Application of 4-hydroxybenzyl alcohol in treatment of hepatic fibrosis and hepatocellular carcinoma

Abstract

The invention discloses application of 4-hydroxybenzyl alcohol in treatment of hepatic fibrosis and hepatocellular carcinoma. According to the application, firstly, it is verified through a TGF-beta induced LX2 fibrosis cell model that 4-hydroxybenzyl alcohol can inhibit TGF-beta induced LX2 cell fibrosis, can inhibit LX2, Huh7 and 97H cell viability and can inhibit the cell proliferation capacity at the same time, and further, in a diethylnitrosamine combined carbon tetrachloride induced hepatocellular carcinoma spontaneous animal model, the 4-hydroxybenzyl alcohol can inhibit TGF-beta induced LX2 fibrosis cell model. The inhibition effect of 4-hydroxybenzyl alcohol on hepatic fibrosis and liver cancer progression is verified. Based on the experimental results, the invention determines the potential value of the compound 4-HBA in preparation of medicines for preventing and treating hepatic fibrosis and / or hepatocellular carcinoma, and the compound 4-HBA has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the application of 4-hydroxybenzyl alcohol in treating liver fibrosis and hepatocellular carcinoma, and belongs to the technical field of biological medicine. BACKGROUND

[0002] Liver fibrosis is one of the key driving factors for the occurrence and development of liver cancer (especially hepatocellular carcinoma, HCC). It is usually induced by chronic liver damage, including viral hepatitis, alcohol abuse and metabolic disorders, etc. These factors can gradually lead to abnormal liver function and even cancer. In fibrotic liver, pathological features such as excessive deposition of extracellular matrix, activation of mesenchymal cells, imbalance of immune microenvironment and abnormal generation of blood vessels, together shape the precancerous microenvironment conducive to tumor occurrence. Therefore, liver fibrosis is not only an early warning signal of liver failure, but also a core high-risk factor for liver cancer.

[0003] Although the research progress in the past decade has greatly deepened the understanding of the mechanism of liver fibrosis, the current treatment methods for liver fibrosis and liver cancer still have significant limitations. Although many anti-fibrosis candidate drugs have shown good efficacy in animal experiments, their clinical anti-fibrosis effect is very small or even ineffective. This suggests that although laboratory research has made breakthroughs, the clinical translation of liver fibrosis treatment still faces great obstacles. In addition, although removing the pathogenic factors (such as control or cure of viral infection) has been proven to reverse liver fibrosis, this reversal is often slow and has a low success rate, making it difficult to avoid the fatal complications faced by patients with advanced fibrosis.

[0004] In terms of treatment, the current intervention measures still focus on removing the damage factors, such as lifestyle adjustment, alcohol abstinence and antiviral therapy. However, such etiology-oriented treatment often has little effect on patients with advanced fibrosis / cirrhosis, so effective anti-fibrosis drug intervention is urgently needed. Currently, some new drugs have been designed to target key pathways in the fibrosis process, covering cell death regulation, inflammation suppression, gut-liver axis regulation and myofibroblast activation. However, effective anti-fibrosis drugs available in the clinic are still extremely rare, highlighting the urgent need to develop new treatment strategies.

[0005] In summary, liver fibrosis is one of the key predisposing factors for the occurrence of liver cancer, and the current treatment methods for liver fibrosis and liver cancer still have obvious deficiencies. Future research needs to further analyze the molecular regulation mechanism of fibrosis and develop more efficient treatment programs to slow down or reverse the fibrosis process and ultimately reduce the risk of liver cancer. SUMMARY

[0006] The application aims to provide an application of 4-hydroxybenzyl alcohol in treating liver fibrosis and hepatocellular carcinoma. The application verifies that 4-hydroxybenzyl alcohol has a significant inhibitory effect on liver fibrosis and hepatocarcinoma progression in a constructed hepatocarcinoma animal model, and can be used for preparing a drug for treating liver fibrosis and / or hepatocellular carcinoma.

