Traditional Chinese medicine composition for improving tumor cachexia muscle atrophy and its preparation method and application

Abstract

The invention discloses a Chinese medicine composition for improving muscle atrophy caused by tumor cachexia, a preparation method and an application thereof. The Chinese medicine composition is mainly composed of the following raw materials by weight: 25-35g of Astragalus, 8-14g of Angelica, 8-14g of White Peony, 6-8g of Licorice, 8-14g of Cinnamomum Cassia, 8-14g of Epimedium, 10-12g of Morinda Officinalis, 18-22g of Agrimoniae, 8-12g of Jujube, 8-14g of Rhizoma Anemarrhenae, 5-8g of Phellodendron, 6-10g of Paris Polygoni Multiflori, 18-22g of Caulis Sargentodoxae, and 18-22g of Caulis Spatholobi. The Chinese medicine composition of the invention uses pure natural Chinese medicine raw materials, has a wide range of raw material sources, has no toxic side effects, can effectively improve the situation of muscle atrophy in patients with tumor cachexia, prevent and treat weight loss in lung cancer cachexia, improve the therapeutic effect and quality of life of tumor patients, and prolong the survival time of patients. It is a major innovation in treating tumor cachexia with Chinese medicine.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine composition for improving muscle atrophy caused by 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 that often manifests as systemic inflammation and involuntary weight loss, which gradually leads to multifunctional metabolic disorders in the body, seriously affecting the prognosis and quality of life of cancer patients. According to statistics, 50%-80% of cancer patients will experience cachexia, leading to a mortality rate of up to 30%. Among them, muscle atrophy is the most important factor leading to weight loss and is also the most common symptom in the late stage of cancer cachexia patients.

[0003] Tumor cachexia belongs to the category of "deficiency" in traditional Chinese medicine. It is believed that tumor cachexia is mostly caused by deficiency of qi and blood and imbalance of yin and yang. Tumor cachexia is mostly seen in the middle and late stages of tumors. The disease consumes human qi and blood over time, and the cancer toxins produced by chemotherapy, radiotherapy or tumors are prone to spleen and stomach weakness in traditional Chinese medicine, and the biochemical state of qi and blood is sourceless, and qi and blood are more prone to weakness. Qi is yang, and blood is yin. If qi and blood are weak for a long time, yin and yang will lose harmony. At the same time, long-term illness and kidneys are also prone to kidney essence deficiency and imbalance of yin and yang. Traditional Chinese medicine believes that solid tumors are mostly blocked by phlegm and blood stasis, which produces cancer toxins for a long time, damages yin and consumes qi, and local stasis is prone to heat for a long time, which further aggravates the disharmony of yin and yang in the body. Therefore, the inventor believes that replenishing qi and blood, harmonizing yin and yang, and promoting blood circulation and detoxification are important methods for treating tumor cachexia. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a Chinese medicine composition (Chinese medicine prescription) for improving muscle atrophy caused by tumor cachexia and its preparation method and application, which can effectively improve the progression of muscle atrophy in patients with tumor cachexia, prevent and treat weight loss in patients with lung cancer cachexia, improve treatment effects and quality of life, and prolong patient survival time.

[0005] In order to achieve the above object, one of the technical solutions of the present invention is:

[0006] The traditional Chinese medicine composition for improving muscle atrophy caused by tumor cachexia is mainly composed of the following raw materials by weight: 25-35g of Astragalus, 8-14g of Angelica, 8-14g of White Peony, 6-8g of Licorice, 8-14g of Cinnamon Twig, 8-14g of Epimedium, 10-12g of Morinda Officinalis, 18-22g of Agrimoniae, 8-12g of Jujube, 8-14g of Anemarrhena, 5-8g of Phellodendron, 6-10g of Paris polyphylla, 18-22g of Sargentodoxa, and 18-22g of Millettia reticulata.

[0007] One of the technical solutions of the present invention is: a method for preparing a traditional Chinese medicine composition for improving muscle atrophy caused by tumor cachexia, which can be made into a decoction, a pill, a tablet, a granule, or an ointment.

