Application of 5beta-cholanic acid in enhancement of NK cell function and preparation of antiviral drugs

Abstract

The invention discloses application of 5 beta-cholanic acid (5 beta-CA) in enhancement of NK cell functions and preparation of antiviral drugs. The NK cell function of a chronic hepatitis B (CHB) patient is depleted, it is found that the intracellular 5beta-CA level is reduced by screening differential metabolites of peripheral blood NK cells of the CHB patient, and the NK cell function of the CHB patient can be enhanced by exogenous supplementation of 5beta-CA; the function of NK cells of HBV persistently infected mice can be enhanced, and the level of virology indexes such as serum HBV related antigens and the like can be reduced. Specifically, the 5beta-CA enhances the NK cell function by activating a TGR5-cAMP-mTOR signal channel so as to play an anti-HBV role. The research finds that the improvement of the 5beta-CA level is expected to become a new thought for enhancing the NK cell function of a CHB patient, and a new technical means is provided for the treatment of CHB.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antiviral technology, and particularly relates to application of 5β-cholanic acid in enhancing NK cell function and preparing antiviral drugs. BACKGROUND

[0002] The 2024 Global Hepatitis Report pointed out that there were still about 254 million chronic hepatitis B virus (HBV) infected people in the world in 2022, and about 1.1 million people died of liver failure, cirrhosis or hepatocellular carcinoma caused by HBV infection. The immune clearance of chronic hepatitis B (CHB) depends on the synergistic action of adaptive immunity such as CD4 + T cells, CD8 + T cells and B cells, and innate immunity such as NK cells and macrophages. Persistent infection of HBV is considered to be due to insufficient immune clearance, including dysfunction of HBV-specific CD4 + and CD8 + T cells, and dysfunction of natural killer cells (NK). Among them, NK cells are the first line of defense against viral infection, and their antiviral effects occur before adaptive immune responses, and even can recognize and kill virus-infected cells before viral replication, playing an important role in early antiviral response, and also participating in the initiation of adaptive immune response.

[0003] Persistent HBV infection leads to NK cell functional exhaustion, showing reduced expression of short-term degranulation marker lysosome-associated membrane glycoprotein-1 (also known as CD107a), reduced secretion of cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and reduced cytotoxic granule release of perforin and granzyme B. CHB patients are in a state of NK cell exhaustion, and it is crucial for the treatment of CHB to find appropriate immunomodulators to reactivate and / or change innate immune responses and restore NK cell function.

[0004] Bile acids are cholesterol-derived molecules that are mainly synthesized in the liver and released into the intestine to aid digestion. In addition to their role in bile formation and nutrient absorption, bile acids also act as ligands for cell membrane and nuclear receptors collectively known as bile acid regulated receptors (BARR). Two typical members of the BARR family, the Farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5), are expressed in neurons, glial cells, endocrine cells (including pancreatic beta cells and L cells), and extracellular matrix (ECM) cells, as well as immune cells such as monocytes, macrophages, dendritic cells, NK cells, and T cells. Under the immune regulation of the intestine and liver, FXR and TGR5 in immune cells activate a negative regulatory signal network through ligand activation, maintaining the immune tolerance homeostasis of the system. The interaction of bile acids with receptors causes a wide range of physiological functions, and the metabolic regulation mechanism provides potential therapeutic targets for various diseases, including infectious diseases, inflammatory bowel disease (IBD), digestive and liver malignancies, nervous system disorders, and metabolic abnormalities.

[0005] The above status indicates that NK cells in CHB patients are in a state of functional exhaustion, and restoring the function of NK cells is crucial for the clearance of HBV; bile acids have been reported to regulate immune cell function in other diseases, but there is no report on the effect on NK cells. SUMMARY

[0006] The purpose of the present application is to provide the application of 5β-cholanic acid (5β-CA) in enhancing the function of NK cells and preparing antiviral drugs. NK cells in CHB patients are in a state of functional exhaustion, and restoring the function of NK cells is crucial for the clearance of HBV. The present application determines the differential metabolites in NK cells of CHB patients, further explores the effect of bile acids on restoring the function of NK cells and its mechanism, and plays its antiviral role. Increasing the level of 5β-CA is expected to become a new idea to enhance the function of NK cells in CHB patients, and to provide a new technical means for the treatment of CHB.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides the application of 5β-cholanic acid in preparing antiviral drugs.

[0008] Further, the virus is hepatitis B virus.

[0009] Furthermore, 5β-cholanoic acid achieves antiviral function by inhibiting HBV DNA replication, hepatitis B virus HBsAg expression, and hepatitis B virus HBeAg expression.

[0010] Furthermore, 5β-cholanoic acid enhances NK cell function by activating the TGR5-cAMP-mTOR signaling pathway to achieve anti-hepatitis B virus effects. The TGR5-cAMP-mTOR signaling pathway is that after 5β-cholanoic acid binds to the TGR5 receptor on the surface of NK cells, it triggers the intracellular cAMP-PKA signaling cascade reaction, promotes PI3K-AKT phosphorylation, and then activates the mTOR pathway.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0012] The present invention also provides the use of 5β-cholanoic acid in preparing a product for enhancing NK cell function.

[0013] Furthermore, 5β-cholanoic acid enhances NK cell function by promoting the secretion of IFN-γ, TNF-α, perforin, and granzyme B.

