Preparation method and application of probiotic and essential amino acid composition for optimizing hepatitis B virus infection immune microenvironment
By using a composition combined with Bacteroides fragile DSM 2151 and branched chain amino acids, the immune microenvironment of hepatitis B virus infection was optimized, and the problems of inefficiency and major side effects of existing HBV treatment methods were solved, and significant HBsAg clearance and improvement of the liver immune microenvironment were achieved.
Patent Information
- Application Number
- CN202510015395.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-06
- Publication Date
- 2025-05-06
AI Technical Summary
The existing hepatitis B virus (HBV) treatments have problems such as inefficiency, long-term use, high side effects and difficult to achieve functional cure. Especially in patients with chronic HBV infection, there is a lack of safe, economical and well-tolerated treatment methods.
Using a composition combined with Bacteroides fragile DSM 2151 and branched chain amino acids, the immune microenvironment of hepatitis B virus infection is optimized by regulating the intestinal flora and immune function, thereby promoting HBsAg clearance and improving liver immune status.
In the mouse model of hepatitis B, Bacteroides fragile DSM 2151 and branched chain amino acid composition significantly improved the clearance of HBsAg, improved the liver immune microenvironment, increased the proportion of CD8+T and CD4+T cells, and increased IFN-γ levels, which was better than the use of probiotics or branched chain amino acids alone.
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Abstract
Description
Technical Field
[0001] The patent of this invention relates to the field of biomedicine technology, and specifically, to the preparation and application of a probiotic and essential amino acid composition that optimizes the immune microenvironment of hepatitis B virus infection. Background Art
[0002] Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family. HBV infection is an important global public health issue and a major pathogenic factor for cirrhosis and hepatocellular carcinoma. Approximately 2 billion people worldwide have been infected with HBV, accounting for about 1 / 3 of the total population. Currently, there are approximately 257 million chronic HBV carriers worldwide. Studies have shown that the lifetime risk of HBV patients developing cirrhosis, liver failure, or hepatocellular carcinoma is as high as 15% to 40%. Clearing chronically infected HBV is the key to fundamentally reducing or preventing HBV-related hepatocellular carcinoma, and is also a difficult point in the current clinical treatment of hepatocellular carcinoma.
[0003] At present, there are two main categories of commonly used hepatitis B antiviral drugs, namely interferons (long-acting interferons, mainly pegylated interferon α-2b) and nucleoside drugs (entecavir, tenofovir disoproxil, tenofovir alafenamide and tenofovir amivir). Long-term antiviral treatment can effectively inhibit HBV replication, reduce the risk of cirrhosis and hepatocellular carcinoma, and slow down the progression of the disease, but there are still many shortcomings. First, the HBsAg serum clearance rate of oral antiviral treatment is low, only about 0.5%, and most patients cannot achieve clinical cure and need long-term use, and its effect in reducing the risk of hepatocellular carcinoma is relatively limited; secondly, although interferon drug treatment can significantly reduce the incidence of liver cancer, its cost is high and the incidence of side effects is high; finally, nucleoside (acid) drugs have been proven to be safe and well tolerated, but long-term use may still lead to cumulative toxicity in some high-risk groups, such as osteoporosis and impaired tubular function in elderly patients and menopausal women when using tenofovir.
[0004] Other treatment strategies for hepatitis B are mainly new agents targeting HBV, which are mainly divided into drugs targeting the hepatitis B virus life cycle (various target drugs, direct-acting antiviral drugs) and drugs targeting host immune regulation. However, the current effects of these treatment strategies are not ideal, and achieving functional cure (such as HBsAg clearance) remains a huge challenge. In high-prevalence areas, universal hepatitis B vaccination and hepatitis B screening strategies are still the main measures to reduce the burden of HBV infection and related liver diseases. Therefore, it is urgent to explore new, safe, economical and well-tolerated treatments.
[0005] To this end, the present application provides the use of a composition of Bacteroides fragilis combined with branched-chain amino acids in the clearance of hepatitis B virus and the improvement of the immune microenvironment of viral infection to solve the above-mentioned problems. Summary of the invention
[0006] The purpose of the present invention is to solve the prior art problems raised in the above background technology and to provide a preparation and application of a probiotic and essential amino acid composition for optimizing the immune microenvironment of hepatitis B virus infection.
[0007] The above-mentioned purpose of the present invention is achieved like this:
[0008] On the one hand, the scheme of the present invention provides the application of Bacteroides fragilis DSM 2151 in optimizing the immune microenvironment of hepatitis B virus infection.
