阅读说明：本技术 一种用于治疗肝癌的中药方剂及用途 (Traditional Chinese medicine prescription for treating liver cancer and application ) 是由 潘战宇 杨银莉 张宇 于 2021-03-26 设计创作，主要内容包括：本发明公开了一种用于治疗肝癌的中药方剂(肝癌协定方GAXD),它是由土贝母5份、防己3份、大黄1份、川芎2-3份和黄芪3-5份组成。制备方法：先将大黄加入水一钟半,再加入土贝母、防己、川芎和黄芪,温火熬制20-30分钟,即可。本发明更进一步公开了用于治疗肝癌中药方剂在制备抑制肝癌肿瘤生长药物中的应用。实验结果显示：本发明中药方剂不仅仅通过STAT3信号通路抑制HBV+肝癌细胞体外生长和病毒复制,在体内实验中GAXD对NK细胞的比例和活性有调节作用。(The invention discloses a traditional Chinese medicine prescription (liver cancer protocol formula GAXD) for treating liver cancer, which consists of 5 parts of rhizoma bolbostemmae, 3 parts of radix stephaniae tetrandrae, 1 part of rheum officinale, 2-3 parts of ligusticum wallichii and 3-5 parts of astragalus membranaceus. The preparation method comprises the following steps: firstly, adding water into rhubarb for half a minute, then adding rhizoma bolbostemmae, radix stephaniae tetrandrae, ligusticum wallichii and astragalus mongholicus, and decocting for 20-30 minutes by slow fire. The invention further discloses application of the traditional Chinese medicine prescription for treating liver cancer in preparing a medicine for inhibiting growth of liver cancer tumors. The experimental results show that: the traditional Chinese medicine preparation disclosed by the invention not only inhibits the in vitro growth and virus replication of HBV + liver cancer cells through STAT3 signal channel, but also has a regulating effect on the proportion and activity of NK cells by GAXD in vivo experiments.)

1. A traditional Chinese medicine prescription for treating liver cancer is characterized by comprising the following raw materials in parts by weight: 5 parts of rhizoma bolbostemmae, 3 parts of radix stephaniae tetrandrae, 1 part of rheum officinale, 2-3 parts of ligusticum wallichii and 3-5 parts of astragalus membranaceus.

2. The Chinese medicinal formula for treating liver cancer according to claim 1, which is characterized by comprising the following raw materials in parts by weight: 5 parts of rhizoma bolbostemmae, 3 parts of radix stephaniae tetrandrae, 1 part of rheum officinale, 2 parts of ligusticum wallichii and 5 parts of astragalus membranaceus.

3. The method for preparing a Chinese medicinal composition for treating liver cancer as claimed in claim 1, wherein the preparation method comprises adding radix et rhizoma Rhei into water for half a minute, adding rhizoma Bolbostematis, radix Stephaniae Tetrandrae, rhizoma Ligustici Chuanxiong and radix astragali, and decocting with slow fire for 20-30 minutes.

4. The use of the Chinese medicinal formulation for treating liver cancer according to claim 1 in the preparation of a medicament for inhibiting the growth of liver cancer tumors.

5. The use of a Chinese medicinal formulation for the treatment of liver cancer as claimed in claim 1 in the preparation of an antiviral medicament.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine treatment, and relates to a traditional Chinese medicine formula (liver cancer protocol formula GAXD) for treating liver cancer and application thereof.

Background

The research of Chinese primary liver cancer registration (CLCS) shows that hepatocellular carcinoma in China is obviously different from that in Europe and America in many aspects, has high heterogeneity, is closely related to hepatitis B virus infection mostly, often reaches middle and late stages in initial diagnosis, loses the chance of surgical operation, has troublesome treatment, bad prognosis and short life cycle, and has the main reason that patients have complicated conditions and basic liver cancer (including hepatitis, liver cirrhosis, liver dysfunction and related complications) and abnormal immune function and other conditions.

