CN115804772B - Application of iron death inhibitor in antiviral infection - Google Patents

Abstract

The invention belongs to the technical field of medicines, and discloses a pharmaceutical composition for resisting viral infection and/or reducing the content of viruses, which comprises Liproxstatin-1 or pharmaceutically acceptable salt thereof and pharmaceutical excipients. The pharmaceutical composition has wide application prospect in the field of treating chronic infection.

Description

Application of a ferroptosis inhibitor in antiviral infection

Technical Field

The present invention relates to the field of biomedicine and particularly to application of a ferroptosis inhibitor, Liproxstatin-1, in antiviral infection.

Background technique

Chronic infection undergoes metabolic adaptation by enhancing lipid uptake or storage, which in turn is associated with their dysfunction. Excessive lipid uptake leads to lipid metabolism dysregulation and increased production of reactive oxygen species (ROS), which in turn causes lipid peroxidation. Lipid peroxidation produces a large amount of harmful lipid peroxides that damage the cell membrane and trigger cell ferroptosis [1], of which GPX4 is considered to be the core regulatory molecule for ferroptosis. The production of cytotoxic cytokines by lipid peroxidation and ferroptosis CD8+ T cells is reduced, resulting in a significant weakening of their anti-tumor ability [2–4]. However, studies on chronic infection leading to ferroptosis of CD8+ cells have not been reported.

According to different mechanisms of action, the currently known ferroptosis inducers can be roughly divided into four types: 1. Type I (Class I FIN) that inhibits the Xc-system; 2. Type II (Class II FIN) that inhibits or degrades GPX4; 3. Type III (Class III FIN) that consumes coenzyme Q10; 4. Type IV (Class IV FIN) that induces lipid peroxidation through iron or polyunsaturated fatty acid (PUFA) overload. These four types have high specificity for the induction of ferroptosis, that is, in the process of inducing ferroptosis, markers of other types of cell death are basically not activated. The mechanisms of action of different types of ferroptosis inducers are completely different and have no reference value.

In existing research, the commonly used ferroptosis inhibitors are Liproxstatin-1, Ferrostatin-1, α-Toc, VitE, DFO, etc. Among them, Liproxstatin-1 and Ferrostatin-1 are aromatic amine antioxidants that inhibit lipid peroxidation by scavenging ROS; α-Toc and VitE are lipophilic natural antioxidants that mainly exert antioxidant capacity by destroying the chain reaction of auto-oxidation; DFO is an iron chelator that inhibits the production of hydroxyl free radicals by blocking the Fenton reaction. Commonly used ferroptosis inducers include Erastin, sulfasalazine, and sorafenib that target System Xc- and affect GSH synthesis; RSL3 that targets GPX4 and reduces antioxidant capacity; and dihydroartemisinin that targets iron ions and promotes cellular ROS accumulation.

Studies have found that ferroptosis inhibitors have different biological effects in different cells, tissues or models. For example, the ferroptosis inhibitor Ferrostatin-1 can reverse the increase in CD8+T cell lipid peroxidation caused by the addition of fatty acids and increase cytokine production [2]. Ferrostatin-1 can also reduce lipid peroxidation of tumor-infiltrating T cells and exert stronger anti-tumor ability [5]. However, another study found that the ferroptosis inhibitor Ferrostatin-1 alone did not improve the killing function of CD8+T cells [6].

Studies have found that different inhibitors have different biological effects. For example, in macrophages infected with the pathogenic yeast Histoplasma capsulatum, Ferrostatin-1 can inhibit the synthesis of sterols, which not only reduces the death of infected macrophages, but also inhibits the growth of Pseudomonas capsulatum; while another aromatic amine antioxidant Liproxstatin-1 cannot play the above role and cannot effectively prevent fungal growth or reduce macrophage death [7]. In addition, in oligodendrocytes treated with RSL3, Liproxstatin-1 can inhibit lipid peroxidation, restore the expression of GSH and GPX4, and thus significantly inhibit ferroptosis, while the effect of other ferroptosis inhibitors DFO is not obvious [8].

