Traditional Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer

Abstract

The invention provides a traditional Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer, and relates to the technical field of traditional Chinese medicine pharmacy. The traditional Chinese medicine composition is prepared from coix seeds, cassia twigs, herba patriniae, oldenlandia diffusa, radix salviae miltiorrhizae, selfheal and pericarpium citri reticulatae, all the medicines are combined to achieve the effects of clearing away heat and toxic materials, promoting blood circulation to remove blood stasis, reducing swelling and removing stasis and strengthening the spleen and excreting dampness, and the curative effect of preventing and treating postoperative recurrence of liver cancer is achieved. The traditional Chinese medicine composition disclosed by the invention has a remarkable inhibiting effect on proliferation and migration of liver cancer cells, and can be used for remarkably inhibiting tumor weight and tumor volume of a liver cancer postoperative recurrent mouse model, reducing the abdominal girth of the model mouse, reducing the number of liver nodules, reducing the AFP level in serum and improving the pathological change condition of liver para-carcinoma tissues. The traditional Chinese medicine composition has the effect of preventing postoperative recurrence of liver cancer and can be used as a clinical treatment medicine for postoperative recurrence of liver cancer.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine pharmaceutical preparations, and in particular to a compound traditional Chinese medicine composition for preventing and treating liver cancer after surgery. Background Art

[0002] Hepatocellular carcinoma is a common malignant tumor, with more than 1 million new cases and more than 500,000 deaths worldwide each year. Liver cancer has become one of the major diseases of global concern, and its mortality rate ranks second among malignant tumors. Currently, surgical treatment is still one of the main treatments for liver cancer, but the recurrence and metastasis rate of liver cancer after surgery is high, and the treatment prospects are not optimistic. The high recurrence and metastasis rates after surgery always affect the long-term efficacy of liver cancer treatment. The recurrence rate 5 years after surgery is about 40% to 70%. Postoperative recurrence is the bottleneck of the 5-year survival rate of liver cancer. Early detection and diagnosis of recurrent liver cancer is an important means to improve survival rate, treatment effect and patient quality of life. Therefore, finding a treatment method to prevent and treat postoperative recurrence of liver cancer and inhibit liver cancer metastasis is of great significance to the clinical postoperative treatment of liver cancer.

[0003] At present, the main treatments for liver cancer recurrence include re-resection, local radiofrequency ablation, percutaneous hepatic arterial chemoembolization, liver transplantation, etc., but the treatment effect is limited. Resection is suitable for patients with few cancer lesions, good liver function, and tolerated body condition. Liver transplantation is suitable for patients with good liver function and within the Milan standard, but there is a shortage of liver sources and high costs. Radiofrequency ablation is suitable for patients with few cancer lesions, small size, and no cancer thrombus. Sorafenib is mainly used for patients with recurrent liver cancer who cannot undergo surgery again, but it is easy to develop drug resistance and has limited clinical effects. Phase III clinical trials of targeted therapeutic drugs such as lenvatinib and regorafenib have made progress, but there are still shortcomings such as low response rates. At present, almost all modern treatments face the bottleneck of recurrence and metastasis.

[0004] Traditional Chinese medicine classifies liver cancer as "liver accumulation", "mass accumulation", "aggregation", etc., and believes that its main cause is the deficiency of vital energy leading to visceral disorders, causing qi stagnation and blood stasis, and obstruction of collaterals. In addition, external factors such as diet, emotions, and exogenous evils promote the endogenous generation of cancer toxins and form liver accumulation. In addition, traditional Chinese medicine believes that insufficient liver yin, qi stagnation and blood stasis, and the inability of the liver and spleen to transport and transform, lead to the stagnation of dampness and heat, and the liquid is refined into phlegm. Over time, phlegm turbidity accumulates toxins, resulting in repeated attacks and metastasis of cancer toxins. Traditional Chinese medicine has the effects of protecting liver cells, inhibiting liver inflammation, and alleviating liver fibrosis. It can effectively improve the clinical symptoms of liver cancer and the body's immune function. The application of traditional Chinese medicine in the treatment of liver cancer is becoming more and more extensive, and it has become an effective complementary and alternative therapy. In addition, a large number of clinical and basic studies in recent years have shown that traditional Chinese medicine has a significant effect in preventing and treating the recurrence and metastasis of liver cancer. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides a traditional Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides a traditional Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-40 parts of coix seeds, 1-15 parts of cassia twigs, 5-30 parts of patrinia herb, 5-30 parts of oldenlandia diffusa, 5-30 parts of salvia miltiorrhiza, 1-15 parts of selfheal and 1-15 parts of tangerine peel.

