CN106063903B - Traditional Chinese medicine preparation and application thereof - Google Patents
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- CN106063903B CN106063903B CN201610452687.1A CN201610452687A CN106063903B CN 106063903 B CN106063903 B CN 106063903B CN 201610452687 A CN201610452687 A CN 201610452687A CN 106063903 B CN106063903 B CN 106063903B
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Abstract
The invention discloses a traditional Chinese medicine preparation and application thereof, wherein the traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 20-30 parts of astragalus membranaceus, 20-30 parts of adenophora tetraphylla, 20-30 parts of radix glehniae, 10-20 parts of lily, 5-15 parts of dendrobe, 10-20 parts of aster, 10-20 parts of loquat leaf, 10-20 parts of bitter almond, 10-20 parts of radix stemonae and 10-20 parts of angelica sinensis. Has effects of clearing lung-heat, relieving cough and asthma, invigorating qi, nourishing yin and promoting blood circulation. After being taken, the medicine can directly act on the pathological change part of the radiation pneumonitis, inhibit exudation of capillary inflammatory cells of alveolar walls, pulmonary interstitial congestion, hemorrhage, edema and alveolar structure damage, and reduce the expression of TLR4 signal molecule signal protein and cell factors of local lung tissue related cells, thereby relieving the lung inflammatory reaction and achieving the aim of delaying or treating the radiation pneumonitis.
Description
(I) technical field
The invention relates to a traditional Chinese medicine preparation, in particular to a traditional Chinese medicine preparation, a preparation method thereof and application thereof in preventing and treating radiation pneumonitis.
(II) background of the invention
The incidence of breast malignant tumors such as lung cancer and the like is on the rising trend year by year and becomes one of the important factors harming health. Radiotherapy is one of the important treatment methods, and radiation pneumonitis is a common complication of breast malignant tumor after radiotherapy, the incidence rate of the radiation pneumonitis reaches 5% to 15%, and serious clinical consequences are caused. The mild patient has no obvious symptoms, and the severe patient can cause extensive fibrosis of the lung, respiratory dysfunction and even respiratory failure.
The existing treatment for radiation pneumonitis mainly uses adrenal cortical hormone which has strong anti-inflammatory effect, but long-term use of the hormone causes dependence and generates larger side effect. The traditional Chinese medicine considers that the radiation belongs to fire-heat toxin pathogen in the traditional Chinese medicine, and the pathogenesis of the radiation is lung heat and blood stasis, qi and yin injury and lung dysfunction. In recent years, the traditional Chinese medicine treatment of the radiation pneumonitis has better effect, no obvious side effect and low cost, so the traditional Chinese medicine has certain prospect in the aspect of treating the radiation pneumonitis.
Disclosure of the invention
The invention aims to provide a traditional Chinese medicine preparation for preventing and treating radiation pneumonitis, which not only can play a role in resisting radiation lung injury, but also has the effects of improving immunity, reducing tumor recurrence and metastasis, and has the advantages of small side effect and low cost, aiming at the limitation of the current medical technology.
The technical scheme adopted by the invention is as follows:
the invention provides a traditional Chinese medicine preparation which is prepared from the following raw materials in parts by weight: 20-30 parts of astragalus membranaceus, 20-30 parts of adenophora tetraphylla, 20-30 parts of radix glehniae, 10-20 parts of lily, 5-15 parts of dendrobe, 10-20 parts of aster, 10-30 parts of loquat leaves, 10-20 parts of bitter apricot seeds, 10-20 parts of radix stemonae and 10-20 parts of angelica sinensis.
Further, the traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 30 parts of astragalus membranaceus, 25-30 parts of adenophora tetraphylla, 25-30 parts of radix glehniae, 10-15 parts of lily, 10-15 parts of dendrobe, 15-20 parts of aster, 15-30 parts of loquat leaves, 10-15 parts of bitter apricot seeds, 10-15 parts of radix stemonae and 15-20 parts of angelica sinensis.
Furthermore, the traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 30 parts of astragalus membranaceus, 30 parts of adenophora tetraphylla, 30 parts of radix glehniae, 15 parts of lily, 10 parts of dendrobe, 15 parts of aster, 15 parts of loquat leaves, 15 parts of bitter apricot seeds, 15 parts of radix stemonae and 15 parts of angelica sinensis.
Furthermore, the traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 30 parts of astragalus membranaceus, 30 parts of adenophora tetraphylla, 30 parts of radix glehniae, 10 parts of lily, 10 parts of dendrobe, 15 parts of aster, 20 parts of loquat leaves, 12 parts of bitter apricot seeds, 15 parts of radix stemonae and 18 parts of angelica sinensis.
Furthermore, the traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 30 parts of astragalus membranaceus, 25 parts of adenophora tetraphylla, 25 parts of radix glehniae, 15 parts of lily, 10 parts of dendrobe, 15 parts of aster, 15 parts of loquat leaves, 15 parts of bitter apricot seeds, 15 parts of radix stemonae and 15 parts of angelica sinensis.