[0014] The present invention discovered that 5β-CA enhances NK cell function by activating the TGR5 receptor-dependent signaling pathway. After 5β-CA binds to the TGR5 receptor on the surface of NK cells, it triggers the intracellular cAMP-PKA signaling cascade, promoting PI3K-AKT phosphorylation, and further activating the mTOR pathway, thereby upregulating the expression of transcription factors Id2 and GATA3, promoting the secretion of effector molecules IFN-γ and TNF-α, and synergistically enhancing the synthesis and release of cytotoxic granules perforin and granzyme B.

[0015] In summary, NK cell function is depleted in patients with chronic hepatitis B (CHB). Screening for differential metabolites in peripheral blood NK cells from patients with CHB revealed decreased intracellular 5β-CA levels. Exogenous supplementation of 5β-CA can enhance NK cell function in patients with CHB and in mice with persistent HBV infection, while also reducing serum levels of HBV-related antigens and other virological markers. Mechanistically, 5β-CA enhances NK cell function by activating the TGR5-cAMP-mTOR signaling pathway, thereby exerting anti-HBV effects. This study found decreased 5β-CA levels in NK cells from patients with CHB and explored the mechanism by which 5β-CA enhances NK cell function. Increasing 5β-CA levels may be a new approach to enhance NK cell function in patients with CHB.

[0016] The beneficial effects of the present invention are: (1) The present application discloses that 5beta-CA significantly improves the function of NK cells through the TGR5 / cAMP / mTOR signaling pathway, specifically, on the one hand, it promotes the synthesis and release of cytotoxic granules such as perforin and granzyme B; on the other hand, it enhances the secretion of IFN-gamma, TNF-alpha and other antiviral cytokines. Therefore, 5beta-CA can activate the cytotoxicity of NK cells, enhance their ability to secrete IFN-gamma and other antiviral cytokines, thereby promoting the clearance efficiency of HBV in CHB patients. The present application shows that 5beta-CA has an enhancing effect on the function of NK cells, and provides a new strategy for anti-HBV replication.

[0017] (2) In addition, the present application has important translational medical significance. Current anti-HBV treatment is mainly based on nucleoside analogues and interferon, but there are problems such as drug resistance, side effects and insufficient immune response. 5beta-CA, as an endogenous bile acid derivative, can achieve more persistent viral control by regulating host innate immunity. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : Function of peripheral blood NK cells of CHB patients and healthy controls; (A) Mean fluorescence intensity of inhibitory receptors and activating receptors; (B) Percentage of peripheral blood NK cells expressing IFN-gamma and its mean fluorescence intensity expression level; (C) Percentage of peripheral blood NK cells expressing TNF-alpha and its mean fluorescence intensity expression level; (D) Percentage of peripheral blood NK cells expressing perforin and its mean fluorescence intensity expression level.

[0019] Figure 2 : Bile acid profile of peripheral blood NK cells and serum of CHB patients and healthy controls; (A) Relative quantitative analysis of the levels of various bile acids in peripheral blood sorted NK cells; (B) Absolute quantitative levels of various bile acids in peripheral blood sorted NK cells; (C) Graph of the absolute quantitative levels of various bile acids in (B); (D) Absolute quantitative levels of 5beta-CA in serum of CHB patients and healthy controls.

[0020] Figure 3: 5β-CA promotes the function of NK cells isolated from peripheral blood of patients with chronic hepatitis (CHB) in vitro; (A) Effects of 0-50 μM 5β-CA on NK92 cell viability; (B-C) Intracellular / extracellular concentrations of 5β-CA and DMSO detected by targeted metabolomics; (D) Levels and mean fluorescence intensity of IFN-γ in NK cells isolated from peripheral blood of patients with CHB after treatment with 25 μM 5β-CA and DMSO; (E) Levels and mean fluorescence intensity of TNF-α in NK cells isolated from peripheral blood of patients with CHB after treatment with 25 μM 5β-CA and DMSO; (F) Levels and mean fluorescence intensity of perforin in NK cells isolated from peripheral blood of patients with CHB after treatment with 25 μM 5β-CA and DMSO; (G) Levels and mean fluorescence intensity of granzyme B in NK cells isolated from peripheral blood of patients with CHB after treatment with 25 μM 5β-CA and DMSO.

[0021] Figure 4 : 5β-CA inhibits HBV replication in vivo; (A) Schematic diagram of mouse model establishment; (B-C) 5β-CA concentrations and percentages in serum and liver tissue after treatment with 5β-CA and control groups; (D) Serum HBsAg, HBeAg, and HBV DNA levels after treatment with 5β-CA and control groups.

[0022] Figure 5 : Effects of 5β-CA on liver function in mice; (A) HE staining analysis of liver pathological structure in the 5β-CA-treated and control groups; (B) Body weight, liver and spleen weights of the two groups of mice after treatment with 5β-CA; (C) Routine liver function analysis of ALT, AST, ALP, LDH, TP, ALB and TBIL in the 5β-CA-treated and control groups.

[0023] Figure 6 : 5β-CA enhances the function of mouse peripheral blood and liver NK cells; (A) Levels and mean fluorescence intensity of cytokines IFN-γ, perforin, and granzyme B secreted by peripheral blood NK cells; (B) Levels and mean fluorescence intensity of cytokines IFN-γ, TNF-α, and perforin secreted by liver NK cells.