[0010] Furthermore, the Chinese medicine composition is made of the following raw materials in parts by weight: 25-35 parts of coix seeds, 3-12 parts of cassia twigs, 10-25 parts of patrinia herb, 10-25 parts of oldenlandia diffusa, 10-25 parts of salvia miltiorrhiza, 3-12 parts of selfheal, and 3-12 parts of tangerine peel.

[0011] Furthermore, the Chinese medicine composition is made of the following raw materials in parts by weight: 30 parts of coix seeds, 6 parts of cassia twigs, 15 parts of patrinia herb, 15 parts of oldenlandia diffusa, 15 parts of salvia miltiorrhiza, 6 parts of selfheal, and 6 parts of tangerine peel.

[0012] The Chinese medicine composition provided by the present invention is used to prepare a compound medicine for preventing and treating liver cancer after surgery. The Chinese medicine combination is prepared into granules, powders, oral liquids, pills, decoctions, lyophilized powders or other pharmaceutically acceptable oral dosage forms according to conventional pharmaceutical preparation methods in pharmacy and with medically acceptable adjuvants.

[0013] (III) Beneficial effects

[0014] After liver cancer surgery, patients suffer from spleen deficiency and dysfunction, imbalance of yin and yang, and often have pathological products such as phlegm, dampness, and blood stasis in their bodies. Cancer toxins and pathological factors such as phlegm, stasis, dampness, and heat interact with each other, hindering the circulation of qi and blood, forming a vicious circle. The invention adopts the Yiyifuzibaijiang powder as a modification, follows the principle of syndrome differentiation and treatment in traditional Chinese medicine, and is prepared with a Chinese medicine composition made of coix seed, cinnamon twig, patrinia, oldenlandia diffusa, salvia miltiorrhiza, herba prunellae and tangerine peel. In the prescription, coix seed is sweet and light in taste, has the effects of detoxifying, dispersing stagnation, promoting diuresis and removing dampness, and is therefore used as the main ingredient; patrinia and herba prunellae have the effects of clearing away heat and detoxifying, and are used as the secondary ingredients; herba prunellae diffusa is an auxiliary medicine, has the functions of clearing away heat and detoxifying, reducing swelling and dispersing stagnation; "Suwen·Qi Sheng Tong Tian Lun" records: "Yang Qi is like the sky and the sun, if it loses its place, it will shorten life and not be obvious", and cinnamon twig is used as a guiding medicine in the prescription, which warms and dredges the meridians, helps yang and transforms qi, guides the medicines in the prescription directly to the diseased area, and works together to expel evil; salvia miltiorrhiza and tangerine peel are also guiding medicines, which can promote blood circulation, dissipate blood stasis, dry dampness and resolve phlegm, and help eliminate tangible real evil. The combination of these medicines has the functions of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis, reducing swelling and dispersing nodules, and strengthening the spleen and eliminating dampness, thereby achieving the therapeutic effect of preventing and treating the recurrence of liver cancer after surgery.

[0015] The Chinese medicine composition of the present invention has a significant inhibitory effect on the proliferation and migration of liver cancer cells, and can significantly inhibit the tumor weight and tumor volume of a mouse model of postoperative recurrence of liver cancer, reduce the abdominal circumference of the model mice, reduce the number of liver nodules, reduce the AFP level in serum, and improve the pathological changes of liver paracancerous tissues. The Chinese medicine composition of the present invention has the effect of preventing postoperative recurrence of liver cancer, and can be used as a therapeutic drug for postoperative recurrence of clinical liver cancer.

