Application of bacteroides acidogenes in treatment of hepatic fibrosis and hepatocellular carcinoma

Abstract

The invention discloses application of acid-producing bacteroides in treatment of hepatic fibrosis and hepatocellular carcinoma. In-vitro cell experiments verify that the Bacteroides acidogenes and the culture medium thereof can inhibit LX2 cell fibrosis induced by TGF-beta, can inhibit the activity of LX2, Huh7 and 97H cells and can inhibit the cell proliferation capacity at the same time, and further, in a hepatocellular carcinoma spontaneous animal model induced by diethylnitrosamine and carbon tetrachloride, the application of the Bacteroides acidogenes and the culture medium of the Bacteroides acidogenes and the culture medium of the Bacteroides acidogenes and the culture medium of the Bacteroides acidogenes in the hepatocellular carcinoma spontaneous animal model has the advantages of being capable of inhibiting the fibrosis of the LX2 cells induced by the TGF-beta and inhibiting the proliferation capacity of the cells. The inhibition effect of the acid-producing bacteroides on hepatic fibrosis and liver cancer progression is verified; the acid-producing bacteroides is recognized as probiotics and has low side effect risk, so that the acid-producing bacteroides has a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to application of Bacteroides acidifaciens in treatment of liver fibrosis and hepatocellular carcinoma and belongs to the technical field of biological medicine. BACKGROUND

[0002] Liver fibrosis is one of the important driving factors for the occurrence and development of hepatocellular carcinoma (HCC). The disease is usually caused by chronic liver damage, and common causes include viral hepatitis, alcohol abuse and metabolic disorders. These factors can gradually cause liver dysfunction and eventually lead to cancer. In fibrotic liver, pathological changes such as excessive deposition of extracellular matrix, activation of mesenchymal cells, disorder of immune microenvironment and abnormal generation of blood vessels, together shape a 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] In recent years, a number of studies have shown that microbial agents have potential value in the prevention and treatment of liver fibrosis and liver cancer. Gut microbiota plays a key role in the mechanism, diagnosis and treatment of HCC, especially in regulating anti-cancer immune response. In addition, intestinal flora imbalance is closely related to the progression of liver cancer in patients with cirrhosis, indicating that intervention of microbiota may become a new strategy for the treatment of liver cancer. In the treatment of liver fibrosis, microbial agents also show broad prospects. Studies have shown that by regulating the gut microbiota, it can affect bile acid metabolism, thereby reversing liver damage and delaying the progression of non-alcoholic steatohepatitis (NASH). In addition, microbial intervention can also alleviate liver fibrosis caused by parasitic infection by inhibiting Th2 cell immune response and restoring intestinal flora balance. These results suggest that the regulation of microbiota not only helps to improve the pathological process of liver fibrosis, but also may provide a new treatment approach for this type of disease. SUMMARY

[0004] The purpose of the present application is to provide application of Bacteroides acidifaciens in treatment of liver fibrosis and hepatocellular carcinoma, and to prepare anti-liver fibrosis / hepatocellular carcinoma drugs by using Bacteroides acidifaciens, so as to inhibit disease progression.

[0005] In order to achieve the above-mentioned purpose, the present application provides application of Bacteroides acidifaciens and / or metabolites thereof in preparation of a drug for prevention and treatment of liver fibrosis and / or hepatocellular carcinoma.

[0006] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient comprises Bacteroides acidifaciens and / or metabolites thereof.

[0007] Preferably, the adjuvant comprises one or more of diluents, wetting agents, binders, disintegrants, lubricants, flavoring agents, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusting agents, isotonizing agents and tonicity adjusting agents.

[0008] Preferably, the dosage form of the drug is a tablet, granules, pills, powder, capsule.

[0009] Preferably, the dosage form of the drug is an injection, enema, oral or tube feeding preparation.

[0010] Compared with the prior art, the present application has the following beneficial effects:

[0011] 1) The present application verifies the inhibitory effect of oral B. acidiproducens on liver fibrosis and liver cancer progression in a diethylnitrosamine (DEN) combined with carbon tetrachloride (CCl4) induced hepatocellular carcinoma spontaneous animal model and in vitro cell model, and has good clinical application prospect.

[0012] 2) The B. acidiproducens proposed in the present application is a recognized probiotic with low side effect risk; oral B. acidiproducens can act on hepatic stellate cells and liver cancer cells at the same time, thereby achieving the dual effect of inhibiting liver fibrosis and liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : The results of western blot detection of the content of alpha-SMA after co-culturing LX2 cells with B. acidiproducens.

[0014] Figure 2 : The results of cell activity detection (A), clonogenicity experiment (B) and CCK-8 experiment (C) after stimulating LX2, Huh7 and 97H with B. acidiproducens.

[0015] Figure 3 : Schematic diagram of the animal experiment process in Example 3.

[0016] Figure 4 : The results of tumor number and volume detection (A) after oral administration of B. acidiproducens to tumor mice, and the results of fibrosis degree detection by HE staining, Masson staining and Sirius red staining, and tumor proliferation ability detection by Ki67 immunohistochemistry (B). DETAILED DESCRIPTION

[0017] In order to make the present application more obvious and easy to understand, the preferred embodiments are described in detail below with the aid of the accompanying drawings.

