Application of Wushan icaritin in preparation of medicine for treating liver cancer diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of Wushan icaritin in preparation of medicines for treating liver cancer diseases. The Wushan icaritin can effectively reduce the expression of lactic acid content in the liver cancer stem cells, reduce the lactic acid modification level of the liver cancer stem cells, inhibit the glycolysis level of the liver cancer stem cells, reduce the number of the liver cancer stem cells in the S phase, and improve the survival rate of the liver cancer stem cells through multiple ways. The compound can inhibit malignant biological behaviors of liver cancer stem cells and slow down the liver cancer disease process, and can be used as a natural preparation for treating liver cancer diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to an application of Wushan icariin in preparing a medicine for treating liver cancer. Background Art

[0002] Liver cancer is a malignant tumor with a high incidence and mortality rate, which seriously endangers human life and health. Due to the hidden early symptoms of liver cancer, it usually progresses to the middle and late stages when diagnosed, missing the best time for radical treatment such as surgical resection or liver transplantation. In this case, chemotherapy has become an important treatment method commonly used in clinical practice. Currently, commonly used chemotherapy drugs in clinical practice include cisplatin, sorafenib, 5-fluorouracil, etc. However, a large number of clinical studies have shown that these traditional drugs have obvious limitations in the treatment of liver cancer: not only do they have significant toxic side effects, but they are also prone to drug resistance, resulting in unsatisfactory overall efficacy. Therefore, exploring new, highly effective and low-toxic anti-liver cancer drugs has become a key scientific issue that needs to be urgently addressed in the current field of liver cancer treatment.

[0003] In traditional Chinese medicine, liver cancer syndrome differentiation believes that the pathogenesis of cancer toxins flowing and stagnation of liver meridians is closely related to the recurrence and metastasis of liver cancer. The "cancer toxin theory" proposed by Zhou Zhongying points out that cancer toxins are caused by multiple pathogenic factors and have multiple characteristics such as complexity and high recurrence and metastasis, emphasizing the important role of cancer toxins flowing in the process of liver cancer recurrence and metastasis. Chen Baogui believes that the characteristics of cancer toxins flowing and invasive can be regarded as the iconic behavior of "flowing toxins", which constitutes a prerequisite for tumor metastasis. Modern medicine believes that liver cancer stem cells (LCSCs) are a subpopulation with stem cell characteristics in liver cancer, with the characteristics of self-renewal, differentiation, promotion of distant metastasis and homing, and can produce all the characteristics of liver cancer. Liver cancer stem cells are the main cause of liver cancer recurrence and metastasis, which can be regarded as the manifestation of cancer toxins flowing and stagnation of liver meridians in traditional Chinese medicine. Therefore, based on the cancer toxin theory of traditional Chinese medicine, exploring Chinese medicinal materials targeting liver cancer stem cells can provide new ideas for the treatment of liver cancer.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Wushan icariin in the preparation of a drug for treating liver cancer. Wushan icariin can inhibit the malignant biological behavior of liver cancer stem cells by reducing the level of histone lactylation modification, thereby playing an anti-hepatocellular carcinoma role.

[0006] In order to achieve the above-mentioned object of the present invention, the first aspect of the present invention provides the use of wushanica eccalis in the preparation of a medicine for treating liver cancer.

[0007] In a specific embodiment of the present invention, there is provided the use of icariin in the preparation of a drug for inhibiting the self-renewal of liver cancer stem cells.

[0008] In a specific embodiment of the present invention, the self-renewal of liver cancer stem cells includes at least one of proliferation, differentiation, and colony formation of liver cancer stem cells.

[0009] In a specific embodiment of the present invention, there is provided the use of icariin in the preparation of a drug for inhibiting the proliferation of liver cancer stem cells.