[0016] At the same time, the Chinese medicine composition of the present invention can significantly inhibit the VEGF and bFGF content in the serum of the mouse model of postoperative recurrence of liver cancer, significantly inhibit the expression of VEGF, CD34 and CD44v6 in the homogenate of adjacent cancer tissues, and significantly inhibit the expression of GSK3β, WNT1 and β-catenin. The effect of the Chinese medicine composition of the present invention on preventing and treating postoperative recurrence of liver cancer may be through inhibiting the Wnt-β-catenin signaling pathway to affect tumor microvascular proliferation and play a role in preventing and treating postoperative recurrence of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Results of cell proliferation experiment and Transwell experiment; A is the number of cells perforated by Transwell; B is the cell proliferation inhibition rate; C is the comparison of the cell scratch healing area of ​​each group at 0h, 24h, and 48h after drug administration with that of the control group, *p<0.05, **p<0.01.

[0018] Figure 2 The cell scratch healing areas of each group at 0h, 24h, and 48h after drug administration were compared with those of the control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0019] Figure 3 The effects of the Chinese medicine combination of the present invention on the apparent indicators of model mice; A is the abdominal circumference of mice; B is the weight of the mouse liver; C is the body weight of the mouse; D is the number of liver nodules in the mouse; E is the total tumor weight of the mouse; F is the volume of the largest tumor mass; G is the pathological picture of the recurrent liver tissue of the mouse; among them, the blank group: CG, the model group: MP, the sham operation group: PG, the low-dose group of Yiyifuzibaijiangsan: LG, the medium-dose group of Yiyifuzibaijiangsan: MG, the high-dose group of Yiyifuzibaijiangsan: HG, the tegafur group: TG, compared with the blank group, △△ P<0.01, △ P<0.05; compared with the model group, *p<0.05, **p<0.01, the same below.

[0020] Figure 4 The present invention is an effect of the Chinese medicine composition of the present invention on the levels of AFP (A), bFGF (B) and VEGF (C) in the serum of mice with postoperative recurrence of liver cancer.

[0021] Figure 5 The present invention shows the effect of the Chinese medicine composition on the levels of VEGF (A), CD34 (B) and CD44v6 (C) in the recurrent adjacent cancer tissues of mice with postoperative recurrent liver cancer.

[0022] Figure 6 The present invention shows the effect of the Chinese medicine composition on the levels of GSK3β (A), WNT1 (B) and β-catenin (C) in the recurrent adjacent cancer tissues of mice with postoperative recurrent liver cancer. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] A traditional Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of coix seeds, 2 parts of cassia twigs, 5 parts of patrinia herb, 5 parts of oldenlandia diffusa, 5 parts of salvia miltiorrhiza, 3 parts of selfheal, and 2 parts of tangerine peel.

[0026] The Chinese medicine composition is used to prepare a compound medicine for preventing and treating liver cancer after surgery. The Chinese medicine combination is prepared into granules, powders, oral liquids, pills, decoctions, lyophilized powders or other pharmaceutically acceptable oral dosage forms according to conventional pharmaceutical preparation methods in pharmacy and with medically acceptable excipients.

[0027] Example 2

[0028] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 40 parts of coix seeds, 15 parts of cassia twigs, 30 parts of Patrinia scabra, 30 parts of Hedyotis diffusa, 30 parts of Salvia miltiorrhiza, 15 parts of Prunella vulgaris, and 15 parts of dried tangerine peel.

[0029] Example 3

[0030] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 20 parts of coix seeds, 4 parts of cinnamon twigs, 8 parts of Patrinia scabra, 8 parts of Hedyotis diffusa, 8 parts of Salvia miltiorrhiza, 4 parts of Prunella vulgaris, and 4 parts of dried tangerine peel.

[0031] Example 4

[0032] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 26 parts of coix seeds, 8 parts of cassia twigs, 12 parts of Patrinia scabra, 14 parts of Hedyotis diffusa, 10 parts of Salvia miltiorrhiza, 6 parts of Prunella vulgaris, and 4 parts of dried tangerine peel.

[0033] Example 5

[0034] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 36 parts of coix seeds, 12 parts of cassia twigs, 22 parts of Patrinia scabra, 20 parts of Hedyotis diffusa, 18 parts of Salvia miltiorrhiza, 12 parts of Prunella vulgaris, and 10 parts of dried tangerine peel.