[0008] Furthermore, the decoction is prepared by mixing the raw materials and decocting them in water.

[0009] Furthermore, the preparation method of the ointment is as follows: 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 concentrate it, extract the concentrated drug solution with ethyl acetate, filter the extract solution under negative pressure, rotary evaporate it into a paste, dry it in a vacuum dryer and scrape it for later use.

[0010] Furthermore, the mass ratio of the coarse powder to the ethanol solution is 1:5-10.

[0011] Furthermore, the volume ratio of the concentrated drug solution to ethyl acetate is 1-3:1.

[0012] One of the technical solutions of the present invention is: an application of the traditional Chinese medicine composition in the preparation of a drug for improving tumor cachexia and muscle atrophy.

[0013] Pharmacological effects of each raw material drug of the present invention:

[0014] Astragalus is sweet and slightly warm in nature. It enters the spleen and lung meridians. It has the effects of strengthening the spleen and replenishing the middle, raising yang and lifting the sunken, strengthening the body's defense and strengthening the exterior, promoting urination, and removing toxins and promoting tissue regeneration. It is mainly used to treat spleen qi deficiency syndrome, lung qi deficiency syndrome, qi deficiency and spontaneous sweating syndrome, qi and blood deficiency, sores that are difficult to heal or ulcers that are difficult to heal for a long time, etc.

[0015] Angelica sinensis is sweet and pungent in taste and warm in nature. It enters the liver, heart and spleen meridians. It has the effects of nourishing blood and activating blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relieving constipation. It is often used for blood deficiency and sallow complexion, dizziness and palpitations, irregular menstruation, amenorrhea and dysmenorrhea, abdominal pain due to deficiency and cold, rheumatic arthralgia, injuries from falls, carbuncle, ulcers, and constipation due to dry intestines. Wine Angelica sinensis activates blood circulation and regulates menstruation. It is used for amenorrhea and dysmenorrhea, rheumatic arthralgia, and injuries from falls.

[0016] White peony root has a mild smell, a slightly bitter and sour taste, and a slightly cold nature. It enters the liver and spleen meridians. It has the effects of warming the yang and removing dampness, replenishing the body, and strengthening the spleen and stomach. It is used to treat rheumatic cold pain, rheumatoid arthritis, lumbar muscle strain, physical weakness, limb convulsions, slow convulsions, dog bites, etc.

[0017] Licorice root is sweet and neutral in nature. It enters the heart, lung, spleen and stomach meridians. It has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving pain, and harmonizing various medicines. It is often used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with sputum, abdominal and limb cramps and pain, carbuncle, sore and poison, and relieves drug toxicity and potency.

[0018] Cinnamon twig, pungent, sweet, warm in nature; enters the lung, heart, and bladder meridians. It has the effects of sweating and relieving muscles, warming and unblocking the meridians, assisting yang and transforming qi, and balancing and descending qi. It is commonly used for colds, cold pain in the abdomen, amenorrhea due to cold blood, joint pain, phlegm, edema, palpitations, etc.

[0019] Epimedium is pungent, sweet and warm in nature. It enters the liver and kidney meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is used to treat impotence, dribbling urine, weakness of waist and knees, cold and dampness, and other symptoms.

[0020] Morinda officinalis is sweet and spicy, slightly warm in nature, and enters the kidney and liver meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is often used for impotence and spermatorrhea, uterine coldness and infertility, irregular menstruation, cold pain in the lower abdomen, rheumatic pain, and weakness of tendons and bones.

[0021] Agrimony, bitter, astringent, neutral in nature; enters the heart and liver meridians. It has the effects of astringent hemostasis, antimalarial, antidysentery, detoxification, and tonifying deficiency. It is used for hemoptysis, hematemesis, metrorrhagia, malaria, bloody dysentery, carbuncle, sore, vaginal itching, leucorrhea, and fatigue.

[0022] Jujube, sweet in taste, warm in nature; enters the spleen and stomach meridians. It has the effects of tonifying the spleen and stomach, replenishing qi and promoting body fluid, regulating the body's defense system, and detoxifying drugs. It is used for poor appetite due to weak stomach, loose stools due to weak spleen, insufficient qi, blood and body fluid, disharmony between the body's defense system, palpitations, and impatience in women.