The research on malignant tumors is initiated by a western medicine theoretical system at the earliest, and most researches are based on cytology, prove the existence of protooncogenes and cancer suppressor genes in human cells according to the directions of human gene expression, cell variation and the like, and discover the properties of rapid division, invasion and metastasis and the like of cancer cells. In recent years, the traditional Chinese medicine theory has a great progress in the research of malignant tumors, the traditional Chinese medicine aspect has always paid attention to the prevention of diseases, namely the importance of preventing the diseases is emphasized, and the constitution identification provides a method, a tool and an evaluation system for the prevention of the diseases. The theory of constitutions, the theory of syndrome differentiation and typing, etc. are all practically applied in the research of malignant tumors, and traditional Chinese medicine gradually becomes a research hotspot in the oncology department and occupies a certain position. According to statistics, 45 types of medicaments for treating lung cancer are 295 in total in nearly 30 years by the end of 2012; the traditional Chinese medicine is proved to have the effects of reversing the drug resistance of cancer cells to chemotherapeutic drugs, slowing down the toxic and side effects of malignant tumor patients in the treatment process, improving the life quality of the patients and prolonging the life cycle; the traditional Chinese medicine has definite curative effect in the aspects of inhibiting the progress of tumor diseases and exerting the synergistic attenuation effect, and the reasonable application of the traditional Chinese medicine can greatly improve the prognosis of patients.

The reason of the invention is as follows: when the etiology and pathogenesis of chronic hepatitis B are discussed in traditional Chinese medicine, blood stasis is considered to be both a pathological product and one of the pathogenic factors, for example, Zhang Yi Tong, Ju Jiu, jaundice clouds: "Zhu Huang is mostly damp-heat, but long-term meridian is still ill without blood stasis. The Li Happy professor also considers that qi stagnation and blood stasis are the basic process of the development of the hepatitis B virus cancerogenic lesion. On the basis of syndrome differentiation, the method for promoting blood circulation to remove blood stasis can be used as one of the main principles for treating chronic hepatitis B and liver cancer, and the pharmacological mechanism of the method is mainly to improve microcirculation, facilitate the medicine and immune cells to reach tumor parts to play a role, enhance the immunity of the organism and the like.

Disclosure of Invention

In order to achieve the above purpose, the present invention discloses the following technical contents:

a traditional Chinese medicine prescription (liver cancer agreement formula GAXD) for treating liver cancer is characterized by comprising the following raw materials in parts by weight:

5 parts of rhizoma bolbostemmae, 3 parts of radix stephaniae tetrandrae, 1 part of rheum officinale, 2-3 parts of ligusticum wallichii and 3-5 parts of astragalus membranaceus. The preferred raw material composition is: 5 parts of rhizoma bolbostemmae, 3 parts of radix stephaniae tetrandrae, 1 part of rheum officinale, 2 parts of ligusticum wallichii and 5 parts of astragalus membranaceus.

The invention further discloses a preparation method of a traditional Chinese medicine prescription (liver cancer protocol formula GAXD) for treating liver cancer, which is characterized in that rhubarb is added with water for half a minute, and then bolbostemma paniculatum, radix stephaniae tetrandrae, ligusticum wallichii and astragalus are added, and the mixture is decocted for 20-30 minutes by slow fire.

The invention further discloses application of a traditional Chinese medicine prescription (liver cancer protocol prescription GAXD) for treating liver cancer in preparing a medicine for inhibiting growth of liver cancer tumor, in particular application in preparing an antiviral medicine.

The experimental results show that: after establishing an HBV + HCC transplantation tumor model (namely a nude mouse tumor-bearing HepG2.2.15 cell), the GAXD gastric lavage shows that the tumor proliferation of the GAXD treatment group is obviously inhibited, and the HBV viral load and the HBV antigen (HBsAg and HBeAg) concentration in tumor tissues and peripheral serum are also obviously reduced. In addition, the expression of key molecules HIF-1a and VEGFA associated with tumor angiogenesis was also significantly reduced in tumor tissues of the GAXD treated group. Next, it was found by co-incubation of drug-containing serum prepared from GAXD with hepg2.2.15 cells that GAXD significantly inhibited STAT3 signal pathway, thereby exerting antitumor and antiviral activity. Additional studies have also found that GACD also exerts a regulatory effect on the proportion and functional activity of NK cells.

The invention mainly solves the following problems: the excessive activation of STAT3 plays an important role in the process of liver inflammation carcinogenesis after HBV infection, so STAT3 signal protein becomes an effective target molecule for treating HBV-related liver cancer. We observed that the liver cancer protocol (GAXD) based on STAT3 signal pathway can eliminate HBV virus while inhibiting tumor proliferation, and can positively regulate NK cell function while inhibiting tumor angiogenesis, thereby affecting hepatitis B related liver cancer treatment.

The following points are considered: the liver cancer protocol (GAXD) has comprehensive treatment effects on HBV + HCC: (1) tumor inhibiting effect, (2) antiviral effect, (3) anti-tumor angiogenesis effect, and (4) tumor immunity regulating effect (mainly NK cells).