[0035] Example 6

[0036] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 25 parts of coix seeds, 3 parts of cassia twigs, 10 parts of Patrinia scabra, 10 parts of Hedyotis diffusa, 10 parts of Salvia miltiorrhiza, 3 parts of Prunella vulgaris, and 3 parts of dried tangerine peel.

[0037] Example 7

[0038] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 35 parts of coix seeds, 12 parts of cassia twigs, 25 parts of Patrinia scabra, 25 parts of Hedyotis diffusa, 25 parts of Salvia miltiorrhiza, 12 parts of Prunella vulgaris, and 12 parts of dried tangerine peel.

[0039] Example 8

[0040] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 28 parts of coix seeds, 7 parts of cassia twigs, 20 parts of Patrinia scabra, 20 parts of Hedyotis diffusa, 20 parts of Salvia miltiorrhiza, 10 parts of Prunella vulgaris, and 6 parts of dried tangerine peel.

[0041] Example 9

[0042] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 32 parts of coix seeds, 10 parts of cassia twigs, 18 parts of Patrinia scabra, 22 parts of Hedyotis diffusa, 17 parts of Salvia miltiorrhiza, 7 parts of Prunella vulgaris, and 8 parts of dried tangerine peel.

[0043] Example 10

[0044] The difference between this embodiment and embodiment 1 is that the Chinese medicine composition is made of the following raw materials in parts by weight: 30 parts of coix seeds, 6 parts of cassia twigs, 15 parts of Patrinia scabra, 15 parts of Hedyotis diffusa, 15 parts of Salvia miltiorrhiza, 6 parts of Prunella vulgaris, and 6 parts of dried tangerine peel.

[0045] Test Example 1

[0046] 1 Trial drug

[0047] Drug 1: 30 parts of coix seed, 6 parts of cinnamon twig, 15 parts of patrinia, 15 parts of oldenlandia diffusa, 15 parts of salvia miltiorrhiza, 6 parts of selfheal, 6 parts of dried orange peel

[0048] Drug 2: 6 parts of cinnamon twig, 15 parts of Patrinia scabra, 15 parts of Hedyotis diffusa, 6 parts of Prunella vulgaris, 6 parts of dried orange peel

[0049] Drug 3: 30 parts of coix seed, 6 parts of cinnamon twig, 15 parts of salvia miltiorrhiza, 6 parts of selfheal, 6 parts of tangerine peel

[0050] Drug 4: 30 parts of coix seed, 15 parts of Patrinia scabra, 15 parts of Hedyotis diffusa, 15 parts of Salvia miltiorrhiza

[0051] According to the above recipes, weigh the raw materials, wash them, add 3 times the water and boil them for 40 minutes, repeat the boiling for more than 2 times, collect the decoction, concentrate the decoction to 1g / mL (1mL contains 1g of crude drug), and obtain Chinese medicine extract 1-4, which is stored at 4°C for later use.

[0052] 2 Experimental methods

[0053] 2.1 Preparation of drug-containing serum in rats

[0054] After 3 days of adaptive feeding, SD male rats were randomly divided into a blank group and experimental groups 1-4, with 4 rats in each group. Experimental groups 1-4 were gavaged with Chinese medicine extracts 1-4 respectively, at a dose of 4.4 g / kg. After continuous gavage for 7 days, the rats were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta. The supernatant was obtained by centrifugation at 3500r / min for 10 min to obtain the drug-containing serum of drugs 1-4. The serum was inactivated at 56℃, filtered with a 0.22μm microporous filter membrane, and stored in a refrigerator at -20℃ for later use.

[0055] 2.2 Cell proliferation assay

[0056] HepG2 cells in logarithmic growth phase were selected and 1×10 5 100 μL / well were planted in a 96-well plate. After 24 hours of full cell adhesion, experimental groups 1-4 were set up, and 40 μL of drug-containing serum of drugs 1-4 were added respectively. The rest was supplemented with basal culture medium. The final volume of each well was 200 μL. Blank wells and control wells were set up separately. Each group had 3 replicates. After 48 hours of culture, new culture medium was replaced, MTT was added, and culture was continued for 4 hours. DMSO was added, mixed and absorbance was measured. Calculate the inhibition rate. Drug inhibition rate = 1-(OD value of drug-containing serum group-OD value of PBS group) / (OD value of blank serum group-OD value of PBS group).