[0023] Anemarrhena, bitter in taste, cold in nature; enters the lung, stomach, and kidney meridians. It has the effects of clearing away heat and purging fire, nourishing yin and moistening dryness. It is used for fever, thirst, lung heat and dry cough, bone steaming and tidal fever, internal heat and thirst, and dry intestines and constipation.

[0024] Phellodendron chinense is bitter and cold in nature. It enters the kidney and bladder meridians. It has the effects of clearing away heat and dampness, purging fire and removing steam, detoxifying and curing sores. It is used for damp-heat diarrhea, jaundice and red urine, vaginal itching, hot stranguria, beriberi, bone steaming, fatigue and fever, night sweats, spermatorrhea, sores, swelling and poison, eczema and wet sores.

[0025] Paris polyphylla, bitter, slightly cold; enters the liver meridian. It has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain, cooling the liver and calming the nerves. It is used for furuncle, carbuncle, sore throat, snake and insect bites, falls and injuries, and convulsions.

[0026] Sargentodendron chinense is bitter and neutral in nature. It enters the large intestine and liver meridians. It has the effects of clearing away heat and detoxifying, promoting blood circulation, dispelling wind and relieving pain. It is used for abdominal pain caused by intestinal carbuncle, ulcers caused by heat and toxins, amenorrhea, dysmenorrhea, swelling and pain caused by falls, and rheumatic pain.

[0027] Millettia reticulata is bitter, sweet and warm; it enters the liver and kidney meridians. It has the effects of promoting blood circulation, tonifying blood, regulating menstruation and relieving pain, and relaxing tendons and activating collaterals. It is also effective for rheumatic arthralgia, numbness and paralysis, blood deficiency and sallow complexion, irregular menstruation, dysmenorrhea, amenorrhea and gynecological diseases.

[0028] This prescription has the effects of nourishing qi and blood, harmonizing yin and yang, and promoting blood circulation and detoxification. Among them, Astragalus membranaceus is sweet and warm in nature, which can nourish the qi of the lungs and spleen; Cinnamon twig is pungent, sweet and warm in nature, warms the yang and dredges the collaterals, relieves muscles and expels evil, and both warm and nourish the yang qi, making the yang qi sufficient and the haze disappears by itself, and they are all monarch drugs. Angelica sinensis is pungent and warm, nourishes blood and promotes blood circulation; White peony root is sour and cool in nature, nourishes yin and blood, and relieves cramps; Anemarrhena asphodeloides is bitter, sweet and cold, promotes fluid and moisturizes dryness; Epimedium and Morinda officinalis are pungent, sweet and warm, nourish kidney yang, and strengthen muscles and bones; the combination of the five medicines has the effect of cooperating with the monarch drug to warm yang, nourish blood and nourish yin, and they are all minister drugs. Phellodendron chinense is bitter and cold, clears heat, dries dampness and detoxifies, and is used together with Anemarrhena asphodeloides to prevent and treat excessive warming of yang and dryness; Millettia reticulata nourishes blood and dredges the collaterals, Caulis spatholobi promotes blood circulation and detoxifies, Paris polyphylla detoxifies and reduces swelling, and Herba Agrimoniae astringes, stops bleeding and replenishes qi. The combination of the five medicines not only assists the minister drug in nourishing blood, nourishing yin and replenishing qi, but also has the effect of promoting blood circulation and detoxifying, and they are all adjuvant drugs. Jujubes are sweet and warm, nourishing the spleen and stomach; licorice is sweet and cool, nourishing the spleen and stomach, clearing away heat and detoxifying; both of them nourish the spleen and stomach, prevent and treat drug damage, and make the production of qi and blood active. At the same time, they have the function of harmonizing various medicines, and are collectively called guiding medicines.

