-14 parts of ginger, 10-20 parts of codonopsis pilosula, 30-50 parts of dried rehmannia root, 15-25 parts of prepared rehmannia root, 10-20 parts of donkey-hide gelatin, 5-15 parts of radix ophiopogonis, 6-12 parts of fructus cannabis, 3-9 parts of spina date seed, 20-30 parts of jujube and 3-9 parts of scorpion. And 3-9 parts of tripterygium wilfordii. The traditional Chinese medicine preparation can significantly up-regulate BCL-2 expression, down-regulate BAX and Casp 3 levels, reduce BAX mitochondrial translocation and inhibit mitochondrial apoptosis so as to relieve bortezomib-induced nerve cell damage, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to a traditional Chinese medicine compound for treating bortezomib-induced peripheral neuropathy and an application thereof. Background Art

[0002] As the first clinically used proteasome inhibitor, bortezomib is still the first-line drug of choice for multiple myeloma because of its reliable efficacy and low price. However, bortezomib has significant neurotoxicity and has the potential to damage nerve fibers through various mechanisms, resulting in a 20% to 40% incidence of bortezomib-induced peripheral neuropathy (BiPN) in patients. The occurrence of BiPN severely limits its clinical application. Currently, the treatment of BiPN mainly relies on neuroprotectants and drugs for neuropathic pain, which have limited clinical efficacy. Therefore, more effective methods are urgently needed to alleviate BiPN.

[0003] At present, the methods of TCM for the treatment of bortezomib-related peripheral neuropathy include: 1. Oral administration of Chinese medicine: The current literature focuses on peripheral neuropathy related to chemotherapy such as oxaliplatin, such as Huangqi Guizhi Wuwu Decoction, Buyang Huanwu Decoction, Danggui Sini Decoction, etc., which warm the yang, replenish deficiency and remove blood stasis. There are fewer literatures on the treatment of bortezomib-related peripheral neuropathy, such as Buyang Huanwu Decoction, Zhi Mashuangzi Capsule, etc., and most of them are case reports. 2. External use of Chinese medicine: It can improve the function of peripheral nerves by stimulating the skin. 3. Acupuncture therapy: Acupuncture can adjust the circulation of qi and blood and promote nerve repair by stimulating acupoints or electroacupuncture. 4. Monomers of Chinese medicine: At present, studies have confirmed the in vitro and in vivo experiments of monomers of Chinese medicines such as paeoniflorin, tanshinone, and ginsenoside Rg3 that activate blood circulation and replenish qi, but their clinical application is still a long way off.

[0004] Therefore, how to design a Chinese medicine compound with clear specific mechanism for clinical application needs further research. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a Chinese medicine compound for treating bortezomib-induced peripheral neuropathy and its application. The present invention uses NGF-induced differentiated PC12 cells to construct a BiPN cell model, and administers different concentrations of Chinese medicine compound-containing serum for intervention. It is found that the 20% Chinese medicine compound-containing serum group can significantly improve the viability of PC12 nerve cells and reduce the level of ROS. This concentration was selected for subsequent intervention, and the results showed that bortezomib can induce a decrease in the mitochondrial membrane potential of PC12 cells, and the Chinese medicine compound of the present invention inhibits PC12 cell apoptosis by increasing the mitochondrial membrane potential, and increases the mitochondrial DNA copy number to restore the number of mitochondria. The present invention confirms that bortezomib-induced mitochondrial apoptosis is an important mechanism of its neurotoxicity, and the Chinese medicine compound of the present invention can significantly upregulate BCL-2 expression, downregulate BAX and Casp 3 levels, and reduce BAX mitochondrial translocation, inhibit mitochondrial apoptosis to reduce bortezomib-induced neuronal cell damage. Based on the above research results, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides a traditional Chinese medicine preparation, the active ingredients of which are the following raw materials in parts by weight: 8-14 parts of roasted licorice, 5-12 parts of licorice, 10-20 parts of cassia twig, 8-16 parts of angelica, 5-13 parts of white peony root, 1-5 parts of asarum, 3-9 parts of tongcao, 6-14 parts of ginger, 10-20 parts of codonopsis, 30-50 parts of raw rehmannia, 15-25 parts of cooked rehmannia, 10-20 parts of donkey-hide gelatin, 5-15 parts of ophiopogon, 6-12 parts of hemp seed, 3-9 parts of spinach seed, 20-30 parts of jujube, 3-9 parts of scorpion, and 3-9 parts of tripterygium wilfordii; the traditional Chinese medicine preparation contains pharmaceutically acceptable excipients.

[0008] The second aspect of the present invention provides the use of the above-mentioned Chinese medicine preparation in any one or more of the following:

[0009] A1) for preparing a product for improving bortezomib-induced peripheral neuropathy;

[0010] A2) for preparing a product for promoting neurite outgrowth of neuroendocrine cells;

[0011] A3) for preparing a product for inhibiting mitochondrial apoptosis of neuroendocrine cells;

[0012] A4) is used for preparing a product for reducing BAX ectopic location in mitochondria of neuroendocrine cells.

