CN114984175A - Traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy and application thereof - Google Patents
Traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy and application thereof Download PDFInfo
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Abstract
The invention provides a traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy, which comprises the following components in parts by weight: 25-35 parts of hispid fig, 25-35 parts of astragalus membranaceus, 15-25 parts of codonopsis pilosula, 10-20 parts of bighead atractylodes rhizome, 3-10 parts of angelica sinensis, 5-15 parts of prepared rehmannia root, 5-15 parts of dried orange peel, 5-15 parts of rhizoma pinellinae praeparata, 5-15 parts of pogostemon cablin, 3-10 parts of ginger, 5-15 parts of Chinese date and 5-15 parts of honey-fried licorice root. The compatibility principle of the traditional Chinese medicine composition is as follows: monarch drug: radix fici simplicissimae, radix astragali; ministerial drugs: radix Codonopsis, Atractylodis rhizoma, radix Angelicae sinensis, and radix rehmanniae Preparata; adjuvant drugs: rhizoma Pinelliae Preparata, pericarpium Citri Tangerinae, herba Agastaches, and rhizoma Zingiberis recens; a messenger drug: jujube, radix Glycyrrhizae Preparata. The traditional Chinese medicine composition disclosed by the invention can invigorate spleen and enrich blood, effectively improve the myelosuppression and immunologic function decline state of a patient with myelosuppression after chemotherapy, simultaneously improve the oxidation resistance of the patient, and strengthen the protection function of internal organs, and has the advantages of definite and wide curative effect, no obvious side effect, simplicity, convenience and low price.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy and application thereof.
Background
Chemotherapy is the main means for treating malignant tumor at present, and has certain treatment effect, the tumor focus of the patient is effectively controlled after chemotherapy, the clinical symptoms are improved to a certain extent, and the survival time is prolonged. However, most current chemotherapeutic drugs lack targeting to tumor cells, and can damage normal cells of a human body while inhibiting or killing the tumor cells, so that serious side effects such as nausea, vomiting, bone marrow suppression, alopecia, diarrhea, fatigue and the like are often caused. More than about 90% of chemotherapeutic drugs may cause myelosuppression side effects, resulting in significant reduction of leukocytes in the patient's peripheral blood, requiring repeated leukogenic treatments, thereby seriously affecting the patient's quality of life.
Myelosuppression is one of the serious common side effects of tumor patients after chemotherapy, and is also a toxic reaction which is common in the blood system and can endanger life. The decline of whole blood cells mainly comprising white blood cells in peripheral blood is the main manifestation of bone marrow suppression, and the most common and earliest decline of white blood cells mainly comprises the decrease of white blood cells, and the second is thrombocytopenia, and the decline of red blood cell number and hemoglobin amount is relatively less, so that severe adverse reactions such as anemia, bleeding, decline of anti-infection capability and the like are caused, and the treatment and prognosis of tumor patients are influenced to a great extent. The current research suggests that the mechanism of bone marrow suppression induced by chemotherapeutic drugs is mainly direct damage to hematopoietic stem cells, as well as damage to the structure or function of bone marrow stromal cells. The chemotherapy drug can inhibit the proliferation of cancer cells at different stages and inhibit the division and proliferation capacity of DNA, thereby playing a role in treating tumors, but can kill a large number of tumor cells and also kill a plurality of normal bone marrow cells, thereby increasing the incidence rate of bone marrow inhibition and immunologic function reduction.
The traditional Chinese medicine researches believe that radiotherapy and chemotherapy are external toxic factors, and on the basis of the deficiency of vital qi of malignant tumors, the 'toxic factors' invade the body and further consume the vital qi of the human body, so that the deficiency of the five zang-organs and the six fu-organs and the deficiency of qi, blood, yin and yang are caused. The 'toxin' injures the spleen and stomach internally, which causes the dysfunction of the spleen in transportation, the dysfunction of the stomach in descending and the dysfunction of food essence in distribution, thus causing the source of qi and blood deficiency; if the liver and kidney are damaged, the liver cannot store blood, and the kidney essence is insufficient, which results in insufficiency of essence and blood, and the "toxin" is in conflict with qi and blood in the body, which results in abnormal circulation of qi and blood, aggravating the deficiency of yin, yang, qi and blood of the viscera, and causing this disease. Because the pathogenesis of the disease is relatively complex, the understanding of the pathogenesis of the disease is inconsistent, and the deficiency syndrome is considered to be the root of the disease. For the disease symptoms, the deficiency of spleen and kidney is considered as the main pathogenesis. In clinical treatment, patients with malignant tumor often suffer from diseases caused by internal deficiency of vital qi, internal invasion of pathogenic qi and failure of vital qi to surpass pathogenic factors, and often cause further deficiency syndrome due to radiotherapy, chemotherapy and other treatment means, thereby generating malignant circulation. Most patients with malignant tumors have the symptoms of deficiency of qi and blood or deficiency of spleen and kidney, and compared with normal people, the immune function of the body of the patients is obviously reduced.
Clinically, the selection of effective drugs to increase the number of leucocytes is an important link for ensuring the successful implementation of chemotherapy. The currently effective method is to apply colony stimulating factors, but because the price of the colony stimulating factors is high, the medicines easily cause a patient to have a plurality of side reactions such as bone pain, fever, muscle pain, hypodynamia, allergy and the like, the duration of the curative effect is short, the expected benefit is poor, and the medical cost is increased. Therefore, how to effectively prevent and treat myelosuppression and immunologic function reduction is one of the important means for improving the curative effect of chemotherapy and is also an important prerequisite for matching the treatment of a clinician for a tumor patient.
Disclosure of Invention
Based on the above, the invention aims to provide the traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy, which can effectively treat bone marrow suppression after chemotherapy, enhance the immune function of an organism and improve the oxidation resistance.
In order to achieve the purpose, the invention adopts the following technical scheme.
A traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy comprises the following components in parts by weight: 25-35 parts of hispid fig, 25-35 parts of astragalus membranaceus, 15-25 parts of codonopsis pilosula, 10-20 parts of bighead atractylodes rhizome, 3-10 parts of angelica sinensis, 5-15 parts of prepared rehmannia root, 5-15 parts of dried orange peel, 5-15 parts of rhizoma pinellinae praeparata, 5-15 parts of pogostemon cablin, 3-10 parts of ginger, 5-15 parts of Chinese date and 5-15 parts of honey-fried licorice root.
In some preferred embodiments, the traditional Chinese medicine composition comprises the following components in parts by weight: 30-35 parts of hispid fig, 30-35 parts of astragalus membranaceus, 20-25 parts of codonopsis pilosula, 15-20 parts of bighead atractylodes rhizome, 6-8 parts of angelica sinensis, 10-15 parts of prepared rehmannia root, 10-15 parts of dried orange peel, 10-15 parts of rhizoma pinellinae praeparata, 10-15 parts of pogostemon cablin, 6-8 parts of ginger, 10-15 parts of Chinese date and 10-15 parts of honey-fried licorice root.
More preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: 30 parts of radix fici simplicissimae, 30 parts of astragalus membranaceus, 20 parts of codonopsis pilosula, 15 parts of bighead atractylodes rhizome, 6 parts of angelica sinensis, 10 parts of prepared rehmannia root, 10 parts of dried orange peel, 10 parts of rhizoma pinellinae praeparata, 10 parts of pogostemon cablin, 6 parts of ginger, 10 parts of Chinese date and 10 parts of honey-fried licorice root.