Ferroptosis inhibitors/inducers have different effects on different immune cells. For example, in macrophages, the ferroptosis inducer RSL3 can induce ferroptosis in M2 macrophages, but cannot induce ferroptosis in M1 macrophages[9]. In the tumor microenvironment, a certain concentration of ferroptosis inducers Erastin and RSL3 can effectively induce ferroptosis in tumor cells, but cannot induce ferroptosis in CD4+T cells and CD8+T cells[10]. In the LCMV-infected mouse model, feeding vitamin E significantly increased the absolute cell number of CD8+T cells, but had no effect on the absolute cell number of CD4+T cells; however, in GPX4-knockout infected mice, feeding vitamin E can restore the absolute cell number of CD4+T cells, but cannot completely restore the absolute cell number of CD8+T cells[1].

Hepatitis B virus (HBV) is the pathogen that causes hepatitis B (abbreviated as HBV) and belongs to the Hepadnaviridae family. The virus in this family includes two genera, Orthohepadnavirus and Avian Hepadnavirus. The Orthohepadnavirus is the one that causes human infection. The degree of liver cell damage is related to the strength of the body's immune response. The mechanisms of HBV-induced immunopathological damage include: low immune response caused by the virus, drug resistance caused by viral mutation, antibody-mediated immunopathological damage, and cell-mediated immunopathological damage. Commonly used drugs include interferon, lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoproxil, etc. Since the current first-line antiviral drugs cannot meet the clinical cure needs of most patients, it is urgent to continue to develop new antiviral drugs and study new treatment options to improve the clinical cure of HB within a limited course of treatment. The new antiviral drugs currently under development are mainly divided into direct antiviral drugs (DAAs) targeting the virus life cycle and indirect antiviral drugs involved in immune regulation. DAAs mainly include small interfering RNA, antisense oligonucleotides, gene editing silencing, capsid assembly regulators, cell entry inhibitors, and HBsAg release inhibitors; indirect antiviral drugs involved in immune regulation mainly include TLR7/8 innate immune regulators, therapeutic vaccines, monoclonal antibodies, immune checkpoint inhibitors, etc.

In chronic HBV infection, the body's innate and acquired immune responses are seriously impaired. HBV replication in hepatocytes is not detected by the innate immune system in the cells, resulting in impaired antigen presentation function of DCs [11]. In addition, NK cells and HBV-specific CD4+ and CD8+ T cells also suffer from severe dysfunction and exhaustion. HBV can produce and secrete a large amount of viral antigens, gradually changing and exhausting the function of HBV-specific T cells, resulting in reduced production of antiviral cytokines such as IFN-γ and TNF-α, and ultimately causing HBV to persist in the body for a long time and be difficult to eliminate [12,13]. The current research consensus is that the immune response mediated by CD4+ and CD8+ T cells determines the outcome of HBV treatment. However, even with the current standard of care, defects in the immune response make it difficult for patients to restore the function of HBV-specific immune cells to normal levels, and thus the infection cannot be effectively controlled.

The mechanism of viral infection is extremely complex. Different viral infections cause very different clinical symptoms. The immunological mechanisms and principles of different treatment strategies are very different. It is difficult to predict whether a single mechanism inhibitor can inhibit viral infection, and there are significant differences in treatment effects. For example, the new anti-HBV drug IAP antagonist APG-1387 can induce apoptosis of hepatocytes expressing HBV antigens and enhance HBV-specific T cell responses [14]; the anti-HBV IL-2 immunotherapy drug AB359 selectively activates the IL-2 pathway in CD8+T cells, reverses the deficiency of CD8+T cells, but has no significant effect on other immune cells [15]; the TLR8 agonist GS-9688 can improve the cytolytic and non-cytolytic effector functions of NK cells in anti-HBV treatment, increase the number of HBV-specific CD8+T cells and cytokine production in some patients, increase the number of follicular helper T cells, and reduce the number of Tregs and mononuclear MDSCs [16]; the mitochondrial antioxidants MitoQ and MitoTempo can significantly improve the effector function of HBV-specific CD8+T cells, while only very weak effects were observed on the total T cell population [17]. In view of this, the mechanisms by which different antiviral drugs regulate antiviral immune responses are relatively complex.