[0057] 2.3 Transwell invasion assay

[0058] HepG2 cells in logarithmic growth phase were taken and 1×10 510 cells / well were seeded in a 96-well plate. After 24 hours of full attachment of the cells, experimental groups 1-4 were set up, and 40 μL of drug-containing serum of drugs 1-4 was added respectively. Blank wells were also set up, and each group had 3 replicates. The matrix gel was mixed with the basal culture medium, and the cells were placed in a serum-free culture medium and cultured for 24 hours to make 2.5×10 5 / mL cell suspension, add 100μL cell suspension to the upper chamber, add 500μL complete medium to the lower chamber, let it stand for 15min, and then put it into the incubator for 24h. Add 4% paraformaldehyde to fix the cells for 15min, wash them twice with PBS, stain them with 0.1% crystal violet for 10min, wash them with ultrapure water, observe them under a microscope, and analyze them with ImageJ software.

[0059] 2.4 Cell scratch assay

[0060] HepG2 cells in logarithmic growth phase were taken and 1×10 5 100 μL / well were planted in a 96-well plate. After 24 hours of full cell adhesion, experimental groups 1-4 were set up, and 40 μL of drug-containing serum of drugs 1-4 were added respectively, and the rest was supplemented with basal culture medium. The final volume of each well was 200 μL, and blank wells were set up separately, with 3 replicates in each group. After the cells covered the bottom of the cell culture dish, a 200 μL pipette tip was used to scratch a mark perpendicular to the central symmetry axis of the cell culture dish. The scratched cells were washed with PBS, and the scratched cells were observed under a microscope and photographed by group at 0h, 24, and 48h, respectively. The scratch position of the cells in the photos was recorded, and the uncovered area at 48h was analyzed using ImageJ software. The area change was used to evaluate the healing efficiency.

[0061] 2.4 Statistical analysis

[0062] SPSS 21.0 for Windows was used to analyze the data, and the experimental results were expressed as mean ± standard deviation. Data were expressed in the form of , and one-way ANOVA was used for comparison among groups. Dunnett's T3 was used for comparison among groups when variance was unequal, and LSD method was used for equal variance. Ridit test was used for rank data, and GraphPad Prism v.8.0 (GraphPad Software Inc., San Diego, CA, USA) was used for drawing. P < 0.05 was considered statistically significant.

[0063] 3 Results

[0064] 3.1 Effects on HepG2 cell proliferation and perforation number

[0065] The effects of different drug combinations on the number of cell perforations and cell proliferation inhibition rates were observed using HepG2 cells. Figure 1AB shows. Compared with the control group, the number of cell perforations in drug 1-4 groups decreased to varying degrees, among which the number of perforations in drug 1 group was significantly lower than that in the control group (p<0.01), and the number of perforations in drug 1 group was also significantly less than that in drug 2-4 groups. Compared with the control group, drugs 1-4 had different degrees of inhibitory effects on HepG2 cell proliferation, among which drug 1 group had the highest inhibition rate on HepG2 cell proliferation, and its inhibitory effect was significantly better than that of drugs 2-4.

[0066] 3.2 Effects on HepG2 cell migration

[0067] The results of cell scratch assay are as follows Figure 2 As shown in the figure, at 0-48h, the HepG2 cells in the control group showed obvious migration. After 24h of administration, compared with the control group, the migration area of ​​HepG2 cells in drug 1 group was significantly smaller than that in the control group (P<0.01), and the migration area of ​​HepG2 cells in drug 2-4 groups was also smaller than that in the control group, but there was no significant difference (P>0.05). After 48h of administration, the migration area of ​​HepG2 cells in drug 1 group was significantly smaller than that in the control group, and the difference was extremely significant (P<0.0001), and the migration area of ​​HepG2 cells in drug 2-4 groups was also significantly smaller than that in the control group (P<0.05). Drug 1 had the most obvious inhibitory effect on HepG2 cell migration, and its inhibitory effect was significantly better than that of drugs 2-4.