[0013] One or more of the above technical solutions have the following beneficial effects:

[0014] The present invention confirms that bortezomib-induced mitochondrial apoptosis is an important mechanism of its neuronal cytotoxicity, and the Chinese medicinal compound of the present invention can significantly upregulate BCL-2 expression, downregulate BAX and Casp 3 levels, and reduce BAX mitochondrial translocation, thereby inhibiting mitochondrial apoptosis and alleviating bortezomib-induced neuronal cell damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 To differentiate the syndromes of patients according to Traditional Chinese Medicine and its relationship with PN grading.

[0017] Figure 2 The Chinese herbal preparation of the present invention improves the symptoms of peripheral neuropathy in patients; A is the PN classification before and after the application of the Chinese herbal preparation of the present invention; B is the effect of the Chinese herbal preparation of the present invention on neuropathic pain in BiPN patients; C is the effect of the Chinese herbal preparation of the present invention on the neuroelectrophysiology of BiPN patients, **P<0.01.

[0018] Figure 3 The chromatograms are for identifying the active ingredients in serum of traditional Chinese medicine preparations using ultra-performance liquid chromatography-high-resolution mass spectrometry, wherein (A) is the chromatogram of the active ingredients in serum of mice in the control group that did not take traditional Chinese medicine preparations, and (B) is the chromatogram of the active ingredients in serum of mice that took traditional Chinese medicine preparations.

[0019] Figure 4 This is the network pharmacology analysis of the Chinese medicine preparation of the present invention intervening in bortezomib-related peripheral neuropathy, A is the Venn diagram of the intersection target of the Chinese medicine preparation of the present invention and BiPN; B is the PPI network diagram of the intersection target, the darker the color and the larger the shape, the higher the node degree value; C is the GO biological function enrichment diagram of the intersection target; D is the KEGG pathway enrichment diagram of the intersection target; E is the docking mode diagram of the monomer components of the Chinese medicine preparation of the present invention and the core target molecule.

[0020] Figure 5 The Chinese medicine serum of the present invention improves bortezomib-induced PC12 neuronal cell injury (x±s, n=5), A shows the effect of different concentrations of the Chinese medicine serum of the present invention on PC12 cell viability; B and C show the effect of different concentrations of the Chinese medicine serum of the present invention on PC12 cell ROS levels. Compared with the control group, *P<0.05, **P<0.01; compared with the model group, ##P<0.01.

[0021] Figure 6The serum containing the traditional Chinese medicine preparation of the present invention promoted the growth of neurites in PC12 cells. Compared with the control group, *P<0.05, **P<0.01; compared with the model group, ##P<0.01.

[0022] Figure 7 The Chinese medicine serum of the present invention inhibits mitochondrial apoptosis of PC12 neurons (x±s, n=5). A and B are the effects of the Chinese medicine serum of the present invention on PC12 cell apoptosis; C and D are the effects of the Chinese medicine serum of the present invention on the mitochondrial membrane potential of PC12 cells; E is the effect of the Chinese medicine serum of the present invention on the mitochondrial DNA copy number of PC12 cells; F and G are the effects of the Chinese medicine serum of the present invention on the apoptotic protein level of PC12 cells. Compared with the control group, *P<0.05, **P<0.01; compared with the model group, ##P<0.01.

[0023] Figure 8 This is the effect of the Chinese medicine preparation of the present invention on BAX mitochondrial translocation in PC12 cells (x±s, n=5). Compared with the control group, **P<0.01; compared with the model group, ##P<0.01. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0025] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0026] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0027] In a typical specific embodiment of the present invention, a traditional Chinese medicine preparation is provided, wherein the active ingredients are the following raw materials in parts by weight: 8-14 parts of roasted licorice, 5-12 parts of licorice, 10-20 parts of cinnamon twig, 8-16 parts of angelica, 5-13 parts of white peony root, 1-5 parts of asarum, 3-9 parts of tongcao, 6-14 parts of ginger, 10-20 parts of codonopsis pilosula, 30-50 parts of raw rehmannia root, 15-25 parts of prepared rehmannia root, 10-20 parts of donkey-hide gelatin, 5-15 parts of ophiopogon japonicus, 6-12 parts of hemp seed, 3-9 parts of spinach seed, 20-30 parts of jujube, 3-9 parts of scorpion, and 3-9 parts of tripterygium wilfordii; the traditional Chinese medicine preparation contains pharmaceutically acceptable excipients.

[0028] In another specific embodiment of the present invention, the active ingredients are the following raw materials in parts by weight: 10 parts of roasted licorice, 10 parts of licorice, 15 parts of cinnamon twig, 12 parts of angelica, 9 parts of white peony root, 3 parts of asarum, 6 parts of tongcao, 10 parts of ginger, 15 parts of codonopsis, 40 parts of raw rehmannia, 20 parts of prepared rehmannia, 15 parts of donkey-hide gelatin, 10 parts of ophiopogon, 9 parts of hemp seed, 6 parts of spinach seed, 25 parts of jujube, 6 parts of whole scorpion, and 6 parts of tripterygium wilfordii.