[0007] In order to achieve the above-mentioned purpose, the application provides an application of 4-hydroxybenzyl alcohol in preparing a drug for preventing and treating liver fibrosis and / or hepatocellular carcinoma.

[0008] Preferably, the drug comprises an active ingredient comprising 4-hydroxybenzyl alcohol and a pharmaceutically acceptable carrier or excipient.

[0009] In some embodiments, the drug takes 4-hydroxybenzyl alcohol as the only drug active ingredient.

[0010] Preferably, the dosage form of the drug is a tablet, granules, pills, powder or capsule.

[0011] Preferably, the dosage form of the drug is an injection, enema, oral agent or tube feeding preparation.

[0012] Compared with the prior art, the application has the following beneficial effects:

[0013] The application provides an application of 4-hydroxybenzyl alcohol in a drug for treating liver fibrosis and hepatocellular carcinoma (HCC). The application first verifies that the compound can inhibit the activity and proliferation ability of hepatic stellate cells and tumor cells, and can inhibit the activation of hepatic stellate cells at the molecular level, and verifies that 4-hydroxybenzyl alcohol can significantly inhibit the degree of liver fibrosis and the progression of HCC in a diethylnitrosamine combined with carbon tetrachloride induced spontaneous animal model of hepatocellular carcinoma, and can be used for preparing a new drug for treating liver fibrosis and HCC. The 4-HBA proposed in the application has low toxicity, good solubility and stability at room temperature, and has been used as a synthetic intermediate of various drugs. Oral 4-HBA can act on hepatic stellate cells and hepatoma cells at the same time, so as to achieve the dual effects of inhibiting liver fibrosis and hepatoma, and has a good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 In a co-culture system of LX2 cells and 4-HBA, TGF-β, the content of fibrosis marker α-SMA was detected by western blot to detect the inhibitory effect of 4-HBA on TGF-β induced LX2 fibrosis.

[0015] Figure 2: LX2, Huh7, 97H cell activity detection results (A), clonogenic assay results (B), CCK-8 assay results (C) after 4-HBA stimulation; *P<0.05, **P<0.01, ***P<0.001 indicate that there is a significant difference between the two groups after statistical analysis.

[0016] Figure 3 : Schematic diagram of animal experiment process in Example 3.

[0017] Figure 4 : Tumor pictures, quantitative detection results of tumor number and size (A) after oral administration of 4-HBA in mice with hepatocellular carcinoma, and staining pictures and quantitative statistical analysis results of fibrosis degree detected by HE staining, Masson staining, Sirius red staining and tumor proliferation ability detected by Ki67 immunohistochemistry (B); *P<0.05, **P<0.01, ***P<0.001 indicate that there is a significant difference between the two groups after statistical analysis. DETAILED DESCRIPTION

[0018] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail below with the help of the accompanying drawings.

[0019] Example 1

[0020] Implementation materials:

[0021] LX2 hepatic stellate cells, Huh7 hepatocarcinoma cells, LM3 hepatocarcinoma cells and human TGF-β1 (PCK354) are purchased from Wuhan Pons Life Science Co., Ltd. The cells are cultured in a 37℃ culture box containing 5% CO2, and the culture medium is DMEM culture medium containing 10% fetal bovine serum, 1% penicillin / streptomycin. 4-HBA (4-Hydroxybenzyl alcohol, 4-HBA, CAS number: 623-05-2) is purchased from MCE company. α-SMA antibody (#AF1032, 1:1000 dilution) is purchased from Kinbio, and β-actin antibody (66009-1-Ig, 1:20000 dilution) is purchased from Wuhan Sanyou Biotechnology Co., Ltd.

[0022] Implementation method:

[0023] LX2 cells were cultured in culture medium containing 0 (blank control), 0.1, 1, 10, 100 μM 4-HBA and 5 ng / mL TGF-β, respectively, for 24 h. The cells were lysed with RIPA lysis buffer, and the supernatant was collected and added to loading buffer. After boiling in a 95℃ metal bath for 5 minutes, the α-SMA content was detected by western blot.