[0068] The above experimental results show that the components of the Chinese medicine composition of the present invention synergize with each other, can significantly reduce the number of perforations of HepG2 cells, and have a significant inhibitory effect on the proliferation and migration of HepG2 cells, indicating that the Chinese medicine composition of the present invention is expected to be developed into a drug for preventing and treating postoperative recurrence of liver cancer.

[0069] Test Example 2

[0070] Effect of the Chinese medicine composition of the present invention on the recurrence model of H22 mouse orthotopic transplanted tumor

[0071] 1. Experimental Materials

[0072] 1.1 Trial Drugs

[0073] The Chinese medicine composition of the present invention comprises: 30 parts of coix seeds, 6 parts of cinnamon twigs, 15 parts of patrinia herb, 15 parts of oldenlandia diffusa, 15 parts of salvia miltiorrhiza, 6 parts of selfheal and 6 parts of tangerine peel. The raw materials are weighed, washed, decocted for 40 minutes with 3 times of water, and the decocting is repeated for more than 2 times. The decoction is collected, and the decoction is concentrated to 1g / mL (1mL contains 1g of crude drug), and stored at 4°C for standby use.

[0074] 1.2 Experimental animals

[0075] A total of 130 male KM mice, weighing 18–20 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Animal Certificate No.: 370726231100983763, Ethics Approval No.: DWLLGZR202202217).

[0076] 2 Experimental methods

[0077] 2.1 Animal modeling and grouping

[0078] Mouse liver cancer H22 cells were routinely cultured and inoculated into the livers of adult male mice to establish a nude mouse model of liver orthotopic transplanted tumors. A nude mouse model of liver orthotopic carcinoma in situ was established by surgical resection. Subsequently, continuous traumatic stimulation was given to induce postoperative recurrence of liver cancer in nude mice. The mice were randomly divided into a model group, a sham operation group (laparotomy only but no surgery), a tegafur group (10 mg / mL), a high, medium, and low (13.2, 6.6, 3.3 g / kg) dose group of the Chinese medicine composition of the present invention, and a blank control group, with 12 mice in each group. Except for the model, sham operation, and blank groups, which were given normal saline, each medication group was gavaged once a day for 14 consecutive days.

[0079] 2.2 Observation indicators

[0080] 2.2.1 Apparent indicators

[0081] During the administration period, the general condition of the mice, their weight, hair cleanliness, diet, water intake, activity level, and mental state were observed. After the administration, the changes in the abdominal circumference of the mice were observed, the tumors were peeled off under a sterile environment, the number of liver nodules was counted, the total weight of the tumors was measured, and the volume of the largest tumor was detected.

[0082] 2.2.2 Biochemical index detection

[0083] 2 hours after the last administration, blood was collected from mice and centrifuged at 4°C, 3000r / min for 15min to separate serum, which was stored in a -40°C refrigerator for later use. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of AFP, bFGF and VEGF in serum.

[0084] 2.2.3 Pathological indicators

[0085] After the administration, the mice were killed by cervical dislocation, and the liver tumor and adjacent tissues were removed. After fixation, they were dehydrated, embedded, transparent, sliced, dewaxed, and stained with hematoxylin-eosin. The pathological sections were observed using a digital pathological section scanner. [13-14] For the pathological condition of liver cancer recurrence, the pathological status of mice was graded and evaluated according to Table 1.

[0086] Table 1 Comprehensive scoring criteria for pathology combined with physical liver status

[0087]

[0088] 2.2.4 Immunofluorescence detection of changes in microvessels and their regulatory pathways

[0089] Tissue sections were prepared from paraffin-embedded liver tissue samples, dewaxed, hydrated, repaired, blocked, and treated with bovine serum albumin. The primary antibody working solution and the corresponding secondary antibody working solution were added, and then the nucleus was stained with DAPI. Finally, the sections were sealed with anti-fluorescence quenching sealing agent, and the expression of CD34, GSK-3β, Wnt1, β-catenin, VEGF, and CD44v6 was detected by immunofluorescence. Five fields of view were randomly selected under the microscope, and the average fluorescence intensity in the area was calculated using imagej software.