The invention also provides a preparation method of the traditional Chinese medicine preparation, which comprises the following steps: mixing the components according to the formula amount, adding 3-4 times of water by weight, soaking for 20min, decocting for 20min at 120 ℃, then adjusting to 80 ℃, decocting for 20min, filtering to obtain a primary filtrate and a primary filter cake, repeatedly decocting the primary filtrate once, filtering to obtain a secondary filter cake and a secondary filtrate, combining the primary filtrate and the secondary filtrate, and concentrating under reduced pressure to 1.5-2 times of volume to obtain the traditional Chinese medicine preparation.
Further, the concentration under reduced pressure is carried out at 0.097MPa and 50-60 deg.C.
In addition, the invention also provides application of the traditional Chinese medicine preparation in preparation of a medicine for preventing and treating radiation pneumonitis.
The pharmacological actions of the traditional Chinese medicine raw materials of the invention are as follows:
radix astragali has effects of invigorating lung and spleen, strengthening middle warmer and invigorating qi. Astragalus root can promote the hemopoietic function of bone marrow, stimulate the hemopoiesis to increase leukocytes, improve and enhance the phagocytosis and sterilization functions of neutrophils and macrophages to different degrees, also can obviously stimulate the division and proliferation of NK cells, and has the function of resisting infection by inducing lymphocytes to generate interferon. The astragalus polysaccharide contained in the astragalus polysaccharide can effectively accelerate the repair of damaged tissues, enhance the immune regulation function of organisms and play a role in resisting radioactive radiation.
Radix Adenophorae has effects of nourishing yin, clearing lung-heat, clearing stomach, promoting fluid production, and eliminating phlegm. The radix adenophorae can regulate immune balance, increase leukocyte number and T, B cell value, enhance phagocytic ability of macrophage, prolong the in vivo maintenance time of antibody, and has positive and effective effects on body treatment of hypoimmunity after radiotherapy and chemotherapy.
The lily has the effects of nourishing yin, moistening lung, clearing heart fire and soothing nerves, and is most suitable for patients with deficiency of both qi and yin caused by radiotherapy and chemotherapy. It can be used for preventing leukopenia caused by radiotherapy and chemotherapy, and its main ingredient colchicine can increase cAMP level in cancer cell, prevent mitosis, and inhibit tumor cell proliferation.
Herba Dendrobii has effects of benefiting stomach, promoting fluid production, nourishing yin and clearing heat. Mainly enhances the immunity of the organism and induces various cell factors to enhance the immunity of the organism so as to play a role in resisting tumors, and has good auxiliary treatment efficacy on patients with low immunity caused by radiotherapy, chemotherapy and other reasons.
Radix Asteris has effects of moistening lung, eliminating phlegm and relieving cough. Experimental studies such as luyanhua and the like find that shionone and episuberol in the extract have remarkable inhibitory effect on cough caused by various reasons.
Pi Pa Ye, bitter in flavor and slightly cold, enters lung and stomach meridians. Has the effects of clearing lung-heat, relieving cough, lowering adverse qi and arresting vomiting. Mainly contains flavonoid, phenols, terpenes, amygdalin and other components, and has the functions of resisting oxidation, resisting inflammation, relieving cough, inhibiting bacteria, resisting tumor and the like.
Radix Stemonae has effects of moistening lung and relieving cough. Modern pharmacological research shows that radix stemonae mainly has the effects of resisting oxidation activity, relieving cough and asthma, resisting tumor, resisting bacteria and the like.
The almond has the effects of depressing qi, reducing phlegm, relieving cough and asthma. Has antioxidant, hypertension preventing and treating, antitumor, antitussive, analgesic, anticoagulant, and toxic materials clearing away effects.
The angelica has the effects of enriching blood and promoting blood circulation. The angelica has obvious inhibition effect on capillary permeability increase, tissue edema and chronic injury caused by various inflammatory factors; the Angelica sinensis polysaccharide can activate macrophage, lymphocyte, etc. in bone marrow through related ways, and also has functions of correcting hematopoiesis and resisting anemia, and in addition, Angelica sinensis can dilate blood vessel, improve microcirculation blood flow, inhibit platelet aggregation, resist thrombosis, promote fibrinolysis, and promote blood circulation to remove blood stasis.
Compared with the prior art, the invention has the following beneficial effects: the invention takes the principle of clearing lung, relieving cough and asthma, tonifying qi, nourishing yin and activating blood circulation as the principle, and various medicaments mutually form synergistic action. Is prepared from astragalus root, root of straight ladybell, lily bulb, dendrobium stem, aster root, apricot kernel, stemona root, Chinese angelica root and the like by adding and subtracting, wherein the astragalus root and the root of straight ladybell are monarch drugs for tonifying qi, nourishing lung and moistening lung; dendrobe, almond and lily are used as ministerial drugs for clearing lung heat; radix Asteris and radix Stemonae are added to relieve cough and asthma; and the angelica sinensis is used as a messenger for supplementing blood and regulating blood circulation, and promoting blood circulation to remove blood stasis, and all the medicines supplement each other. Has effects of clearing lung-heat, relieving cough and asthma, invigorating qi, nourishing yin and promoting blood circulation. After being taken, the medicine can directly act on the pathological change part of the radiation pneumonitis, inhibit exudation of capillary inflammatory cells of alveolar walls, pulmonary interstitial congestion, hemorrhage, edema and alveolar structure damage, and reduce the expression of TLR4 signal molecule signal protein and cell factors of local lung tissue related cells, thereby relieving the lung inflammatory reaction and achieving the aim of delaying or treating the radiation pneumonitis. 114 cases (67.86%) are cured clinically, 41 cases (24.40%) are effective, 13 cases (7.74%) are ineffective, and the total effective rate is 92.26%. The invention has the advantages of obvious curative effect, no obvious side effect, good safety and low cost. Not only can improve the symptoms of the patients, but also can improve the life quality of the patients, relieve the pain and prolong the life cycle.