[0024] Figure 7: 5β-CA promoted the increase of the level of bile acid receptor TGR5 on the surface of NK cells; (A) mRNA of bile acid receptor related genes was detected after 5β-CA and DMSO were respectively treated with CHB patient peripheral blood sorted NK cells; (B) mRNA expression level of TGR5 of NK92 cells treated with 5β-CA and INT777 was detected; (C) Western blot was used to detect the expression level of bile acid receptor TGR5 and FXR of CHB patient peripheral blood sorted NK cells treated with 5β-CA and DMSO respectively; (D) Western blot was used to detect the expression level of bile acid receptor TGR5 and FXR of mouse spleen sorted NK cells treated with 5β-CA and DMSO respectively; (E) Western blot was used to detect the expression level of bile acid receptor TGR5 of NK92 cells treated with 5β-CA and INT777 respectively; (F) Western blot was used to detect the expression level of bile acid receptor TGR5 of NK92 cells treated with 5β-CA and SBI-115 respectively.

[0025] Figure 8 : 5β-CA activated the TGR5 / cAMP / mTOR signaling pathway of NK cells; (A) Direct immunofluorescence was used to detect the cAMP concentration of NK92 cells treated with 5β-CA and DMSO respectively; (B) Western blot was used to detect the phosphorylation level of mTOR protein of CHB patient peripheral blood NK cells treated with 5β-CA and DMSO respectively; (C) Western blot was used to detect the phosphorylation level of mTOR, PI3K, AKT and PKA proteins of NK92 cells treated with 5β-CA and DMSO respectively; (D) Western blot was used to detect the expression level of transcription factors Id2 and GATA3 of NK92 cells treated with 5β-CA and DMSO respectively; (E) The phosphorylation level of mTOR and PKA proteins of mouse spleen NK cells sorted and treated with 5β-CA and DMSO respectively; (F) The phosphorylation level of mTOR protein of NK92 cells treated with rapamycin (Rapamycin, Rapa), 5β-CA, INT777, Rapa+5β-CA, Rapa+INT777 and DMSO respectively; (G-I) The levels and mean fluorescence intensity of IFN-γ, TNF-α and granzyme B of NK92 cells treated with TGR5 inhibitor SBI-115+5β-CA, mTOR inhibitor Rapa+5β-CA, 5β-CA and DMSO respectively. DETAILED DESCRIPTION

[0026] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited to this.

[0027] The materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially. In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0028] The experiments related to the following examples are described as follows: Study population: The application inventors obtained the informed consent of patients and collected 25 cases of peripheral whole blood specimens from healthy controls (HC) and CHB patients who had not received any antiviral treatment, and collected detailed clinical data of the patients at the same time. The above research process conforms to the ethical principles of the Helsinki Declaration, and each patient and healthy person has signed the informed consent form.

[0029] Inclusion criteria: The diagnostic criteria of all selected patients meet the 2022 edition of "Guidelines for the Prevention and Treatment of Chronic Hepatitis B" or "EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection", HBsAg and / or HBV DNA positive for more than 6 months; no interferon or nucleotide analog treatment in the past or within half a year.

[0030] (1) HBeAg-positive chronic HBV infection (also known as immune tolerance period, chronic HBV carrier state): HBsAg > 1 x 10 4 IU / mL, HBeAg positive, HBV DNA > 2 x 10 7 IU / mL, ALT < upper limit of normal, liver pathology has no obvious inflammation necrosis and fibrosis; (2) HBeAg-positive CHB patients (also known as immune clearance period, immune active period): HBsAg positive, HBeAg positive, HBV DNA positive, ALT persistent or repeated elevation, liver pathology has obvious inflammation necrosis and / or fibrosis; (3) HBeAg-negative chronic HBV infection (non-active period, immune control period, non-active HBsAg carrier state): HBsAg < 1 x 10 3 IU / mL, HBeAg negative, HBV DNA negative, ALT < upper limit of normal, liver pathology has no or only mild inflammation, and may have varying degrees of fibrosis; (4) HBeAg-negative CHB patients (reactivation period): HBsAg positive, HBeAg negative, HBV DNA positive, ALT persistent or repeated elevation, liver pathology has obvious inflammation necrosis and / or fibrosis.

[0031] Exclusion criteria (satisfy one of the following conditions): (1) patients with acute hepatitis B and severe hepatitis B; (2) patients with HCV, HIV and HDV infection; (3) patients receiving immunosuppressive agents, chemotherapy and hormone therapy; (4) patients with other liver diseases such as alcoholic hepatitis, non-alcoholic fatty liver, cirrhosis, liver cancer, autoimmune liver disease and drug-induced liver injury; (5) patients who have used antibiotics, probiotics or immunosuppressive agents within 3 months before sample collection; (6) patients with a history of alcohol abuse for more than 2 years; (7) patients with gastrointestinal diseases; (8) patients with metabolic diseases such as obesity, diabetes, and stones; (9) patients with mental illness.

[0032] Experimental animals: wild type C57BL / 6N mice (Wild type, WT) female mice, weighing 18-20 g, about 6-8 weeks old, purchased from Zhejiang Vintech Lihua Experimental Animal Technology Co., Ltd., and raised in the SPF (Specific Pathogen Free) level laboratory of Fujian Medical University Experimental Animal Center. The mice used in this experiment are all SPF level experimental animals. Animal experiments are strictly in accordance with the Laboratory Animal Care and Use Guidelines issued by the Ministry of Science and Technology of the People's Republic of China, and in accordance with the management regulations of the Fujian Medical University Experimental Animal Ethics Committee. Experimental animal license number: SCXK (Zhe) 2019-0001.

