A traditional Chinese medicine with the effects of strengthening spleen, soothing liver, tonifying kidney and strengthening tendons for improving muscle atrophy caused by tumor cachexia, and its preparation method and application

Abstract

The present invention discloses a Chinese medicine for improving muscle atrophy caused by tumor cachexia, which has the effects of strengthening spleen, soothing liver, tonifying kidney and strengthening tendons, as well as a preparation method and application. The Chinese medicine includes the following raw materials: Chinese yam, white atractylodes, Pseudostellaria baicalensis, citron, bergamot, malt, liquorice, dendrobium, Chinese cyperus, papaya, millennium jian, and rhizoma dioscoreae. The results of in vivo and in vitro experiments show that the Chinese medicine prescription of the present invention has an improving effect on muscle atrophy in cachectic tumor-forming mice, and the mechanism may be that the level of inflammatory factors in myoblasts is inhibited, and the IL-6 / STAT3 / ubiquitin-protein autophagosome signaling pathway is affected, thereby reducing the degradation of muscle protein and the occurrence of autophagy, increasing the weight of mice, and effectively improving the symptoms of muscle atrophy caused by tumor malignancy; in terms of composition and proportion, the Chinese medicine prescription is adjusted according to the pathogenesis characteristics of tumor malignancy, making up for the limitations of Liujunzi Decoction, and effectively improving the muscle atrophy phenomenon in tumor malignancy.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine with the effects of strengthening spleen, soothing liver, tonifying kidney and strengthening tendons for improving tumor cachexia, a preparation method and application thereof, and belongs to the field of traditional Chinese medicine. Background Art

[0002] Tumor cachexia is a paraneoplastic syndrome, which is often manifested as systemic inflammation and involuntary weight loss, gradually leading to multifunctional metabolic disorders in the body, seriously affecting the prognosis and quality of life of cancer patients. Systemic inflammation is common in cancer cachexia, and the enhanced activity of proinflammatory cytokines in the body is a significant feature of cachexia in advanced cancer patients; the inflammatory response can lead to muscle protein degradation, the occurrence of anorexia, affect the signaling pathways in cells, induce abnormal muscle protein metabolism, and then induce the occurrence of muscle atrophy in tumor cachexia. Traditional Chinese medicine believes that tumor cachexia belongs to the category of "deficiency and fatigue". At present, there is no drug specifically for the treatment of tumor malignancy in the fields of traditional Chinese and Western medicine. The present invention effectively improves tumor malignancy and its muscle atrophy symptoms based on the pathogenesis characteristics of tumor malignancy.

[0003] Interleukin-6 (IL-6) and interleukin-10 (IL-10) are typical inflammatory factors of skeletal muscle atrophy, which can regulate the growth and differentiation of various cells. Their high expression plays an important role in muscle atrophy. Inhibiting the secretion of both can reduce muscle atrophy. Many studies on muscle atrophy caused by tumor cachexia have shown that skeletal muscle protein degradation is the main cause of muscle atrophy caused by tumor cachexia, and the activation of the ubiquitin-proteasome system (UPS) is one of the main ways of skeletal muscle protein degradation. UPS is the main pathway for proteolysis in the body, and more than 80% of protein degradation in the human body is carried out with the participation of UPS. IL-6 / STAT3 signaling mediates UPS activation and plays an important role in cachexia muscle atrophy.

[0004] Another important pathway leading to muscle atrophy in tumor cachexia is the autophagy-lysosome pathway (ALP). When cells are under stress, in hypoxia, starvation, or infection by exogenous factors, autophagy will be activated, accelerating muscle protein degradation. Excessive activation of UPS and ALP can produce a synergistic effect in the pathological process of protein degradation in tumor cachexia muscle atrophy. Therefore, the two may have a consistent regulatory mechanism. In recent years, some research reports have shown that the activation of UPS and ALP is closely related to the activation of STAT3 signaling. By inhibiting the STAT3 signaling pathway, the expression of key molecules in the ubiquitin-proteasome pathway can be mediated, effectively improving muscle atrophy in lung cancer cachexia; when STAT3 is activated, it promotes cell autophagy, and the IL-6 / STAT3 / autophagy pathway plays an important role in cachexia muscle atrophy. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a traditional Chinese medicine with the effects of strengthening the spleen, soothing the liver, tonifying the kidney and strengthening the tendons for improving muscle atrophy caused by tumor cachexia, as well as a preparation method and application, which can effectively improve muscle atrophy caused by tumor cachexia.