(IV) description of the drawings
FIG. 1 staining electron microscopy of rat lung tissue sections HE (HE X400), A (irradiated rat + TCM 1) (400X), B (irradiated rat + TCM 2) (400X), C (irradiated rat + TCM 3) (400X), D (irradiated rat + dexamethasone) (400X), E (irradiated rat + distilled water) (400X), F (healthy rat + distilled water) (400X), G (healthy rat + TCM 2) (400X).
FIG. 2 is a signal molecule protein Western blot, wherein A is MyD88Western blot, B is TRAF-6Western blot, C is TRIF Western blot, and D is TLR4Western blot.
FIG. 3 shows the gray value of Myd88TRAF-6, wherein A irradiates a mouse and Chinese medicine 1, B irradiates a mouse and Chinese medicine 2, C irradiates a mouse and Chinese medicine 3, D irradiates a mouse and dexamethasone, E irradiates a mouse and distilled water, F healthy mouse and distilled water, and G healthy mouse and Chinese medicine 2.
FIG. 4 shows the grey level of TRIF TLR4, A irradiating mice and Chinese medicine 1, B irradiating mice and Chinese medicine 2, C irradiating mice and Chinese medicine 3, D irradiating mice and dexamethasone, E irradiating mice and distilled water, F healthy mice and distilled water, and G healthy mice and Chinese medicine 2.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the components used in the embodiment of the invention are collected and prepared according to the Chinese pharmacopoeia 2015.
Example 1:
30g of astragalus membranaceus, 30g of adenophora tetraphylla, 30g of radix glehniae, 10g of lily, 10g of dendrobe, 15g of aster, 20g of loquat leaf, 12g of bitter apricot kernel, 15g of radix stemonae and 18g of angelica.
The preparation method comprises the following steps: mixing the components according to the formula amount, adding 3 times of water by weight, soaking for 20min, decocting at 120 ℃ for 20min, adjusting to 80 ℃ for 20min, filtering, repeatedly decocting filter cakes once, filtering, combining filtrates, and concentrating under reduced pressure (0.097Mpa, 50-60 ℃) to 1.5 times of volume to obtain decoction with the concentration of crude drug of the traditional Chinese medicine of 2 g/ml.
Example 2:
30g of astragalus membranaceus, 25g of adenophora tetraphylla, 25g of radix glehniae, 15g of lily, 10g of dendrobe, 15g of aster, 15g of loquat leaf, 15g of bitter apricot seed, 15g of radix stemonae and 15g of angelica sinensis.
The preparation method comprises the following steps: mixing the components according to the formula amount, adding 4 times of water by weight, soaking for 20min, decocting at 120 ℃ for 20min, adjusting to 80 ℃ and decocting for 20min, filtering, repeatedly decocting filter cakes once, filtering, combining filtrates, and concentrating under reduced pressure (0.097Mpa, 50-60 ℃) to 2 times of volume to obtain decoction with the concentration of crude drug of 2 g/ml.
Example 3:
30g of astragalus membranaceus, 30g of adenophora tetraphylla, 30g of radix glehniae, 15g of lily, 10g of dendrobe, 15g of aster, 15g of loquat leaf, 15g of bitter apricot seed, 15g of radix stemonae and 15g of angelica sinensis.
The preparation method comprises the following steps: mixing the components according to the formula amount, adding 3.5 times of water by weight, soaking for 20min, decocting at 120 ℃ for 20min, then adjusting to 80 ℃ for 20min, filtering, repeatedly decocting filter cakes once, filtering, combining filtrates, and concentrating under reduced pressure (0.097Mpa, 50-60 ℃) to 1.8 times of volume to obtain decoction with the concentration of crude drug of the traditional Chinese medicine of 2 g/ml.
Example 4
30g of astragalus membranaceus, 20g of adenophora tetraphylla, 20g of radix glehniae, 15g of lily, 15g of dendrobe, 20g of aster, 25g of loquat leaf, 12g of bitter apricot seed, 15g of radix stemonae and 18g of angelica.
The preparation method is the same as example 1, and decoction with the concentration of the traditional Chinese medicine crude drug of 2g/ml is obtained.
Example 5
30g of astragalus membranaceus, 20g of adenophora tetraphylla, 20g of radix glehniae, 10g of lily, 15g of dendrobe, 20g of aster, 30g of loquat leaf, 10g of bitter apricot kernel, 15g of radix stemonae and 20g of angelica sinensis.