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2Ma X, Xiao L, Liu L, Ye L, Su P, Bi E, et al. CD36-mediated ferroptosis dampens intratumoral CD8+T cell effector function and impairs their antitumorability. Cell Metab 2021;33:1001-1012.e5.

3Xu S,Chaudhary O,Rodríguez-Morales P,Sun X,Chen D,Zappasodi R,etal.Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8+T cells in tumors.Immunity 2021；54:1561-1577.e7.

4Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, et al. Regulation of Ferroptotic Cancer Cell Death by GPX4. Cell 2014; 156: 317-331.

5Xiao L, Ma X, Ye L, Su P, Xiong W, Bi E, et al. IL-9/STAT3/fatty acidoxidation-mediated lipid peroxidation contributes to Tc9 cell longevity and enhanced antitumor activity. J Clin Invest;132:e153247.

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7Horwath MC, Bell-Horwath TR, Lescano V, Krishnan K, Merino EJ, Deepe GS. Antifungal Activity of the Lipophilic Antioxidant Ferrostatin-1. Chembiochem 2017;18:2069-2078.

8Fan B-Y, Pang Y-L, Li W-X, Zhao C-X, ZhangY, Wang X, et al. Liprpxstatin-1 is an effective inhibitor of oligodendrpcyte ferroptosis induced by inhibition of glutathione peroxidase 4. Neural Regen Res 2020;16:561-566.

9Kapralov AA, Yang Q, Dar HH, Tyurina YY, Anthonymuthu TS, Kim R, et al. Redpx Lipid Reprogramming Commands Susceptibility of Macrophages and Micropglia to Ferroptotic Death. Nat Chem Biol 2020;16:278-290.

10Wang W, Green M, Choi JE, Gijón M, Kennedy PD, Johnson JK, et al. CD8+T cells regulate tumor ferroptosis during cancer immunptherapy. Nature 2019; 569: 270-274.

11Papatheodoridis GV, Manolakopoulos S, Dusheiko G, Archimandritis AJ. Therapeutic strategies in the management of patients with chronic hepatitis B virus infection. Lancet Infect Dis 2008;8:167-178.

12 Guidotti LG, Chisari FV. Noncytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001; 19: 65-91.

13Iannacone M, Guidotti LG. Immunobiology and pathogenesis of hepatitis B virus infection. Nat Rev Immunol 2022; 22: 19-32.

14Aseentage Pharma Group Inc. A Phase I Study of the Safety, Pharmacokinetic and Pharmacodynamic Properties of APG-1387 in Patients With Chronic Hepatitis B. clinicaltrials.gov; 2021. https://clinicaltrials.gov/ct2/show/NCT03585322 (accessed 9Nov2022).

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16Amin OE, Colbeck EJ, Daffis S, Khan S, Ramakrishnan D, Pattabiraman D, et al. Therapeutic Potential of TLR8 Agonist GS-9688 (Selgantolimod) in Chronic Hepatitis B: Remodeling of Antiviral and Regulatory Mediatprs. Hepatology 2021;74:55-71.

17Fisicaro P, Barili V, Montanini B, Acerbi G, Ferracin M, Guerrieri F, et al. Targeting mitpchondrial dysfunction can restpre antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B. Nat Med 2017;23:327-336.