[0009] Another aspect of the present invention provides the use of branched-chain amino acids in optimizing the immune microenvironment of hepatitis B virus infection.
[0010] Another aspect of the present invention provides the use of a composition of Bacteroides fragilis DSM 2151 and branched-chain amino acids in optimizing the immune microenvironment of hepatitis B virus infection.
[0011] The difficulty and significance of the technical problem solved by the present invention are:
[0012] Healthy intestinal flora is essential for maintaining liver metabolism and immune function. Studies have shown that intestinal microorganisms interact with the liver through the gut-liver axis, affecting HBV replication and immune response; the composition of the intestinal flora may affect the efficacy and side effects of antiviral drugs. Current research focuses on the relationship between the overall composition of the intestinal flora and the development and prognosis of hepatitis. Branched-chain amino acids (BCAA) can regulate immune function, including increasing the energy source of immune cells and enhancing innate and adaptive immunity. Studies have shown that intestinal flora can take up dietary BCAA from the host intestine, and its metabolites can directly regulate intestinal immune homeostasis. In addition, there are currently no reports on the combination of BCAA with intestinal flora to regulate the immune status of the liver and apply it to the treatment of hepatitis B. Therefore, the scheme of the present invention provides a new, safe, economical and well-tolerated treatment for the current difficulties in the clinical treatment of chronic hepatitis B.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] 1. The Bacteroides fragilis DSM 2151 and branched-chain amino acid composition provided by the present invention can promote the clearance of serum HBsAg, and experiments have confirmed that the HBsAg negative conversion rate in the hepatitis B mouse model is about 92.86%, while the negative conversion rate of the PBS control group is only 12.5%, which is 80.36% higher than that of the control group.
[0015] 2. The effect of the Bacteroides fragilis DSM 2151 and branched-chain amino acid composition provided by the present invention on promoting serum HBsAg clearance is better than that of the probiotic Bacteroides fragilis DSM 2151 and branched-chain amino acids alone. Experiments have confirmed that in the hepatitis B mouse model, the serum HBsAg negative conversion rate of the Bacteroides fragilis DSM 2151 group was about 66.67%, while that of the branched-chain amino acid group was 35.71%. The negative conversion rates of the Bacteroides fragilis and branched-chain amino acid composition group were 26.19% and 57.15% higher, respectively.
[0016] 3. The Bacteroides fragilis DSM 2151 and branched-chain amino acid composition provided by the present invention can improve the immune microenvironment of the hepatitis B liver, and experiments have confirmed that the average value of the effector CD8+T and effector CD4+T cell ratios in the hepatitis B mouse model is 2 times higher than that of the PBS control group.
[0017] 4. The Bacteroides fragilis DSM 2151 and branched-chain amino acid composition provided by the present invention can increase the level of the effector factor IFN-γ in serum. Experiments have confirmed that the average level of serum IFN-γ in the hepatitis B mouse model is 2 times higher than that in the PBS control group. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG1 is a diagram showing the effect of Bacteroides fragilis DSM 2151 and a branched-chain amino acid composition in promoting the clearance of serum HBsAg in a hepatitis B mouse model according to an embodiment of the present invention ((a) baseline value of serum HBsAg in a hepatitis B mouse model, (b) dynamic monitoring of serum HBsAg levels during the test, and (c) negative conversion of serum HBsAg during the test);
[0019] FIG2 is a diagram showing the effect of Bacteroides fragilis DSM 2151 and a branched-chain amino acid composition on improving the liver immune microenvironment of a hepatitis B mouse model according to an embodiment of the present invention ((a) ratio of effector CD8+T cells (CD8+IFN-γ+) in liver tissue, (b) ratio of effector CD4+T cells (CD4+IFN-γ+) in liver tissue);
[0020] Figure 3 This is a diagram showing the effect of Bacteroides fragilis DSM 2151 and a branched-chain amino acid composition on improving the serum effector factor IFN-γ in an embodiment of the present invention (the figure shows the serum IFN-γ level in a hepatitis B mouse model). DETAILED DESCRIPTION
[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0022] The implementation of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.
[0023] The Bacteroides fragilis DSM 2151 used in the following examples was deposited in the German Collection of Biological Resources (DSMZ, address: Braunschweig-Süd Science Park, Germany) on September 14, 2009 with the deposit number DSM 2151.