[0029] It should be noted that the above-mentioned raw materials can be traditional Chinese herbal medicines; at the same time, in order to facilitate drug quality control, the above-mentioned raw materials can also be Chinese herbal formula granules made by extracting and concentrating Chinese herbal medicines according to traditional standards, and no specific limitation is made here.

[0030] In another specific embodiment of the present invention, the content of the adjuvant in the Chinese medicine preparation is 1% by weight-99% by weight. Specifically, in addition to the above-mentioned active pharmaceutical ingredients, the Chinese medicine preparation may also contain a pharmaceutically acceptable adjuvant, and the content of the adjuvant in the Chinese medicine preparation may be 1% by weight-98% by weight, including but not limited to 5% by weight, 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, 95% by weight, 98% by weight, usually accounting for about 80% by weight.

[0031] The Chinese medicine preparation can be in the form of a liquid or solid dosage form. The liquid dosage form can be a true solution (such as a decoction), a colloid, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form. The solid dosage form can be a tablet, a capsule, a pill, a powder, a granule, a freeze-dried powder injection, an inclusion compound, a landfill, etc.

[0032] The Chinese medicine preparation of the present invention may contain commonly used auxiliary materials, and the auxiliary materials described herein include but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbitan, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium trisilicate, polyvinyl pyrrolidone, etc.

[0033] Tablets and capsules for oral administration may contain excipients such as syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone.

[0034] Pyrrolidone; fillers such as dextrin, lactose, sucrose, corn starch, calcium phosphate, sorbitol, aminoacetic acid; lubricants such as magnesium stearate, talc, polyethylene glycol, silica; disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. Oral tablets can be coated by pharmaceutically known methods.

[0035] Oral liquid can be made into water and oil suspension, solution, emulsion, syrup, etc., or it can be made into dry product.

[0036] Filled with water or other suitable medium. Such liquid preparations may contain conventional additives, such as suspending agents, sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils and fats, emulsifiers, such as lecithin, sorbitan monooleate, gum arabic; or non-aqueous carriers, including almond oil, oils such as glycerol, ethylene glycol, or ethanol; preservatives, including methyl parahydroxybenzoate, propyl parahydroxybenzoate or sorbic acid.

[0037] The specific preparation method of the above-mentioned traditional Chinese medicine preparation is not specifically limited, and those skilled in the art can prepare the preparation method according to the corresponding dosage form.

[0038] Production and preparation. In a specific embodiment of the present invention, the Chinese medicine preparation, the active ingredients of which can be prepared by conventional decoction, such as mixing the above-mentioned raw materials, adding 8-10 times the amount of water, soaking for 10-30 minutes, and then decocting for 10-30 minutes, pouring out the liquid medicine; continue to add 5-8 times the amount of water to the residue, and then decoct for 8-15 minutes, and mix the liquid medicines obtained twice to obtain a decoction. The decoction can be used for patients. To facilitate industrial production, the preparation method also includes decoctioning the decoction to obtain a clear paste, and further mixing it with the above-mentioned excipients to prepare the corresponding Chinese medicine preparations in various dosage forms.

[0039] In another specific embodiment of the present invention, the above-mentioned Chinese medicine preparation is provided for use in any one or more of the following:

[0040] A1) for preparing a product for improving bortezomib-induced peripheral neuropathy;

[0041] A2) for preparing a product for promoting neurite outgrowth of neuroendocrine cells;

[0042] A3) for preparing a product for inhibiting mitochondrial apoptosis of neuroendocrine cells;

[0043] A4) is used for preparing a product for reducing BAX ectopic location in mitochondria of neuroendocrine cells.

[0044] In another specific embodiment of the present invention, the neuroendocrine cells are specifically NGF-induced PC12 cells.

[0045] In the above-mentioned A2, the product for inhibiting mitochondrial apoptosis of neuroendocrine cells inhibits apoptosis of nerve cells by reducing the level of ROS and inhibiting the expression of BAX and Casp 3.

[0046] In A1) to A4), the product is a medicine or a test preparation, and the test reagent is used for basic scientific experimental research.

[0047] Specifically, the product of the Chinese medicine preparation is a concentrated Chinese medicine preparation drink or a drug-containing serum thereof.

[0048] More specifically, the drug-containing serum is obtained by gavage of the above-mentioned Chinese medicine preparation to rats and then collecting blood from the abdominal aorta; the concentration of the drug-containing serum is 20%.

[0049] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0050] Example

[0051] 1 Method

[0052] 1.1 Clinical Data

[0053] 17 patients with myeloma with bortezomib-related peripheral neuropathy (7 females and 10 males; aged 45-85 years, median 65 years). All patients received bortezomib + dexamethasone treatment (bortezomib dose of 1-1.3 mg / m2, days 1, 4, 8, 11 or days 1, 8, 15, 22; dexamethasone 20 mg / d, days 1-2, 4-5, 8-9, 11-12 or days 1-2, 8-9, 15-16, 22-23), once every 4 weeks. In addition to the bortezomib + dexamethasone treatment regimen, some patients also received lenalidomide or cyclophosphamide treatment. All patients were from the Department of Oncology and Hematology of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine and the Department of Hematology of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. After one course of treatment with the Chinese medicine preparation described in the present invention, all patients were evaluated.