Liproxstatin-1 (Lip-1) is an effective ferroptosis inhibitor that can be used in vivo and in vitro. Lip-1 easily penetrates the lipid bilayer and stays inside it, making its active site in close directional contact with the lipid peroxidation site, initiating CH3OO· to extract hydrogen atoms from the aromatic amine site. After scavenging lipid peroxidation, the Lip-1 free radical formed by Lip-1 can be reduced by other antioxidants in the body (such as ubiquinone). Lip-1 is more potent in inhibiting ferroptosis than other ferroptosis inhibitors, such as deferoxamine, vitamin E, and ferrostatin-1. It has been shown that Lip-1 can protect mouse myocardium from ischemia/reperfusion injury by reducing VDAC1 levels and restoring GPX4 levels; it can inhibit RSL3-induced death of human renal proximal tubular epithelial cells and acute renal failure induced by GPX4 deficiency.

To date, the mechanism of action of the ferroptosis inhibitor Liproxstatin-1 in anti-viral infection is still unclear, and there is no report on the anti-viral effect of Liproxstatin-1.

Summary of the invention

In order to overcome the technical problems existing in the prior art, the present invention provides a new application of the ferroptosis inhibitor Liproxstatin-1, especially in improving the effector function of CD8+T cells.

The present invention is achieved through the following technical solutions.

Use of ferroptosis inhibitor Liproxstatin-1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving CD8+T cell effector function or resisting viral infection.

Preferably,

The CD8+T cells are CD8+T cells and antigen-specific CD8+T cells in a chronic hepatitis B model.

As a preferred technical solution of the present invention: the application is to reduce the lipid peroxidation level of CD8+T cells and antigen-specific CD8+T cells.

As a preferred technical solution of the present invention: the application is to increase the production of CD8+T cells and antigen-specific CD8+T cell cytotoxic cytokines.

As a preferred technical solution of the present invention: the application is to inhibit ferroptosis of CD8+T cells and antigen-specific CD8+T cells.

As a preferred technical solution of the present invention: the application is to improve the antiviral ability of CD8+T cells.

Preferably, the virus is HBV virus.

The present invention also provides a drug for antiviral infection, wherein the active ingredient is ferroptosis inhibitor Liproxstatin-1 or a pharmaceutically acceptable salt thereof. The virus is HBV.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A: Effect of co-incubation with the ferroptosis inhibitor Liproxstatin-1 on lipid peroxidation in CD8+ T cells from CHB patients.

Figure 1B: Gating strategy for antigen-specific CD8+ T cells using MHC tetramer markers.

Figure 1C: Effect of co-incubation with the ferroptosis inhibitor Liproxstatin-1 on lipid peroxidation of antigen-specific CD8+ T cells.

Figure 1D: Effect of co-incubation with the ferroptosis inhibitor Liproxstatin-1 on the production of IFN-γ and TNF-α by CD8+ T cells.

Figure 1E: Effect of co-incubation with the ferroptosis inhibitor Liproxstatin-1 on IFN-γ and TNF-α production by antigen-specific CD8+ T cells.

Figure 1F: Effect of co-incubation with the ferroptosis inhibitor Liproxstatin-1 on GPX4-related expression in CD8+ T cells.

Figure 2A: Diagram of mouse treatment schedule.

Figure 2B: Effect of treatment with the ferroptosis inhibitor Liproxstatin-1 on lipid peroxidation in intrahepatic CD8+ T cells.

Figure 2C: Effect of treatment with the ferroptosis inhibitor Liproxstatin-1 on the production of IFN-γ and TNF-α by intrahepatic CD8+ T cells.

Figure 2D: Effects of treatment with the ferroptosis inhibitor Liproxstatin-1 on HBsAg production in mice. Figure 2E: Effects of treatment with the ferroptosis inhibitor Liproxstatin-1 on HBV DNA in mice.

Figure 2F shows the effect of treatment with the ferroptosis inhibitor Liproxstatin-1 on the body weight of mice.

Figure 2G: Effects of treatment with the ferroptosis inhibitor Liproxstatin-1 on pathological changes in the liver of mice.

Detailed ways

The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention.

Definition of Terms

As used herein, the term "CD8+ T cells" refers to cytotoxic T lymphocytes, which are a type of killer T cells that can participate in immune effects by secreting various cytokines.