The preparation method is the same as example 1, and decoction with the concentration of the traditional Chinese medicine crude drug of 2g/ml is obtained.
Example 6
30g of astragalus membranaceus, 25g of adenophora tetraphylla, 25g of radix glehniae, 15g of lily, 15g of dendrobe, 20g of aster, 20g of loquat leaf, 12g of bitter apricot seed, 10g of radix stemonae and 18g of angelica.
The preparation method is the same as example 1, and decoction with the concentration of the traditional Chinese medicine crude drug of 2g/ml is obtained.
Example 7
30g of astragalus membranaceus, 30g of adenophora tetraphylla, 30g of radix glehniae, 10g of lily, 15g of dendrobe, 15g of aster, 20g of loquat leaf, 12g of bitter apricot kernel, 10g of radix stemonae and 18g of angelica.
The preparation method is the same as example 1, and decoction with the concentration of the traditional Chinese medicine crude drug of 2g/ml is obtained.
Example 8
30g of astragalus membranaceus, 30g of adenophora tetraphylla, 30g of radix glehniae, 10g of lily, 10g of dendrobe, 15g of aster, 20g of loquat leaf, 15g of bitter apricot kernel, 15g of radix stemonae and 15g of angelica sinensis.
The preparation method is the same as example 1, and decoction with the concentration of the traditional Chinese medicine crude drug of 2g/ml is obtained. )
Example 9 clinical observations
(1) Clinical data
The inventor carries out clinical observation and treatment on 168 breast malignant tumor patients after radiation therapy forms radiation pneumonia under the informed consent in 2009 till now, and the patients are 20-66 years old.
(2) Method of treatment
The decoction prepared in example 3 was orally administered twice a day, 100ml each time, after meals. The composition is administered continuously for 15-30 days.
(3) Therapeutic results
114 cases (67.86%) are cured clinically, 41 cases (24.40%) are effective, 13 cases (7.74%) are ineffective, and the total effective rate is 92.26%.
Example 10 animal Experimental procedures and conclusions
1. Grouping of laboratory animals
SD rats of healthy males, weighing (200 ± 20) g, 84, provided by the experimental animals center in shanghai, a chinese hospital, were randomly divided into: a (irradiated mice + Chinese medicine 1), B (irradiated mice + Chinese medicine 2), C (irradiated mice + Chinese medicine 3), D (irradiated mice + dexamethasone), E (irradiated mice + distilled water), F (healthy mice + distilled water), G (healthy mice + Chinese medicine 2) are 7 groups, and each group contains 12 mice.
2. Preparation of Experimental drugs
The traditional Chinese medicine 2 is decoction with the crude drug concentration of 2g/ml prepared in the embodiment 3, the traditional Chinese medicine 1 is obtained by concentrating the traditional Chinese medicine 2 to the crude drug concentration of 1g/ml, and the traditional Chinese medicine 3 is obtained by concentrating the traditional Chinese medicine 2 to the crude drug concentration of 4 g/ml. Dexamethasone is prepared by dissolving 5mg dexamethasone (jojoba) in 25ml distilled water to obtain 0.2mg/ml aqueous solution.
3. Model making of experimental animal
Mouse irradiation: according to the equivalent dose conversion between human and rat, 10% chloral hydrate is injected in the abdominal cavity with 0.35ml/100g, after the rat is completely anaesthetized, the rat is fixed on a frame in a supine position, then the rat is placed on an irradiation table, the irradiation field is adjusted to the connecting line of the two armpits of the forelimb of the rat and the lower part of the rat is adjusted to the level of the sternal xiphoid process, so that the irradiation area completely covers the two lungs of the rat. Shielding the rest parts of the rat by lead blocks with the thickness of 2cm, and performing Siemens photon linear accelerator 6MV-X ray irradiation, wherein the absorption dose is 10Gy at a single time, the absorption dose rate is 300cGy/min, the source skin distance is 1m, and the irradiation area is 4X 4 cm. Repeated every other day for 1 time, and the total dose is 20 Gy. After the irradiation is finished, the rats are waited for natural revival and then returned to the experimental animal center of Zhejiang university of traditional Chinese medicine for continuous conventional breeding.
4. Laboratory animal treatment
The rats in each group are adaptively fed for 7 days, and are subjected to the experiment after being fasted and not forbidden for 24 hours. The following day after irradiation, the Chinese medicinal materials (A, B, C, G) or dexamethasone (D) were administered, and the other two groups were administered with equal amount of distilled water (E, F). The drug amount required by each experimental rat is converted according to the equivalent dose between human and rat, namely, the rat is fed by 1ml/100g, the drug is drenched for 1 time each day, the rat is killed after being continuously fed for 14 days, the two lungs are cut out under the aseptic condition, the upper left lung lobe of each rat is placed in an embedding box and is placed in formaldehyde for subsequent pathological section staining after being observed by naked eyes, the residual lung tissue is cut into pieces, and the pieces are respectively placed in liquid nitrogen according to the upper right lung lobe, the middle lung lobe and the lower lung lobe and are transferred to a refrigerator at the temperature of minus 80 ℃ to be stored for subsequent Elisa and Western blot experiments.