[0033] Example 1 Functional detection of peripheral blood NK cells of CHB patients and healthy controls In this example, 10 CHB patients and 10 healthy controls (Healthy control, HC) were collected, and flow cytometry was used to analyze the surface receptors and functions of NK cells: (1) Take 100 μL of fresh ethylenediaminetetraacetic acid tri-potassium (EDTA-3K) anticoagulant whole blood and add it to a flow tube; (2) Take 3 μL of anti-human CD3-FITC / CD(16+56)-PE, 1 μL of NKG2D-PE-CY7, 1 μL of NKG2A-APC, 1 μL of NKp30-PE-CY7, 1 μL of 2B4-APC, 1 μL of TIM3-APC and 1 μL of TIGIT-APC flow cytometry antibodies, mix well, and incubate at 4 ℃ in the dark for 30 min; (3) Add 250 μL of OptiLyse C Lysing Solution, vortex immediately for 1-2 s, and incubate at room temperature in the dark for 15 min; (4) Add 1 mL of 5% FBS-PBS and wash twice, centrifuge at 2000 rpm for 5 min, and discard the supernatant; (5) Add 400 μL of sheath liquid to resuspend the cells, and detect the cells on a flow cytometer. Analyze the results using FlowJo software.

[0034] The experimental results are shown in Figure 1 The results show that the average fluorescence intensity of the inhibitory receptors NKG2A and TIM3 in CHB patients is significantly higher than that in HC (A), and the percentage of peripheral blood NK cells expressing IFN-γ, TNF-α and perforin and the average fluorescence intensity expression level in CHB patients are significantly lower than those in HC (B-1D). The above research results show that the NK cell function in CHB patients is in an exhausted state. Figure 1 Figure 1 B-1D). The above research results show that the NK cell function in CHB patients is in an exhausted state.

[0035] Example 2: Detection of the bile acid spectrum of peripheral blood NK cells and serum of CHB patients and healthy controls 1. Isolation of human peripheral blood mononuclear cells by Ficoll gradient method The reagents and instruments used are RNase-free, and the whole process is sterile.

[0036] (1) Take fresh anticoagulated whole blood (EDTA, sodium citrate or heparin anticoagulation can be used), dilute the whole blood with an equal volume of PBS (pH=7.2-7.4), and mix well by inverting; (2) Add Ficoll separation solution to a 15 mL centrifuge tube, and the volume of the separation solution is 1:1 of the volume of the diluted whole blood. Slowly add the diluted whole blood to the Ficoll separation solution, and make sure that the interface between the two liquids is clear; (3) Centrifuge at room temperature, 800 g, 25 min; (4) After centrifugation, a clear layering will appear: the uppermost layer is the diluted plasma layer, the middle layer is the transparent separation solution layer, the white membrane layer between the plasma and the separation solution is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes; (5) Carefully pipette the white membrane layer cells into a 15 mL clean centrifuge tube, and wash the white membrane layer cells with 10 mL of PBS. Centrifuge at 250 g for 10 min; (6) Discard the supernatant, resuspend the cells with 5 mL of PBS, and centrifuge at 250 g for 10 min. Wash twice; (7) Repeat step (6), resuspend the cells in 1640 complete culture medium, and store for use.

[0037] 2. Extensive targeted metabolomics detection of the bile acid spectrum of NK cells UPLC-MS / MS technology is used, and the specific setting parameters are as follows: ​(1) The liquid chromatography conditions mainly include: chromatographic column: ACQUITY HSS T3 (2.1x100mm, 1.8 μm); mobile phase A: 0.1% formic acid / water; mobile phase B: 0.1% formic acid / acetonitrile; column temperature: 40 ℃; flow rate: 0.4 mL / min; injection volume: 5 μL; (2) The mass spectrometry conditions mainly include: positive ion mode (ESI+), negative ion mode (ESI-); (3) The liquid chromatography acquisition conditions mainly include: chromatographic column: Waters ACQUITY UPLC HSS T3 C18 1.8µm, 2.1 mmx100 mm; mobile phase: A phase is ultrapure water (containing 0.1% formic acid), B phase is acetonitrile (containing 0.1% formic acid); elution gradient: 0 min water / acetonitrile (95:5 V / V), 11.0 min (10:90 V / V), 12.0 min (10:90 V / V), 12.1 min (95:5 V / V), 14.0 min (95:5 V / V); flow rate: 0.4 mL / min; column temperature: 40 ℃; injection volume 2 μL; (4) The mass spectrometry acquisition conditions mainly include: Electrospray ionization (ESI) temperature 500 ℃, mass spectrometry voltage 5500 V (positive), -4500 V (negative), ion source gas I (GS I) 50 psi, gas II (GS II) 50 psi, curtain gas (CUR) 25 psi, collision-activated dissociation (CAD) parameter setting is high. In the triple quadrupole (Qtrap), each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0038] The metabolite composition of the peripheral blood sorted NK cells is analyzed by wide-targeted metabolomics, and the specific steps are as follows: (1) The peripheral blood sorted NK cells taken from the-80 ℃ refrigerator are placed on ice for thawing (all subsequent operations are required to be performed on ice); (2) 200 μL of PBS buffer is added, and vortexed for 3 min to make the sample completely suspended; (3) 600 μL of pure methanol water internal standard extractant is added, and vortexed for 3 min to make the sample completely suspended; (4) Put the centrifuge tube into liquid nitrogen for quick freezing for 5 min, take it out and thaw on dry ice for 5 min, and then thaw on ice for 5 min (note that prevent the cover from exploding due to large temperature difference, and the sample from being sprayed out), vortex for 2 min, mix well, and repeat the process of putting into liquid nitrogen, thawing, and vortexing for 3 times; (5) Centrifuge at 12000 rpm for 10 min at 4°C, and transfer 300 μL of supernatant to another corresponding numbered centrifuge tube, and stand in a -20°C refrigerator for 30 min; (6) Centrifuge at 12000 rpm for 3 min at 4°C, and transfer 200 μL of supernatant to a corresponding sample bottle inner tube for machine analysis.