The invention also provides a medicine for treating bone marrow suppression and/or immune function suppression after chemotherapy, which comprises the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials.
In some embodiments, the pharmaceutical dosage form is a tablet, solution, granule, pill, capsule.
The invention also provides application of the traditional Chinese medicine composition in preparing a medicine for treating bone marrow suppression after chemotherapy.
In some embodiments, the medicament has any one of the following effects: (1) increasing the leukocyte content in peripheral blood after chemotherapy; (2) reducing the content of red blood cells in peripheral blood after chemotherapy; (3) reduce the content of platelets in peripheral blood after chemotherapy.
In some embodiments, the medicament has any one of the following effects: (1) increasing the level of hematopoietic factors in plasma after chemotherapy; preferably, the hematopoietic factor is selected from at least one of GM-CSF, TPO, EPO, IL-2; (2) increasing the number of hematopoietic stem cells after chemotherapy, and improving the hematopoietic function of bone marrow; (3) reducing the differentiation activity of hematopoietic stem cells after chemotherapy.
The invention also provides application of the traditional Chinese medicine composition in preparing a medicine for enhancing the body immune function after chemotherapy.
The invention also provides application of the traditional Chinese medicine composition in preparing a medicine for improving the oxidation resistance of an organism after chemotherapy.
The invention provides a traditional Chinese medicine composition for treating bone marrow suppression and/or immunologic function suppression after chemotherapy, which comprises the following components according to the compatibility principle: monarch drug: radix fici simplicissimae, radix astragali; ministerial drugs: radix Codonopsis, Atractylodis rhizoma, radix Angelicae sinensis, and radix rehmanniae Preparata; adjuvant drugs: rhizoma Pinelliae Preparata, pericarpium Citri Tangerinae, herba Agastaches, and rhizoma Zingiberis recens; a messenger drug: jujube, radix glycyrrhizae preparata. Radix codonopsitis and rhizoma atractylodis macrocephalae are used as ministerial drugs, and the prepared radix glycyrrhizae preparata is used as a four-monarch-drug decoction, so that the effect of strengthening the spleen and replenishing qi of monarch drugs is enhanced; the prepared rehmannia root and the angelica form a four-ingredient soup base, and the emphasis is on enriching and nourishing blood; the astragalus and the angelica are matched according to a specific proportion to form the angelica blood-enriching soup, which has the efficacy of tonifying qi and generating blood; rhizoma Pinelliae Preparata and pericarpium Citri Tangerinae are adjuvant drugs, and used together with radix Codonopsis, Atractylodis rhizoma and radix Glycyrrhizae Preparata to make Chenxia Liujunzi decoction, which has effects of invigorating spleen and invigorating qi, eliminating dampness and regulating stomach; the dried orange peel has the effects of promoting the circulation of qi and eliminating dampness, the wrinkled gianthyssop herb has the effects of eliminating dampness by virtue of aroma, regulating the flow of qi and regulating the middle warmer, and the ginger is matched with the vomiting family holy medicine, so that the effects of harmonizing the stomach and stopping vomiting are enhanced; rhizoma pinelliae preparata: eliminating dampness and phlegm, lowering adverse qi and relieving vomiting, relieving distension and fullness and resolving masses; dried orange peel: regulating qi-flowing, invigorating spleen, eliminating dampness, and eliminating phlegm; the two are used in combination to prevent the deficiency of nourishing, greasy and qi-stagnation of tonics; ginger: relieving exterior syndrome, dispelling cold, warming middle energizer, relieving vomiting, warming lung, and relieving cough; ginger is a heaver herb for vomiting and pinellia tuber, when combined, it can be used as a powder to both help check adverse rise of qi and arrest vomiting; the Chinese dates and the honey-fried licorice roots are used as guiding drugs, the honey-fried licorice roots are used for strengthening the spleen, the Chinese dates are used for enriching the blood, and the spleen and the blood are strengthened to harmonize the drug property. Through the compatibility of medicines, the traditional Chinese medicine composition disclosed by the invention can invigorate spleen and enrich blood, effectively improve the myelosuppression and immunologic function reduction states of myelosuppressed patients after chemotherapy, simultaneously improve the oxidation resistance of the patients, and strengthen the protection function of internal organs, and has the advantages of definite and wide curative effect, no obvious side effect, simplicity, convenience and low cost.
Drawings
FIG. 1 is a fingerprint of the decoction of the Chinese herbs of example 2.
FIG. 2 shows the change in thymus (0.73X) in each group of mice.
FIG. 3 shows the change in spleen (1.0X) in each group of mice.
FIG. 4 shows HE staining of femur (10X 40), spleen (10X 40), and liver (10X 40) of each group of mice.
FIG. 5 is a comparison of Nqo1 and Ho1 (10X 40) immunohistochemistry for the spleens of various groups of mice.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or apparatus that comprises a list of steps is not limited to only those steps or modules recited, but may alternatively include other steps not recited, or may alternatively include other steps inherent to such process, method, article, or apparatus.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The following description is given with reference to specific examples.
Example 1
The embodiment provides a traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy, which comprises the following components in parts by weight: 30 parts of radix fici simplicissimae, 30 parts of astragalus membranaceus, 20 parts of codonopsis pilosula, 15 parts of bighead atractylodes rhizome, 6 parts of angelica sinensis, 10 parts of prepared rehmannia root, 10 parts of dried orange peel, 10 parts of rhizoma pinellinae praeparata, 10 parts of pogostemon cablin, 6 parts of ginger, 10 parts of Chinese date and 10 parts of honey-fried licorice root.
Example 2
This example investigated the efficacy of the Chinese medicinal composition described in example 1 for post-chemotherapy treatment.
First, experimental animal
Healthy female C57BL/6 mice, purchased from the laboratory animal center of Guangzhou university of traditional Chinese medicine, 6-8 weeks old, and having a weight of 18-22 g. Animals are raised in 12h of light/12 h of dark, the temperature is controlled to be 20-26 ℃, the temperature difference is not lower than 3 ℃, the humidity is 40-70% of Specific Pathogen Free (SPF) grade environment, the animals are freely drunk and ingested, and the animal experiment is started after 1 week of adaptive raising.
Second, prescription composition and preparation
Taking the traditional Chinese medicine composition in the embodiment 1: 30 parts of radix fici simplicissimae, 30 parts of astragalus, 20 parts of codonopsis pilosula, 15 parts of bighead atractylodes rhizome, 6 parts of angelica, 10 parts of prepared rehmannia root, 10 parts of dried orange peel, 10 parts of rhizoma pinellinae praeparata and patchouli (rear lower) 10 parts of ginger, 6 parts of Chinese date and 10 parts of honey-fried licorice root, and decocting to obtain a traditional Chinese medicine decoction, wherein the preparation process of the traditional Chinese medicine decoction is as follows: soaking the above Chinese medicinal materials in 8 times of warm water for 0.5 hr, boiling, decocting with slow fire for 0.5 hr, and keeping the decoction uniformStirring, collecting juice, and filtering with three layers of gauze; the second decoction is carried out by decocting 6 times of water for 1 hour with slow fire, taking juice by the same method, combining the liquid medicines, putting the liquid medicines into a rotary evaporator at 60 ℃, concentrating to 2.5g/ml which is a high-dose traditional Chinese medicine, diluting to 2 times which is 1.25g/ml which is a medium-dose traditional Chinese medicine, and diluting to 4 times which is 0.62g/ml which is a low-dose traditional Chinese medicine. Cooling the liquid medicine at normal temperature, and storing in a refrigerator at 4 deg.C for use.