[0054] The National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE) 4.0 was used to grade BIPN. Neurotoxicity symptoms and neuropathic pain, as well as neurophysiological evaluation were assessed using the Functional Assessment of Neurotoxicity by Oncology Therapy / Gynecologic Oncology Group (FACT / GOG-Ntx) score and the visual analog scale (VAS) score. All participants provided informed consent in accordance with the Declaration of Helsinki. Ethics number: (2020) Ethics Review No. (010)-KY.

[0055] 1.2 Experimental Materials

[0056] SD rats were half male and half female, SPF grade, 6-8 weeks old, and weighed 180-220 g. They were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006. They were kept in the animal room of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine and were approved by the Animal Experiment Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (AWE-2019-038). PC12 cell line (SCSP-517) was purchased from the Cell Bank of the Chinese Academy of Sciences. Bortezomib was purchased from Dalian Meilun Biotechnology Co., Ltd. (Batch number: MB1040).

[0057] CCK-8 kit and mitochondrial membrane potential detection kit were purchased from Beijing Biotech Co., Ltd. (Batch No.: E-BC-K138-F, C2006). Reactive oxygen species (ROS) detection kit was purchased from Wuhan Beiyinlai Biotechnology (Batch No.: BTK024). Anti-Bcl-2, BAX, and Caspase-3 antibodies were purchased from Santa Cruz Biotechnology, USA.

[0058] 1.3 Preparation of drug-containing serum

[0059] The Chinese medicine preparation of the present invention is composed of 10 grams of roasted licorice root, 10 grams of licorice root, 15 grams of cinnamon twig, 12 grams of angelica root, 9 grams of white peony root, 3 grams of asarum, 6 grams of tongcao, 10 grams of ginger, 15 grams of codonopsis pilosula, 40 grams of raw rehmannia root, 20 grams of prepared rehmannia root, 15 grams of donkey-hide gelatin, 10 grams of ophiopogon japonicus, 9 grams of hemp seed, 6 grams of spiny jujube seed, 25 grams of jujube, 6 grams of scorpion, and 6 grams of tripterygium wilfordii. The decoction pieces were purchased from the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, and the Chinese medicine decoction was prepared according to the process specified in the Chinese Pharmacopoeia (2010 edition). According to the equivalent dose conversion between rats and humans, SD rats were fed with 1 ml / 100 g of the Chinese medicine preparation of the present invention, containing 10.5 g / kg / time of crude drug, twice a day for 3 consecutive days. After the last feeding of the rats, blood samples were collected from the abdominal aorta under anesthesia, centrifuged at 3000 rpm for 10 minutes, and then the serum containing the drug was separated. The low, medium and high concentrations of the Chinese medicine preparation of the present invention were set to contain 5%, 10% and 20% of the drug respectively. The samples were stored in a refrigerator at 4°C for later use.

[0060] 1.4 Network pharmacology analysis of the Chinese medicine preparation of the present invention in the treatment of bortezomib-related peripheral neuropathy

[0061] Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to identify the active ingredients of the serum containing the Chinese medicine preparation of the present invention, and the serum of mice in the same batch that did not take the Chinese medicine preparation was set as the control group. The Vanquish UHPLC (ThermoScientific, Waltham, MA) ultra-high performance liquid chromatography system combined with ACQUITY UPLC HSS T3 (2.1mm X100mm, 1.8μm) chromatographic column separated the samples, the column temperature was 35°C; the flow rate was 0.3mL / min, the mobile phase composition A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution. The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. Subsequently, the TCMSP (https: / / old.tcmsp-e.com / tcmsp.php), TCMID (http: / / www.megabionet.org / tcmid / ), and CTD (http: / / ctdbase.com / ) databases were used to screen the potential targets of the compounds. BiPN targets were obtained through OMIM (https: / / www.omim.org / ) and GeneCards (https: / / www.genecards.org / ) using "Bortezomib-induced peripheral neuropathy", "Peripheral Nerve Injuries", and "Inherited Peripheral Neuropathy" as keywords. The drug and disease targets were intersected using R language, imported into the STRING website to obtain the PPI network, and analyzed using the Cytoscape 3.10.2 software Network Analyer module. The target proteins were enriched and visualized for GO and KEGG pathways using the R clusterProfiler package, and the "target-pathway" network was constructed using Cytoscape. Finally, Discovery Studio was used to verify the binding activity of the core components of the traditional Chinese medicine preparation described in the present invention with the target.

[0062] 1.5 PC12 cell grouping and treatment

[0063] PC12 cells were cultured for 5 days in 1640 complete medium containing 50 ng / mL rat β nerve growth factor (NGF, R&D Systems, USA) to differentiate into neuron-like cells. The differentiated PC12 neuron-like cells were inoculated into 6-well plates and cultured in a 37°C, 5% CO2 incubator.