[0018] Example 1

[0019] Implementation materials:

[0020] LX2 liver stellate cells, Huh7 hepatoma cells, LM3 hepatoma cells and human TGF-β1 (PCK354) were purchased from Wuhan Pons 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. Bacteroides acidifaciens B98300 (other numbers: DSM 15896 / BCRC 81230 / JCM 10556) was purchased from Ningbo Mingzhou Biological Technology Co., Ltd. and cultured in an anaerobic environment at 37°C. The culture medium was blood agar medium (peptone 10.0 g, glucose 2.0 g, NaCl 5.0 g, NaH2PO4 2.5 g, sodium citrate 5.0 g, brain infusion 800.0 ml, pig heart infusion 200.0 ml, 10% sheep blood, pH 7.2). α-SMA antibody (#AF1032, 1:1000 dilution) was purchased from Kinbio, and β-actin antibody (66009-1-Ig, 1:20000 dilution) was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0021] Implementation method:

[0022] LX2 cells were co-cultured with blank control, heat-inactivated Bacteroides acidifaciens (MOI = 100), live Bacteroides acidifaciens (MOI = 100), and Bacteroides acidifaciens culture medium for 24 hours, and were stimulated with 5 ng / mL TGF-β at the same time. The cells were lysed with RIPA lysis buffer, and the supernatant was collected and added to loading buffer. After boiling in a 95°C metal bath for 5 minutes, the α-SMA content was detected by western blot.

[0023] Implementation results:

[0024] Live Bacteroides acidifaciens and its culture medium can inhibit TGF-β-induced LX2 fibrosis, which is manifested as a decrease in α-SMA protein levels, as shown in Figure 1 .

[0025] Example 2

[0026] Implementation materials:

[0027] LX2 hepatic stellate cells, Huh7 hepatoma cells, 97H hepatoma cells were purchased from Wuhan Ponsay Life Technology 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. Bacteroides acidiproducens B98300 was purchased from Ningbo Mingzhou Biological Technology Co., Ltd. and cultured in an anaerobic environment at 37°C. The culture medium was blood agar medium (peptone 10.0 g glucose 2.0 g, NaCl 5.0 g, NaH2PO4 2.5 g, sodium citrate 5.0 g, brain infusion 800.0 ml, pig heart infusion 200.0 ml, 10% sheep blood, pH 7.2). CCK-8 reagent (PF00004) was purchased from Wuhan Sany Biological Technology Co., Ltd.

[0028] Implementation method:

[0029] Cell activity detection: LX2, Huh7, 97H cells were inoculated in 96-well plates (2000 cells / well), and co-cultured with blank control, heat-inactivated Bacteroides acidiproducens (MOI=100), live Bacteroides acidiproducens (MOI=100), and Bacteroides acidiproducens culture medium for 24h. After adding CCK-8 for 2h, the OD450 value was detected by spectrophotometer.

[0030] Cloning experiment: LX2, Huh7, 97H cells were co-cultured with blank control, heat-inactivated Bacteroides acidiproducens (MOI=100), live Bacteroides acidiproducens (MOI=100), and Bacteroides acidiproducens culture medium for 24h, and then inoculated in 6-well plates (LX2: 1000 cells / well, Huh7, 97H: 500 cells / well). After 9-14 days of culture, crystal violet staining was performed.

[0031] CCK-8 experiment: LX2, Huh7, 97H cells were co-cultured with blank control, heat-inactivated Bacteroides acidiproducens (MOI=100), live Bacteroides acidiproducens (MOI=100), and Bacteroides acidiproducens culture medium for 24h, and then inoculated in 96-well plates (1000 cells / well). CCK-8 was added on the 1st, 2nd, 3rd, 4th, and 5th days of culture, respectively. After 2h of culture, the OD450 value was detected by spectrophotometer.

[0032] Implementation results:

[0033] Live Bacteroides acidiproducens and its culture medium can inhibit the activity of LX2, Huh7, 97H cells, and also inhibit the proliferation ability of the cells, which is 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.

[0034] Example 3

[0035] Implementation materials:

[0036] DEN (73861) and CCl4 (319961) were purchased from Sigma-Aldrich. C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Bacteroides acidogeneticus B98300 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and cultured in an anaerobic environment at 37℃ on blood agar medium (10.0g peptone, 2.0g glucose, 5.0g NaCl, 2.5g NaH2PO4, 5.0g sodium citrate, 800.0ml brain extract, 200.0ml pig heart extract, 10% sheep blood, pH 7.2). Ki67 antibody (27309-1-AP, 1:15000 dilution) was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0037] Implementation method:

[0038] DEN was dissolved in physiological saline and administered intraperitoneally at 25 mg / kg to female 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, Bacteroides acidogenis was administered via gavage (2 × 10⁻⁶). 8 CFU / time, every other day, for 16 weeks) was collected at 24 weeks of age for observation. Figure 3 HE, Masson's red, Sirius red, and Ki67 immunohistochemistry were performed.

[0039] Implementation results:

[0040] Oral administration of Bacteroides acidogenae can significantly reduce the number and size 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).

[0041] 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 Bacteroides acidifaciens and / or metabolites thereof 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 Bacteroides acidifaciens and / or metabolites thereof 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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