The dosage of the medicine is as follows: referring to the equivalent dose conversion between animals and human bodies in pharmacological tests (Chinese clinical pharmacology and therapeutics, 9 months 2004) compiled by the King of Huang-Ji Han, the dosage of a specific experimental animal is calculated according to the standard that the dosage of a mouse (20g) is equal to the total amount of traditional Chinese medicines (167 g)/the amount of human body (60 kg). times.9 multiplied by 20g is equal to 0.501g/d, and the dosage of 0.501/0.4ml is equal to 1.25g/ml/d, which is the medium dosage; 1.25g/ml × 2 ═ 2.5g/ml/d is the high dose; the dosage is low when 1.25g/ml/2 is 0.62 g/ml/d.
Thirdly, establishing an animal model
According to the dose-time relation of the cyclophosphamide induced bone marrow suppression, a mouse bone marrow suppression model is established by adopting a cyclophosphamide intraperitoneal injection method. The injection is administered by intraperitoneal injection 1 time per day at a dose of 25mg/kg/d for 10 days.
Fourth, animal grouping and treatment
72C 57BL/6 female mice were bred for 1 week, and then divided into 6 groups at random, each group containing 12 mice, including a normal Control group (Control), a Model group (Model), a Positive drug group (Positive), a Low-dose Chinese medicine group (Low dose), a Medium-dose Chinese medicine group (Medium dose) and a High-dose Chinese medicine group (High dose). Normal control group mice are normally raised without any intervention except for the intragastric administration of normal saline; carrying out intraperitoneal injection on model mice for 10 days and 1 time per day according to the molding method; the low, medium and high dose Chinese medicine group mice are modeled according to the method, the low, medium and high dose Chinese medicine group mice are respectively treated with the gavage Chinese medicine decoction according to three doses of 0.62g/ml/d, 1.25g/ml/d and 2.5g/ml/d, 1 time per day and the treatment lasts for 12 days until the experiment is finished; the mice in the positive drug group were injected with colony stimulating factor (GM-CSF) subcutaneously every day starting on day 1 of intraperitoneal cyclophosphamide injection, and GM-CSF was injected subcutaneously at a dose of 5. mu.g/kg 1 time a day in a conversion of a clinically usual dose to an animal dose for up to 12 days to the end of the experiment.
Fifth, sample collection and index observation
1. The analysis of the components of the traditional Chinese medicine formula: to clarify the main compound components in the Chinese medicinal preparation in this study, 2.5g/ml of the high-dose group of Chinese medicinal materials was diluted to 5mg/ml with 50% methanol, 400ul of the diluted solution was taken at 13500rpm for 25min, and 80ul of the supernatant was taken for High Performance Liquid Chromatography (HPLC) analysis.
2. Dynamic observations of gross condition of mice: during the experiment, the weight change, hair condition, drinking and eating change condition, liveness and response condition to external stimulation of each group of mice are observed. Before the experiment, the weight of each group of mice was measured, the weight, the amount of water and the amount of food were measured once a day, and the weight, the drinking water and the eating changes of each group of mice were dynamically observed.
3. Measurement of thymus, spleen and liver of mice: weighing the weight of the mice before taking materials by using a balance, dislocating the cervical vertebra after blood collection to kill the mice, fixing the mice in a supine position on a foam box cover by using pins and coarse cotton threads, cutting off the skin of the chest and abdomen on ice, taking the thymus, spleen and liver in the mice, sucking the thymus, spleen and liver by using filter paper, weighing the weight of the thymus, spleen and liver and organ indexes of the mice in each group, specifically comparing the weight of the thymus, spleen and liver of the mice in each group and observing and comparing the morphological change of the thymus and spleen of the mice in each group by using a stereoscope.
4. Collecting and preserving peripheral blood cells of mice: after the administration of each group of mice, the weight of the mice is weighed, peripheral blood of the mice is extracted by an eyeball-picking method, the mice are placed in a heparin EP tube and are frozen and stored at 4 ℃ to be tested, and the number of the peripheral blood cells of each group is calculated by a blood analyzer.
5. Collecting and storing mouse plasma and detecting GM-CSF, TPO and EPO contents
After the administration of each group of mice is completed, peripheral blood of the mice is extracted by an eyeball-picking method, the mice are placed in a heparin EP tube and centrifuged for 20min at 3000rpm, and supernatant is taken to be blood plasma, and the blood plasma is frozen and stored at minus 80 ℃ to be detected.
(1) Taking out the serum sample from a refrigerator at the temperature of-80 ℃, naturally thawing the serum sample on ice, and fully and uniformly mixing the serum sample and the sample diluent according to the volume ratio of 1: 1. (2) The panel was removed from the sealed bag returned to room temperature, leaving a blank well. (3) Respectively adding the specimen and the standard substance with different concentrations into the corresponding design holes (100 ul/hole), sealing the reaction holes with a sealing plate film, and gently shaking and placing in an incubator at 37 ℃ for incubation for 90 min. (4) Biotinylated antibody working solution was prepared 30min in advance. (5) The plate was washed 5 times. (6) Except for blank wells, the remaining wells were filled with antibody working solution (100 ul/well). The reaction well to which the reagent was added was sealed with a sealing plate film, and incubated in an incubator at 37 ℃ for 60 min. (7) The enzyme conjugate working solution was prepared 30min in advance and left at room temperature in the dark. (8) The plate was washed 5 times. (9) The remaining reaction wells, except the blank wells, were loaded with enzyme conjugate (100 ul/well). Sealing the reaction hole with sealing film, placing in incubator at 37 deg.C, and incubating for 30min in dark. (10) The plate was washed 5 times. (11) 100ul of chromogenic substrate per well is added into all the wells, and the mixture is placed in an incubator at 37 ℃ and protected from light for 15 min. (12) 100ul of stop solution is added into all the working holes, and after the working holes are uniformly mixed and lightly shaken, the OD450 value (within 10 min) of each hole is immediately detected. (13) And (4) calculating a result: subtracting the OD of the blank well from the OD of each standard and specimen; making a standard curve: taking the concentration of the standard substance as an abscissa and the OD value as an ordinate to prepare a standard curve equation; according to the standard curve equation, the OD value of each sample (the OD value of the specimen should be subtracted by the OD value of the blank well) is input, the concentration is calculated, and then the concentration is multiplied by the corresponding dilution factor.