As used herein, the term "ferroptosis" is a novel cell death mode first proposed by Dr. Brent R Stockwell's laboratory in 2012. Ferroptosis is different from pyroptosis, apoptosis, necrosis, and autophagic cell death. The process of ferroptosis is characterized by the accumulation of lethal ROS and lipid peroxidation products (LPO) during iron metabolism.

As used herein, the term "Lip-1", i.e., Liproxstatin-1, a ferroptosis inhibitor, is a potent ferroptosis inhibitor that inhibits lipid peroxidation and ferroptosis by inhibiting the generation of lipid free radicals.

As used herein, the term "Med" is cell culture medium, ie, primary cell culture medium.

As used herein, the term "Pep" is an HBV antigen peptide, and the sequence is HBV-core (aa 18-27: FLPSDFFPSV).

As used herein, the term "MHC tetramer" is a combination of soluble MHC protein and polypeptide, and is a simple and effective method for identifying, isolating and studying antigen-specific T cells from a variety of biological samples.

As used herein, the term "IFN-γ" is interferon gamma, which is an important cytokine that can perform immunomodulatory functions. It was discovered for its antiviral activity. IFN-γ plays a key role in host defense through antiviral, antiproliferative and immunomodulatory functions. It can reflect immune cell effector functions.

As used herein, the term "TNF-α" refers to tumor necrosis factor α, which participates in normal inflammatory and immune responses and can coordinate the homeostasis of tissues by coordinating the production of other cytokines, cell survival and death. It is cytotoxic to various tumor cells and is an important factor in mediating the immune response to viral infection. It can reflect the effector function of immune cells.

As used herein, the term "GPX4" refers to glutathione peroxidase-4, which plays a key role in degrading harmful lipid peroxides to maintain homeostasis as a core regulatory molecule of ferroptosis.

The present invention is further described in detail below in conjunction with the accompanying drawings and specific experiments. Unless otherwise specified, the reagents, instruments, equipment and methods used in this patent are all conventional commercially available reagents, instruments, equipment and methods in the art.

Example 1

In vitro experiments.

Isolation of PBMCs: PBMCs were isolated from fresh heparinized blood using the Ficoll-Hypaque density gradient method.

Specific T cell treatment: PBMCs isolated from CHB patients were cultured in RPMI1640 containing 10% fetal bovine serum. PBMCs were stimulated with HBV peptides (1 μM) for 10 days in the presence or absence of ferroptosis inhibitor Liproxstatin-1 (50 nM), and HBV-specific CD8+ T cells were further analyzed. Sorting and activation of CD8+ T cells: CD8+ T cells were isolated from PBMCs using a negative selection kit (Biolegend) and stimulated with human T-activating antibodies CD3/CD28 (10 μg/ml, Biolegend) and IL-2 (10 ng/ml). CD8+ T cells were stimulated with HBV peptides (1 μM) for 10 days in the presence or absence of ferroptosis inhibitor Liproxstatin-1.

Lipid peroxidation measurement: The experiment was performed according to the manufacturer's protocol. Briefly, cells were incubated with lipid peroxidation sensor C11 BODIPY 581/591 (Invitrogen) in cell culture medium in a 37°C incubator containing 5% carbon dioxide for 30 minutes. After incubation, cells were washed and flow cytometry was performed using FACSCelesta (BD) within 2 hours after staining.

MHC tetramer staining: 1 μL MHC tetramer dye (HelixGen) was added to 25 μL peripheral blood mononuclear cell suspension (2×10 ⁷ /mL) and incubated on ice in the dark for 60 minutes. After incubation, FACS Buffer (PBS + 2% calf serum + 0.1% sodium azide) was added for washing, and then resuspended in 200 μL fixative (PBS + 1% paraformaldehyde) for fixation.