5. Observation index
(1) General conditions in rats
(2) Body weight change in rats
(3) Pathological changes of lung tissue
(4) Lung pathology scoring: observing pathological section lung tissue damage degree
(5) Signaling pathway molecule protein expression: western blot method for measuring expression level of MyD88, TRAF-6, TRIF and TLR4
(6) Partial cytokine expression results: elisa method for detecting expression levels of TNF-alpha, IL-17 and TGF-beta in lung tissue homogenate supernatant
6. HE staining Lung tissue pathological step
(1) Fixing
The solution was fixed in 10% neutral formaldehyde for 24h at room temperature. Washing the specimen with running water for 30 min;
(2) dewatering
The sample is subjected to gradient dehydration according to the following processes in sequence: 75% ethanol (1h), 95% ethanol I (1h), 95% ethanol II (1h), 100% ethanol I (25min), 100% ethanol II (25 min);
(3) is transparent
Carrying out secondary xylene transparency for 12 min;
(4) wax dipping
Soaking the sample in 60-degree paraffin for three times for 2 hours;
(5) embedding
Opening an embedding machine in advance for preheating, precooling by a freezing table, and embedding by a conventional method to prepare a wax block;
(6) slicing
Continuously slicing the specimen, wherein the thickness of the sliced specimen is 5mm, fishing the sliced specimen in a constant-temperature water bath at 40 ℃, and roasting the sliced specimen for 3 hours at 65 ℃;
(7) hematoxylin-eosin staining (HE staining) was performed as follows:
1) naturally drying the specimen, and soaking the specimen in 4% paraformaldehyde for 30 min;
2) washing with distilled water, and soaking in PBS (pH 7.2-7.4) for 5 min;
3) degreasing with dimethylbenzene for 10min 2 times;
4) absolute ethyl alcohol, 3min x 2 times;
5) descending 95%, 85% and 75% alcohol to distilled water for 2min respectively;
6) harris hematoxylin for 5 min;
7) 10% acidified alcohol differentiation (glacial acetic acid), 5-l0 sec;
8) washing with tap water for 30 min;
9) washing with distilled water for 20 min;
10) ascending 75%, 85% and 95% alcohol for 2min respectively;
11) eosin 0.5-1% (prepared with 95% ethanol) for 5-l0 min;
12) color separation with 95% ethanol for 1 min;
13) absolute ethyl alcohol, 3min x 2 times;
14) xylene, 10min x 2 times;
15) and (6) sealing the gum.
The above operations are completed by laboratory staff of the experimental animal center of Zhejiang university of traditional Chinese medicine.
(8) Pathological specimen scoring
Scoring was performed according to the following 5 criteria: 1) pulmonary interstitial and alveolar edema; 2) alveolar hemorrhage; 3) neutrophil infiltration or aggregation in the interstices or vessel walls; 4) forming a transparent film; 5) the lung is atelectasis. According to the semi-quantitative analysis of the light and heavy degree of each index, the damage is 0 point, the lesion range accounts for 1 point less than 25 percent, 2 points 25-50 percent, 3 points 50-75 percent and 4 points more than 75 percent under each high-power visual field. For double-blind assessment, the total lung injury score is the average of the above scores.
7. Experimental step for detecting MyD88 expression by Western blot
(1) Extraction of Total protein from Lung tissue
1) Taking out the right lung middle lobe of each sample from a refrigerator at the temperature of-80 ℃, placing the right lung middle lobe in liquid nitrogen, weighing, and placing each sample with the weight of about 20mg in a 1.5ml EP tube;
2) RIPA cell lysate and PMSF (lysate: PMSF volume ratio of 99: 1, generally 20mg lung tissue is added with 200ul liquid), and then 2 steel balls are added;
3) grinding in a grinder for 60sec 2 times;
4) centrifuging at 12000rpm at 4 deg.C for 10min, collecting supernatant, and storing in refrigerator at-80 deg.C.
(2) BCA method for determining total protein concentration of lung tissue
1) According to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and the mixture is fully mixed. The BCA working solution is stable within 24 hours at room temperature;
2) dilution of standard by multiple times: 8 small EP tubes of 150ul, numbered S1-S8, S1 were added with 100ul of 1mg/ml protein standard, and the rest were added with 50ul of pure water. 50ul of liquid is taken from an EP tube of S1 and added into an EP tube of S2 to be uniformly blown and beaten, and then 50ul of liquid is taken from an S2 tube and added into an S3 tube to be uniformly blown and beaten. And so on to S7. Transferring 20ul of the S1-S8 standard substance to a 96-well plate, and repeating the steps;
3) transferring 20ul of sample to a 96-well plate, and repeating the steps;
4) adding 200ul of BCA working solution into each hole, and placing in a constant temperature box at 37 ℃ for 30 min;
5) the data were read with a microplate reader 595nm wavelength. Drawing a standard curve according to the concentration of the standard substance and the optical density (OD value), and then calculating the protein concentration of the sample according to the curve;
6) protein denaturation: adding a corresponding amount of protein electrophoresis loading buffer solution and lysis solution according to the concentration of the protein to be detected to ensure that the final concentration is 3ug/ul, shaking and uniformly mixing, and carrying out constant temperature water bath at 95 ℃ for 5min to denature the protein.