The main difficulties are that: china is a high incidence area of liver cancer, and particularly, liver cancer patients caused by HBV are numerous. The liver of the HBV-related liver cancer patients has two diseases with different properties, namely malignant tumor and viral hepatitis, which are often mutually influenced and in vicious circle, so the HBV-related liver cancer patients have the characteristics of high vicious degree, rapid development and easy relapse and transfer after treatment.

Therefore, for HBV-related liver cancer patients, antiviral treatment and anticancer treatment must be performed simultaneously, which cannot be partial to waste. However, the current clinical treatment options (including liver transplantation, hepatectomy, interventional therapy, etc.) for these patients are not ideal due to the high recurrence rate; the benefit of chemotherapeutic drugs (oxaliplatin combined with fluorouracil) and targeted drugs (sorafenib and the like) to liver cancer patients is very limited; meanwhile, the research shows that the practical effect of the antiviral nucleic acid medicament in the treatment is not obviously increased even if the antiviral nucleic acid medicament is combined in the treatment. Therefore, there is an urgent need to find new therapeutic targets, therapeutic methods and protocols for HBV-related liver cancer.

In recent years, the traditional Chinese medicine has the characteristics of more components and targets, obvious regulation effect on an immune system, small side effect and good superposition and synergistic effect by taking the whole treatment idea and dialectical treatment mode, and has attracted wide attention of the medical field and more researchers. Therefore, the treatment of the traditional Chinese medicine is actively applied to the hepatitis B related liver cancer, and the action mechanism of the traditional Chinese medicine is gradually and deeply clarified.

The invention is described in more detail below:

the medicine composition of the invention is as follows: the liver cancer protocol (GAXD) is prepared from Fritillaria paniculata (5X), Stephania tetrandra (3X), Rheum officinale (1X), Ligusticum chuanxiong (2X) and Astragalus membranaceus (5X) in combination. Wherein:

dose 1: rhizoma Bolbostematis 5, radix Stephaniae Tetrandrae 3, radix et rhizoma Rhei 1, rhizoma Ligustici Chuanxiong 2 and radix astragali 5

Dose 2: rhizoma Bolbostematis 5, radix Stephaniae Tetrandrae 3, radix et rhizoma Rhei 1, rhizoma Ligustici Chuanxiong 2 and radix astragali 3

Dose 3: 3 pieces of rhizoma bolbostemmae, 3 pieces of radix stephaniae tetrandrae, 1 piece of rhubarb, 2 pieces of rhizoma ligustici wallichii and 5 pieces of astragalus

Experiments show that the anti-tumor effect of the dosage 1 in HBV-related liver cancer is the best.

The effects of the medicines are as follows:

paniculate Bolbostemma rhizome: clear heat and resolve phlegm, dissipate nodulation and remove toxicity.

Stephania tetrandra: induce diuresis to alleviate edema, dispel wind to alleviate pain.

Rhubarb: purging heat-toxin, breaking stagnation and removing blood stasis. Purge heat and unblock intestines, cool blood and remove toxicity, dispel stasis and dredge meridians.

Ligusticum wallichii: promote blood circulation and move qi, dispel wind and alleviate pain.

Astragalus root: invigorating qi, invigorating yang, consolidating superficial resistance, removing toxic substance, relieving swelling, inducing diuresis, and promoting granulation,

the boiling method comprises the following steps: firstly, adding water into rhubarb for half a minute, then adding rhizoma bolbostemmae, radix stephaniae tetrandrae, ligusticum wallichii and astragalus, and decocting with slow fire.

The basic experimental results of the invention are as follows:

the applicant teams detect the treatment effect of the liver cancer agreement (GAXD) on the hepatitis B related liver cancer transplantation tumor model and preliminarily detect the influence of the liver cancer agreement (GAXD) on the proportion and the activity of NK cells, and the main results are as follows:

(1) effective inhibition of tumor growth by the liver cancer protocol (GAXD) on transplanted tumor models

The applicant killed 2 weeks later by inoculating a tumor mass, i.e., cells HepG2.2.15 bearing tumors in the axilla of nude mice, collected the tumor mass, and divided the tumor mass into 1 '-1' 1mm³The small tumor blocks with the size are inoculated to the armpit of a new nude mouse, and a transplantation tumor model (FIG. 1A) of an HBV-related hepatoma cell line HepG2.2.15 is established. After 10 days of tumor mass inoculation, 4 groups were randomized: control group (Untreated group), positive control group (S31-201 group), GAXD low dose group (6 g/Kg), and GAXD medium dose group (12 g/Kg). The tumor mass was treated 13 days after inoculation according to the grouping. As a result, the transplanted tumor model of the liver cancer agreement (GAXD) treatment group has obvious tumor inhibition effect, and the treatment effect is related to the dosage (FIG. 1B)&C) In that respect This suggests that the liver cancer protocol (GAXD) plays a therapeutic role in hepatitis B-related liver cancer.

