Application of N-acetylcysteine in preparation of medicine for enhancing immune effect

Abstract

The invention discloses application of N-acetylcysteine in preparation of a medicine for enhancing an immune effect, and relates to the technical field of biology. On the basis of the existing vaccine immunomodulator technology, the invention provides an application of N-acetylcysteine or pharmaceutically acceptable salt thereof as an active ingredient in preparation of drugs for enhancing the vaccine immune effect and / or for adjuvant treatment of infection, and the N-acetylcysteine can be used for enhancing the immune effect of the vaccine by directly protecting the mitochondrial respiratory chain function of plasma cells. According to the present invention, with the application of the traditional immunomodulator, the low-level intracellular active oxygen can be maintained, and the life in the bone marrow can be prolonged so as to provide the unique advantages in immune reaction and memory enhancement, such that the long-term immune protection effect can be improved, and the safety problem such as local or whole body adverse reaction possibly caused by the traditional immunomodulator can be avoided; a new safe and effective source is provided for the existing vaccine immunomodulator and the medicine for treating infection, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of N-acetylcysteine ​​in the preparation of drugs that enhance immune effects. Background Technology

[0002] With the rapid development of biotechnology and genetic engineering, various novel vaccines have been developed to target different infectious diseases. However, these vaccines generally suffer from limitations such as small molecular size, limited immunogenicity, difficulty in sustaining an effective immune response, and short duration of protection. Therefore, a suitable adjuvant is needed to better enhance their immune effect. Current vaccine immunomodulators mainly include adjuvants and immune enhancers, which can activate the host's immune system, enhance the immune response to the vaccine, and improve its efficacy. Commonly used adjuvants include aluminum glue, mineral oil, inactivated bacteria, and chemically synthesized substances. Different vaccine immunomodulators have different mechanisms of action, including antigen storage effect (i.e., after local injection of antigen, the adjuvant recruits and stores excess antigen and releases it slowly and continuously, prolonging the continuous stimulation time and increasing antibody titer), increasing antigen surface area (i.e., for antigens that are small in size and have low relative molecular mass, which are insufficient to induce an immune response, encapsulating them increases the molecular weight and surface area of ​​the antigen, thereby enhancing its immunogenicity), carrier function, and promoting the activation and maturation of dendritic cells. However, vaccine immunomodulators also have many problems, such as safety issues and local side effects. Absolutely safe vaccine immunomodulators do not exist. The goal is to adjust the immunomodulator's mechanism of action to maximize immune stimulation while minimizing side effects. Some immunomodulators can induce strong antibody responses and cellular immunity, potentially leading to local or systemic adverse reactions such as inflammation and allergic reactions. Furthermore, some immunomodulators have formulation incompatibilities; for example, oil-based immunomodulators are too viscous for injection, and liposome-based immunomodulators have poor stability. Therefore, it is essential to develop vaccine immunostimulants that can improve vaccine immunogenicity, prolong protective duration, and optimize the quality of the immune response while ensuring safety. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of N-acetylcysteine ​​in the preparation of drugs that enhance immune effects.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is the application of N-acetylcysteine ​​or its pharmaceutically acceptable salt as an active ingredient in the preparation of vaccines that enhance immune efficacy and / or in adjuvant treatment of infectious diseases.

[0005] Studies have found that N-acetylcysteine ​​(NAC), as a potent antioxidant, can promote glutathione synthesis, thereby reducing intracellular reactive oxygen species (ROS) and free radicals. High levels of ROS can cause oxidative stress damage to cells, especially plasma cells. Excessively high ROS levels can lead to mitochondrial dysfunction, affecting their survival and function. NAC protects the mitochondrial respiratory chain function of plasma cells by maintaining low ROS levels, thereby enhancing the retention and long-term survival of plasma cells in the bone marrow. The inventors of this application, through extensive research, have demonstrated that N-acetylcysteine ​​can enhance vaccine efficacy, particularly in applications that enhance humoral immune responses and memory. Experiments have verified that N-acetylcysteine ​​reduces intracellular ROS and free radicals in plasma cells by promoting glutathione synthesis, protecting plasma cells from oxidative damage, thereby significantly promoting plasma cell production and prolonging plasma cell survival time, ultimately enhancing long-term antibody immune responses. Furthermore, based on the unique mechanism by which N-acetylcysteine ​​protects plasma cell mitochondrial function and prolongs plasma cell lifespan, the inventors of this application have discovered through experimental research that when pathogen infection occurs, the body needs plasma cells to rapidly produce a large number of antibodies to clear the pathogen. At this time, the administration of N-acetylcysteine ​​can also protect these effector plasma cells, enhance the body's immediate humoral immune response, thereby assisting in the treatment of infection, accelerating pathogen clearance, and alleviating clinical symptoms.