[0064] The mice were divided into a control group, a model group (based on our previous research and related literature, 50nmol / L bortezomib was given to treat the model), and a Chinese medicine preparation group of the present invention (different concentrations of the Chinese medicine preparation of the present invention + 50nM bortezomib treatment). Different concentrations of the Chinese medicine preparation of the present invention containing serum (5%, 10% and 20%) were treated for 12h.

[0065] 1.6 Cell viability assay

[0066] The cell density of PC12 cells was adjusted to 2×105 / mL and inoculated in a 96-well plate at 100ul / well. 10μLCK-8 solution was added at 24 and 48 hours respectively. After incubation in a 37℃, CO2 incubator for 2 hours, the cells were detected at 450nm using an ELISA reader to calculate the cell activity of each group.

[0067] 1.7 DCFH-DA fluorescent probe to detect ROS levels in PC12 cells

[0068] After each group of cells were rinsed with PBS, DCFH-DA was added at a final concentration of 10 μmol / L according to the instructions, and incubated at 37°C for 30 min. After rinsing with PBS, the cells were counterstained with DAPI and rinsed three times. The cells were imaged using a laser confocal microscope to measure the distribution and expression of green fluorescence (indicating ROS) (excitation wavelength: 488 nm; emission wavelength: 525 nm), and the immunofluorescence intensity was evaluated using Image J software.

[0069] 1.8 PC12 neuron length determination

[0070] After intervention, PC12 cells in each group were fixed with 4% paraformaldehyde at room temperature and blocked with 5% bovine serum albumin at room temperature for 30 minutes. Acti-stain 488phalloidin (Cytoskeleton, Inc., USA) was added to bind to F-actin, incubated overnight at 4°C, washed with PBS, and embedded in 80% glycerol. DAPI was then used for nucleus staining. ImageXpressMicro XL (Molecular Devices, USA) was used to analyze neurite length.

[0071] 1.9 Detection of apoptosis in PC12 cells by flow cytometry

[0072] Each group collected 1×10 5 For each cell, add 100 μL 1×Binding Buffer to resuspend the cells, then add 5 μL Annexin V-FITC and 10 μL PI Staining Solution and mix gently. Incubate at room temperature in the dark for 10 to 15 min. Add 400 μL 1×Binding Buffer, mix well and place on ice. Detect the apoptosis rate of PC12 cells by flow cytometry within 1 h.

[0073] 1.10 Mitochondrial membrane potential detection

[0074] The mitochondrial membrane potential (MMP) was measured using a mitochondrial membrane potential detection kit. The treated cells were incubated with JC-1 staining solution for 20 minutes and then washed twice with PBS. Fluorescence was measured using a flow cytometer.

[0075] 1.11 qPCR detection of relative copy number of mitochondrial DNA

[0076] Total RNA was extracted from each group of cells, and reverse transcribed into cDNA after RNA concentration and purity testing, and amplified using SYBR Green qPCR kit. 16S rRNA in the mitochondrial genome was used as the gene to be detected, and ACTB was used as the internal reference. -ΔΔCt The relative expression of 16S rRNA mRNA was analyzed by the method.

[0077] Table 1 RT-qPCR primer sequences

[0078]

[0079] 1.12 Western blot detection of mitochondrial apoptosis protein levels

[0080] After 48h of cell culture in each group, 5×105 cells were collected, total protein was extracted, and quantitative analysis was performed using the BCA protein detection kit. Separation gel was prepared, and primary antibodies were added and incubated overnight at 4°C after electrophoresis, transfer, and washing. The dilution ratio was: BCL-2 (1:1000), BAX (1:1000), Casp 3 (1:2000). After washing, the membrane was reacted with HRP-labeled secondary antibody (1:5000). After ECL color development, the images were exposed and analyzed using ImageJ 8.0 software. GAPDH was used as an internal reference to calculate the relative expression of the target protein.

[0081] 1.13 Mitochondrial BAX co-localization detection

[0082] Cells from each group were taken, 500 nM Mito-Tracker Red CMXRos working solution was added to label mitochondria, and Bax antibody (1:100) was added and incubated overnight at 4°C. The co-localization of mitochondrial BAX was observed by laser confocal microscopy at a wavelength of 550 nm.

[0083] 1.14 Statistical methods

[0084] The experimental results were statistically analyzed using SPSS26. The measurement data were expressed as mean ± standard deviation (x ± s). The differences between the two groups were analyzed using Student's t test, and the differences between multiple groups were analyzed using one-way analysis of variance. P < 0.05 was considered statistically significant.