(2) GAXD effectively cleared the virus on a model of transplanted tumors;

the applicant detects the HBV virus titer in tumor mass tissues of different treatment groups and peripheral serum of nude mice, and finds that the HBV viral load of the GAXD treatment group is obviously reduced, and the viral load is related to the dose (figures 2A & B). Meanwhile, the expression level of HBV antigens (HBsAg and HBeAg) in peripheral blood serum of nude mice of different treatment groups is also detected. The results showed that the peripheral serum of nude mice in GAXD treated group also had a significant reduction in HBV antigen (FIG. 2.2C & D). These results suggest that GAXD plays an antiviral role in the treatment of hepatitis B-related liver cancer.

(3) GAXD effectively inhibits vascular proliferation in models of transplanted tumors

Angiogenesis is an important factor for tumorigenesis and development, and plays an important role particularly in the process of HBV-induced liver cancer. The applicant detects the expression of tumor angiogenesis related molecules HIF-1a and VEGFA in tumor mass tissues of different treatment groups by an immunohistochemical technology. A significant reduction in HIF-1a and VEGFA expression was found in tumor tissues of the GAXD treated group (FIG. 3A & B). These results suggest that GAXD plays an anti-tumor vascular role in the treatment of hepatitis B-related liver cancer.

(4) GAXD inhibits activation of STAT3 signaling pathway

The applicant observed a similar therapeutic effect of GAXD as STAT3 inhibitor S31-201 in a transplanted tumor nude mouse model of hepg2.2.15 cells, but the molecular mechanism of GAXD is not clear. Therefore, after the applicant co-incubates and cultures the GAXD-containing serum and HepG2.2.15 cells for 6h, protein is extracted, and several signal pathways closely related to the generation and development of hepatitis B-related liver cancer, including PI3K, MAPK and STAT3, are analyzed by using a Western-blot technique. It was found that GAXD significantly inhibited the phosphorylation level of STAT3 protein, while having no significant effect on the expression of key signaling molecules in PI3K and MAPK signaling pathways (fig. 4a & B & C). This suggests that GAXD functions by inhibiting the STAT3 signaling pathway in the treatment of hepatitis B-related liver cancer.

The research results show that clinical meridian GAXD shows similar treatment effect to STAT3 inhibitor S31-201 on a nude mouse transplanted tumor model: both anti-tumor and anti-viral, and GAXD inhibits activation of the STAT3 signaling pathway. It is known that STAT3 in silent liver cancer cells can activate the activity of NK cells in the liver cancer microenvironment to reverse tumor-induced immune tolerance, so we speculate that GAXD may reverse the low function of NK cells in the tumor microenvironment, and then preliminarily detect the influence of GAXD on the proportion and activity of NK cells.

(5) GAXD increases the proportion and activity of peripheral blood NK cells

Applicants isolated nude mouse peripheral blood lymphocytes and specifically labeled NK cells with CD3 and DX5 antibodies. The results found that the proportion of NK cells and the expression level of the activating molecule CD69 were increased in the GAXD treated group (fig.5a & B); the MTT method detects that the killing rate of NK cells to the sensitive cell line YAC-1 is obviously increased (FIG. 5C). This suggests that GAXD has a regulatory effect on the proportion and functional activity of NK cells.

The research also fully proves that the traditional Chinese medicine compound not only inhibits the in vitro growth and virus replication of HBV + liver cancer cells through STAT3 signal channel, but also has the function of regulating the proportion and activity of GAXD to NK cells in vivo experiments.