[0029] The Chinese medicine composition of the present invention uses pure natural Chinese medicine raw materials, which have a wide source and are non-toxic and have no side effects. It can effectively improve the muscle atrophy of patients with tumor cachexia, prevent and treat weight loss caused by lung cancer cachexia, improve the treatment effect and quality of life of tumor patients, and prolong the survival time of patients. It is a major innovation in the treatment of tumor cachexia with Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The myotube differentiation of C2C12 cells was observed under an inverted microscope at different times (100×).

[0031] Figure 2 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.

[0032] Figure 3 Transmission electron microscopy was used to observe the ultrastructure of mitochondria in each group of cells (×30000).

[0033] Figure 4 Flow cytometry was used to observe the effects of drugs on mitochondrial membrane potential of C2C12 cells in each group.

[0034] Figure 5 Immunoblotting was used to detect the expression of PGC-1α / FoxO1 pathway-related proteins in C2C12 cells in each group. In the figure, A is the blank group, B is the model group, C is the Chinese medicine group, D is the inhibitor group, and E is the combined medication group.

[0035] Figure 6 Shown are the appearance pictures of mice in each group.

[0036] Figure 7 The cross sections of the gastrocnemius muscles of mice in each group (400×). DETAILED DESCRIPTION

[0037] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0038] Example 1

[0039] The traditional Chinese medicine composition for improving muscle atrophy caused by tumor cachexia is mainly composed of the following raw materials by weight: 30g of Astragalus, 10g of Angelica, 10g of White Peony, 7g of Licorice, 10g of Cinnamon Twig, 10g of Epimedium, 11g of Morinda Officinalis, 20g of Agrimoniae, 10g of Jujube, 10g of Anemarrhena, 7g of Phellodendron, 8g of Paris polyphylla, 20g of Sargentodoxa, and 20g of Millettia reticulata.

[0040] 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 under negative pressure, rotary evaporating it into a paste, drying it in a vacuum dryer and scraping it for later use.

[0041] Example 2

[0042] The traditional Chinese medicine composition for improving muscle atrophy caused by tumor cachexia is mainly composed of the following raw materials by weight: 25g of Astragalus, 8g of Angelica, 8g of White Peony, 6g of Licorice, 8g of Cinnamon Twig, 8g of Epimedium, 10g of Morinda Officinalis, 18g of Agrimoniae, 8g of Jujube, 8g of Anemarrhena, 5g of Phellodendron, 6g of Paris Polygoni Multiflori, 18g of Sargentodoxae, and 18g of Millettia Reticulata.

[0043] The preparation method comprises the following steps: mixing various raw materials and decocting the mixture in water.

[0044] Example 3

[0045] The traditional Chinese medicine composition for improving muscle atrophy caused by tumor cachexia is mainly composed of the following raw materials by weight: 35g of Astragalus, 14g of Angelica, 14g of White Peony, 8g of Licorice, 14g of Cinnamon Twig, 14g of Epimedium, 12g of Morinda Officinalis, 22g of Agrimoniae, 12g of Jujube, 14g of Anemarrhena, 8g of Phellodendron, 10g of Paris Polygoni Multiflori, 22g of Sargentodoxae, and 22g of Millettia Reticulata.

[0046] The preparation method comprises the following steps: mixing various raw materials and decocting the mixture in water.

[0047] The present invention induces differentiation of myoblasts C2C12 and co-cultures them with Lewis lung adenocarcinoma cells. Based on the myotube atrophy model, the effects of this Chinese medicine prescription on cells and animals are explored through in vitro and in vivo biological methods, in order to provide clinical medication guidance for the prevention and treatment of tumor cachexia and muscle atrophy with traditional Chinese medicine.

[0048] In vitro experiment

[0049] 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, and the paste yield was 1.939%. DMEM culture medium (without serum) was used to prepare the required drug concentration, and filtered for sterilization for subsequent experiments.

[0050] 1 Establishment of myotube differentiation model

[0051] 1.1 Observation of the morphology and transverse diameter of C2C12 cell myotubes using an inverted microscope

[0052] Mouse myoblast C2C12 cells were routinely cultured, and cells in the logarithmic phase were selected and induced to differentiate by adding 2% horse serum differentiation medium. At 0h, 24h, 48h, 72h, and 96h of differentiation, photos were taken with an inverted microscope, and the transverse diameter of the cell myotubes was measured using Image J software. Five fields of view were randomly measured and repeated five times.