[0010] In a specific embodiment of the present invention, icariin inhibits the proliferation of liver cancer stem cells through at least one of the following characteristics:

[0011] (1) Icariin induces cell cycle arrest of liver cancer stem cells in the S phase;

[0012] (2) Icariin reduces the glycolysis level of liver cancer stem cells;

[0013] (3) Icariin reduces the level of lactate modification of liver cancer stem cells.

[0014] In a specific embodiment of the present invention, the subject of the drug is a mammal. Further, the mammal includes at least one of rodents, non-human primates, and humans.

[0015] The second aspect of the present invention provides a drug for treating liver cancer, comprising icariin.

[0016] In a specific embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0017] In a specific embodiment of the present invention, in the drug, the effective cell dose of icariin is 1-100 μM, preferably 5-10 μM.

[0018] In a specific embodiment of the present invention, the dosage form of the drug includes at least one of oral dosage forms and injection dosage forms.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The icariin of the present invention can effectively reduce the expression of lactate content in liver cancer stem cells, down-regulate the level of lactate modification of liver cancer stem cells, and can also inhibit the glycolysis level of liver cancer stem cells, and can also reduce the number of liver cancer stem cells in the S phase. Through multiple ways, it inhibits the malignant biological behavior of liver cancer stem cells and slows down the progression of liver cancer;

[0021] (2) The icariin of Epimedium wushanense in the present invention not only has a good inhibitory effect on liver cancer stem cells even at low concentrations, but also has low toxicity to normal stem cells, featuring high efficiency and low toxicity and being suitable for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 A photograph of Epimedium wushanense provided by the present invention;

[0024] Figure 2 The test results of the inhibition of the proliferation of liver cancer stem cells by icariin of Epimedium wushanense provided in Example 1 of the present invention;

[0025] Figure 3 The test results of the inhibition of the glycolysis level of liver cancer stem cells by icariin of Epimedium wushanense provided in Example 2 of the present invention;

[0026] Figure 4 The test results of the reduction of the lactylation modification level of liver cancer stem cells by icariin of Epimedium wushanense provided in Example 3 of the present invention;

[0027] Figure 5 The influence of icariin on the proliferation of liver cancer stem cells provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the drawings and specific embodiments. However, those skilled in the art will understand that the following described embodiments are some embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0029] Figure 1This is a photo of Epimedium wushanense, which is a local medicinal herb in Wushan County, Chongqing. It mainly grows on the shady and humid slopes at an altitude of about 1000 meters in Wushan County and is a key traditional Chinese medicine for local development. Modern research on traditional Chinese medicine shows that plants of the genus Epimedium and their active ingredients are warm in nature, pungent and sweet in taste, and belong to the liver and kidney meridians. They have the effects of relieving cough, reducing phlegm, relieving asthma, anti-inflammatory, and lowering blood pressure. Moreover, no obvious drug toxicity to normal cells and tissues of the body has been found. Wushanicaritin (WIT) is one of the most important chemical components in Epimedium wushanense, and its structural formula is as follows:

[0030]

[0031] Currently, there is no research report on the treatment of liver cancer with Epimedium wushanense. Based on this, the first aspect of the present invention provides the application of wushanicaritin in the preparation of drugs for treating liver cancer.

[0032] In a specific embodiment of the present invention, there is provided the application of wushanicaritin in the preparation of drugs for inhibiting the self-renewal of liver cancer stem cells.

[0033] Liver cancer stem cells and liver cancer cells play different roles in tumorigenesis, treatment resistance, recurrence, and metastasis. Liver cancer stem cells are the most malignant cell subset in liver cancer, and their characteristics of high invasiveness, strong drug resistance, and easy recurrence constitute the core reasons for the failure of liver cancer treatment. In the present invention, more malignant liver cancer stem cells enriched from HCCLM3 and Hep3B cells are used. It is found that wushanicaritin can effectively reduce the expression of lactate content in liver cancer stem cells, down-regulate the level of lactate modification in liver cancer stem cells, inhibit the glycolysis level of liver cancer stem cells, and reduce the number of liver cancer stem cells in the S phase. Through multiple pathways, it inhibits the malignant biological behavior of liver cancer stem cells and slows down the progression of liver cancer.