[0006] In order to achieve the above object, one of the technical solutions of the present invention is:

[0007] A traditional Chinese medicine for improving tumor cachexia and muscle atrophy with the effects of strengthening the spleen, soothing the liver, tonifying the kidney and strengthening the tendons, comprising the following raw materials in parts by weight: 15-25 parts of yam, 10-15 parts of atractylodes macrocephala, 15-25 parts of pseudostellaria, 6-12 parts of citron, 6-12 parts of bergamot, 6-12 parts of malt, 5-8 parts of liquorice, 6-12 parts of dendrobium, 10-15 parts of cyathula, 10-15 parts of papaya, 10-20 parts of millennium genus, and 10-15 parts of rhizoma dioscoreae.

[0008] Preferably, the traditional Chinese medicine comprises the following raw materials in parts by weight: 20 parts of yam, 12 parts of atractylodes, 20 parts of pseudoginseng, 9 parts of citron, 9 parts of bergamot, 9 parts of malt, 6 parts of licorice, 9 parts of dendrobium, 12 parts of chyranthes bidentata, 12 parts of papaya, 15 parts of millennium jian, and 12 parts of rhizoma schizonepetae.

[0009] One of the technical schemes of the present invention is: a preparation method of a traditional Chinese medicine with the effects of strengthening spleen, soothing liver, tonifying kidney and strengthening tendons for improving tumor cachexia and muscle atrophy, comprising weighing each raw material medicine by weight, mixing, adding water and decocting twice, and combining the decoction.

[0010] One of the technical schemes of the present invention is: a preparation method of a traditional Chinese medicine with the effects of strengthening the spleen, soothing the liver, tonifying the kidney and strengthening the tendons for improving tumor cachexia and muscle atrophy, the raw materials are weighed by weight, ground into coarse powder, refluxed and extracted with 70% ethanol solution, the extracted liquid is filtered and concentrated, the concentrated liquid is extracted with ethyl acetate, the extract is filtered under negative pressure, rotary evaporated into a paste, and then dried and scraped in a vacuum dryer for standby use.

[0011] Furthermore, the mass ratio of the coarse powder to the ethanol solution is 1:5-10.

[0012] Furthermore, the volume ratio of the concentrated drug solution to ethyl acetate is 1-3:1.

[0013] One of the technical solutions of the present invention is: an application of the traditional Chinese medicine in preparing a drug for improving tumor cachexia and muscle atrophy.

[0014] Pharmacological effects of each raw material drug of the present invention:

[0015] Chinese Yam, sweet in taste, neutral in nature. It enters the lung, spleen, and kidney meridians. Functions and indications: Strengthen the spleen, nourish the lungs, strengthen the kidneys, and benefit the essence. Treat spleen deficiency diarrhea, chronic dysentery, consumptive cough, thirst, spermatorrhea, leucorrhea, and frequent urination. It nourishes the spleen and stomach, promotes fluid production and benefits the lungs, and nourishes the kidneys and astringes the essence. It is used for spleen deficiency, poor appetite, chronic diarrhea, asthma and cough due to lung deficiency, spermatorrhea due to kidney deficiency, leucorrhea, frequent urination, and thirst due to deficiency heat. Fried Chinese Yam with Bran Tonifies the Spleen and Strengthens the Stomach. It is used for spleen deficiency, poor appetite, diarrhea, loose stools, and excessive leucorrhea.

[0016] Atractylodes macrocephala, bitter and sweet, warm in nature. Enters the spleen and stomach meridians. Functions and indications: replenishing qi and strengthening the spleen, drying dampness and promoting diuresis, and stopping sweating. Used for spleen deficiency, poor appetite and loose stools, edema caused by phlegm and fluid retention, spontaneous sweating due to deficiency of the exterior, and restless fetal movement.

[0017] Radix Pseudostellariae is sweet, slightly bitter, and neutral in nature. It enters the spleen and lung meridians. Functions and indications: replenishing qi and strengthening the spleen, promoting fluid production and moistening the lungs. It is used for spleen deficiency, fatigue, loss of appetite, weakness after illness, insufficient qi and yin, spontaneous sweating and thirst, and dry cough due to lung dryness.