[0006] As a preferred embodiment of the application described in this invention, the drug enhances the long-term immune response of antibodies by protecting the mitochondrial function of plasma cells, promoting plasma cell production, and / or prolonging the survival time of plasma cells.

[0007] In a preferred embodiment of the application described in this invention, the drug is administered to a subject.

[0008] In a preferred embodiment of the application described in this invention, the subject includes a human or an animal.

[0009] As a preferred embodiment of the application described in this invention, the route of administration includes oral or injectable administration.

[0010] As a preferred embodiment of the application described in this invention, the drug is administered to the subject simultaneously with and / or after the subject is vaccinated and / or exposed to a pathogen.

[0011] The present invention also provides a pharmaceutical composition for enhancing the immune effect of a vaccine and / or for adjuvant treatment of an infection, comprising N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof.

[0012] In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0013] As a preferred embodiment of the composition of the present invention, the dosage form of the pharmaceutical composition includes oral preparations and injections.

[0014] The present invention also provides a method for enhancing the protective effect of antibodies, comprising administering N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof concurrently and / or after vaccination of a subject and / or exposure of the subject to a pathogen.

[0015] Compared with existing technologies, this invention has the following beneficial effects: Based on existing immunomodulatory agent technologies, this invention provides the application of N-acetylcysteine ​​or its pharmaceutically acceptable salts as active ingredients in the preparation of vaccines to enhance immune efficacy and / or in adjuvant therapy for infections. Unlike traditional vaccine adjuvants, which mainly regulate the early immune response of B cells and Tfh cells, N-acetylcysteine ​​directly protects the mitochondrial respiratory chain function of plasma cells, maintains low levels of intracellular reactive oxygen species, and prolongs their lifespan in the bone marrow, thus providing a unique advantage in enhancing immune response and memory. Furthermore, based on the same mechanism, N-acetylcysteine ​​can also protect these effector plasma cells during pathogen infection, enhancing the body's immediate humoral immune response, thereby adjuvant therapy for infection, accelerating pathogen clearance, and alleviating clinical symptoms. Therefore, N-acetylcysteine ​​not only improves long-term immune protection but also avoids the safety issues that traditional immunomodulators may cause, such as local or systemic adverse reactions, providing a new, safe, and effective source for existing vaccine immunomodulators and drugs for treating infections, with broad application prospects. Attached Figure Description

[0016] Figure 1 A schematic diagram of the experimental method for investigating the effect of drinking water containing NAC on the immune response to model antigens in mice.

[0017] Figure 2 The level of anti-NP IgG1 antibody in the serum of mice immunized with a model antigen from drinking water containing NAC.

[0018] Figure 3 A schematic diagram of the experimental method used to investigate the effect of drinking water containing NAC on the immune response of mice to a vaccine.

[0019] Figure 4 The level of anti-NP IgG1 antibody in the serum of mice that received a vaccine after drinking water containing NAC.

[0020] Figure 5 Neutralizing antibody activity against influenza virus in the serum of mice that received a vaccine after drinking water containing NAC.

[0021] Figure 6A shows the weight change of the experimental mice; B shows the survival rate of the experimental mice; C shows the RNA load of influenza HA virus in mice; D shows the acute lung injury score of mice. Detailed Implementation

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available.

[0023] The C57BL / 6J mice used in this embodiment of the invention were purchased from Guangdong Yaokang, catalog number N000013.

[0024] The N-acetylcysteine ​​(NAC) used in this embodiment of the invention was purchased from MCE, catalog number HY-B0215.

[0025] The immune model antigen NP-KLH used in this embodiment of the invention was purchased from Biosearch Technologies, catalog number N-5060-5. NP-KLH is a commonly used model antigen in vaccine immunization research, which can simulate the T cell-dependent antibody response during the vaccine immunization process.

[0026] The inactivated influenza vaccine in this embodiment of the invention is laboratory-made.

[0027] Example 1: Effect of drinking water containing NAC on immune responses to mouse model antigens This embodiment investigates the effect of drinking water containing NAC on the immune response to mouse model antigens. Specific experimental methods are described below. Figure 1 This includes the following steps: 1. Inject C57BL / 6J mice with the immunization pattern antigen NP-KLH to simulate the vaccine immunization process; 2. Mice were divided into two groups: the NAC group was given drinking water containing NAC (0.1%) for 4 consecutive weeks, and the Vehicle group (control group) was given ordinary drinking water for 4 consecutive weeks. 3. At weeks 2, 4, and 8 post-vaccination, mouse serum was collected to measure changes in antibody levels (anti-NP IgG1).

[0028] The results are as follows Figure 2 As shown, the serum anti-NP IgG1 antibody level in mice in the NAC group was consistently significantly higher than that in the Vehicle group throughout the observation period. This indicates that NAC can effectively enhance humoral immune responses and strengthen the sustained production of antibodies.