[0034] In a specific embodiment of the present invention, the self-renewal of liver cancer stem cells includes at least one of the proliferation, differentiation, and colony formation of liver cancer stem cells.

[0035] In a specific embodiment of the present invention, there is provided the application of wushanicaritin in the preparation of drugs for inhibiting the proliferation of liver cancer stem cells.

[0036] In a specific embodiment of the present invention, wushanicaritin inhibits the proliferation of liver cancer stem cells through at least one of the following characteristics:

[0037] (1) Wushanicaritin induces cell cycle arrest of liver cancer stem cells in the S phase;

[0038] (2) Wushanicaritin reduces the glycolysis level of liver cancer stem cells;

[0039] (3) Icariin reduces the level of lactate modification in liver cancer stem cells.

[0040] In a specific embodiment of the present invention, the object of the drug is a mammal. Further, the mammal includes at least one of rodents, non-human primates, and humans. Among them, rodents include but are not limited to rats, mice, etc.

[0041] The second aspect of the present invention provides a drug for treating liver cancer diseases, including icariin.

[0042] In a specific embodiment of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0043] Among them, the excipients can be routinely adjusted according to the actual dosage form of the drug. For example, they can include but are not limited to diluents, buffers, suspensions, emulsions, excipients, fillers, binders, wetting agents, disintegrants, and so on.

[0044] In a specific embodiment of the present invention, in the drug, the effective cell dose of icariin is 1 - 100 μM, preferably 5 - 10 μM. In different embodiments, in the drug, the effective cell dose of icariin can be 1 μM, 3 μM, 5 μM, 8 μM, 10 μM, 20 μM, 50 μM, 60 μM, 80 μM, 100 μM, or the range composed of any two of them.

[0045] Icariin of the present invention can significantly inhibit the proliferation of liver cancer stem cells at a low concentration of 5 - 10 μM, has the potential of natural anti - liver cancer diseases, and can be used as a natural preparation for treating liver cancer diseases.

[0046] In a specific embodiment of the present invention, the dosage form of the drug includes at least one of oral dosage forms and injection dosage forms.

[0047] The liver cancer stem cells used in the following examples are liver cancer stem cells M3L and 3BL enriched from HCCLM3 and Hep3B cells.

[0048] Example 1

[0049] Icariin inhibits the proliferation of liver cancer stem cells

[0050] The CCK8 method was used to detect the effect of icariin on the proliferation of liver cancer stem cells (M3L and 3BL), and the results are as follows Figure 2As shown in Figure a. The liver cancer stem cells were divided into three groups, namely the control group (DMSO group), the 5 μM group (5 μM icariin), and the 10 μM group (10 μM icariin). The colony formation assay and flow cytometry were performed on these three groups of cells, and flow cytometry was used to detect the cell cycle. The colony formation assay detected that compared with the control group, after adding icariin, the number of cell colonies decreased significantly, and the decrease in the 10 μM group was more than that in the 5 μM group, indicating that icariin can inhibit the proliferation of liver cancer stem cells and shows a concentration-dependent trend to a certain extent. The results are as shown in Figure 2 Figure b. The flow cytometry assay for cell cycle detection detected that compared with the control group, after adding icariin, the number of cells entering the S phase decreased significantly, and the decrease in the 10 μM group was more than that in the 5 μM group, indicating that icariin can inhibit the cell cycle arrest of liver cancer stem cells and thus inhibit cell proliferation. The results are as shown in Figure 2 Figure c. The above results indicate that icariin can inhibit the proliferation of liver cancer stem cells.