[0018] Citron tastes pungent, bitter, sour, and warm in nature. It enters the liver, spleen, and lung meridians. Functions and indications: soothing the liver and regulating qi, relieving fullness, and resolving phlegm. It is used for liver and stomach qi stagnation, chest and flank pain, abdominal distension, vomiting, belching, and coughing with excessive phlegm.

[0019] Citron tastes pungent, bitter, sour, and warm in nature. It enters the liver, spleen, stomach, and lung meridians. Functions and indications: Soothes the liver and regulates qi, harmonizes the stomach and relieves pain, and dries dampness and resolves phlegm. It is used for liver and stomach qi stagnation, chest and flank pain, fullness in the stomach, loss of appetite and vomiting, and cough with excessive phlegm.

[0020] Malt is sweet and neutral in nature. It enters the spleen and stomach meridians. Functions and indications: promotes qi and digestion, strengthens the spleen and stimulates appetite, relieves milk and distension. It is used for indigestion, abdominal distension and pain, spleen deficiency and loss of appetite, milk and sweat accumulation, breast pain, and weaning of women. Raw malt strengthens the spleen and stomach and relieves milk. It is used for spleen deficiency and loss of appetite, milk and sweat accumulation.

[0021] Licorice root, sweet in taste, neutral in nature. It enters the heart, lung, spleen, and stomach meridians. Functions and indications: tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving pain, and harmonizing various medicines. It is commonly used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with sputum, abdominal and limb cramps and pain, carbuncle, sore and ulcer, and relieving drug toxicity and potency.

[0022] Dendrobium officinale is sweet and slightly cold in nature. It enters the stomach and kidney meridians. Functions and indications: nourishes the stomach and produces body fluid, nourishes yin and clears away heat. It is used for damage to body fluid caused by febrile diseases, dry mouth and thirst, insufficient stomach yin, poor appetite and dry retching, persistent deficiency heat after illness, yin deficiency and excessive fire, bone steaming and fatigue fever, blurred vision, and weakness of tendons and bones.

[0023] Rhizoma Cynanchum, slightly bitter and sour in taste, cold in nature. Enters the liver and kidney meridians. Functions and indications: promoting blood circulation and dispersing blood stasis, removing dampness and promoting urination, clearing away heat and detoxifying. Treats gonorrhea, hematuria, amenorrhea, abdominal masses, rheumatism, joint pain, beriberi, edema, dysentery, malaria, diphtheria, carbuncle, and traumatic injuries.

[0024] Papaya is sour and warm in nature. It enters the liver and spleen meridians. Functions and indications: relaxes muscles and activates collaterals, harmonizes the stomach and eliminates dampness. It is used to treat dampness, stiffness and cramps, soreness and pain in the waist and knee joints, vomiting and diarrhea due to summer heat and dampness, cramps and pain, and beriberi and edema.

[0025] Qiannianjian, bitter, pungent, warm in nature. Enters the liver and kidney meridians. Functions and indications: dispel rheumatism, strengthen muscles and bones. Used for wind-cold-dampness arthralgia, cold pain in the waist and knees, cramps and numbness, and weakness of muscles and bones.

[0026] Rhizoma Dioscoreae, sweet and bitter in taste, warm in nature. Enters the liver, kidney and lung meridians. Functions and indications: dispel wind and dampness, relax muscles and tendons, promote blood circulation and relieve pain, relieve cough and asthma. Used for rheumatism, joint numbness, injuries from falls, sprains, shortness of breath, cough and asthma.