[0024] The solution provided in this embodiment is the use of a combination of Bacteroides fragilis DSM 2151 and branched-chain amino acids in promoting the clearance of hepatitis B virus, that is, combining branched-chain amino acids (BCAA) with intestinal flora to regulate the immune status of the liver for the treatment of hepatitis B.
[0025] The following is a specific test of the embodiment of the present invention:
[0026] Experimental Example 1: Investigation of the HBV-clearing effect of the Bacteroides fragilis DSM 2151 and branched-chain amino acid composition of the present invention
[0027] 1. Experimental methods
[0028] The prepared Bacteroides fragilis DSM 2151 and branched-chain amino acid composition was diluted with sterile PBS and then gavaged at a certain dose into the tail vein high pressure pAAV / HBV1.2 plasmid constructed HBV mice, and the HBsAg and HBeAg levels in serum were detected to investigate the HBV clearance effect of the Bacteroides fragilis DSM 2151 and branched-chain amino acid composition of the present invention. The specific method is as follows:
[0029] (1) Prepare Bacteroides fragilis DSM 2151 suspension and branched-chain amino acid solution:
[0030] In a clean hood, dilute Bacteroides fragilis DSM 2151 to 1 × 10 10 CFU / ml, divided into EP tubes and stored at 4°C for future use. The amount of branched-chain amino acids supplemented is equivalent to an increase of 10% of the total calories. For mice weighing 25 grams, the daily dose of branched-chain amino acids supplemented is about 75 mg.
[0031] (2) Animal experiments
[0032] 1) Four-week-old C3H / HeN mice were housed together for one week to reduce differences in intestinal flora between individual mice. Dissolve 10 μg of recombinant plasmid pAAV / HBV 1.2 (pAAV-HBV 1.2 plasmid is highly safe and stable and not pathogenic to humans) in saline equivalent to 10% of the mouse body weight, mix well and transfer to a syringe for use. Inject the HBV recombinant plasmid into the terminal 1 / 3 of the mouse tail vein, and complete the injection within 5 to 8 seconds. 24 hours after high-pressure tail vein injection, detect the hepatitis B virus surface antigen HBsAg in the mouse peripheral blood serum to determine that the mouse model was successfully constructed.
[0033] 2) The hepatitis B mice were then divided into four groups. One group was the PBS group, which was gavaged with 400 μL PBS as the control group; the second group was the Bacteroides fragilis DSM 2151 group, which was gavaged with 200 μL bacterial solution (2×10 9 CFU / mouse)+200μL PBS; the third group was the branched-chain amino acid (BCAA) group, which was gavaged with 200μL BCAA+200μL PBS; the fourth group was the Bacteroides fragilis DSM 2151+BCAA group, which was gavaged with 200μL bacterial solution (2×10 9 CFU / mouse) + 200 μL BCAA. The mice were gavaged daily according to the grouping until serum HBsAg turned negative (<0.05 IU / mL) or until the end of observation (17 weeks), and serum was collected every week.
[0034] 5) Serum collection method: About 100 μL of blood was collected from the inner canthus of the mouse, and the blood was allowed to stand at room temperature for 30 minutes. The blood was centrifuged at 4-6000 rpm for 10 minutes. The serum was transferred to a new 1.5 mL EP tube and stored in a -80 refrigerator to avoid repeated freezing and thawing of the serum.
[0035] (3) Detection of HBsAg in mouse serum
[0036] The Abbott ARCHITECT instrumental HBsAg assay is a chemiluminescent microparticle immunoassay that quantifies HBsAg levels in mouse sera collected weekly.
[0037] (4) Liver non-parenchymal cell extraction and flow cytometry
[0038] 1) The liver of the hepatitis B mouse was removed, and the gallbladder was removed. The liver was digested in situ in 5 mL collagenase, poured into a C tube, and ground in a GentleMax instrument. After grinding, 5 mL of DMEM culture medium was immediately added to the dissociated tissue.
[0039] 2) Transfer to a 70 μm filter to filter single cells, rinse the digestive tube with 5 mL of culture medium and sieve, centrifuge the filtered single cell suspension at 50 g, 3 times up and 3 times down, 4°C, for 10 min to allow the liver tissue to settle, take the supernatant and part of the precipitated tissue, and transfer them to a new 50 mL centrifuge tube.