(3) Glue making
1) 2 sets of clean glass plates (one thick and one thin) are taken and wiped clean by alcohol;
2) carefully checking whether the glass plate has cracks or not, and placing thick plate letters upwards;
3) respectively fixing the 2 sets of glass plates on a glue making frame, and adding double distilled water to verify whether leakage exists or not;
4) inclining the glue making frame and pouring out water;
5) preparing SDS-PAGE gel;
a, preparing a lower layer glue: ammonium persulfate is prepared into 10 percent solution and then is subpackaged into small tubes to be stored at the temperature of-20 ℃. The gel fractions (i.e.subgel) of 10% SDS-PAGE were selected based on rat MyD88 molecular weight (33-35kD) and formulated as in Table 1. The mixture was poured into the gap between the two glass plates (about 4.5ml) with a pipette, carefully adding distilled water to the liquid surface to seal the gap with water, taking care to avoid the formation of bubbles. Standing for 30min until the gel is solidified, and pouring out the upper water layer after layering;
b, preparing a top glue: a5% SDS-PAGE gel concentrate (i.e., supernatant) was prepared as in Table 1, added to a glass plate, degassed, quickly inserted into a comb, and allowed to stand for 15 min.
TABLE 1 Western blot separation gel and concentrated gel configuration
(4) Electrophoresis
1) Diluting 10 × electrophoresis solution with distilled water by 10 times to prepare 1 × electrophoresis solution;
2) taking out the gel plate from the gel making frame, fixing the two plates in the electrophoresis tank with the concave surfaces facing inwards, putting the tank into the electrophoresis box, and paying attention to the direction of the electrodes;
3) adding the electrophoresis solution into an electrophoresis tank, wherein the inner flow is flush with the mouth, and the outer flow is about 1cm higher than the metal wire;
4) loading: taking out a comb by balancing two hands, and loading the Mark and the protein sample into an SDS-PAGE gel loading hole; adding 10ul of Mark and 13ul of protein sample;
5) and (5) connecting a power supply, adjusting the voltage to be 100V and the time to be 90 min. Electrophoresis is carried out until the bromophenol blue line reaches the bottom of the separation gel.
(5) Semi-dry rotor
a, preparing an electrotransformation liquid: preparing 400ml of electrotransformation liquid by using 40ml of 10X electrotransformation liquid, 80ml of methanol and 280ml of distilled water;
b, taking out the gel after the electrophoresis is finished, placing the gel in an electrotransfer solution, cutting gel, filter paper and a PVDF membrane with proper sizes according to molecular weights, soaking the PVDF membrane in methanol for 3min, sequentially placing the filter paper, the PVDF membrane, the gel and the filter paper in the electrotransfer solution, removing bubbles, placing the gel in a semi-dry rotating device, and adjusting the voltage to 20V for 40min of semi-dry rotation.
(6) Sealing of
Diluting 10 times of TBST with distilled water to prepare 1 times TBST; weighing 2.5g of skimmed milk powder, dissolving in 50ml of 1 × TBST, and preparing into 5% skimmed milk powder confining liquid; immediately after the semi-dry transfer, the PVDF membrane is placed in a TBST (Tunnel boring machine) washing solution prepared in advance, and the membrane is rinsed for 10min to wash off the membrane transfer solution on the membrane. Adding 5% skimmed milk powder sealing solution, shaking slowly on shaking table, sealing at room temperature for 40min, and washing with TBST washing solution for 10min × 3 times.
(7) Primary antibody incubation
The primary antibody was diluted with the primary antibody diluent at the appropriate ratio (1:500) with reference to the specifications for the MyD88 primary antibody. After 4 ℃ refrigerator incubation overnight, TBST wash was added for 10min x 3 times.
(8) Incubation with secondary antibody
Referring to the secondary antibody specification, the secondary antibody was diluted with a dilution at a ratio of 1:5000, incubated for 1 hour at room temperature with a shaker, and washed with TBST wash for 10min x 3 times.
(9) Exposure and analysis
Adding a developer for 3-4min for color development, and processing the Image collected by a bioluminescent imaging system by using Image J software. And (3) taking the gray value of the beta-actin as 1, and representing a quantitative value by the ratio of the gray value of each sample to the gray value of the internal reference beta-actin for analysis.
The detection steps of TRAF-6(58kD), TRIF (66kD) and TLR4(75-80kD) Western blot are basically the same as those of MyD88Western blot and are carried out according to the instruction.