[0027] In this prescription, Chinese yam can invigorate the spleen and benefit the kidney, white atractylodes can invigorate the spleen and benefit the qi, and can eliminate dampness and dispel wind. The two can invigorate the spleen and benefit the qi, and are the main drugs; citron and bergamot can soothe the liver, regulate qi and harmonize the stomach, dendrobium can benefit the stomach, nourish yin and eliminate numbness, and Pseudostellariae can benefit the qi and invigorate the spleen. The four drugs have the functions of soothing the liver, invigorating the spleen and harmonizing the stomach, and are the minister drugs; papaya can harmonize the stomach and eliminate dampness, soothe the liver and relax the tendons, Achyranthes bidentata can nourish the liver and kidney, Qiannianjian can eliminate rheumatism, strengthen the tendons and bones, and Rhizoma Dioscoreae can eliminate wind, dampness, activate blood circulation and dredge the collaterals. The four drugs are adjuvant drugs, malt can invigorate the spleen, soothe the liver and harmonize the stomach, and licorice can invigorate the spleen, benefit the qi and detoxify, and harmonize the drugs. The drugs are used together to play the effects of invigorating the spleen and soothing the liver, and strengthening the kidney and strengthening the tendons.

[0028] The invention is composed of pure Chinese medicine, has significant efficacy and few side effects, and the Chinese medicine raw materials of the invention are abundant, the preparation process is simple, and the cost is low. It is an innovation in drugs for treating tumor cachexia and has a good market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Western blotting was used to detect the protein expression of MyOD in C2C12 cells in each group. In the figure, A is the blank group and B is the model group.

[0030] Figure 2 is the expression level of key proteins in the IL-6 / STAT3 signaling pathway in C2C12 cells. In the figure, A is the blank group, B is the model group, C is the Liujunzi Decoction group, and D is the Chinese medicine prescription group.

[0031] Figure 3 Comparison of the general conditions of mice in each group.

[0032] Figure 4 The effect of Chinese herbal medicine on the cross-sectional area of ​​gastrocnemius muscle fibers (400×).

[0033] Figure 5 This is the effect of the Chinese medicine prescription on the gastrocnemius muscle fibers (the arrow points to the dark band in the gastrocnemius muscle fibers, and the light band is between two adjacent dark bands).

[0034] Figure 6This is the effect of the Chinese medicine prescription on the autophagosome of gastrocnemius muscle (the arrow indicates the autophagosome). DETAILED DESCRIPTION

[0035] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Example 1

[0037] A traditional Chinese medicine for improving tumor cachexia and muscle atrophy with the effects of invigorating the spleen, soothing the liver, tonifying the kidney and strengthening the tendons, comprising the following raw materials in parts by weight: 20 parts of yam, 12 parts of atractylodes macrocephala, 20 parts of pseudostellaria, 9 parts of citron, 9 parts of bergamot, 9 parts of malt, 6 parts of liquorice, 9 parts of dendrobium, 12 parts of cyathula capitata, 12 parts of papaya, 15 parts of millennium ginseng and 12 parts of rhizoma dioscoreae.

[0038] The preparation method comprises the following steps: weighing each raw material drug by weight, grinding it into coarse powder, adding 70% (v / v) ethanol solution of 8 times the weight of the coarse powder for reflux extraction, filtering the extracted drug solution and concentrating it, adding ethyl acetate into the concentrated drug solution in a volume ratio of 2:1 and mixing it evenly in a separating funnel for extraction, filtering the extract liquid under negative pressure, rotary evaporating it into a paste, drying it in a vacuum dryer and scraping it for later use.

[0039] Example 2

[0040] A traditional Chinese medicine for improving tumor cachexia and muscle atrophy with the effects of invigorating the spleen, soothing the liver, tonifying the kidney and strengthening the tendons, comprising the following raw materials in parts by weight: 15 parts of yam, 10 parts of atractylodes macrocephala, 15 parts of pseudostellaria, 6 parts of citron, 6 parts of bergamot, 6 parts of malt, 5 parts of liquorice, 6 parts of dendrobium, 10 parts of cyathula, 10 parts of papaya, 10 parts of millennium genus, and 10 parts of rhizoma dioscoreae.

[0041] The preparation method is as follows: weigh each raw material medicine according to weight, mix them, add water and boil them twice, and combine the decoctions.

[0042] Example 3

[0043] A traditional Chinese medicine for improving tumor cachexia and muscle atrophy with the effects of invigorating the spleen, soothing the liver, tonifying the kidney and strengthening the tendons, comprising the following raw materials in parts by weight: 25 parts of yam, 15 parts of atractylodes macrocephala, 25 parts of pseudostellaria, 12 parts of citron, 12 parts of bergamot, 12 parts of malt, 8 parts of liquorice, 12 parts of dendrobium, 15 parts of cyathula, 15 parts of papaya, 20 parts of millennium ginseng and 15 parts of rhizoma dioscoreae.