[0053] The results showed that mouse myoblasts C2C12 were spindle-shaped and adherent to the wall. 2% horse serum was used to induce differentiation of C2C12 cells. The myotube morphology at different time periods was observed and the diameter of myotubes was measured. When the differentiation was induced for 48 hours, C2C12 cells exited the cell proliferation cycle, and most of the cells changed from spindle-shaped to myotube-shaped, with a beaded binucleate or multinucleate structure. The myotubes became thicker and longer, and the transverse diameter increased significantly. Muscle fiber fusion occurred, and differentiation was obvious. Therefore, 48 hours of induction of C2C12 cell differentiation was the best differentiation period, which was suitable for inducing the establishment of a tumor cachexia myotube atrophy model (see Table 1 and Figure 1 ).

[0054] Table 1 Transverse diameter of C2C12 cell myotubes observed under inverted microscope ( n=10)

[0055]

[0056] Note: * Compared with the 72h and 96h groups, P < 0.05; # Compared with the 96h group, P<0.05

[0057] 1.2 Model evaluation: Western immunoblotting was used to detect the expression of MyOD, a marker protein for differentiation of C2C12 cells in the model group. C2C12 cells were inoculated into the lower chamber of a six-well Transwell co-culture plate, and 2% horse serum was used to induce differentiation. A blank group and a model group were set up. In addition to the blank group, the same number of Lewis cells were inoculated in the upper chamber of each group. 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.

[0058] Table 2 Protein expression of MyOD in C2C12 cells in each group detected by Western blotting ( n=4)

[0059]

[0060] Note: * Compared with the blank control group, P<0.05

[0061] 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 2 and Figure 2 ).

[0062] 2Indicator detection

[0063] 2.1 Grouping and Dosing

[0064] C2C12 cells were inoculated in the lower chamber of the Transwell co-culture plate, and 2% horse serum was added to induce differentiation when they grew to about 60%-80%. At the same time, Lewis cells were inoculated in the upper chamber of the co-culture plate at a cell number of 1:1 with C2C12 cells, and grouped. The specific grouping and drug addition are shown in Table 3. The final volume was 2.6mL in the lower chamber and 1.5mL in the upper chamber. In this study, the final concentration of the Chinese medicine was 100μg / mL, and the final concentration of FoxO1 inhibitor was 1μmol / L. After 48h of culture, the cell detection indicators were collected.

[0065] Table 3 Fluid addition to upper and lower chambers of Transwell co-culture

[0066]

[0067]

[0068] 2.2 Transmission electron microscopy observation of the number and structure of mitochondria in co-cultured C2C12 cells in each group

[0069] Lewis cells were co-cultured with C2C12 cells (same as above), drugs were added according to Table 3 and cultured for 48 h. Then the slides were taken out, fixed, collected, and fixed again overnight. Changes in the number and ultrastructure of mitochondria in each group of C2C12 cells were observed using a transmission electron microscope.

[0070] Table 4 Transmission electron microscopy observation of the number of mitochondria in each group under co-culture conditions ( n=4)

[0071]

[0072] Note: * Compared with the blank control group, P < 0.05; # Compared with the model group, P<0.05

[0073] Proteomic analysis of animal experiments showed that mitochondria in the muscles of tumor cachexia mice showed ultrastructural and functional changes, such as reduced mitochondrial numbers, impaired oxidative capacity, increased secretion of inflammatory factors, and abnormal expression of proteins involved in energy homeostasis and mitochondrial function in muscles; similarly, pathological changes in mitochondrial phagocytosis and mitochondrial dynamics markers were also found in patients with tumor cachexia. Table 4 and Figure 3 The results showed that compared with the blank group, the boundaries of the mitochondrial double membrane in the model group were unclearly wrinkled and even C-shaped or swollen, the number of mitochondrial cristae decreased, the arrangement was disordered, they were broken or dissolved and disappeared into vacuoles; after drug intervention, the number of mitochondria in the C2C12 cells of each medication group increased significantly, and the inner and outer membrane structures were significantly restored, indicating that the Chinese medicine prescription of the present invention increased the number of mitochondria, improved the mitochondrial morphology, and played a role in restoring the mitochondrial function of the cells; it effectively maintained the energy homeostasis in the muscle cells of mice, restored the normal expression of proteins, reduced myotube atrophy, and thus improved the malignant degeneration of tumors in tumor mice.