[0085] 2 Results

[0086] 2.1 Results of syndrome differentiation of MMPN patients

[0087] The Sankey diagram was used to show the results of syndrome differentiation of 17 patients and the relationship between PN grade and syndrome type. Figure 1 A shows that BiPN patients were mainly diagnosed with yang deficiency and cold stagnation, qi and blood deficiency, and blood stasis, with 8, 6, and 3 cases, respectively. Among them, yang deficiency and cold stagnation were the most common PN patients, followed by qi and blood deficiency. Further evaluation of the relationship between TCM syndrome differentiation and PN severity in BiPN patients showed that most patients with grade 1 PN were diagnosed with qi and blood deficiency, while most patients with grade 2 and 3 PN were diagnosed with yang deficiency and cold stagnation, blood stasis, and internal resistance. Figure 1 B). It indicates that BiPN is a disease of deficiency in the root and excess in the side. The deficiency in the root is manifested in deficiency of qi and blood, and weakness of yang qi, while the excess in the side is manifested in internal obstruction of cold and blood stasis. The deficiency in the root is not nourishing, while the excess in the side is blocked, and the obstruction of qi and blood circulation causes pain. This provides a specific idea for our clinical application of TCM intervention in BiPN, and also provides clinical evidence for the effective application of the Chinese medicine preparation for strengthening the body and removing blood stasis of the present invention.

[0088] 2.2 The Chinese medicine preparation of the present invention improves the clinical symptoms of BiPN patients

[0089] The PN grading evaluation, pain score, and electrophysiological motor nerve testing were performed on BiPN patients before and after treatment with the Chinese medicine preparation of the present invention. The results showed that the Chinese medicine preparation of the present invention can not only alleviate the progression of the patient's disease, but also significantly reduce the PN grading of some patients ( Figure 2 A), significantly reducing the pain scores of patients ( Figure 2 B, P<0.01), and significantly improved the conduction velocity of motor nerves and sensory nerves ( Figure 2 C, P<0.01).

[0090] 2.3 Network pharmacology analysis of the Chinese medicine preparation of the present invention in the intervention of bortezomib-related peripheral neuropathy

[0091] Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to identify the active ingredients of traditional Chinese medicine preparations. A total of 3432 compounds were identified in positive and negative ion modes, including 457 from licorice, 226 from cinnamon twigs, 228 from angelica sinensis, 176 from white peony root, 102 from asarum, 57 from tongcao, 285 from ginger, 306 from codonopsis pilosula, 254 from rehmannia root, 189 from donkey hide gelatin, 206 from ophiopogon japonicus, 132 from hemp seed, 187 from spiny jujube seed, 423 from jujube, 79 from scorpion, and 125 from tripterygium wilfordii. The peaks with higher abundance were confirmed by the base peak chromatogram and the secondary spectrum was checked. The chromatographic peaks of the control group were Figure 3 (A), chromatogram of drug-containing serum group is shown in Figure 3 (B), compared with the control group, the identified compounds in the drug-containing serum included isoliquiritigenin, naringenin, DL-3-indole lactic acid, formononetin, arjuna acid, apigenin isoliquiritigenin, 6,7-dihydroxyligustilide, glycyrrhetinic acid, cryptotanshinone, etc. (as shown in Table 2), all of which were not detected in the control group.

[0092] Table 2 Chemical composition identification results of BPC icon peaks Top 20 compounds

[0093]

[0094]

[0095] Table Notes: m / z: parent ion mass-to-charge ratio; RT / min: retention time / min; ppm: primary mass deviation; Adduct: adduct; Score: secondary mass spectrum matching score.

[0096] Potential targets of compounds were screened by using TCMSP (https: / / old.tcmsp-e.com / tcmsp.php), TCMID (http: / / www.megabionet.org / tcmid / ), and CTD (http: / / ctdbase.com / ) databases. A total of 3741 targets of the Chinese medicine preparations of the present invention and 19836 BiPN-related targets were obtained. After taking the intersection, 3541 potential targets of Chinese medicine were obtained ( Figure 4 A). Protein interaction PPI network topology analysis showed that apoptotic proteins such as BAX, P53, BCL-2, and CASP3 are important targets for the Chinese medicine preparation of the present invention to intervene in BiPN ( Figure 4B). GO and KEGG pathway enrichment revealed that the intersection targets were mainly involved in biological processes such as "regulation of apoptotic process", "response to oxidative stress", "occurs in mitochondrion" and PI3K-Akt, MAPK, suggesting that mitochondrial apoptosis is an important pathway for the Chinese medicine preparation of the present invention to intervene in BiPN ( Figure 4 C, D). Further analysis of the components of the Chinese medicine preparation of the present invention and its mechanism of action revealed that a variety of monomer components can act on core mitochondrial apoptosis targets such as BAX, BCL-2, and CASP3. Isoliquiritigenin, naringenin, formononetin, glycyrrhetinic acid, cryptotanshinone, etc. can all target core mitochondrial apoptosis proteins such as BAX, BCL-2, and CASP3. Taking isoliquiritigenin and naringenin as examples, molecular docking technology was used to demonstrate that the monomers of the Chinese medicine preparation of the present invention have good docking activity with BAX, BCL-2, and CASP3 ( Figure 4 E).