Drawings

FIG.1 shows that GAXD inhibits the growth of HepG2.2.15 transplantable tumors; wherein (A) the cells of the axillary tumor-bearing HepG2.2.15 of the nude mice are killed after 2 weeks, tumor masses are taken and divided into 1' 1mm³The tumor mass with the size is inoculated to the armpit of a new nude mouse, and HBV is established⁺Transplantation tumor model of HCC. (B)&C) 13 days after tumor mass inoculation, mice were treated randomly in 4 groups: a control group (Untreated group), a positive control group (S31-201 group), a GAXD low dose group (6 g/Kg), and a GAXD medium dose group (12 g/Kg); as a result, the transplanted tumor model of the GAXD treatment group has obvious tumor inhibition effect, and the treatment effect is related to the dosage. This indicates that GAXD is in HBV⁺Has therapeutic effect on HCC.

FIG. 2 GAXD inhibits HBV viral titer and antigen expression; wherein the HBV virus titer is measuredThe detection kit respectively detects the HBV DNA content in the tumor tissue grinding fluid (A) and the mouse peripheral serum (B), and finds that the HBV DNA in the GAXD treatment group is obviously reduced, and the viral load is related to the dosage (A)&B) In that respect Next, ELISA was performed to examine the expression levels of s antigen (C) and e antigen (D) in peripheral serum, and it was found that HBsAg and HBeAg were also significantly reduced in the GAXD-treated group (C)&D) In that respect These results indicate that GAXD is in HBV⁺Plays an antiviral role in the treatment of HCC.

FIG.3 GAXD decreases expression of the angiogenesis-related molecules HIF1a and VEGFA; wherein angiogenesis is an important factor for the development of tumor, and immunohistochemical technology detects the expression of HIF-1a (A) and VEGFA (B) which are closely related to tumor angiogenesis in tumor tissues. As a result, the expression of HIF-1a and VEGFA was found to be significantly reduced in the GAXD-treated group. The results show that GAXD is in HBV⁺The HCC plays a role in inhibiting tumor angiogenesis.

FIG.4 GAXD inhibits STAT3 activation in HepG2.2.15 cells; wherein rats are GAXD perfused, prepared drug-containing serum and HepG2.2.15 cells are co-incubated for 6h, and protein extraction-Western-blot analysis is carried out to analyze the expression of key pathway protein molecules of HBV-related HCC-related signal pathways (A) PI3K, and (B) AMPK and (C) STAT3 and the like. The results found that GAXD significantly inhibited the phosphorylation level (C) of STAT3 protein, but had no significant effect on the expression of key signal molecules in PI3K and MAPK pathways (a)&B) In that respect This indicates that GAXD is in HBV⁺Function in the treatment of HCC by inhibiting the STAT3 signaling pathway.

FIG.5 GAXD increases the proportion and activity of peripheral blood NK cells; peripheral blood lymphocytes of the mice are separated, and flow cytometry detection shows that in the GAXD treatment group: (A) the proportion of NK cells of CD3-DX5+ is increased, and (B) the expression of an NK cell surface activation molecule CD69 is also increased. (C) The MTT method detects that the killing rate of NK cells of the GAXD treatment group on the sensitive cell line YAC-1 is obviously increased. This suggests that GAXD has a positive immunomodulatory effect on NK cells.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

(1) The medicine comprises the following components: the liver cancer protocol (GAXD) is prepared from Fritillaria paniculata (5X), Stephania tetrandra (3X), Rheum officinale (1X), Ligusticum chuanxiong (2X) and Astragalus membranaceus (5X) in combination.

(2) The boiling method comprises the following steps: firstly, adding water into rhubarb for half a minute, then adding rhizoma bolbostemmae, radix stephaniae tetrandrae, ligusticum wallichii and astragalus, and decocting with slow fire.

Example 2

(1) The medicine comprises the following components: the liver cancer protocol (GAXD) is prepared by combining Fritillaria paniculata (5), Stephania tetrandra (3), Rheum officinale (1), Ligusticum wallichii (3), Astragalus membranaceus (5) and five traditional Chinese medicines.

(2) The boiling method comprises the following steps: firstly, adding water into rhubarb for half a minute, then adding rhizoma bolbostemmae, radix stephaniae tetrandrae, ligusticum wallichii and astragalus, and decocting with slow fire.

Example 3

(1) The medicine comprises the following components: the liver cancer protocol (GAXD) is prepared from Fritillaria paniculata (5X), Stephania tetrandra (3X), Rheum officinale (1X), Ligusticum chuanxiong (2X) and Astragalus membranaceus (3X) in combination.

(2) The boiling method comprises the following steps: firstly, adding water into rhubarb for half a minute, then adding rhizoma bolbostemmae, radix stephaniae tetrandrae, ligusticum wallichii and astragalus, and decocting with slow fire.