[0074] 2.3 Flow cytometry observation of mitochondrial membrane potential of C2C12 cells in each group

[0075] The grouping and drug addition were the same as above. The C2C12 cells in each group were collected and cultured at 1×10 6 The cells were treated according to the instructions of the Mitochondrial Membrane Potential Assay Kit (JC-1), and the flow cytometer was used for detection. The data were acquired and analyzed by CELLQuest software.

[0076] Changes in mitochondrial structure and function are the key links that determine cell apoptosis. When the cell mitochondrial membrane potential decreases, the early apoptosis rate detected by the kit will increase. The results of detecting changes in cell membrane potential by flow cytometry are reliable. This study reflects the changes in mitochondrial membrane potential by analyzing the early apoptosis rate of cells. The results showed that the early apoptosis rate in the model group was significantly different from that in the blank control group (P < 0.05). Compared with the model group, the early apoptosis rates in the FoxO1 inhibitor group and the Chinese medicine group were significantly reduced, indicating that the cell mitochondrial membrane potential increased significantly, and the difference was statistically significant (P < 0.05). This shows that the Chinese medicine has a good repair effect on mitochondrial structure and function, significantly improves the cell mitochondrial membrane potential, and limits the process of cell apoptosis (see Tables 5 and Figure 4).

[0077] Table 5 Effects of drugs on mitochondrial membrane potential of C2C12 cells in each group observed by flow cytometry ( n=5)

[0078]

[0079] Note: * Compared with the blank control group, P < 0.05; # Compared with the model group, P<0.05

[0080] 2.4 Detection of mitochondrial-related proteins in C2C12 cells of each group by immunoblotting

[0081] PGC-1α participates in the biological activity of skeletal muscle and adipose tissue. It activates transcription factors to initiate mitochondrial biogenesis, maintains mitochondrial function, increases mitochondrial mass and activity, improves muscle function, delays muscle atrophy, and improves muscle endurance. FoxO1 is a member of the forkhead box transcription factor family, which intervenes in the normal function of skeletal muscle mitochondria. Increased expression of FoxO1 can further activate the ubiquitin proteasome system and promote protein degradation. PGC-1α can resist muscle atrophy by inhibiting FoxO to block the transcription of muscle RING finger gene 1 (MuRF1) and E3 ligase-mediated ubiquitin ligase. Therefore, the function of mitochondria is closely related to the PGC-1α / FoxO1 pathway.

[0082] In this study, Western blotting was used to detect the expression of PGC-1α / FoxO1 pathway-related proteins in C2C12 cells of each group. The method was the same as 1.2 “Western blotting was used to detect the expression of MyOD in C2C12 cells of the model group”. The results are shown in Tables 6 and Figure 5 .

[0083] Table 6 Immunoblotting to detect the expression of PGC-1α / FoxO1 pathway-related proteins in C2C12 cells of each group ( n=5)

[0084]

[0085] Note: * Compared with the blank control group, P < 0.05; # Compared with the model group, P<0.05; ▲Compared with the Chinese medicine group, P<0.05

[0086] The above data show that after Lewis cells were co-cultured with C2C12 cells, compared with the cells in the model group, the expression of PGC-1α protein in the Chinese medicine prescription group and the FoxO1 inhibitor group was increased, and the expression of FoxO1 protein was decreased, which indicates that the Chinese medicine combination can effectively affect the expression of two key proteins, PGC-1α and FoxO1, maintain mitochondrial function, improve myotube atrophy, and correct muscle atrophy caused by tumor cachexia.