6. Mouse bone marrow cell Collection and surface markers Sca1, C-kit, Lamin, CD48 + CD150 + Flow detection of
After the mice are killed by dislocation of cervical vertebrae, the mice are soaked in alcohol for several minutes, the skins of the two lower limbs of the mice are cut off in a sterile super clean bench, the muscles of the lower limbs are separated, and the thighbones at the two sides are taken out and soaked in 1640 serum-free cell culture solution. The femoral head and the femoral shaft are separated by sterile scissors, and a 5ml syringe sucks 1640 serum-free cell culture solution to flush out bone marrow cells in the femur until the color of the femur is whitened. The cell culture fluid was aspirated by a 1ml pipette, passed through a 75mm cell sieve and aliquoted in sterile EP tubes at 4 ℃ and 1800rpm for 5 min. Bone marrow cells were fixed in 70% ethanol at 4 ℃ overnight in a refrigerator at 4 ℃ at 3000rpm for 3 min. The supernatant was discarded and 1ml of PBS was resuspended at 3000rpm for 3 min. Discarding the supernatant, resuspending 1% BSA100ul, blocking in a refrigerator at 4 ℃ for 1h, diluting Sca1, C-kit, and Lami with PBSn、CD48 + CD150 + Antibody (1: 100), mixed well and placed in a refrigerator at 4 ℃ overnight. 3000rpm, 3min, recover primary antibody, 200ul PBS heavy suspension, 3000rpm, 3 min. Discard the supernatant, resuspend the cells in 100ul PBS, incubate 0.5ul Rabbit-PE in the dark at room temperature for 1 h. 3000rpm, 3min, abandoning the supernatant, resuspending 100ul PBS, 3000rpm, 3min, abandoning the supernatant, resuspending 200ul PBS, filtering through a 70mm filter screen, and detecting by a flow machine.
7. Collection of mouse Peripheral Blood Mononuclear Cells (PBMCs) and detection of CD3, CD4 and CD8a
After blood is taken from a heparin EP tube, supernatant plasma is sucked at 2000rpm for 20min, then 100ul of balanced salt is used for re-suspending red blood cells, the red blood cells are diluted to 4ml, the red blood cells are gently added to 3ml of lymphocyte separation liquid at 20 ℃, 400g and 30min or 2000rpm for 30min, the plasma supernatant is discarded, and a second white membrane layer is sucked to the EP tube. Then, the mixture is diluted by balanced salt in an equal time at 3000rpm for 5min, supernatant is discarded, cells at the bottom are left, part of the cells are fixed by 100ul of 75% ethanol, and the cells are stored at 4 ℃ for preparing flow detection. Refrigerator at 4 ℃ overnight, 3000rpm, 3 min. The supernatant was discarded, and 1ml of PBS was used to resuspend the cells at 3000rpm for 3 min. The supernatant was discarded, resuspended in 1% BSA100ul, blocked at 4 ℃ for 1h, and the CD3, CD4 and CD8a antibodies (1: 100) diluted in PBS were mixed and mixed overnight at 4 ℃. 3000rpm, 3min, recover primary antibody, 200ul PBS heavy suspension, 3000rpm, 3 min. Discard the supernatant, resuspend 100ul PBS, incubate 0.5ul Rabbit-PE/Rat-Cy5.5 at room temperature in the dark for 1 h. 3000rpm, 3min, abandoning the supernatant, resuspending 100ul PBS, 3000rpm, 3min, abandoning the supernatant, resuspending 200ul PBS, filtering through a 70mm filter screen, and detecting by a flow machine.
8. Collection of mouse thymus and detection of CD3, CD8a, Nrf2, Nqo1 and Ho1 proteins
And (3) detecting by adopting a western blot method. Approximately 100mg of tissue was harvested, minced, and placed in 400. mu.l cold nuclear lysis buffer A (10mM HEPES-KOH pH7.9, 2mM MgCl) 2 0.1mM EDTA, 10mM KCl, 1mM DTT, 1. mu.g/ml Leuteptin, 1mM PMSF) with a 1ml glass homogenizer, vortexed at 0 ℃ for 10min, centrifuged at 4 ℃ for 15,000 g for 30s, and the supernatant was cytoplasmic protein. The pellet was resuspended in 50. mu.l cold nuclear lysis buffer B (50mM HEPES-KOH pH7.9, 10% glycerol, 300mM NaCl, 1.5mM KCl, 0.1mM EDTA, 1mM DTT, 1. mu.g/ml Leuteptin, 1mM PMSF) and vortexed at 0 ℃ for 30minCentrifugation at 15,000 Xg for 10min at 4 ℃ was carried out, the supernatant was collected, the protein concentration was determined using BCA protein quantification kit, and the supernatant was stored at-80 ℃ for further use.
(1) Preparing 12% of separation glue and 5% of concentrated glue according to the formula; protein samples were mixed with 5x protein loading buffer at 4: 1 in a volume ratio; (2) after the gel is well solidified, placing the gel in an electrophoresis tank, filling electrophoresis buffer solution in the inner tank, and adding 60 mu g of protein into each hole according to the quantitative result of the protein; (3) and (3) connecting an electrode, adjusting the voltage to 80V to start electrophoresis, adjusting the voltage to 120V when the bromophenol blue passes through the interface of the concentrated gel and the separation gel, keeping constant voltage until the bromophenol blue runs to the bottom, and stopping electrophoresis. (4) And immersing the PVDF membrane into a methanol solution for 1-2min for activation. Cutting off the separation gel, placing the gel into a membrane transferring buffer solution for balancing for 30min, preparing a membrane transferring sandwich in the order of an anode carbon plate, filter paper, a PVDF membrane, gel, filter paper and a cathode carbon plate from bottom to top, and removing bubbles by using a glass rod; (5) placing the film transferring groove in an ice bath, and transferring the film for 100min at a constant current of 300 mA; (6) and (3) sealing: after the membrane transfer is finished, carefully taking out the PVDF membrane, washing the membrane with TBST for 5min multiplied by 3 times, adding 5% BSA/skimmed milk blocking solution, and slowly shaking for 1.5h on a decoloring shaking table at room temperature; (7) incubating the primary antibody: after blocking was completed, the membrane was washed with TBST 5min × 3 times, slightly blotted with filter paper to remove excess TBST, and then placed in a position as 1: in 1000 diluted primary antibody solution, shaking table incubation overnight in a refrigerator at 4 ℃; (8) incubation of secondary antibody: the next day, the PVDF membrane was removed, washed with TBST 10min × 3 times, and then the membrane was slightly blotted with filter paper to remove excess TBST, and then placed in a position of 1: diluting a secondary antibody with 2000%, preparing the secondary antibody by using 5% BSA or skim milk, and incubating for 1.5h at room temperature; (9) taking out PVDF, washing the membrane by 1 xTBST for 5min x 3 times, coating the membrane with ECL luminous liquid (luminous liquid A: liquid B are prepared according to the proportion of 1: 1) prepared in advance in a dark room, ensuring that the whole PVDF membrane is covered by the luminous liquid, absorbing the redundant luminous liquid, covering a preservative film, and starting tabletting, wherein the time for tabletting each time is determined according to the actual situation; (10) taking out the film in a dark room and putting the film in a developing solution, taking out the film after observing an image, immediately putting the film in a fixing solution after rinsing with clear water, then washing the film clean and airing the film. (11) And (3) analysis results: scanning the film into electronic pictures by a scanner and storing the electronic pictures; the integrated optical density of each protein band was analyzed using the Quantity one image analysis system from Bio-rad.
9. Collection of mouse spleen tissue and detection of Nrf2, Nqo1 and Ho1 proteins and genes
Collecting tissue specimens: cutting off spleen tissues, washing with PBS, dividing into three parts, fixing one part in 4% paraformaldehyde solution, and standing at room temperature for histological detection; immersing a part of the extract in RNAlater preservation solution, and preserving the extract at the temperature of minus 80 ℃ for freezing and reserving the extract for RT-PCR detection; one part of the sample was directly put into a 1.5ml EP tube and stored frozen at-80 ℃ for Western blot detection.