[0040] 3) The supernatant was centrifuged at 500g, 9°C, 4°C, for 10 min. The supernatant was removed and the precipitate was kept. The precipitate was resuspended with 3mL of 42% Percoll and 1mL of 70% Percoll to the bottom to separate the two clearly. The supernatant was centrifuged at 1260g, 6°C, 2°C, for 25 min.
[0041] 4) Take the middle layer, keep the tip of the gun 2-3mm above the middle layer, slowly extract the cells in the middle layer, put them into a new 15mL centrifuge tube, resuspend the extracted cells in pre-cooled PBS to 12mL, and resuspend them thoroughly by turning them upside down. 9 up and 9 down, centrifugal force 1060g, 10min, centrifuge at 4 degrees, discard PBS, and resuspend the precipitate in 1mL DMEM medium.
[0042] 5) Thaw the Leukocyte Activation Cocktail quickly in a 37°C water bath, add 2 μL of the Cocktail to 1 mL of cell suspension, and incubate in a cell culture incubator for 4-6 hours.
[0043] 6) After cell culture, gently blow and centrifuge at 300-500g, 4℃, for 5 minutes to wash the cells.
[0044] 7) The cells were incubated with the corresponding flow cytometry antibodies and then the data were read using a flow cytometer.
[0045] (4) Detection of mouse serum IFN-γ
[0046] 1) According to the instructions of the Luminex X-20 kit, 50 μL of beads, standards, and samples were added in sequence at room temperature and 800 rpm, and incubated for 1 hour.
[0047] 2) Add 200 μL of cleaning solution to rinse the beads three times, then add 50 μL of IFN-γ antibody and incubate at room temperature, 800 rpm, for 1 hour.
[0048] 3) Add 200 μL of cleaning solution to rinse the beads three times, then add 50 μL of PE-streptavidin and incubate at room temperature, 800 rpm, for 30 minutes.
[0049] 4) Add 200 μL of cleaning solution to rinse the beads three times, then add 150 μL of sheath solution at room temperature, 800 rpm, incubate for 2 minutes, and detect on the machine.
[0050] 2. Experimental results
[0051] (1) Detection results of serum HBsAg levels in hepatitis B mice and serum HBsAg clearance
[0052] The baseline value of serum HBsAg in hepatitis B mice is shown in Figure 1a, the dynamic monitoring of serum HBsAg level is shown in Figure 1b, and the serum HBsAg negative conversion (HBsAg < 0.05 IU / mL) is shown in Figure 1c.
[0053] It can be seen from the above test results that compared with the use of Bacteroides fragilis DSM 2151 and branched-chain amino acids alone, the composition of Bacteroides fragilis DSM 2151 and branched-chain amino acids in the embodiment of the present invention more effectively promotes the decrease and negative conversion of serum HBsAg levels, and the composition can be used to accelerate the clearance of HBsAg.
[0054] (2) Flow cytometry results of effector T cells in the liver of hepatitis B mice
[0055] The proportion of effector CD8+T cells (CD8+IFN-γ+) in liver tissue of hepatitis B mice is shown in FIG2 a, and the proportion of effector CD4+T cells (CD4+IFN-γ+) in liver tissue is shown in FIG2 b.
[0056] It can be seen from the above monitoring results that the combination of Bacteroides fragilis DSM 2151 and branched-chain amino acids in the embodiment of the present invention increases the proportion of effector CD8+T and CD4+T cells in the liver tissue of hepatitis B mice, and can improve the liver immune microenvironment. Its effect is better than the use of Bacteroides fragilis DSM 2151 or branched-chain amino acids alone, and is more helpful in clearing HBV-infected hepatocytes.
[0057] (3) Detection results of serum IFN-γ effector factor levels in hepatitis B mice
[0058] Serum IFN-γ levels in hepatitis B mice Figure 3 As shown by Figure 3 It can be seen that compared with the use of Bacteroides fragilis DSM 2151 and branched-chain amino acids alone, the combination of Bacteroides fragilis DSM 2151 and branched-chain amino acids can more effectively increase the level of effector IFN-γ in serum, which helps to clear HBV.
[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. Application of Bacteroides fragilis in products that promote the clearance of hepatitis B virus and optimize the immune microenvironment of hepatitis B virus infection.
2. Application of branched-chain amino acids in products that promote the clearance of hepatitis B virus and optimize the immune microenvironment of hepatitis B virus infection.
3. Application of a combination of Bacteroides fragilis and branched-chain amino acids in products that promote the clearance of hepatitis B virus and optimize the immune microenvironment of hepatitis B virus infection.