[0039] 3. Targeted metabolomics detection of bile acid spectrum of NK cells (1) Sample pretreatment method The NK cells sorted from peripheral blood are slowly thawed at 4°C (if it is a solid sample, it needs to be weighed and ground after thawing), an appropriate amount of sample (grinding liquid) is added to methanol / acetonitrile (1:1, v / v) containing an internal standard, vortex mixed, -20°C for 10 min, 4°C, 14000 rpm centrifugation for 10 min, nitrogen blowing of the supernatant, then 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) is added for re-dissolution, vortex, 4°C, 14000 rpm centrifugation for 10 min, and the supernatant is taken for sample analysis.

[0040] (2) Chromatographic conditions The sample is separated by CalQuant-S high performance liquid system and C18 chromatographic column (Phenomenex, 100×2.1 mm, 1.7 μm); the column temperature is 60°C; the flow rate is 0.3 mL / min; the sample amount is 2 μL; the mobile phase composition A: pure water containing 1 mM ammonium acetate, B: acetonitrile / isopropanol (2:1, v / v); the gradient elution program is as follows: 0-0.5 min, 20% B; 0.5-12.0 min, B from 20% linearly changes to 80%; 12.0-13.5 min, 80% B; 13.5-14.5 min, B from 80% linearly changes to 100%; 14.5-15.5 min, 100% B; 15.5-15.9 min, B from 100% linearly changes to 20%; 15.9-17 min, 20% B. During the whole analysis process, the sample is placed in a 4°C automatic sample injector.

[0041] (3) Mass spectrometry conditions Mass spectrometry was performed using a CalQuant-S mass spectrometer (Calibra, Hangzhou, China). The ESI source negative ion mode conditions were as follows: desolvation gas (GS1) 50 psi, heating gas (GS2) 50 psi, desolvation gas temperature 450 °C, curtain gas (CUR) 20 psi, collision gas (CAD) 10 psi; spray voltage -4000 V. The scanning mode was multiple reaction monitoring (MRM) mode.

[0042] (4) Data analysis The MRM raw data were extracted using MultiQuant 3.0.3 software to obtain the peak area ratio of each substance and internal standard peak area, and the content was calculated according to the standard curve.

[0043] The relative quantitative analysis of various bile acid levels in NK cells sorted from the peripheral blood of 25 CHB patients and HC was performed by ultra-high performance liquid chromatography tandem mass spectrometry technology, and the relative expression of 5β-CA in NK cells sorted from the peripheral blood of CHB patients was significantly lower than that of HC (P < 0.05). Figure 2 A). To further confirm the above results, the absolute quantitative verification of the targeted bile acid spectrum was then performed on 6 CHB patients and HC samples, and the results showed that the absolute quantitative levels of 5β-CA and UDCA in NK cells sorted from the peripheral blood of CHB patients were significantly lower than those of HC (P < 0.05) (B), and the absolute quantitative level of 5β-CA decreased the most (C). The serum bile acid components were also quantitatively analyzed, and it was found that the absolute quantitative level of 5β-CA in the serum of CHB patients was significantly lower than that of HC (D). Figure 2 B). The serum bile acid components were also quantitatively analyzed, and it was found that the absolute quantitative level of 5β-CA in the serum of CHB patients was significantly lower than that of HC (D). Figure 2 C). The serum bile acid components were also quantitatively analyzed, and it was found that the absolute quantitative level of 5β-CA in the serum of CHB patients was significantly lower than that of HC (D). Figure 2 D).

[0044] Example 3 5β-CA promotes the function of NK cells sorted from the peripheral blood of CHB patients in vitro The levels of cytokine secretion and cytotoxic granules of NK cells were detected by flow cytometry: The detected cytokines include IFN-γ and TNF-α, and the detected cytotoxic particles include granulysin B and perforin. The experimental steps refer to the previous research reports and reagent instructions (see literature Tim-3 blockade promotes iNKT cell function to inhibit HBV replication. J Cell Mol Med, 2018. 22(6):3192-3201; Dysfunction of Natural Killer Cells by FBP1-Induced Inhibition of Glycolysis during Lung Cancer Progression. Cell Metab, 2018. 28(2): 243-255.e5).

[0045] 1. Flow cytometry detection (1) In a 96-well cell culture plate, add 0.4 μL Cell Stimulation Cocktail (plus protein transport inhibitors) (500x) (diluted 10 times and add 4 μL) to each well of PBMC cells, and stimulate culture at 37°C, 5% CO2 for 5 h; (2) Collect the cells in an EP tube, wash once with pre-cooled 1 mL 5% FBS-PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend the cells with 100 μL PBS; (3) Add anti-CD8-PE-CY7 1 μL, anti-CD3-FITC / CD(16+56)-PE 3 μL to each sample, and incubate at 4°C in the dark for 30 min, mix once at 15 min; (4) Wash once with 1 mL 5% FBS-PBS, centrifuge at 3000 rpm for 5 min, and discard the supernatant after centrifugation; (5) Add 100 μL BD fixation / permeabilization membrane breaking solution, and incubate at 4°C in the dark for 30 min; (6) Add 1 mL 1x BD perm / wash Buffer, centrifuge at 3000 rpm for 5 min, discard the supernatant after centrifugation, and wash twice; (7) Resuspend the cells in 100 μL PBS, add anti-IFN-γ-PerCP-CY5.5 and anti-TNF-α-APC, anti-granzyme B-APC and anti-perforin-PerCP-CY5.5 each 1 μL, mix, incubate at 4 ℃ for 30 min, mix once every 15 min; (8) Add 1 mL 5% FBS-PBS in the EP tube, mix, centrifuge at 3000 rpm for 5 min, discard the supernatant after centrifugation; (9) Add 400 μL sheath fluid to resuspend, move the cells to the flow tube, detect on the flow cytometer, and analyze the results using FlowJo software.