[0087] PGC-1α can also activate mitochondrial transcription factor A (tFAM), which translocates to mitochondria and participates in the replication and transcription of mitochondrial DNA, protecting it from reactive oxygen species (ROS) and degradation, and maintaining the normal physiological function of mitochondria. In this experiment, the expression level of tFAM protein showed an upward trend compared with the model group, indicating that the Chinese medicine composition can improve mitochondrial function, which is consistent with the observed phenomenon that the mitochondrial membrane potential of the Chinese medicine group increased, the number of mitochondria increased, and the structure was relatively complete.

[0088] FTO regulates mitochondrial biogenesis through the mTOR-PGC1α pathway in C2C12 cells, increases myotube differentiation, reduces oxidative stress, endoplasmic reticulum stress and cell apoptosis through demethylation function, affects fat decomposition in vivo and in vitro, and reduces muscle atrophy. Mammalian target of rapamycin (mTOR) is a conserved serine / threonine kinase that is involved in regulating mitochondrial metabolic function, controlling cytoskeleton and protein synthesis. During muscle atrophy, the mTOR pathway is inhibited. Compared with the model group, the expression levels of FTO and mTOR proteins in this Chinese medicine prescription were significantly increased. The mechanism is that it inhibits the damage of cell mitochondria, reduces early cell apoptosis and fat loss, which is consistent with the changes in mitochondrial morphology and quantity observed under electron microscopy, and is also consistent with the mitochondrial membrane potential (premature apoptosis rate) detected by flow cytometry.

[0089] In summary, this Chinese medicine prescription increased the expression of PGC-1α, FTO, tFAM and mTOR proteins, decreased the expression of FoxO1 protein, and effectively regulated the PGC-1α / FoxO1 signaling pathway in in vitro cell experiments, thereby improving the mitochondrial function and number in myoblasts, thereby intervening in myoblast differentiation and proliferation, and improving muscle atrophy caused by tumor cachexia.

[0090] 2. Animal Experiments on Mice

[0091] 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 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.

[0092] 2.1 Preparation of Lewis lung cancer cachexia muscle atrophy mouse model

[0093] When collecting Lewis cells in the logarithmic growth phase, adjust the cell concentration to 10 7 The mice were randomly numbered, 8 mice were used as the blank group, and the remaining 32 mice were placed in the right anterior armpit to establish the animal model of muscle atrophy caused by lung cancer cachexia.

[0094] 2.2 Animal Experiment Grouping Design and Processing

[0095] The tumor volume and body weight of mice were recorded weekly. On the 8th day of modeling, 32 mice were randomly divided into model group, Chinese medicine group, and positive drug group, with 8 mice in each group. The administration route of each experimental group was: on the 8th day of modeling, the blank group and model group were given normal saline, the Chinese medicine group was given Chinese medicine liquid (same as in vitro experiment), and the positive drug group was given L-carnitine by gavage, once a day, for three weeks. The dosage was calculated according to the following formula and compared with the blank group.

[0096] 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.

[0097] The positive drug group is L-carnitine, which is a common drug used in modern medicine to improve tumor cachexia and slow down the process of muscle loss. In this experiment, it was intragastrically administered at a concentration of 10 mg / kg.

[0098] 2.3 Experimental Results

[0099] 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 0.712±0.413cm 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 6 ).

[0100] After 3 weeks of administration, the weight of the gastrocnemius muscles of both hind limbs was accurately weighed on an analytical balance. Part of 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.

[0101] Mouse gastrocnemius HE sections were placed under an inverted microscope (Olympus, CX31) and photographed. The Image proplus6.0 image acquisition system was used to circle the muscle fiber cross-section and calculate the total area of ​​the unit section under the 400x field of view. The mean and standard deviation of the muscle fiber cross-section were calculated based on the number of muscle fibers.

[0102] Table 7 Effects of Chinese herbal medicine on body weight, gastrocnemius muscle and cross-sectional area of ​​mice with lung cancer cachexia ( n=6)

[0103]

[0104] Note: # Compared with the blank group, P < 0.05; *Compared with the model group, P < 0.05; ^Compared with the positive drug group, P < 0.05.