8. Experimental procedure for measuring content of TNF-alpha by Elisa method
(1) Preparing a reagent and a standard dilution according to the introduction of the instruction;
(2) removing the redundant microporous plate strips from the plate frame, putting the box bag back, including the drying agent, and sealing again;
(3) the wells were marked, 100ul of standards and samples (each duplicate) were added to the center of each well and a seal was applied. Incubating for 2.5h at room temperature;
(4) taking out the plate frame, discarding liquid in each hole, drying the liquid on clean absorbent paper, adding 300ul 1x Wash Buffer into each hole, and standing for 1 min; discarding the liquid in each hole, drying the liquid on clean absorbent paper, and repeating the operation for 4 times;
(5) add l00ul 1x Biotinylated TNF-. alpha.detection Antibody per well; covering a new sealing strip, and incubating for 1h in a shaking table at room temperature;
(6) repeating the process of (4);
(7) adding 100ul of 1x HRP-Streptavidin Solution to each well, and performing shake culture at room temperature for 45 min;
(8) repeating the process of (4);
(9) adding l00ul TMB One-Step Substrate Reagent into each well, and incubating for 30min at room temperature in a light-proof shaking table;
(10) to each well was added l00ul Stop Solution, immediately placed in a microplate reader to determine the optical density (OD value) of each well, with the wavelength set at 450 nm. According to OD value and standard substance concentrationPlotting a standard curve (y is 116.71 e)3.1608xY is the standard concentration and x is the OD value), and then the concentration of TNF- α in the sample is calculated from the curve.
The procedure for the Elisa detection of IL-17, TGF-. beta.was essentially the same as that for the TNF-. alpha.Elisa detection, as described herein.
9. Results
(1) General conditions in rats
The normal group of rats which are not irradiated has excellent mental state, activity, better appetite and smooth and moist hair color, and the rats of each irradiated group begin to appear symptoms of listlessness, decreased appetite, dull hair color, yellowing, dryness, erection and even hair removal and the like at about 5 days after irradiation, with the incidence rate of 90%. The more obvious the symptoms are caused by the accumulation of rats filled with the traditional Chinese medicine 1 and the distilled water along with the time; the rats taking the traditional Chinese medicine 2, the traditional Chinese medicine 3 and the dexamethasone have better general conditions of mental state, appetite, hair color and the like from 8 days.
(2) Body weight change in rats
The initial body weight and the body weight after 2 weeks of the rats are shown in Table 2. There was no significant difference in initial body weight between rats in each group before the experiment (p >0.05), and the body weight change of rats in each group after the experiment was entered was significant: the rats in the two normal groups (F and G) which are not irradiated have the weight gain rapidly compared with the rats in each irradiated group, and the difference has the statistical significance (p is less than 0.01); however, there was no significant difference in weight gain between the groups of normal rats receiving distilled water and the traditional Chinese medicine 2 (p > 0.05). In each irradiation group, the weight of the rats treated by the traditional Chinese medicine 2, the traditional Chinese medicine 3 and the dexamethasone is obviously increased compared with that of the rats treated by the gastric perfusion traditional Chinese medicine 1 and the distilled water. P <0.05 for a versus B, p <0.01 for a versus C, D; B. p <0.05 for C compared to E and p <0.01 for D compared to E. However, the differences in weight gain between groups B and C, B and D, C and D were not statistically significant (p > 0.05); similarly, there was no significant difference in the body weight gain between A and E (p > 0.05).
TABLE 2 rat body weight
Note: in comparison with the group a,*p<0.05,**p<0.01; in comparison with the group E,△p<0.05,△△p<0.01; in comparison with the group F,□p<0.05,□□p<0.01; in comparison with the group G,#p<0.05, ##p<0.01
(3) pathological changes of the lung
The rats in each irradiated group showed acute inflammation, and the lesions mainly appeared exudation. The main pathological manifestations are: thickening of lung space, widening of alveolar wall, congestion of capillary vessels in alveolar wall, congestion and edema of pulmonary interstitium, and infiltration of neutrophils in the alveolar space and partial alveolar cavities. F. G two normal groups of rats have clear alveolar structures under a lung tissue section electron microscope and have no obvious inflammatory exudation; obvious alveolar wall thickening, pulmonary interstitial capillary congestion, edema, alveolar structural disorder and even transparent film formation can be observed under an optical microscope of the pathological sections of the groups A and E; while a small amount of congestion, edema and inflammatory exudation of pulmonary interstitial tissues can be observed under a light microscope of B, C, D groups of rats, but the phenomenon is not obvious; the alveolar structure is clearer, the alveolar wall is thinner, and the inflammatory response is lighter than that of A, E groups. See fig. 1.
(4) Lung injury scoring
F. G, pathological sections of rats in two normal unirradiated groups show that most of the inflammatory exudation area of lung tissues is less than 25 percent, and the score is 0-1; B. c, D groups of rats had lung lesions with areas mostly between 25% and 50%, few over 75%, scored mainly on 1-3 points, and no difference between the three groups (p > 0.05); A. the group E can obviously observe that large-area lung tissues are damaged, the vast majority of damaged areas reach more than 50 percent, the score is 2-4, and the two groups have no difference (p is more than 0.05); however, there were significant statistical differences in scores between B, C, D and A, E groups, and B, C, D compared to a for p <0.05, B compared to E for p <0.05, C, D compared to E for p < 0.01. Specific scores are shown in table 3.