[0044] The preparation method is the same as that of Example 1.

[0045] The present invention explores the influence of Chinese medicinal prescriptions on the secretion of inflammatory factors in vitro, and at the same time explores the influence of Chinese medicinal prescriptions on the malignant transformation of mouse tumors through mouse animal experiments.

[0046] In vitro experiment

[0047] 1 Cell experiment

[0048] The Chinese medicine slices of the Chinese medicine prescription of the present invention were purchased from Beijing Tong Ren Tang, and were all authentic products identified by the Chinese Medicine Laboratory of Henan University of Traditional Chinese Medicine. The Chinese medicine prescription was prepared according to the method of Example 1. DMEM medium (without serum) was used to prepare the required drug concentration, and filtered and sterilized for subsequent experiments. Liujunzi Decoction composition: 9 grams of ginseng, 9 grams of Atractylodes macrocephala, 9 grams of Poria, 6 grams of roasted licorice, 3 grams of dried tangerine peel, and 4.5 grams of Pinellia tuber.

[0049] 1.1 Establishment of myotube differentiation model

[0050] C2C12 cells were induced to differentiate for 48 h in DMEM medium containing 2% horse serum to establish a tumor cachexia myotube atrophy model.

[0051] 1.2 Model evaluation: Western blotting was used to detect the expression of MyOD (myogenic differentiation protein), a marker protein for C2C12 cell differentiation in the model group

[0052] C2C12 cells were inoculated into the lower chamber of a six-well Transwell co-culture plate, and induced to differentiate with 2% horse serum DMEM medium. A blank group and a model group were set up. In addition to the blank group, the model group was inoculated with the same number of Lewis cells in the upper chamber. After 48 hours of continuous culture, the total protein of C2C12 cells in each group was extracted, and the protein concentration was detected using a BCA protein quantification kit. Electrophoresis, transfer, blocking, incubation with primary antibody (MyOD 1: 4000) and secondary antibody (goat anti-rabbit GAPDH 1: 10000) were performed, and the gray value of the target band was developed and analyzed.

[0053] Table 1 Protein expression of MyOD in C2C12 cells in each group detected by Western blotting

[0054]

[0055] Note: * Compared with the blank control group, P < 0.05, # Compared with the model group, P<0.05

[0056] Myocyte regulatory factor 1 (MyoD), as a member of the myogenic factor (MRFs) family, is a driving transcription factor for myogenesis and a marker protein for early skeletal muscle differentiation. It plays an important role in skeletal muscle hypertrophy and growth, and can induce myocyte proliferation and differentiation. The level of MyoD increases most significantly during proliferation. In this study, when C2C12 cells were co-cultured with lung adenocarcinoma Lewis cells at a ratio of 1:1, the protein level of MyoD in the model group was significantly reduced compared with the blank group, indicating that the model was successfully constructed (see Table 1 and Figure 1 ).

[0057] 2. Index detection

[0058] 2.1 Grouping and Dosing

[0059] C2C12 cells were inoculated in the lower chamber of the Transwell co-culture plate, and when they grew to about 60%-80%, 2% horse serum DMEM medium was added to induce differentiation, and then grouped. Each group was added with drugs according to the dosing method in Table 2. Three duplicate wells were set up in each of the four groups. Lewis cells were inoculated in the upper chamber of the co-culture plate at a cell number of 1:1 with C2C12 cells. The final concentration of the Chinese medicine prescription and Liujunzi Decoction group was 100μg / mL, and the final volume was 2.6mL in the lower chamber and 1.5mL in the upper chamber. After 48h of culture in this study, the indicators were detected.

[0060] Table 2 Transwell co-culture upper and lower chamber fluid addition

[0061]

[0062] 2.2 Effect of Chinese herbal medicine on the contents of IL-6 and IL-10 in Lewis / C2C12 co-culture supernatant

[0063] A Lewis / C2C12 cell co-culture system was constructed (same method as 2.1). After culturing for 48 h, the cell supernatant was collected. IL-6 and IL-10 ELISA kits were used respectively according to the instructions of the ELISA kits. The microplate reader was adjusted to 450 nm to detect the OD value.