Histological examination of mouse femur, spleen and liver tissues
After the mouse lung tissue fixing solution is cleaned, the slices with the size of 2-3 mu m are made after dehydration, transparency and wax immersion and embedding. The cut slices were placed in heated water to be ironed, stuck on an anti-peeling glass slide, and baked in an oven at 45 ℃.
(1) HE staining of femur, spleen and liver tissues
1) Dewaxing: xylene I5 min → xylene II 5min → 100% alcohol I1 min → 100% alcohol II 30s → 95% alcohol 30s → 90% alcohol 30s → tap water for 10-30 s.
2) Dyeing: hematoxylin staining solution 10-15min → water washing 20s → 1% hydrochloric acid alcohol differentiation section (three times up and down, color changing from blue to red) → tap water washing 15min → 1% eosin alcohol staining 3min → water washing 1 min.
3) Dehydrating, transparent and sealing: 95% alcohol I30 s → 95% alcohol II 30s → 100% alcohol I30 s → 100% alcohol II 30s → xylene I30 s → xylene II 30s → xylene I clear 30s → xylene II clear 30s → neutral gum is used for sealing at room temperature after taking out.
4) And (3) dyeing results: and (3) nucleus: bluish purple, cytosol: different degrees of pink color.
(2) Immunohistochemical method for detecting Nqo1 and Ho1 expression in spleen tissues of mice
1) Dewaxing: dewaxing the tissue slices by a conventional method, and washing the tissue slices for 3 times for 3min by PBS (phosphate buffer solution); 2) antigen retrieval: performing high-pressure repair by using EDTA antigen repair solution (pH 8.0), cooling to room temperature, and washing with PBS for 3 times for 3min each time; 3) blocking endogenous peroxidase: adding 1% TritonX-100 solution to promote permeation, washing, and treating with 0.3% hydrogen peroxide solution for 10min to eliminate the effect of endogenous catalase; 4) and (3) sealing: washing with PBS, adding 10% bovine serum albumin solution, and sealing for 20 min; 5) incubating the primary antibody: after PBS washing, sucking water nearby the tissues by using filter paper, drawing a circle nearby the tissues by using an immunohistochemical pen, putting a slide on a shelf in an incubation cassette, respectively dropwise adding anti-mouse Nqo1 and Ho1 antibodies diluted by 1:200 into the circle, and incubating for 1h at 37 ℃; 6) incubation of HRP-labeled complex: washing with PBS for 3 times, each for 5min, dripping solution A in DAKO immunohistochemical detection kit into the circle, and incubating at 37 deg.C for 30 min; 7) color development, lining dyeing and piece sealing: temporarily preparing DAB color developing solution. And (3) after the section is washed, spin-drying the section, dripping DAB (DAB) color solution, observing under a microscope from time to time, immediately washing when the cells are obviously colored and the bottom surface is relatively light in color, stopping the dyeing reaction, after hematoxylin counterstaining, dehydrating by gradient alcohol, sealing the section, and observing and shooting under a common microscope.
Detection of mouse spleen tissue Nrf2, Nqo1 and Ho1 mRNA level
(1) Extraction of total RNA from tissue by Trizol
The method comprises the following specific operation steps:
1) 100mg of mouse lung tissue was placed in a 2ml EP tube, 1ml Trizol was added, a steel ball was added, and homogenization was performed in a grinder. Splitting on ice for 1 h. 2) Centrifuging at 4 deg.C for 10min at 12,000 Xg, and removing precipitate; 3) transferring the supernatant into another tube, adding 200 mu l of chloroform into each tube, violently shaking and uniformly mixing for 15s, and standing for 2-3 min at room temperature; 4) centrifuging at 4 deg.C for 15min at 12,000 Xg, sucking upper water phase 200ul, and transferring to another centrifuge tube; 5) adding 0.5ml of isopropanol into each tube, mixing uniformly, and standing at room temperature for 10 min; 6) centrifuging at 4 deg.C for 10min at 12,000 Xg, discarding supernatant with 200ul pipette gun, and precipitating RNA at the bottom of the tube; 7) adding 900ul of 75% ethanol into each tube, gently shaking the centrifuge tube, suspending and precipitating, and scattering lumps; 8) centrifuging at 4 deg.C for 5min at 8,000 Xg, discarding supernatant as carefully as possible, and air drying at room temperature for 10 min; 9) dissolving RNA sample in 50 μ l DEPC-treated water per tube, heating at 55 deg.C for 10min, and taking out on ice for 3 min; 10) the OD at A260 and A280 was measured to determine the purity and quantity of RNA.
(2) Reverse transcription of RNA into cDNA
The method comprises the following specific operation steps:
1) thawing the RNA sample on ice; 2) DEPC water 18ul diluted RNA sample 2 ul; 3) re-loading the RNA to quantify the RNA concentration before detection; 4) the operation is performed according to the reverse transcription kit instruction: 5Xprime Script Buffer 2ul, Prime Script RI Enzyme Mix 0.5ul, Oligo Dt Prime (50mM)0.5ul, Random 6mers 0.5ul, adding each hole according to the proportion and mixing evenly; 5) 3.5ul of the above mixture was added per EP tube, and the volume of DEPC water plus RNA sample was 6.5ul, based on RNA concentration and loading volume, for a total of 10 ul. 6) Heating at 37 deg.C for 15min, terminating the reaction, and placing on ice; 7) heating at 85 deg.C for 5s to inactivate enzyme;
8) the reaction was diluted to 50. mu.l with DEPC water, and 2 to 5. mu.l was taken for PCR amplification reaction.
(3) Fluorescent real-time quantitative PCR
1) Primer design and Synthesis
Mouse Nrf2, Nqo1 and Ho1 primer sequences were designed autonomously according to the authoritative literature (synthesized by the great gene science co.
TABLE 13 primer sequence information
2) Real-time PCR amplification
TABLE 14 amplification System
And (3) PCR reaction conditions: 95 ℃ for 30 sec; 5sec at 95 ℃; 34sec at 60 ℃; 30sec at 72 ℃ for 120 min.
(3) Analysis of PCR results
In the amplification power curve, the Ct value represents the number of amplification cycles that pass when the fluorescence signal of the amplification product reaches a set threshold value; 2 -ΔΔCt The expression of the target gene in the experimental group was expressed as a fold change from the control group, and Δ Δ Ct ═ Ct (Ct) Object(s) to –Ct Internal reference ) Treatment group –(Ct Purpose(s) to –Ct Internal reference ) Control group 。
Sixthly, statistical processing method
Data from the experiment are mean. + -. standard deviation
The SPSS 19.0 statistical software is adopted, a one-factor variance analysis method is used for comparison among multiple groups, if the variances are equal, the two-two comparison adopts LSD (least squares) test, the difference is P less than 0.05, and the difference is significant. If the variance is not uniform, the two-by-two comparison is performed by Dunnett T3 test, and the difference is shown as P < 0.05 and has significant statistical significance when the P < 0.01.
Seventh, experimental results
1. Analysis of Chinese medicinal prescription composition
As can be seen from figure 1 and table 1, the results of the detection of the main components of the Chinese herbal compound suggest that the compounds detected in the compound are mainly the compounds in astragalus, dried orange peel, ginger and liquorice.