[0090] 2.3 Statistical analysis

[0091] SPSS 21.0 for Windows was used to analyze the data, and the experimental results were expressed as mean ± standard deviation. Data were expressed in the form of , and one-way ANOVA was used for comparison among groups. Dunnett's T3 was used for comparison among groups when variance was unequal, and LSD method was used for equal variance. Ridit test was used for rank data, and GraphPad Prism v.8.0 (GraphPad Software Inc., San Diego, CA, USA) was used for drawing. P < 0.05 was considered statistically significant.

[0092] 3 Results

[0093] 3.1 Effects of the Chinese medicine composition of the present invention on the epidemiological parameters of mice with postoperative recurrence of liver cancer

[0094] 3.1.1 Growth status and abdominal circumference

[0095] The mice in the blank group grew well, their hair was shiny and lively, the mice in the model group had obvious huddling and messy hair, and the mice in the drug-treated group were in good spirits. Compared with the blank group, the abdominal circumference of the mice in the model group increased significantly (p<0.01); compared with the model group, the abdominal circumference of the mice in the low-dose group and the tegafur group decreased significantly (p<0.05), and the abdominal circumference of the mice in the medium- and high-dose groups decreased significantly (p<0.01). For specific results, see Figure 3 A.

[0096] 3.1.2 Liver weight and body weight

[0097] Compared with the blank group, the liver weight and body weight of the model group were significantly increased (p<0.01); compared with the model group, the liver weight and body weight of the mice in the sham operation group, low-dose group, medium-dose group and tegafur group were significantly decreased (p<0.05), and the liver weight and body weight of the high-dose group were significantly decreased (p<0.01). The specific results are shown in Figure 3 BC.

[0098] 3.1.3 Number of liver nodules, total tumor weight, and maximum tumor volume

[0099] Compared with the model group, the maximum tumor volume of mice in the low-dose group was significantly reduced (p<0.05); the maximum tumor volume of mice in the medium-dose group was significantly reduced (p<0.01), the number of liver nodules and the total tumor weight were significantly reduced (p<0.05); the maximum tumor volume of mice in the high-dose group was significantly reduced (p<0.01), the number of liver nodules and the total tumor weight were significantly reduced (p<0.05); the maximum tumor volume of mice in the tegafur group was significantly reduced (p<0.01), the number of liver nodules and the total tumor weight were significantly reduced (p<0.05). For specific results, see Figure 3 DF.

[0100] 3.1.4 Pathological changes of recurrent liver tissue

[0101] The liver tissue of the blank group was normal, and no recurrent nodules were formed. Compared with the blank group, the recurrent foci of the mice in the model group were obvious, with more atypical nuclei in the tumor tissue, obvious nuclear division, and infiltrative growth of tumor cells. Compared with the model group, the treatment groups were significantly improved. All doses of Yiyifuzibaijiangsan and tegafur could improve the pathological recurrence of liver cancer after surgery. The specific results of the grading evaluation are shown in Table 2. Figure 3 .

[0102] Table 2 Effect of the Chinese medicine composition of the present invention on liver pathology in mice with postoperative recurrence of liver cancer

[0103]

[0104] Note: Compared with the blank group, △△ P<0.01, △ P<0.05; compared with the model group, *p<0.05, **p<0.01.

[0105] 3.2 Effect of the Chinese medicine composition of the present invention on the levels of AFP, bFGF and VEGF in the serum of mice with postoperative recurrence of liver cancer

[0106] Compared with the blank group, the serum AFP, bFGF and VEGF levels of mice in the model group were significantly increased (p<0.01); compared with the model group, the bFGF level of the low-dose group was significantly decreased (p<0.05), and the bFGF levels of the high, medium and tegafur groups were significantly decreased (p<0.01); the AFP level of the high, medium, low-dose groups and tegafur group was significantly decreased (p<0.01); the VEGF level of the high, medium and tegafur groups was significantly decreased (p<0.01). Figure 4 .