[0064] The test results showed (Table 3) that compared with the blank group, the IL-6 and IL-10 levels in the model group were significantly increased (P<0.05); compared with the model group, the IL-6 and IL-10 levels in the Liujunzi decoction group and the Chinese medicine prescription group were significantly decreased (P<0.05), indicating that both Liujunzi decoction and the Chinese medicine prescription can inhibit and reduce the content of inflammatory factors in cells; compared with the Liujunzi decoction group, the IL-6 and IL-10 levels in the Chinese medicine prescription group were significantly reduced (P<0.05), indicating that the Chinese medicine prescription group has a more significant inhibitory effect on the secretion of inflammatory factors by tumor cells than Liujunzi decoction.

[0065] Table 3 Contents of IL-10 and IL-6 in the supernatant of each group (unit: pg / mL, n=3)

[0066]

[0067] Note: *Compared with the blank group, P < 0.05, # Compared with the model group, P<0.05, ▲ Compared with Liujunzi decoction group, P<0.05

[0068] 2.3 Effects of the Chinese medicine prescription group on the expression of STAT3, Beclin1, P62, and LC3B / LC3A proteins in the IL-6 / STAT3 pathway in C2C12 cells under co-culture conditions

[0069] In cells, when the expression of LC3B protein exceeds the expression level of LC3A, that is, when the ratio of the two is greater than 1, the cell initiates downstream autophagy. The larger the LC3B / LC3A ratio, the more obvious the degree of autophagy in the cell. The Western blot experiment was used to detect the expression of STAT3, Beclin1, P62, and LC3B / LC3A proteins in the IL-6 / STAT3 pathway in C2C12 cells under co-culture conditions. The results showed that compared with the blank group, the expression levels of STAT3, Beclin1, P62, and LC3B / LC3A proteins in the model group were increased (P<0.05), indicating that autophagy was activated in the tumor cachexia muscle atrophy model and could promote the muscle protein degradation process of tumor cachexia; compared with the model group, the expression levels of STAT3, P62, and LC3B / LC3A proteins in the Liujunzi decoction group were significantly decreased (P<0.05), and the expression levels of STAT3, Beclin1, P62, and LC3B / LC3A proteins in the Chinese medicine prescription group were significantly decreased (P<0.05), indicating that both the Liujunzi decoction and the Chinese medicine prescription groups reduced the occurrence of autophagy, indicating that the overactivated autophagy-lysosome system in tumor cells can be reversed under drug treatment. Combined with the above IL-6 and STAT3 content and expression results, it shows that Liujunzi decoction and Chinese medicine prescription group can reduce the content of IL-6, IL-10 and other inflammatory factors in cells, reduce the activation of STAT3 signal, thereby reducing the expression of key molecules in the autophagy signaling pathway, reducing muscle protein degradation caused by autophagy, and playing a role in treating tumor cachexia muscle atrophy; compared with the Liujunzi decoction group, the expression of STAT3 and LC3B / LC3A in the Chinese medicine prescription group was significantly reduced (P<0.05), and Beclin1 and P62 also showed a downward trend, indicating that the Chinese medicine prescription group has a more obvious inhibitory effect on the occurrence of autophagy (see Table 4 and Figure 2 ).

[0070] Table 4 STAT3, Beclin1, P62, LC3B / LC3A contents under co-culture conditions

[0071]

[0072] Note: *Compared with the blank group, P < 0.05, # Compared with the model group, P<0.05, ▲ Compared with Liujunzi decoction group, P<0.05

[0073] 2. Animal Experiments on Mice

[0074] The two main characteristics of tumor cachexia are weight loss and muscle atrophy. Weight is an important observation indicator in the clinical diagnosis of tumor cachexia and is related to the degree of muscle atrophy in patients. In this experiment, Lewis cells were subcutaneously injected into the right anterior armpit of mice to replicate the classic animal model of muscle atrophy caused by lung cancer cachexia, and the effect of Chinese herbal medicine on the body weight and gastrocnemius muscle of tumor-bearing mice was observed.

[0075] 2.1 Preparation of Lewis lung cancer cachexia muscle atrophy mouse model

[0076] Collect Lewis cells in the logarithmic growth phase and adjust the cell concentration to 10 7 The mice were randomly numbered, 8 mice were used as the blank group, and the other 24 mice were injected with Lewis cells in the right anterior axilla to prepare the lung cancer cachexia muscle atrophy model, 0.2 mL / mouse.