TABLE 1 major assay Components
2. Effect of Chinese medicinal formulation on general conditions of mice
TABLE 2 weight changes in the groups of mice
Note: compared with the normal control group, * P＜0.05， ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## P＜0.01。
as can be seen from Table 2, before modeling, all groups of mice are healthy, have normal food intake and water drinking, lively and active hair, glossy hair and no symptoms such as weight loss and hair loss, and the weights of all groups of mice have no obvious difference (P is more than 0.05) through statistical analysis. After the molding was started, the general condition of each group of mice was continuously observed, and the weight and food intake of the mice were regularly measured. The results show that: the normal control group, the positive drug group and the traditional Chinese medicine group of the mice always present a healthy state, the weight is stable, and no obvious abnormal fluctuation exists. Mice molded by the chemotherapeutics show obvious anorexia at the initial molding stage, the food intake and water intake are obviously reduced compared with other groups, the hair gradually loses luster, even falls off, the spirit is listened, the activity is obviously reduced, and therefore, the weight is also obviously reduced compared with other groups. From the weight change curves of the mice in each group, the weight of the molded mice is progressively reduced in the first 9 days of the start of chemotherapy, the weight change curves of the mice in the normal control group, the positive drug group and the traditional Chinese medicine dosage groups are relatively flat, which indicates that the chemotherapy drugs have obvious inhibition effect on the weight and diet of the mice, and the weight change curves of the mice in the traditional Chinese medicine dosage groups and the positive drug group are relatively flat, which prompts that the weight and diet of the mice subjected to the intervention of the treatment drugs are not obviously reduced. The Chinese medicinal composition can improve the life quality of tumor patients and has the attenuation function.
3. Effect of Chinese medicinal formulation on thymus, spleen and liver of mice
TABLE 3 Net weight of thymus, spleen and liver of each group of mice
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## p is less than 0.01; compared with the positive drug group, the drug group, && P＜0.01; compared with the high-dose traditional Chinese medicine group, the ^ P is less than 0.05 and the ^ P is less than 0.01.
TABLE 4 organ index comparison of thymus, spleen and liver of each group of mice
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05, && p is less than 0.01; compared with the high-dose traditional Chinese medicine group, the ^ P is less than 0.05.
As can be seen from FIGS. 2 and 3 and tables 3 and 4, the net weight and organ index of thymus and spleen in the model group are both significantly reduced (P < 0.01) and the net weight and organ index of liver are significantly increased (P < 0.01) compared with the normal control group. Compared with the model group, the net weight of thymus of the positive drug group is increased (P is less than 0.05), the net weight of thymus of the traditional Chinese medicine group with high, middle and low dosages is decreased (P is less than 0.05), the organ index of thymus of the positive drug group is increased (P is less than 0.01), and the organ index of thymus of each traditional Chinese medicine dosage group is decreased (P is less than 0.01). The net weight of spleen in the positive medicine group and each dosage of the traditional Chinese medicines is increased (P is less than 0.01), and the organ index of spleen in the traditional Chinese medicines in the middle dosage is increased (P is less than 0.01). The net weight of the liver and the organ index of the positive medicine group and the traditional Chinese medicine of each dosage are reduced (P is less than 0.01). The results show that the traditional Chinese medicine composition has obvious protection effect on the viscera after chemotherapy.
4. Effect of Chinese medicinal formulation on mouse peripheral blood cell count
TABLE 5 changes in the number of leukocytes, platelets and erythrocytes in the peripheral blood of mice in each group
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## p is less than 0.01; compared with the positive drug group, the drug group, & P＜0.05， && p is less than 0.01; compared with the traditional Chinese medicine group with the medium dose, the ^ P is less than 0.01; compared with the low-dose traditional Chinese medicine group, ％％ P＜0.01。
as can be seen from Table 5, the number of leukocytes in the model group was significantly decreased (P < 0.01) and the number of platelets and erythrocytes was significantly increased (P < 0.01) compared to the normal control group, which may be related to the stress response of the chemotherapeutic agent to the body. Compared with the model group, the positive medicine group and each traditional Chinese medicine dosage group can obviously improve the number of leucocytes (P is less than 0.01), and the positive medicine and each traditional Chinese medicine dosage group can improve the number of leucocytes in peripheral blood cells of mice and reduce the occurrence of leucocyte reduction caused by chemotherapy medicines. The positive medicine group and the traditional Chinese medicine with the middle dose can obviously increase the number of the platelet (P is less than 0.01), and the traditional Chinese medicine with the low dose can obviously decrease (P is less than 0.05). The red blood cell number of the traditional Chinese medicine in high and medium dosage is obviously reduced (P is less than 0.01). The positive drug group and the low dose traditional Chinese drug group have no obvious change in the number of red blood cells (P is more than 0.05). The positive medicine and the traditional Chinese medicines in each dosage can up-regulate the white blood cell count, and have obvious effect in high and medium dosages. The Chinese medicinal composition has certain regulation effect on white blood cells, platelets and red blood cells.
5. Influence of Chinese medicinal preparation on concentration of mouse plasma factors GM-CSF, TPO, EPO and IL-2
TABLE 6 plasma factor GM-CSF, TPO, EPO and IL-2 concentrations in peripheral blood of groups of mice
Note: and isCompared with the common control group, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05， && p is less than 0.01; ^ P is less than 0.05 and ^ P is less than 0.01 compared with a high-dose traditional Chinese medicine group; compared with the traditional Chinese medicine group with the medium dosage, ％％ P＜0.01。
the concentrations of hematopoietic factors GM-CSF, TPO and EPO in the plasma of mice (Table 6) are obviously reduced (P is less than 0.01) compared with the concentration of mice in a normal control group in a model group, and the concentrations of the hematopoietic factors (P is less than 0.01) can be obviously improved to different degrees in a positive medicament group and each traditional Chinese medicine dosage group, so that the positive medicament and the low-dosage traditional Chinese medicine can improve the concentration of GM-CSF in the plasma of the mice and reduce the occurrence of leukocyte decrease after chemotherapy. Meanwhile, the positive medicine group and the high and medium dosage traditional Chinese medicines can obviously improve the TPO concentration (P is less than 0.01) in the blood plasma of mice. Compared with the model group, the positive medicine group and the traditional Chinese medicines with various dosages can obviously improve the concentration of EPO (P is less than 0.01, P is less than 0.05) in the plasma of mice after the mice are cured. The Chinese medicinal composition has certain regulation effect on the concentrations of hematopoietic factors GM-CSF, TPO and EPO in the plasma of mice. In addition, the IL-2 concentration in the plasma of the mice also changes, compared with the normal control group, the IL-2 concentration in the model group is obviously reduced (P is less than 0.01), and the IL-2 concentration in the plasma of the positive drug group and the high, medium and low dose traditional Chinese medicine groups is obviously increased (P is less than 0.01). The results show that the high and low dose traditional Chinese medicines can regulate the expression of plasma factors GM-CSF, TPO and EPO of mice, and the traditional Chinese medicine composition can play a role in regulating peripheral blood cells by regulating the concentration of various hematopoietic factors.
6. Sca1, C-kit, Lamin and CD48 of traditional Chinese medicine prescription on mouse bone marrow cells + CD150 + Influence of (2)
TABLE 7 Sca1, C-kit, Lamin, CD48 of bone marrow cells of groups of mice + CD150 + Expression of
Note: compared with the normal control group, the preparation method has the advantages that, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, the drug group, & P＜0.05， && p is less than 0.01; compared with the group of high-dose traditional Chinese medicines, ％％ p is less than 0.01; compared with the traditional Chinese medicine group with the middle dosage, the ^ P is less than 0.01.