T cell cytokine production detection: Prepare cell activation medium (100IU/mL IL-2+1μg/mL ionomycin+50ng/mL PMA+10μg/mL brefeldin A), add 1ml activation medium for every 1×10 ⁶ PBMC cells, and culture in a 24-well plate for 4 hours. After labeling with CD3-Percp-Cy5.5 (Biolegend), CD8-BV421 (Biolegend) and MHC tetramer-APC, fix, permeabilize and block, then label with IFN-γ-PE (Biolegend) and TNF-α-PE-CF594 (Biolegend), wash and test. GPX4 labeling: After fixed, permeabilized and blocked cells, add 1/400 dilution of recombinant Anti-Glutathione Peroxidase 4 antibody (Abcam) at 4℃ for 2h, add goat serum for blocking, and goat anti-rabbit IgG-FITC (Abclonal) at a dilution of 1/2000 is used as the secondary antibody.

As shown in Figure 1A , Lip-1 significantly reduced the lipid peroxidation level of CD8 + T cells in PBMCs of CHB patients.

As shown in Figure 1B, the main cell population was circled in the sorted CD8+T cells, and after diagonal de-adhesion, the single cell population was gated, and finally the tetramer-positive cell population, i.e., HBV-specific CD8+T cells, was circled.

As shown in Figure 1C , Lip-1 significantly reduced the lipid peroxidation level of HBV-specific CD8 + T cells in PBMCs of CHB patients.

As shown in Figure 1D , Lip-1 significantly enhanced the production of cytokines (IFN-γ and TNF-α) by CD8 + T cells in PBMCs of CHB patients.

As shown in Figure 1E , Lip-1 was able to enhance the production of cytokines (IFN-γ and TNF-α) by HBV-specific CD8 + T cells in PBMCs of CHB patients.

As shown in Figure 1F , Lip-1 significantly increased the level of GPX4 in CD8 + T cells and HBV-specific CD8 + T cells in PBMCs of CHB patients.

(“ns” means no significant difference, “*” means p<0.05, and “**” means p<0.01)

Example 2

In vivo experiments.

Construction of HBV-carrier mouse model: 5-6 week old C57BL/6J mice were injected with 8 μg pAAV/HBV1.2 plasmid via tail vein high pressure. Peripheral blood was collected 5-6 weeks later to separate serum and detect HBsAg level in serum. Mice with serum HBsAg concentration higher than 500 ng/mL were considered as successful HBV-carrier mice.

Treatment strategy: The mice with successful modeling were intraperitoneally injected with 30 mg/kg of the ferroptosis inhibitor Liproxstatin-1 every day, and the body weight changes were monitored every two days. The treatment was continued for 15 days.

Cut the tail of the mouse to collect blood and separate serum: fix the mouse in a fixator under sterile conditions, exposing the mouse tail; gently wipe the mouse tail with 70% alcohol for disinfection; cut the tail about 2mm from the end of the mouse tail, massage from the base of the tail to the tip of the tail, and collect the blood flowing from the tip of the tail. Let it stand at room temperature for 30 minutes, centrifuge at 3000rpm for 15 minutes, and take the supernatant as the mouse peripheral blood serum, which is stored at -80℃.

Obtaining liver mononuclear cells: dissect and separate mouse liver, grind and collect into 15mL centrifuge tube; centrifuge at 100g for 1 minute, collect supernatant; continue centrifugation at 400g for 5 minutes, discard supernatant and retain precipitate; add 4mL 40% Percoll solution (cytiva) to resuspend cells; centrifuge at 800g for 25 minutes, discard supernatant and retain precipitate; add red blood cell lysis solution to resuspend cells, place at 4℃ for 10 minutes to stop red blood cell lysis, filter and divide into tubes.