[0105] Compared with the blank group, the body weight, gastrocnemius muscle weight and muscle fiber cross-sectional area of ​​the mice in the model group were significantly decreased (P < 0.05); compared with the model group, the body weight, gastrocnemius muscle weight and muscle fiber cross-sectional area of ​​the mice in the Chinese medicine group were significantly increased, and were statistically significant (P < 0.05). There were significant differences in body weight and gastrocnemius muscle weight between the positive drug group (P < 0.05), and no significant differences in other indicators (P > 0.05); compared with the positive drug group, there were no significant differences in body weight and gastrocnemius muscle weight between the Chinese medicine group and the positive drug group (P > 0.05), but there were differences in muscle fiber cross-sectional area (P < 0.05) (see Table 7 and Figure 7 ).

[0106] The results showed that the Chinese herbal prescription and L-carnitine had similar intervention effects on cachectic tumor-forming mice, increasing the body weight and intestinal muscle weight of mice. The Chinese herbal prescription could also significantly increase the cross-sectional area of ​​muscle fibers, effectively improving the symptoms of muscle atrophy caused by tumor malignancy.

Claims

1. A Chinese medicine composition for improving muscle atrophy caused by tumor cachexia, characterized in that: It is made from the following raw materials by weight: 25-35 g of Astragalus, 8-14 g of Angelica, 8-14 g of White Peony, 6-8 g of Licorice, 8-14 g of Cinnamon Twig, 8-14 g of Epimedium, 10-12 g of Morinda officinalis, 18-22 g of Agrimoniae, 8-12 g of Jujube, 8-14 g of Anemarrhena, 5-8 g of Phellodendron, 6-10 g of Paris polyphylla, 18-22 g of Sargentodoxa, and 18-22 g of Millettia reticulata.

2. The Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 1, characterized in that: It is made from the following raw materials by weight: 30 g of Astragalus, 10 g of Angelica, 10 g of White Peony Root, 7 g of Licorice, 10 g of Cinnamon Twig, 10 g of Epimedium, 11 g of Morinda Officinalis, 20 g of Agrimoniae, 10 g of Jujube, 10 g of Anemarrhena, 7 g of Phellodendron, 8 g of Paris polyphylla, 20 g of Sargentodoxa, and 20 g of Millettia reticulata.

3. The Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 1, characterized in that: It is made from the following raw materials by weight: 25 g Astragalus, 8 g Angelica, 8 g White Peony, 6 g Licorice, 8 g Cinnamon Twig, 8 g Epimedium, 10 g Morinda Officinalis, 18 g Agrimoniae, 8 g Jujube, 8 g Anemarrhena, 5 g Phellodendron, 6 g Paris polyphylla, 18 g Sargentodoxae, and 18 g Millettia reticulata.

4. The Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 1, characterized in that: It is made from the following raw materials by weight: 35 g of Astragalus, 14 g of Angelica, 14 g of White Peony, 8 g of Licorice, 14 g of Cinnamon Twig, 14 g of Epimedium, 12 g of Morinda Officinalis, 22 g of Agrimoniae, 12 g of Jujube, 14 g of Anemarrhena, 8 g of Phellodendron, 10 g of Paris polyphylla, 22 g of Sargentodoxa, and 22 g of Millettia reticulata.

5. A method for preparing a Chinese medicine composition for improving muscle atrophy caused by tumor cachexia as claimed in any one of claims 1 to 4, characterized in that: Made into decoction, pills, tablets, granules and ointment.

6. The method for preparing the Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 5, characterized in that: The decoction is prepared by mixing the raw materials and decocting them in water.

7. The method for preparing the Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 5, characterized in that: The preparation method of the ointment is as follows: 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 solution under negative pressure, rotary evaporate it into a paste, dry it in a vacuum dryer and scrape it for use.

8. The method for preparing the Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 7, characterized in that: The mass ratio of the coarse powder to the ethanol solution is 1:5-10.

9. The method for preparing the Chinese medicine composition for improving muscle atrophy caused by tumor cachexia according to claim 7, characterized in that: The volume ratio of the concentrated drug solution to ethyl acetate is 1-3:

1.

10. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a drug for improving tumor cachexia and muscle atrophy.

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