[0046] 2. Sorting of human peripheral blood NK cells (1) The same as the steps of Ficoll gradient method for isolating human peripheral blood mononuclear cells, discard the supernatant, and determine the cell number; (2) Add 40 μL pre-cooled sorting buffer per 10 7 total number of cells to resuspend the cells; (3) Add 10 μL NK cell biotin antibody mixture per 10 7 total number of cells; (4) Mix well, try not to generate bubbles, and incubate at 4 ℃ in the refrigerator for 5 min; (5) Add 30 μL pre-cooled sorting buffer per 10 7 total number of cells; (6) Add 20 μL NK cell microspheres per 10 7 total number of cells; (7) Mix well, try not to generate bubbles, and incubate at 4 ℃ in the refrigerator for 10 min; (8) The magnetic selection amount needs at least 500 μL, and 400 μL pre-cooled sorting buffer can be added to the new EP tube to resuspend the cells; (9) Rinse the column with 3 mL pre-cooled sorting buffer; (10) Infuse 500 μL of cell suspension into the column, and start collecting the unlabelled cells in the washing liquid; (11) Wash the column with 3 mL pre-cooled sorting buffer, and collect the unlabelled cells from step 10. The collected liquid represents the sorted NK cells.

[0047] 3. Evaluation of the effect of 5β-CA on the viability of NK92 cells Use a 96-well plate, inoculate 200 μL, 2.5×10 5NK92 cells suspension, and cultured for 24 h. 5β-CA powder 7.2114 mg was weighed by a micro-electronic balance, dissolved in 1 mL DMSO to prepare a stock solution of 20 mmol / L, and then diluted 20, 10, 4 and 2 times with DMSO to prepare working solutions of 1 mM, 2 mM, 5 mM and 10 mM, respectively. Then, 1 μL of each working solution was added to 200 μL of culture medium to obtain 5β-CA with final concentrations of 0 μM, 5 μM, 10 μM, 25 μM and 50 μM, respectively. After 24 h of treatment, 20 μL of CCK-8 solution was added to each well, and the cells were further incubated in a cell incubator for 2 h. The absorbance was measured at 450 nm. The cell viability was calculated according to the OD value, and the toxicity of 5β-CA to NK cells was inversely proportional to the cell viability.

[0048] The calculation formula was: cell viability (%) = [A (drug) - A (blank)] / [A (0 drug) - A (blank)] x 100%. A (drug): the absorbance of the well containing cells, CCK-8 solution and drug solution; A (blank): the absorbance of the well containing culture medium and CCK-8 solution without cells; A (0 drug): the absorbance of the well containing cells and CCK-8 solution without drug solution.

[0049] The experimental results showed that 25 μM 5β-CA had the greatest effect on the cell viability of NK92 cells Figure 3 A). Meanwhile, by detecting the intracellular / extracellular concentrations of 5β-CA after treatment of NK92 cells with 5β-CA, it was found that the intracellular and supernatant concentrations of 5β-CA in NK92 cells treated with 25 μM 5β-CA were significantly higher than those in the DMSO treatment group Figure 3 B-3C). Finally, NK cells sorted from the peripheral blood of CHB patients were treated with 25 μM 5β-CA or DMSO in vitro, and the levels and mean fluorescence intensity of intracellularly secreted cytokines (IFN-γ and TNF-α) and perforin were detected by FCM. It was found that the levels and mean fluorescence intensity of IFN-γ, TNF-α and perforin secreted by NK cells sorted from the peripheral blood of CHB patients treated with 25 μM 5β-CA were significantly higher than those in the DMSO treatment group Figure 3 D-3F), and the mean fluorescence intensity of granzyme B was significantly higher than that in the DMSO treatment group Figure 3 G). The above results showed that 5β-CA treatment of NK cells increased the secretion of cytokines and enhanced degranulation, and 5β-CA could enhance the function of NK cells in the peripheral blood of CHB patients.

[0050] Example 4 Inhibition of HBV replication by 5β-CA in vivo 1. Establishment and evaluation of a mouse model of persistent HBV infection (1) Establishment of HBV persistent infection mouse model: 200 μL containing 1.34×10 11 The rAAV8-1.3HBV (recombinant adeno-associated virus 8 carrying 1.3 copies of the full-length HBV genome) with a high viral load was injected into the tail vein of 12 C57BL / 6N mice (reference: A mouse model for HBV immunotolerance and immunotherapy. Cell Mol Immunol, 2014. 11(1): 71-78). The tail vein blood of the mice was collected every two weeks to measure the levels of HBsAg, HBeAg and HBV DNA. (2) Evaluation method and standard: Six weeks after tail vein injection of rAAV8-1.3HBV virus, the serum HBV DNA level of mice was 10 5 ~10 7 IU / mL, HBsAg level is 10 2 ~10 3.5 IU / mL, HBeAg level is 10 2 ~10 3.5 Between S / CO; (3) Oral gavage of 5β-CA: Each mouse was gavaged with 100 mg / kg / day 5β-CA (if the mouse weighed 20 g, 200 μL of 10 mg / ml 5β-CA dissolved in DMSO was orally gavaged every day). The mice were killed after six weeks. The control group was gavaged with DMSO.