[0077] 2.2 Animal Experiment Grouping Design and Processing

[0078] The tumor volume and body weight of mice were recorded weekly. On the eighth day of modeling, 24 mice were randomly divided into model group, Liujunzi Decoction group, and Chinese medicine prescription group, with 8 mice in each group. The administration route of each experimental group was: on the eighth day of modeling, the blank group and model group were given normal saline, the Chinese medicine prescription group was given Chinese medicine liquid, and the Liujunzi Decoction group was given Liujunzi Decoction by gavage, once a day, for three weeks. The dosage was calculated according to the following formula and compared with the blank group.

[0079] The dosage for mice = the commonly used clinical dosage for humans × the animal equivalent dosage coefficient 9.1 (based on body surface area), each mouse weighs 20 g, and the daily gavage volume is 0.2 mL / time.

[0080] Three weeks after administration, mice were killed by dislocating the cervical vertebrae, and the weight of the gastrocnemius muscles of both hind limbs was accurately weighed on an analytical balance. The gastrocnemius muscles of 6 mice in each group were placed in a test tube containing 4% paraformaldehyde solution and stored at room temperature. After tissue sampling, dehydration, paraffin embedding and sectioning, HE staining was performed and observed under a microscope; photos were taken under an inverted microscope (Olympus, CX31), and the Image pro plus6.0 image acquisition system was used to circle the cross section of the muscle fibers to calculate the total area of ​​the unit section under the 400-fold field of view, and the average and standard deviation of the cross section of the muscle fibers were calculated according to the number of muscle fibers. Electron microscope sample processing: Quickly cut a thin slice with a thickness of less than 1 mm from the gastrocnemius muscle that had just been separated from the body, fix it with a 4-5°C fixative, and observe it under an electron microscope after dehydration, infiltration, embedding polymerization, sectioning and staining.

[0081] 2.3 Experimental Results

[0082] Observation of general condition of mice: On the 7th day of modeling, an obvious mass could be felt under the right armpit skin of the mice, and the tumor volume was 0.725±0.398cm 3 Compared with the blank group, the model mice showed obvious dull hair, weak limbs, reduced activity, and weight loss, showing symptoms of tumor malignancy ( Figure 3 ).

[0083] Body weight test: Compared with the blank group, the body weight of mice in the model group was significantly decreased (P < 0.05); compared with the model group, the body weight of mice in the Liujunzi Decoction group and the Chinese medicine group was significantly increased (P < 0.05), and there was no significant difference in the body weight of mice between the Chinese medicine group and the Liujunzi Decoction group (P > 0.05), but the effect of the Chinese medicine group was better (Table 5).

[0084] Detection of gastrocnemius muscle weight and muscle fiber cross-sectional area in mice: Compared with the blank group, the gastrocnemius muscle weight and muscle fiber cross-sectional area in the model group were significantly reduced (P < 0.05), the gastrocnemius muscle fiber morphology became smaller and thinner, and the number of cell nuclei decreased; Compared with the model group, the gastrocnemius muscle weight and muscle fiber cross-sectional area in the Liujunzi Decoction group and the Chinese medicine group were significantly increased (P < 0.05), and there was no significant difference in gastrocnemius muscle weight and muscle fiber cross-sectional area between the Chinese medicine group and the Liujunzi Decoction group (P > 0.05), but the effect of the Chinese medicine group was better (see Table 5 and Figure 4 ).

[0085] Table 5 Effects of Chinese herbal medicine on body weight, gastrocnemius muscle and cross-sectional area of ​​mice with lung cancer cachexia

[0086]

[0087] Note: *Compared with the blank group, P < 0.05, # Compared with the model group, P<0.05, ▲ Compared with Liujunzi decoction group, P<0.05

[0088] Electron microscopic examination of gastrocnemius muscle of mice: Compared with the blank group, the arrangement of muscle fibers in the gastrocnemius muscle of the model group was scattered under the 6000x field of view, the boundary between cells was widened, the distribution of light and dark bands was uneven, and the arrangement of muscle fiber stripes was irregular; compared with the model group, the arrangement of muscle fibers in the Liujunzi decoction group and the inhibitor group was more orderly under the 6000x field of view, the cell gap was tighter, the distribution of light and dark bands was more uniform, and the arrangement of muscle fiber stripes was more orderly ( Figure 5 ).