[0024] Implementation results:

[0025] 4-HBA can inhibit TGF-β-induced LX2 fibrosis, manifested as a decrease in the level of a-SMA protein, as shown in Figure 1 .

[0026] Example 2

[0027] Materials for implementation:

[0028] LX2 hepatic stellate cells, Huh7 hepatoma cells, 97H hepatoma cells were purchased from Wuhan Ponsay Life Science Co., Ltd. The cells were cultured in a 37°C incubator containing 5% CO2, and the culture medium was DMEM medium containing 10% fetal bovine serum, 1% penicillin / streptomycin. 4-HBA (4-Hydroxybenzyl alcohol, 4-HBA, CAS No.: 623-05-2) was purchased from MCE Company. CCK-8 reagent (PF00004) was purchased from Wuhan Sany Biological Technology Co., Ltd.

[0029] Method for implementation:

[0030] Cell activity detection: LX2, Huh7, 97H cells were seeded in 96-well plates (2000 cells / well), and 0 (blank control), 0.1, 1, 10, 100 μM 4-HBA containing medium was added respectively for 24 h, CCK-8 was added for 2 h, and then the OD450 value was detected by spectrophotometer.

[0031] Cloning experiment: LX2, Huh7, 97H cells were cultured in medium containing 0 (blank control), 1, 100 μM 4-HBA for 24 h, and then seeded in 6-well plates (LX2: 1000 cells / well, Huh7, 97H: 500 cells / well). After 9-14 days of culture, crystal violet staining was performed.

[0032] CCK-8 experiment: LX2, Huh7, 97H cells were cultured in medium containing 0 (blank control), 1, 100 μM 4-HBA for 24 h, and then seeded in 96-well plates (1000 cells / well). CCK-8 was added on days 1, 2, 3, 4, and 5 of culture, respectively, and then incubated for 2 h. The OD450 value was detected by spectrophotometer.

[0033] Results of implementation:

[0034] 4-HBA can inhibit the activity of LX2, Huh7, 97H cells, and also inhibit the proliferation ability of the cells, manifested as a decrease in the number of colony formation and a decrease in the OD450 value in the CCK-8 experiment, as shown in Figure 2 A-C.

[0035] Example 3

[0036] Materials for implementation:

[0037] DEN (73861) and CCl4 (319961) were purchased from Sigma-Aldrich. C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 4-HBA (HY-Y0892) was purchased from MCE. Ki67 antibody (27309-1-AP, 1:15000 dilution) was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0038] Implementation method:

[0039] DEN was dissolved in physiological saline and administered intraperitoneally at a dose of 25 mg / kg to male C57BL / 6 mice at 2 weeks of age. At 6 weeks of age, CCl4 was administered intraperitoneally (2 mL / kg, twice weekly for 18 weeks). At 8 weeks of age, 4-HBA was administered by gavage (200 mg / kg, every other day for 16 weeks). Samples were collected at 24 weeks of age for observation. Figure 3 HE, Masson's red, Sirius red, and Ki67 immunohistochemistry were performed.

[0040] Implementation results:

[0041] Oral administration of 4-HBA can significantly reduce the number and volume of tumors. Figure 4 A) inhibits the degree of fibrosis and tumor proliferation, manifested as reduced intrahepatic collagen deposition and a decrease in the number of Ki67-positive cells within the tumor. Figure 4 B).

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Use of 4-hydroxybenzyl alcohol in the preparation of a drug for preventing and / or treating liver fibrosis and / or hepatocellular carcinoma.

2. Use according to claim 1, characterized in that, The drug comprises an active ingredient comprising 4-hydroxybenzyl alcohol and a pharmaceutically acceptable carrier or excipient.

3. Use according to claim 1, characterized in that, The dosage form of the drug is a tablet, granules, pills, powder, or capsules.

4. Use according to claim 1, characterized in that, The dosage form of the drug is an injection, enema, oral agent, or tube feeding preparation.

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