[0097] Table 3 Docking activity of monomer components of the traditional Chinese medicine preparation of the present invention with apoptotic proteins

[0098]

[0099] 2.4 The Chinese medicine preparation containing serum of the present invention improves bortezomib-induced PC12 neuronal cell injury

[0100] NGF was used to induce PC12 cells to differentiate into neurons, a BiPN cell model was constructed, and different concentrations of the Chinese medicine preparation serum of the present invention were administered for intervention. The results showed that 20% of the Chinese medicine preparation serum of the present invention could significantly improve the decrease in PC12 neuronal cell viability induced by bortezomib ( Figure 5 A, P<0.01) and reduced ROS levels ( Figure 5 B, C, P<0.01). Based on the above results, 20% serum containing the Chinese medicine preparation of the present invention was selected as the optimal concentration for subsequent index detection.

[0101] 2.5 The Chinese medicine preparation containing serum of the present invention promotes the growth of neurites in PC12 cells

[0102] The effect of the Chinese medicine preparation of the present invention on the growth of neurites of PC12 cells after bortezomib intervention was further tested. The results showed that bortezomib significantly inhibited the growth of neurites of PC12 cells, and the Chinese medicine preparation of the present invention could significantly reverse the inhibitory effect of bortezomib on the growth of neurites of PC12 cells ( Figure 6 , P<0.01).

[0103] 2.6 The Chinese medicine preparation containing serum of the present invention inhibits mitochondrial apoptosis of PC12 cells

[0104] Compared with the control group, the apoptosis level of PC12 cells in the model group was significantly increased, while the serum containing the Chinese medicine preparation of the present invention significantly reduced the apoptosis of neurons induced by bortezomib ( Figure 7 A, B, P<0.01). Then, the mitochondrial membrane potential, mitochondrial DNA copy number, and mitochondrial apoptosis protein levels were detected. It was found that the mitochondrial membrane potential level of PC12 cells treated with bortezomib ( Figure 7 C, D, P<0.01), mtDNA ( Figure 7 E, P<0.01) and BCL-2 / BAX protein levels ( Figure 7 F, G, P<0.01) decreased significantly, indicating the occurrence of mitochondrial apoptosis, and the Chinese medicine preparation of the present invention can significantly increase mitochondrial membrane potential, mitochondrial DNA copy number and Bcl-2 / BAX ratio, and reverse bortezomib-induced PC12 cell apoptosis.

[0105] 2.7 Effect of the Chinese medicine preparation containing serum on the co-localization of BAX in PC12 mitochondria

[0106] Compared with the control group, the co-localization of BAX and mitochondria in the model group increased significantly ( Figure 8 , P<0.01), indicating the activation of mitochondrial apoptosis pathway. After intervention with the serum containing the Chinese medicine preparation of the present invention, BAX mitochondrial translocation was significantly reversed.

[0107] 3 Discussions

[0108] According to the clinical manifestations of BiPN, it should be classified into the categories of "paralysis" and "blood stasis" in traditional Chinese medicine. There is no definite consensus on the pathogenesis of this disease in traditional Chinese medicine, but studies have found that it is often related to factors such as yang deficiency, qi deficiency, and blood stasis. We first retrospectively classified 17 patients with BiPN and used the Sankey diagram to display the results of TCM syndrome differentiation and its relationship with PN grading ( Figure 1 ), found that BiPN patients were mainly diagnosed as Yang deficiency and cold coagulation, Qi and blood deficiency, and blood stasis. Among them, patients with Yang deficiency and cold coagulation accounted for the highest proportion, and the PN grade was higher, which was consistent with previous studies, suggesting the pathological nature of BiPN as a mixture of deficiency and excess. According to the Huangdi Neijing, "Blood and qi like warmth and hate cold, and tears cannot flow when cold, and warmth dissipates and removes them", "Cold should be treated with heat... knots should be dispersed... deficiency should be supplemented", the rational use of Chinese medicines such as strengthening the body, warming the yang, removing blood stasis, and relieving pain in the treatment of BiPN may achieve certain therapeutic effects.

[0109] The present invention uses NCI-CTCAE grading, FACT / GOG-Ntx scale, and VAS scale to evaluate the PN grading and pain score of BiPN patients before and after the application of the Chinese medicine preparation of the present invention, and detects the patient's neuroelectrophysiology. The results show that the Chinese medicine preparation of the present invention can delay the progression of PN in patients and reduce the PN grading of patients, significantly relieve the pain of patients, and significantly increase the motor and sensory nerve conduction velocity ( Figure 2 ).

[0110] The present invention uses network pharmacology analysis to obtain 3541 targets for the intervention of BiPN by the Chinese medicine preparation of the present invention, among which mitochondrial apoptosis proteins such as BAX, BCL-2, and CASP3 are its core targets ( Figure 4 GO and KEGG pathway enrichment indicated that mitochondrial apoptosis was an important pathway for the Chinese medicine preparation of the present invention to intervene in BiPN, and molecular docking verified that the monomer components of the Chinese medicine preparation of the present invention had good docking activity with mitochondrial apoptosis proteins BAX, BCL-2, and CASP3.