[0089] Electron microscopic examination of autophagosomes in gastrocnemius muscle of mice: compared with the blank group, the number of autophagosomes in gastrocnemius muscle of model group increased significantly at 50,000 times field of view (P < 0.05); compared with the model group, the number of autophagosomes in gastrocnemius muscle of Liujunzi decoction group and Chinese medicine prescription group decreased significantly at 50,000 times field of view (P < 0.05), and the number of autophagosomes in gastrocnemius muscle of Chinese medicine prescription group was also significantly lower than that of Liujunzi decoction group (P < 0.05) (Table 6 and Figure 6 ).

[0090] Table 6 Effects of Chinese herbal medicine on gastrocnemius autophagosomes

[0091]

[0092] Note: * Compared with the blank group, P < 0.05; # Compared with the model group, P < 0.05; ▲ Compared with Liujunzi decoction group, P<0.05

[0093] The results of in vivo mouse experiments showed that the Chinese medicine prescription can effectively reduce the occurrence of autophagy in the gastrocnemius muscle of mice, improve the mass of the gastrocnemius muscle of mice, increase the weight of mice, and improve the malignant degeneration and muscle atrophy symptoms of tumor-bearing mice.

[0094] Conclusion

[0095] The above in vivo and in vitro experimental results show that the Chinese herbal medicine formula and Liujunzi Decoction both have an improving effect on muscle atrophy in cachectic tumor-bearing mice. The mechanism may be that it inhibits the level of inflammatory factors in myoblasts and affects the IL-6 / STAT3 / ubiquitin-protein autophagosome signaling pathway, thereby reducing the degradation of muscle protein and the occurrence of autophagy, increasing the weight of mice, and effectively improving the symptoms of muscle atrophy caused by tumor malignancy. In terms of composition and ratio, the Chinese herbal medicine formula has been adjusted according to the pathogenesis characteristics of tumor malignancy, which makes up for the limitations of Liujunzi Decoction and effectively improves the muscle atrophy phenomenon in tumor malignancy.

Claims

1. A Chinese medicine for improving muscle atrophy caused by tumor cachexia, which has the effects of strengthening the spleen, soothing the liver, tonifying the kidney and strengthening the tendons, characterized in that: The invention is prepared from the following raw materials in parts by weight: 15-25 parts of yam, 10-15 parts of atractylodes macrocephala, 15-25 parts of pseudoginseng, 6-12 parts of citron, 6-12 parts of bergamot, 6-12 parts of malt, 5-8 parts of liquorice, 6-12 parts of dendrobium, 10-15 parts of cyathulae, 10-15 parts of papaya, 10-20 parts of millennium scutellaria, and 10-15 parts of rhizoma dioscoreae.

2. The Chinese medicine according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 20 parts of yam, 12 parts of atractylodes macrocephala, 20 parts of pseudoginseng, 9 parts of citron, 9 parts of bergamot, 9 parts of malt, 6 parts of licorice, 9 parts of dendrobium, 12 parts of cyathulae, 12 parts of papaya, 15 parts of millennium jian, and 12 parts of rhizoma schizonepetae.

3. A method for preparing the Chinese medicine according to any one of claims 1 or 2, characterized in that: Weigh each raw material according to weight, mix, add water and boil twice, and combine the decoctions.

4. A method for preparing the Chinese medicine according to any one of claims 1 or 2, characterized in that: Weigh each raw material drug by weight, grind it into coarse powder, add 70% ethanol solution to reflux extraction, filter the extracted drug solution and then concentrate it, extract the concentrated drug solution with ethyl acetate, filter the extract under negative pressure, rotary evaporate it into a paste, dry it in a vacuum dryer and scrape it for later use.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the coarse powder to the ethanol solution is 1:5-10.

6. The preparation method according to claim 4, characterized in that: The volume ratio of the concentrated drug solution to ethyl acetate is 1-3:

1.

7. Use of the traditional Chinese medicine according to any one of claims 1 or 2 in the preparation of a drug for improving tumor cachexia and muscle atrophy.

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