As can be seen from Table 7, the expression of the hematopoietic cell surface marker Sca1 in the bone marrow cells of each group of mice is different, the expression of the model group is obviously reduced (P is less than 0.01) compared with that of the normal control group of mice, and the expression of Sca1 (P is less than 0.01) can be obviously improved by the traditional Chinese medicine with the medium dosage, which suggests that the traditional Chinese medicine composition can enhance the expression of Sca1 in the bone marrow cells of the mice and reduce the occurrence of bone marrow suppression. Compared with a normal control group, the C-kit and Lamin in the bone marrow cells of the model group are remarkably reduced (P is less than 0.01). Compared with the model group, the high-dose traditional Chinese medicine can obviously enhance the expression of C-kit in bone marrow cells (P is less than 0.01), the expression of Lamin in the positive-dose traditional Chinese medicine group and the medium-low-dose traditional Chinese medicine group is obviously reduced (P is less than 0.01), and the expression of Lamin in the high-dose traditional Chinese medicine group is not obviously changed (P is more than 0.05). The positive medicine group and the traditional Chinese medicines in each dose can obviously enhance CD48 + CD150 + Thereby reducing the activity expression of the hematopoietic stem cells and relieving the stimulation of the chemotherapy drugs on the bone marrow hematopoietic stem cells. The results show that the expression of Sca1 and C-kit can be obviously improved and the expression of Lamin can be reduced by the dosage of the traditional Chinese medicine, so that the traditional Chinese medicine composition can improve the hematopoietic function by increasing the number of hematopoietic stem cells and reducing cell rupture and apoptosis.
7. Effect of Chinese medicinal preparations on CD3, CD4 and CD8a expression of mouse peripheral blood mononuclear cells
TABLE 8 expression of CD3, CD4, and CD8a in groups of mouse PBMCs
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05， && p is less than 0.01; compared with the group of high-dose traditional Chinese medicines, ％％ p is less than 0.01; compared with the traditional Chinese medicine group with the medium dosage, the ^ P is less than 0.01.
As can be seen from table 8, compared with the normal control group, the expressions of CD3, CD4 and CD8a in the peripheral blood mononuclear cells of the model group mice are all significantly reduced (P is less than 0.01), and the expressions of CD3, CD4 and CD8a in the peripheral blood mononuclear cells of the positive drug group and the traditional Chinese medicines in all doses can be significantly enhanced (P is less than 0.01), which suggests that the traditional Chinese medicine composition of the invention has the effects of enhancing the expressions of CD3, CD4 and CD8a in the peripheral blood mononuclear cells of the mice, thereby reducing the occurrence of immune function inhibition after chemotherapy and enhancing the immune function of the organism.
8. Influence of Chinese medicinal preparation on expression of CD3 and CD8a proteins of thymus of mouse
TABLE 9 expression of CD3 and CD8a in thymus of groups of mice
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05， && P＜0.01。
it can be seen from table 9 that the expression of CD3 and CD8a proteins in the thymus of mice is significantly reduced (P < 0.01) in the model group compared with the normal control group, and the positive drug group and the traditional Chinese medicines in each dose can significantly increase the expression of CD3 and CD8a (P < 0.01) in the thymus to different extents, which suggests that the traditional Chinese medicine composition of the present invention has the effect of enhancing the expression of CD3 and CD8a in the thymus of mice, thereby participating in enhancing the immune function of the organism.
9. Influence of Chinese medicinal preparation on expression of Nrf2, Nqo1 and Ho1 proteins of thymus of mouse
TABLE 10 expression of Nrf2, Nqo1 and Ho1 proteins from thymus of groups of mice
Note: compared with the normal control group, the preparation method has the advantages that, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05， && p is less than 0.01; compared with the group of high-dose traditional Chinese medicines, ％ P＜0.05， ％％ p is less than 0.01; compared with the traditional Chinese medicine group with the medium dosage, the ^ P is less than 0.01.
As can be seen from Table 10, the expression of Nrf2, Nqo1 and Ho1 proteins was significantly increased in the thymus of the model group mice as compared with the normal control group (P < 0.01). Compared with a model group, the expression of the Nrf2 and Nqo1 proteins in the positive drug group is obviously reduced (P is less than 0.01), the expression of the Nrf2 protein in thymus tissue (P is less than 0.01) can be obviously reduced by the traditional Chinese medicines in each dosage, and the expression of the Nqo1 and Ho1 proteins (P is less than 0.01) is obviously increased, which indicates that the traditional Chinese medicine composition of the invention can enhance the expression of antioxidant proteins in the thymus of mice, thereby reducing the occurrence of in-vivo oxidation reaction after chemotherapy and enhancing the body resistance.
10. Effect of Chinese medicinal preparation on Nrf2, Nqo1 and Ho1 protein expression of mouse spleen
TABLE 11 expression of Nrf2, Nqo1 and Ho1 proteins from the spleens of groups of mice
Note: comparing with normal control group， ** P is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, & P＜0.05， && p is less than 0.01; compared with the group of high-dose traditional Chinese medicines, ％ P＜0.05， ％％ p is less than 0.01; compared with the traditional Chinese medicine group with the medium dosage, the ^ P is less than 0.01.
As can be seen from Table 11, the expression of Nrf2, Nqo1 and Ho1 proteins was significantly increased in the spleen of the model group mice as compared with the normal control group (P < 0.01). Compared with the Nrf2 protein expression of the model group, the positive drug group and the high and low dose traditional Chinese medicines have no obvious change, and the Nrf2 protein expression of the traditional Chinese medicine with the medium dose is obviously increased (P is less than 0.01); compared with Nqo1 protein expression of the model group, the positive drug group and the high and low dosage traditional Chinese medicine groups are obviously increased (P is less than 0.01), and Nqo1 protein expression in the traditional Chinese medicine group with the medium dosage is not obviously changed (P is more than 0.05); compared with Ho1 protein expression of the model group, the positive drug group and the high, medium and low dose traditional Chinese medicine group are obviously reduced (P is less than 0.01). The results show that the traditional Chinese medicine can improve the effect of Nrf2, the traditional Chinese medicine group with the medium dosage is most obvious, Nqo1 is most obvious in the high and low traditional Chinese medicine groups, and the expression of Ho1 is obviously reduced due to the antioxidation effect of the traditional Chinese medicine.
11. Effect of Chinese medicinal preparation on mRNA expression of Nrf2, Nqo1 and Ho1 in spleen of mouse
TABLE 12 expression of Nrf2, Nqo1 and Ho1 mRNA from spleens of groups of mice
Note: compared with the normal control group, ** p is less than 0.01; in comparison with the set of models, ## p is less than 0.01; compared with the positive drug group, the drug group, & P＜0.05， && p is less than 0.01; with high agentsCompared with the traditional Chinese medicine group, the medicine is prepared, ％ P＜0.05， ％％ p is less than 0.01; compared with the traditional Chinese medicine group with the medium dosage, the ^ P is less than 0.01.