CLIA method for detecting HBsAg levels in mouse peripheral blood serum: dilute serum samples according to experimental needs; add 50 μL of samples or standards (0, 0.05, 0.8, 10, 85, 250 ng/mL) to the corresponding coated wells; add 50 μL of enzyme conjugate to each well, gently shake and mix, apply the sealing film, and incubate at 37°C for 1 hour; after the incubation, remove the sealing film, shake off the liquid in the well, and wash the coated plate with HBsAg washing solution, repeat the washing 5 times, and pat dry as much as possible for the last time; mix luminescent substrate A and luminescent substrate B in a 1:1 ratio, add 50 μL of the mixed luminescent substrate to the coated wells, shake gently, and stand at room temperature in the dark for 10 minutes. Use Synergy 2 multifunctional microplate reader to detect the luminescence intensity, make a standard curve, and calculate the HBsAg concentration in the sample. Serum HBV DNA detection: Add 450μl DNA extraction solution I and 4μL internal standard solution to 200μL sample, shake and mix for 15s, and centrifuge to remove water droplets on the cap and tube wall. Treat at 100℃ for 10±1min, centrifuge at 12000rpm for 5min, and set aside. Take the corresponding amount of PCR reaction solution and Taq enzyme (HBV PCR reaction solution 27μL/tube + Taq enzyme 3μL/tube) in proportion, mix thoroughly and dispense into 30μL/tube into empty PCR reaction tubes suitable for each instrument, and set aside. Use a pipette with a filter to add 20μL each of the extracted sample nucleic acid to be tested, HBV negative quality control product, HBV strong positive quality control product, HBV critical positive quality control product and positive quantitative reference product to the above HBV reaction tube, centrifuge at 8000rpm for a few seconds, and transfer to the amplification detection area. The PCR reaction conditions are as follows (DA7600):

Table 1 PCR reaction conditions for peripheral blood serum DNA detection

As shown in Figure 2A, HBV-carrier mice were injected intraperitoneally with 30 mg/kg of the ferroptosis inhibitor Liproxstatin-1 every day 5 weeks after successful modeling, and their body weight was measured every two days. The treatment continued for 15 days.

As shown in Figure 2B , the lipid peroxidation level of CD8 + T cells in the liver of HBV-carrier mice was significantly higher than that of wild-type mice, and Lip-1 treatment significantly reduced the lipid peroxidation level of HBV-carrier mice.

As shown in Figure 2C, Lip-1 treatment significantly improved the production of cytokines (IFN-γ and TNF-α) by CD8 + T cells in the liver of HBV-carrier mice.

As shown in Figure 2D, Lip-1 treatment was able to reduce the level of HBsAg in the serum of HBV-carrier mice.

As shown in Figure 2E, Lip-1 treatment was able to reduce the level of HBV DNA in the serum of HBV-carrier mice.

As shown in Figure 2F, Lip-1 treatment reduced the body weight of HBV-carrier mice on day 5 and gradually returned to normal on day 10.

As shown in Figure 2G, Lip-1 treatment did not show damage to the mouse liver.

(“ns” means no significant difference, “*” means p<0.05, “**” means p<0.01, and “****” means p<0.0001)

This study also explored the effects of other ferroptosis inhibitors on HBV-infected antigen-specific CD8+T cells and found that edaravone, ferrostatin-1, vitamin E, UAMC-3203 and defetoxamine had no obvious activation/stimulation effects. In the in vivo anti-HBV infection study, no significant therapeutic effects were observed.

Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims (2)

1. A method for preparing antigen-specific CD8+T cells, characterized in that the method comprises the following steps: Step 1) PBMCs isolated from patients with chronic hepatitis B were cultured in RPMI 1640 containing 10% fetal bovine serum, and the PBMCs were stimulated with HBV peptides in the presence of ferroptosis inhibitor Liproxstatin-1 for 10 days; Step 2) CD8+T cells were isolated from PBMCs and stimulated with human T-activating antibodies CD3/CD28 and IL-2; CD8+T cells were stimulated with HBV peptides for 10 days in the presence of ferroptosis inhibitor Liproxstatin-1; The HBV peptide is FLPSDFFPSV. 2. The method according to claim 1, characterized in that the concentration of Liproxstatin-1 used in step 1) and step 2) is 50 nM.