[0051] Example 2

[0052] Icariin inhibits the proliferation of liver cancer stem cells

[0053] The third-generation liver cancer stem cells M3L enriched from HCCLM3 cells were taken and cultured in a low-attachment 6-well plate with an inoculation density of 1×10 6 cells / cm 2 . The cells were divided into two groups, namely the control group (DMSO group) and the 10 μM group, with 10 replicates in each group. After treatment for 48 hours, the cells were collected, and after adding lysis buffer, GC-MS metabolomics detection was performed to evaluate the effect of icariin treatment on the primary metabolites of liver cancer stem cells. Figure 3 The heatmap in Figure a shows that icariin affects the production of many primary metabolites in liver cancer stem cells. OPLS-DA was used to exclude possible established variables unrelated to the evaluation of the statistical significance of these signals, such as Figure 3 Figure b. In addition, the metabolic pathway impact analysis showed that these differential metabolites were mainly enriched in the glycolysis / gluconeogenesis and pyruvate metabolic pathways, such as Figure 3 Figure c. The above results indicate that icariin can regulate the glycolysis metabolic pathway to control the proliferation of liver cancer stem cells.

[0054] Example 3

[0055] Icariin reduces the level of lactate modification in liver cancer stem cells

[0056] Hepatocellular carcinoma stem cells (M3L and 3BL) were divided into three groups, namely the control group (DMSO group), the 5 μM group, and the 10 μM group. Western blotting experiments were performed on these three groups of cells to detect the expression levels of lactic acid-modified proteins in hepatocellular carcinoma stem cells. The results are as Figure 4 shown in Figure 4 Figures a and

[0057] b. The detection results showed that compared with the control group, after adding icariin, the level of lactic acid modification was significantly reduced, and the number of reductions in the 10 μM group was more than that in the 5 μM group, indicating that icariin can inhibit the expression level of lactic acid-modified proteins in hepatocellular carcinoma stem cells and shows a concentration-dependent trend to a certain extent. The above results showed that icariin can regulate the proliferation of hepatocellular carcinoma stem cells by regulating the level of lactic acid modification.

[0058] Comparative Example 1

[0059] The structural formula of icariin is as follows:

[0060]

[0061] The CCK8 method was used to detect the effect of icariin on hepatocellular carcinoma stem cells (M3L and 3BL). The method was referred to Example 1, and the detection results are as Figure 5 shown. 50 μM icariin can inhibit the proliferation of hepatocellular carcinoma stem cells; comparing with the results of Figure 2 Figure a of the present invention, it can be seen that icariin in the present invention can significantly inhibit the proliferation of hepatocellular carcinoma stem cells at a lower concentration. Icariin and icariin belong to different subclasses and are different substances, showing significantly different effects on inhibiting the proliferation of hepatocellular carcinoma stem cells.

[0062] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of icariin in the preparation of a drug for treating liver cancer diseases.

2. The application according to claim 1, wherein Application of icariin in the preparation of a drug for inhibiting the self-renewal of liver cancer stem cells.

3. The application according to claim 2, wherein The self-renewal of the liver cancer stem cells includes at least one of proliferation, differentiation and colony formation of the liver cancer stem cells.

4. The application according to claim 1, wherein Application of icariin in the preparation of a drug for inhibiting the proliferation of liver cancer stem cells.

5. The application according to claim 4, wherein The icariin inhibits the proliferation of liver cancer stem cells through at least one of the following characteristics: (1) The icariin induces cell cycle arrest of liver cancer stem cells in the S phase; (2) The icariin reduces the glycolysis level of liver cancer stem cells; (3) The icariin reduces the lactylation modification level of liver cancer stem cells.

6. The application according to claim 1, wherein The drug is targeted at mammals; Preferably, the mammals include at least one of rodents, non-human primates and humans.

7. A drug for treating liver cancer, characterized in that, The drug includes icariin.

8. The drug according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

9. The drug according to claim 7, characterized in that, In the drug, the effective cell dose of the icariin is 1-100 μM; Preferably, the effective cell dose of the icariin is 5-10 μM.

10. The drug according to claim 7, characterized in that, The dosage form of the drug includes at least one of oral dosage forms and injection dosage forms.

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