[0107] 3.3 Effect of the Chinese medicine composition of the present invention on the levels of VEGF, CD34 and CD44v6 in the recurrent adjacent cancer tissues of mice with postoperative recurrent liver cancer

[0108] Compared with the blank group, the expression of VEGF, CD34 and CD44v6 in the model group mice adjacent to the cancer was significantly increased (p<0.01); compared with the model group, the expression of CD34 and CD44v6 in the dose group was significantly decreased (p<0.05), and the expression of VEGF in the low dose group was significantly decreased (p<0.01); the expression of VEGF, CD34 and CD44v6 in the high and medium dose groups was significantly decreased (p<0.01); the results are shown in Figure 5 .

[0109] 3.4 Effect of the Chinese medicine composition of the present invention on the levels of GSK3β, WNT1 and β-catenin in the recurrent adjacent tumor tissues of mice with postoperative recurrent liver cancer

[0110] Compared with the blank group, the expression of GSK3β, WNT1 and β-catenin in the model group mice was significantly increased (p<0.01); compared with the model group, the expression of GSK3β, WNT1 and β-catenin in the low-dose group was significantly decreased (p<0.05), the expression of GSK3β, WNT1 and β-catenin in the medium-dose group was significantly decreased (p<0.01), the expression of GSK3β in the high-dose group was significantly decreased (p<0.01), the expression of WNT1 and β-catenin was significantly decreased (p<0.05); the expression of VEGF in the dose group was significantly decreased (p<0.01). Figure 6 .

[0111] 4 Conclusion

[0112] According to the above experimental results, the Chinese medicine composition of the present invention can significantly inhibit the tumor weight and tumor volume of the recurrent mice in the mouse model of liver cancer recurrence after surgery, reduce the abdominal circumference (ascites volume) of the model mice, reduce the number of liver nodules, reduce the AFP level in serum, and improve the pathological changes of liver paracancerous tissues, indicating that the Chinese medicine composition of the present invention has the effect of preventing the recurrence of liver cancer after surgery and can be used as a therapeutic drug for clinical liver cancer recurrence after surgery.

[0113] At the same time, the Chinese medicine composition of the present invention can significantly inhibit the content of VEGF and bFGF in the serum of the mouse model of liver cancer recurrence after surgery, and significantly inhibit the expression of VEGF, CD34 and CD44v6 in the adjacent cancer tissues. The effect of the Chinese medicine composition of the present invention on preventing and treating liver cancer recurrence after surgery may be related to its effect of inhibiting tumor microvascular proliferation.

[0114] Furthermore, the Chinese medicine composition of the present invention can significantly inhibit the expression of GSK3β, WNT1 and β-catenin, indicating that the Chinese medicine composition of the present invention may play a role in preventing and treating postoperative recurrence of liver cancer by inhibiting the Wnt-β-catenin signaling pathway and affecting tumor microvascular proliferation.

[0115] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for preventing and treating postoperative recurrence of liver cancer, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-40 parts of coix seeds, 1-15 parts of cassia twigs, 5-30 parts of patrinia herb, 5-30 parts of oldenlandia diffusa, 5-30 parts of salvia miltiorrhiza, 1-15 parts of selfheal and 1-15 parts of tangerine peel.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 25-35 parts of coix seeds, 3-12 parts of cassia twigs, 10-25 parts of patrinia herb, 10-25 parts of oldenlandia diffusa, 10-25 parts of salvia miltiorrhiza, 3-12 parts of selfheal, and 3-12 parts of tangerine peel.

3. The Chinese medicine composition according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: 30 parts of coix seeds, 6 parts of cassia twigs, 15 parts of patrinia herb, 15 parts of oldenlandia diffusa, 15 parts of salvia miltiorrhiza, 6 parts of selfheal and 6 parts of tangerine peel.

4. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a compound medicine for preventing and treating postoperative liver cancer, characterized in that: The Chinese medicine combination is prepared into granules, powders, oral liquids, pills, decoctions, lyophilized powders or other pharmaceutically acceptable oral dosage forms according to conventional pharmaceutical preparation methods in pharmacy, together with medically acceptable excipients.

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