[0111] PC12 cells can differentiate into neurons under NGF stimulation and are a commonly used cell model in peripheral neuropathy research. We used NGF-induced differentiation of PC12 cells to construct a BiPN cell model and administered different concentrations of the Chinese medicine preparation-containing serum of the present invention for intervention. We found that the 20% Chinese medicine preparation-containing serum group could significantly improve the viability of PC12 neurons and reduce the level of ROS ( Figure 5 This concentration was selected for subsequent intervention, and the results showed that the serum containing the Chinese medicine preparation of the present invention can significantly promote the growth of neurites in PC12 cells ( Figure 6 The apoptosis level of PC12 neurons, mitochondrial membrane potential, mitochondrial DNA copy number, mitochondrial apoptosis protein expression, and BAX mitochondrial translocation were further evaluated ( Figure 7 , 8). The decrease of mitochondrial membrane potential is a key event in initiating mitochondrial-dependent apoptosis. This study found that bortezomib can induce a decrease in mitochondrial membrane potential of PC12 cells, and the Chinese medicine preparation of the present invention inhibits PC12 cell apoptosis by increasing mitochondrial membrane potential, and increases mitochondrial DNA copy number to restore the number of mitochondria. In order to clarify whether the Chinese medicine preparation of the present invention exerts its neuroprotective effect by regulating mitochondrial apoptosis through BCL-2 / BAX, the present invention evaluates the expression of mitochondrial apoptosis-related proteins BCL-2, BAX, and Casp 3 and BAX mitochondrial translocation by Western Blot and mitochondrial immunofluorescence co-localization. Consistent with previous studies, this study confirmed that bortezomib-induced mitochondrial apoptosis is an important mechanism of its neurotoxicity, and the Chinese medicine preparation of the present invention can significantly upregulate BCL-2 expression, downregulate BAX and Casp 3 levels, and reduce BAX mitochondrial translocation, inhibiting mitochondrial apoptosis to alleviate bortezomib-induced neuronal cell damage.

[0112] In summary, the present invention describes the TCM syndrome differentiation of BiPN patients and its relationship with PN grading. At the same time, it is confirmed that the Chinese medicine preparation of the present invention improves BiPN by inhibiting the mitochondrial apoptosis pathway, providing a basic basis for preventing and treating tumor complications.

[0113] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A Chinese medicine preparation, characterized in that: The active ingredients of the medicine are the following raw materials in parts by weight: 8-14 parts of roasted liquorice, 5-12 parts of liquorice, 10-20 parts of cassia twig, 8-16 parts of angelica, 5-13 parts of white peony root, 1-5 parts of asarum, 3-9 parts of tongcao, 6-14 parts of ginger, 10-20 parts of codonopsis, 30-50 parts of raw rehmannia, 15-25 parts of cooked rehmannia, 10-20 parts of donkey-hide gelatin, 5-15 parts of ophiopogon, 6-12 parts of hemp seed, 3-9 parts of spinach seed, 20-30 parts of jujube, 3-9 parts of scorpion and 3-9 parts of tripterygium wilfordii; the traditional Chinese medicine preparation contains pharmaceutically acceptable excipients.

2. The Chinese medicine preparation according to claim 1, characterized in that: The active ingredients are the following raw materials in parts by weight: 10 parts of roasted licorice, 10 parts of licorice, 15 parts of cinnamon twig, 12 parts of angelica, 9 parts of white peony root, 3 parts of asarum, 6 parts of tongcao, 10 parts of ginger, 15 parts of codonopsis, 40 parts of raw rehmannia, 20 parts of prepared rehmannia, 15 parts of donkey-hide gelatin, 10 parts of ophiopogon, 9 parts of hemp seed, 6 parts of spiny jujube seed, 25 parts of jujube, 6 parts of whole scorpion, and 6 parts of tripterygium wilfordii.

3. The Chinese medicine preparation according to claim 1, characterized in that: The content of the auxiliary material in the traditional Chinese medicine preparation is 1% by weight to 99% by weight.

4. Application of a product containing the Chinese medicine preparation according to any one of claims 1 to 3 in any one or more of the following: A1) for preparing a product for improving bortezomib-induced peripheral neuropathy; A2) for preparing a product for promoting neurite outgrowth of neuroendocrine cells; A3) for preparing a product for inhibiting mitochondrial apoptosis of neuroendocrine cells; A4) is used for preparing a product for reducing BAX ectopic location in mitochondria of neuroendocrine cells.

5. The use according to claim 4, characterized in that: The neuroendocrine cells are specifically NGF-induced PC12 cells.

6. The use according to claim 4, characterized in that: In A2, the product for inhibiting mitochondrial apoptosis of neuroendocrine cells inhibits apoptosis of nerve cells by reducing ROS levels and inhibiting the expression of BAX and Casp 3.

7. The use according to claim 4, characterized in that: In A1) to A4), the product is a drug or a test preparation, and the test reagent is used for basic scientific experimental research.

8. The use according to claim 4, characterized in that The product containing the Chinese medicine preparation according to any one of claims 1 to 3 is a concentrated solution of the Chinese medicine preparation or a drug-containing serum thereof.

9. The use according to claim 7, characterized in that: The drug-containing serum is obtained by gavage of the Chinese medicine preparation according to any one of claims 1 to 3 to rats and then collecting blood from the abdominal aorta.

10. The use according to claim 7, characterized in that The concentration of the drug-containing serum is 20%.

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