[0051] 2. Flow cytometry analysis of cytokine and cytotoxic granule secretion by NK cells in mouse peripheral blood and liver The cytokines detected include IFN-γ and TNF-α, and the cytotoxic granules detected include granzyme B and perforin.

[0052] (1) In a 96-well cell culture plate, PBMCs were isolated from mouse peripheral blood, spleen, and liver, and 1×10 6 0.4 μL Cell Stimulation Cocktail (plus protein transport inhibitors) (500×) was added to the PBMC suspension (4 μL was added after diluting 10-fold) and stimulated and cultured at 37°C in 5% CO2 for 5 h. (2) Collect cells in EP tube, wash twice with pre-cooled 1 mL 5% FBS-PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant, resuspend the cells with 100 μL PBS; (3) Add anti-CD3-FITC, anti-CD8-PE-CY7 and anti-NK1.1-PE each 1 μL to each sample, incubate at 4°C in the dark for 30 min; (4) Wash once with 1 mL 5% FBS-PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant after centrifugation; (5) Add 100 μL BD fixation / permeabilization solution, incubate at 4°C in the dark for 30 min; (6) Add 1 mL 1x BD perm / wash Buffer, centrifuge at 3000 rpm for 5 min, discard the supernatant after centrifugation, wash twice; (7) Resuspend the cells in 100 μL PBS, add anti-IFN-γ-PerCP-CY5.5, anti-TNF-a-APC, anti-Granzyme B-APC and anti-perforin-APC each 1 μL, mix well; (8) Incubate at 4°C in the dark for 30 min; (9) Add 1 mL 5% FBS-PBS to the EP tube, mix well, centrifuge at 3000 rpm for 5 min, discard the supernatant after centrifugation; (10) Resuspend with 400 μL sheath fluid, move the cells to a flow tube, detect on a flow cytometer, and analyze the results using FlowJo software.

[0053] The specific experimental procedure is shown in Figure 4 A, it was found that after 6 weeks of gavage, the serum and liver tissue 5β-CA concentration and percentage of 5β-CA treatment group were significantly higher than those of DMSO control group (P < 0.05) Figure 4 B-4C), the serum HBsAg, HBeAg and HBV DNA levels of 5β-CA treatment group were significantly lower than those of DMSO control group (P < 0.05) Figure 4 D). The above results show that 5β-CA can inhibit viral replication in HBV persistently infected mouse model.

[0054] Example 5 Effect of 5β-CA on liver function in mice After 6 weeks of gavage of HBV persistently infected mice with 5β-CA, the pathological structure of liver was analyzed by HE staining, about 50 mg of liver tissue was soaked in 4% paraformaldehyde for fixation, and the subsequent fixation and staining steps were as follows: (1) Tissue paraffin embedding and sectioning: tissue blocks were dehydrated, transparent, and immersed in wax, and then continuously sectioned by a paraffin rotary sectioning machine. The thickness of the section was 4 μm. The prepared wax slices were taken by a glass slide and placed in a 37 ℃ incubator overnight, and then placed in a 4 ℃ refrigerator for standby; (2) Paraffin section deparaffinization to water: sequentially place the section in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and tap water; (3) Hematoxylin staining: place the section in hematoxylin dyeing solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, return to blue with return blue solution, and rinse with running water; (4) Eosin staining: sequentially place the section in 85% and 95% gradient alcohol for 5 min, and then place in eosin dyeing solution for 5 min; (5) Dehydration and mounting: sequentially place the section in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min for transparency, and then mount with neutral balsam; (6) Microscope examination, image collection and analysis.

[0055] It was found that the basic morphological structure of the liver tissues of the two groups of mice was similar, and there was no obvious liver damage or inflammation Figure 5 A). There was no significant difference in the body weight, liver weight and spleen weight of the two groups of mice Figure 5 B). There was also no significant difference in the levels of ALT, AST, ALP, LDH, TP, ALB and TBIL and other indicators in the routine liver function analysis Figure 5 C). The above research results showed that there was no obvious change in liver function after the mice were treated with 5β-CA, and there was no hepatotoxicity.

[0056] Example 6: 5β-CA enhances the function of peripheral blood and liver NK cells of mice After the HBV persistently infected mice were gavaged with 5β-CA for 6 weeks, the function of the peripheral blood and liver NK cells of the mice was analyzed. By FCM, it was found that the level of the cytokine IFN-γ secreted by the peripheral blood NK cells was significantly higher than that of the DMSO control group, and the average fluorescence intensity of the cytotoxic granules (perforin and granzyme B) was significantly higher than that of the DMSO control group Figure 6 A). The levels of the cytokines IFN-γ, TNF-α and cytotoxic granules perforin secreted by the liver NK cells were significantly higher than those of the DMSO control group Figure 6 B). The above in vivo research results showed that 5β-CA enhanced the function of the peripheral blood and liver NK cells of mice, which was basically consistent with the results of the in vitro experiment that 5β-CA enhanced the function of the peripheral blood NK cells of CHB patients.