As can be seen from Table 12, the expression of Nrf2 and Ho1 mRNA was significantly increased in the model group (P < 0.01), and the expression of Nqo1 mRNA was significantly decreased (P < 0.01) compared to the normal group. Compared with the expression of Nrf2 mRNA in the model group, the positive drug group and the high and medium dose groups are both obviously increased (P is less than 0.01), and the low dose traditional Chinese medicine group is obviously decreased (P is less than 0.01). Compared with the expression of Nqo1 mRNA in the model group, the positive drug group and the high and low dose groups are both obviously improved (P is less than 0.01), and the traditional Chinese medicine group in the medium dose has no obvious change (P is more than 0.05). Compared with the expression of Ho1 mRNA in the model group, the positive drug group is remarkably increased (P is less than 0.01), the medium dose group is remarkably reduced (P is less than 0.01), and the high and low dose groups have no obvious change (P is more than 0.05). The results show that the traditional Chinese medicine can improve the expression of Nrf2 and Nqo1 mRNA, and is consistent with protein expression.
12. Effect of Chinese medicinal preparation on HE staining of femur, spleen and liver of mouse
Hematoxylin-eosin staining (HE staining for short) is one of the staining methods commonly used in paraffin section technology. The phosphate groups on both strands of deoxyribonucleic acid (DNA) are outward, negatively charged, acidic, and easily ionically bound to the positively charged hematoxylin basic dye to be stained. Hematoxylin is blue in alkaline solution, so the nuclei are stained blue. Eosin is a chemically synthesized acidic dye that dissociates in water into negatively charged anions that bind to the positive cations of the amino group of proteins staining the cytoplasm, and cytoplasm, erythrocytes, muscle, connective tissue, eosin particles, etc. are stained in different degrees of red or pink, in sharp contrast to blue nuclei.
As shown in fig. 4, HE staining can stain nuclei blue in femoral tissue sections. The nucleus in the femur tissue of the normal control group mouse is stained more, and multiple cell divisions can be seen in the nucleus, while the nucleus in the femur tissue of the model group mouse is stained less, and the number of cell divisions can be seen in the nucleus is reduced. Compared with the mice of a model group, the femur tissue cell nucleus staining of the mice treated by the traditional Chinese medicines with high, medium and low doses is obviously increased, and the number of the cell nucleus divisions in the cells is also obviously increased, wherein a plurality of the cell nucleus divisions in the cells can be seen by the traditional Chinese medicines with low doses. The cell nuclei in the spleen tissues of the mice in the normal control group are more stained and deepened, while the cell nuclei in the spleen tissues of the mice in the model group are less stained and the number of the staining in the nuclei is reduced. Compared with the mice of a model group, the nucleus staining of the femoral tissue of the mice treated by the traditional Chinese medicines with high, medium and low doses is obviously increased, and the nucleus staining number is also obviously increased. The normal control group mice have more nucleus staining in liver tissues, deepened nucleus staining, deformed and irregular nuclei and less vacuole, and are accompanied with peripheral cell infiltration, while the model group mice have less nucleus staining in spleen tissues, reduced number of staining in nuclei, less vacuole and no obvious peripheral cell infiltration. Compared with the mouse of the model group, the hepatic tissue cell nucleus staining of the mouse treated by the traditional Chinese medicines with high, medium and low doses is obviously increased, the vacuole number is obviously reduced, and the hepatic tissue cell nucleus staining is obviously shown by the traditional Chinese medicines with medium and low doses.
13. Effect of Chinese medicinal formulation on mouse spleen Nqo1 and Ho1 immunohistochemistry
From the immunohistochemical results in fig. 5, Nqo1 and Ho1 were positively expressed in normal mouse spleen tissue, as indicated by a uniform staining of pale brownish yellow in the cytoplasm and intercellular spaces. In spleen tissue of model mice, Nqo1 and Ho1 exhibited weakly positive expression, as indicated by a small amount of tan staining in the cytoplasm and intercellular spaces. The contents of Nqo1 and Ho1 in spleen tissues of mice treated by low, medium and high doses of traditional Chinese medicines are obviously increased, and the contents are obviously increased by high and medium doses of traditional Chinese medicines, and the contents are shown as dark brown staining in cytoplasm and intercellular spaces of the cells. The positive drug group also significantly increased Nqo1 and Ho1 expression in mouse spleen tissue. Nqo1 and Ho1 show that the traditional Chinese medicine composition and colony stimulating factors have obvious up-regulating effects on the expression of Nqo1 and Ho1 in mouse spleen tissues, and the traditional Chinese medicine composition is suggested to possibly participate in antioxidant reaction and enhance the expression of antioxidant factors Nqo1 and Ho 1.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1. A traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy is characterized by comprising the following components in parts by weight: 25-35 parts of hispid fig, 25-35 parts of astragalus membranaceus, 15-25 parts of codonopsis pilosula, 10-20 parts of bighead atractylodes rhizome, 3-10 parts of angelica sinensis, 5-15 parts of prepared rehmannia root, 5-15 parts of dried orange peel, 5-15 parts of rhizoma pinellinae praeparata, 5-15 parts of pogostemon cablin, 3-10 parts of ginger, 5-15 parts of Chinese date and 5-15 parts of honey-fried licorice root.
2. The traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy according to claim 1, which is characterized by comprising the following components in parts by weight: 30-35 parts of hispid fig, 30-35 parts of astragalus membranaceus, 20-25 parts of codonopsis pilosula, 15-20 parts of bighead atractylodes rhizome, 6-8 parts of angelica sinensis, 10-15 parts of prepared rehmannia root, 10-15 parts of dried orange peel, 10-15 parts of rhizoma pinellinae praeparata, 10-15 parts of pogostemon cablin, 6-8 parts of ginger, 10-15 parts of Chinese date and 10-15 parts of honey-fried licorice root.
3. The traditional Chinese medicine composition for treating bone marrow suppression and/or immune function suppression after chemotherapy according to claim 1, which is characterized by comprising the following components in parts by weight: 30 parts of radix fici simplicissimae, 30 parts of astragalus membranaceus, 20 parts of codonopsis pilosula, 15 parts of bighead atractylodes rhizome, 6 parts of angelica sinensis, 10 parts of prepared rehmannia root, 10 parts of dried orange peel, 10 parts of rhizoma pinellinae praeparata, 10 parts of pogostemon cablin, 6 parts of ginger, 10 parts of Chinese date and 10 parts of honey-fried licorice root.
4. A medicine for treating bone marrow suppression and/or immune function suppression after chemotherapy, which is characterized by comprising the traditional Chinese medicine composition of any one of claims 1-3 and pharmaceutically acceptable auxiliary materials.
5. The medicament of claim 4, wherein the medicament is in the form of tablets, solutions, granules, pills, capsules.
6. The use of a Chinese medicinal composition according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of post-chemotherapy myelosuppression.
7. The use of claim 6, wherein the medicament has any one of the following effects: (1) increasing the leukocyte content in peripheral blood after chemotherapy; (2) reducing the content of red blood cells in peripheral blood after chemotherapy; (3) reduce the content of platelets in peripheral blood after chemotherapy.
8. The use of claim 6, wherein the medicament has any one of the following effects: (1) increasing the content of hematopoietic factors in plasma after chemotherapy; preferably, the hematopoietic factor is selected from at least one of GM-CSF, TPO, EPO, IL-2; (2) increasing the number of hematopoietic stem cells after chemotherapy, and improving the hematopoietic function of bone marrow; (3) reducing the differentiation activity of hematopoietic stem cells after chemotherapy.
9. Use of the Chinese medicinal composition of any one of claims 1 to 3 in the preparation of a medicament for enhancing the immune function of a body after chemotherapy.
10. Use of the Chinese medicinal composition of any one of claims 1 to 3 in the preparation of a medicament for improving the oxidation resistance of an organism after chemotherapy.
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