TABLE 3 Lung tissue injury pathology score
Note: in comparison with the group a,*p<0.05,**p<0.01; in comparison with the group E,△p<0.05,△△p<0.01; in comparison with the group F,□p<0.05,□□p<0.01; in comparison with the group G,#p<0.05,##p<0.01
(5) changes in the signaling pathway molecules MyD88, TRAF-6, TRIF, TLR4
Molecular proteins are detected through a western blot, and images are acquired through a bioluminescent imaging system, so that the method is known as follows: compared with two groups of normal groups which are not irradiated, the expression of the TLR4 signaling pathway molecular proteins MyD88, TRAF-6, TRIF and TLR4 of the irradiated group is obviously increased; however, the expression of MyD88, TRAF-6, TRIF and TLR4 in B, C, D group rats was lower than that in A, E group. See fig. 2. And (3) displaying a result after gray value analysis: F. g, the gray value of the two normal groups is obviously lower than that of the rats irradiated with the groups (p is less than 0.01); between irradiation groups, B, C, D groups had differences in signal molecule proteins from A, E, and the gray value for MyD88 in B, C group compared to group A was p <0.05, D compared to A was p <0.01, TRAF-6 in B group compared to A was p <0.05, C, D compared to A was p < 0.01; B. c, D p <0.01 in comparison to the E signal molecule proteins. See table 4, table 5, fig. 3, fig. 4.
TABLE 4 MyD88TRAF-6 Gray values
Note: in comparison with the group a,*p<0.05,**p<0.01; in comparison with the group E,△p<0.05,△△p<0.01; in comparison with the group F,□p<0.05,□□p<0.01; in comparison with the group G,#p<0.05, ##p<0.01
TABLE 5 TRIFTLR4 grayscale values
Note: in comparison with the group a,*p<0.05,**p<0.01; in comparison with the group E,△p<0.05,△△p<0.01; in comparison with the group F,□p<0.05,□□p<0.01; in comparison with the group G,#p<0.05, ##p<0.01
(6) partial cytokine assay results
The results of Elisa measurements of the expression levels of TNF-. alpha.IL-17 and TGF-. beta.in each group are shown in Table 6. F. G, the expression levels of the cytokines TNF-alpha, IL-17 and TGF-beta of the two normal non-irradiated groups are obviously in a low level state; compared with the normal group, the expression level of TNF-alpha, IL-17 and TGF-beta of each irradiation group is obviously increased, and the difference has statistical significance (p is less than 0.01); and the expression conditions of the B, C, D group TNF-alpha, IL-17 and TGF-beta are obviously reduced compared with the A, E group, and the statistical significance is achieved (p is less than 0.01); however, there was no difference between the B, C, D groups, A, E groups (p > 0.05).
TABLE 6 Elisa test results
Note: in comparison with the group a,*p<0.05,**p<0.01; in comparison with the group E,△p<0.05,△△ p<0.01; in comparison with the group F,□p<0.05,□□p<0.01; in comparison with the group G,#p<0.05, ##p<0.01
10. conclusion
(1) The self-made proved formula (traditional Chinese medicine 2) can obviously improve the general conditions of the radiation pneumonitis rats such as mental state, hair color, appetite and the like, obviously increase the weight of the rats and improve the quality of life;
(2) the self-prepared empirical formula (traditional Chinese medicine 2) can obviously relieve the local inflammatory reaction of the lung of a rat with radiation pneumonitis, the interstitial congestion of the lung under a microscope, the exudation, the hemorrhage, the edema and the damage of the alveolar structure, and the curative effect of the radiation pneumonitis resistance is equivalent to that of hormone;
(3) the self-prepared empirical formula (traditional Chinese medicine 2) can inhibit the conduction of a downstream MyD 88-dependent signaling pathway and a downstream non-MyD 88-dependent signaling pathway of TLR4 by inhibiting an upstream signaling pathway of TLR4, thereby inhibiting the expression of cytokines of lung tissue-related cells and preventing the occurrence and development of radiation pneumonitis.
Claims (3)
1. A traditional Chinese medicine preparation for inhibiting TLR4 signal conduction is characterized by being prepared from the following raw materials in parts by weight: 30 parts of astragalus membranaceus, 30 parts of adenophora tetraphylla, 30 parts of radix glehniae, 15 parts of lily, 10 parts of dendrobe, 15 parts of aster, 15 parts of loquat leaves, 15 parts of bitter apricot seeds, 15 parts of radix stemonae and 15 parts of angelica sinensis.
2. A method for preparing a traditional Chinese medicine preparation for inhibiting TLR4 signaling according to claim 1, characterized in that the method comprises: mixing the components according to the formula amount, adding 3-4 times of water by weight, decocting at 120 ℃ for 20min, adjusting to 80 ℃ and decocting for 20min, filtering to obtain a primary filtrate and a primary filter cake, repeatedly decocting the primary filter cake once, filtering to obtain a secondary filter cake and a secondary filtrate, combining the primary filtrate and the secondary filtrate, and concentrating under reduced pressure to 1.5-2 times of volume to obtain the traditional Chinese medicine preparation.
3. The method of preparing a traditional Chinese medicine preparation for inhibiting TLR4 signaling according to claim 2, wherein the concentration under reduced pressure is performed under the conditions of 0.097MPa and 50-60 ℃.
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