[0057] Example 7: 5β-CA promotes the increase of the level of bile acid receptor TGR5 on the surface of NK cells CHB patient peripheral blood sorted NK cells were treated with 25 μM 5β-CA and DMSO respectively, and the mRNA expression levels of bile acid receptor related genes (TGR5, FXR, S1PR2, PXR) were detected by qPCR. It was found that only the mRNA expression level of TGR5 was significantly increased after 5β-CA treatment 6 Figure 7 A). INT777 is an agonist of TGR5 receptor. NK92 cells were treated with 25 μM 5β-CA, 30 μM INT777 and DMSO, and it was found that the mRNA expression level of TGR5 in the 5β-CA and INT777 treatment groups was significantly higher than that in the DMSO control group Figure 7 B). At the protein level, CHB patient peripheral blood sorted NK cells were treated with 25 μM 5β-CA and DMSO respectively, and the expression levels of bile acid receptors TGR5 and FXR were detected by Western blot. It was found that the expression level of TGR5 in the 5β-CA treatment group was significantly higher than that in the DMSO control group, while the expression level of FXR was significantly lower than that in the DMSO control group Figure 7 C). Mouse spleen sorted NK cells were treated with 25 μM 5β-CA and DMSO respectively, and the expression levels of bile acid receptors TGR5 and FXR were detected by Western blot. It was found that the expression level of TGR5 in the 5β-CA treatment group was significantly higher than that in the DMSO control group, while the expression level of FXR showed no significant difference between the two groups Figure 7 D). On the cell line, NK92 cells were treated with 25 μM 5β-CA, 30 μM INT777, 5 μM SBI-115 and DMSO, and the expression level of bile acid receptor TGR5 was detected by Western blot. It was found that the expression level of TGR5 in the 5β-CA and INT777 treatment groups was significantly higher than that in the DMSO control group Figure 7 E), the expression level of TGR5 in the SBI-115 treatment group was significantly lower than that in the DMSO control group Figure 7 F). The above research results show that 5β-CA can up-regulate the expression level of TGR5, suggesting that 5β-CA may induce NK cell activation by binding to TGR5 on the surface of NK cells.

[0058] Example 8: 5β-CA activates the TGR5 / cAMP / mTOR signaling pathway of NK cells The cAMP concentration of NK92 cells treated with 25 μM 5β-CA and DMSO respectively was detected by direct immunofluorescence method, and it was found that the cAMP concentration of cells in the 5β-CA treatment group was significantly higher than that in the DMSO control group Figure 8 ​A). The mTOR protein phosphorylation level of the NK cells sorted from the peripheral blood of CHB patients in the 5β-CA treatment group was significantly higher than that in the DMSO control group (P < 0.05) Figure 8 B). Western blot detection of 5β-CA treated NK92 cells found that the mTOR, PI3K, AKT and PKA protein phosphorylation levels of the 5β-CA treatment group were significantly higher than those of the DMSO control group (P < 0.05) Figure 8 C). The expression levels of transcription factors Id2 and GATA3 were also significantly higher than those of the DMSO control group (P < 0.05) Figure 8 D). After the HBV persistently infected mice were gavaged with 5β-CA for 6 weeks, the NK cells in the spleen of the mice were sorted, and Western blot detection found that the mTOR and PKA protein phosphorylation levels of the NK cells in the 5β-CA treatment group were significantly higher than those in the DMSO control group (P < 0.05) Figure 8 E). NK92 cells were also treated with 1 μM mTOR inhibitor rapamycin (Rapa), and the results showed that the mTOR protein phosphorylation level after Rapa treatment was significantly lower than that of the DMSO control group; after 1 μM Rapa treatment combined with 25 μM 5β-CA and 30 μM INT777 co-treatment, the effect of 5β-CA or INT777 on up-regulating the mTOR protein phosphorylation level disappeared, and was significantly lower than that of the DMSO control group (P < 0.05) Figure 8 F). Finally, after NK92 cells were co-treated with 5 μM TGR5 inhibitor SBI-115 and 1 μM mTOR inhibitor Rapa, FCM was used to detect the levels and mean fluorescence intensity of cytokines (IFN-γ and TNF-α) and cytotoxic granules (granzyme B) of NK92 cells, and it was found that the levels and mean fluorescence intensity of IFN-γ and TNF-α secreted by 5β-CA treated NK92 cells were significantly higher than those of the DMSO control group; after SBI-115 or Rapa treatment combined with 5β-CA co-treatment, the effect of 5β-CA on up-regulating the levels of IFN-γ, TNF-α and granzyme B disappeared, and was significantly lower than that of the DMSO control group (P < 0.05) Figure 8 G-8I). The above research results all show that 5β-CA can activate the TGR5 / cAMP / mTOR signaling pathway.

[0059] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. 5β-Cholanic acid for use in the preparation of an antiviral drug.

2. Use according to claim 1, characterized in that: The virus is hepatitis B virus.

3. Use according to claim 1 or 2, characterized in that: 5β-Cholanic acid achieves antiviral function by inhibiting HBV DNA replication, hepatitis B virus HBsAg expression and hepatitis B virus HBeAg expression.

4. Use according to claim 1, characterized in that: 5β-Cholanic acid achieves anti-hepatitis B virus effect by activating TGR5-cAMP-mTOR signaling pathway to enhance NK cell function.

5. Use according to claim 4, characterized in that: The TGR5-cAMP-mTOR signaling pathway is that after 5β-cholanic acid binds to TGR5 receptor on the surface of NK cells, it triggers intracellular cAMP-PKA signal cascade reaction, promotes PI3K-AKT phosphorylation, and then activates the mTOR pathway.

6. Use according to claim 1, characterized in that: The drug also includes a pharmaceutically acceptable carrier. 7.5β-Cholanic acid for use in the preparation of a product for enhancing NK cell function.

8. Use according to claim 7, characterized in that: 5β-Cholanic acid enhances NK cell function by promoting the secretion of IFN-γ, TNF-α, perforin and granzyme B.

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