[0029] Example 2: Effect of drinking water containing NAC on the immune response to a mouse vaccine. This embodiment investigates the effect of drinking water containing NAC on the immune response of mice to a vaccine. Specific experimental methods are described below. Figure 3 This includes the following steps: 1. Each C57BL / 6J mouse was injected intramuscularly with 50 μL of inactivated influenza vaccine, which included 6 μg of inactivated influenza virus and 25 μL of AddaVax adjuvant; 2. Mice were divided into two groups: the Vehicle group drank ordinary drinking water for 8 consecutive weeks, and the NAC group drank drinking water containing NAC (0.1% dose) for 8 consecutive weeks. 3. At weeks 2, 4, 8 and 12 post-vaccination, mouse serum was collected to measure changes in antibody levels (anti-NP IgG1).

[0030] The results are as follows Figure 4 , Figure 5 As shown, throughout the observation period, the serum anti-HA IgG1 antibody levels and hemagglutination inhibition antibody titers in the NAC group mice were significantly higher than those in the Vehicle group, and the difference continued to increase. This indicates that oral administration of NAC can effectively enhance the immune response and maintain long-term antibody levels, while significantly enhancing the activity of neutralizing antibodies against influenza virus in the serum.

[0031] Example 3 This embodiment investigates the effect of drinking water containing NAC on the response of mice to viral infection. The specific experimental method includes the following steps: 1. Each C57BL / 6J mouse was infected with a lethal dose of influenza A virus (H1N1, PR8) via intranasal instillation. 2. Mice were divided into two groups: the Vehicle group drank ordinary drinking water, and the NAC group drank drinking water containing NAC (0.1%). 3. After infection, the weight and survival of the two groups of mice were continuously tracked (ethical death was defined as a weight loss of less than 75%).

[0032] 4. Lung tissue was collected from mice 7 days after death or infection to detect viral load and damage in the lungs. Specific experimental methods for detecting viral load in the lungs: Lung tissue was collected from mice and homogenized using a MACS dissociator. Total RNA was extracted from the homogenate using TRIzol reagent (Invitrogen). The concentration and purity of the extracted RNA were determined spectrophotometrically using NanoDrop. Subsequently, cDNA was synthesized using a reverse transcription kit (TaKaRa) with 500 ng of total RNA as a template, following the manufacturer's instructions. The hemagglutinin protein (HA) gene of influenza virus (H1N1, PR8) was cloned into the pcDNA3.1 expression plasmid and transcribed in vitro to obtain RNA and cDNA as standards. Standard curves were generated by serially diluting the samples tenfold. Real-time PCR analysis was performed using a universal SYBR qPCR Mix (Vazyme) and a CFX 96 touchscreen real-time PCR instrument. The viral RNA copies per mg were calculated for each tissue and expressed as log10. Lung Injury Severity Scoring Criteria: To investigate lung injury in mice attacked with influenza virus, a standardized lung injury scoring system was used, with independent scoring by two researchers. At least 20 random fields (magnified 400x) were analyzed from each sample. Five histological parameters were assessed: (A) alveolar neutrophils (0: none; 1: 1–5; 2: 5), (B) interstitial neutrophils (0: none; 1: 1–5; 2: >5), (C) hyaline membrane (0: none; 1: 1; 2: >1), (D) protein debris (0: no; 1: 1; 2: >1), and (E) alveolar septal thickening (0: <2x; 1: 2×–4×; 2: >4×). Score = [(20×A) + (14×B) + (7×C) + (8×D) + (2×E)] / (number of fields × 100).

[0033] The results are as follows Figure 6 As shown, after 7 days, the body weight of mice in the NAC group showed a downward trend followed by an increase, and the survival rate of the mice was high; while the body weight of mice in the Vehicle group showed a continuous decrease, and the survival rate was low. The influenza HA virus load in the lungs and the acute lung injury score of mice in the NAC group were significantly lower than those in the Vehicle group.

[0034] The above experimental results indicate that N-acetylcysteine ​​can significantly enhance antibody persistence after vaccination. Its mechanism of action involves protecting the mitochondrial function of plasma cells and reducing intracellular reactive oxygen species, thereby prolonging the survival time of plasma cells in the bone marrow. During infection, when the body needs plasma cells to rapidly produce large amounts of antibodies to clear pathogens, administration of N-acetylcysteine ​​can also protect these effector plasma cells and enhance the body's immediate humoral immune response. Therefore, N-acetylcysteine ​​not only has the potential to be used as a vaccine immunostimulant but also as an adjunct therapy for infections. Administration of N-acetylcysteine ​​when patients are exposed to pathogens or develop infections can help enhance the body's own antibody defense capabilities and accelerate the recovery process.

[0035] Finally, it should be noted that the above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The use of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of vaccines to enhance their immunogenicity and / or as an adjunct therapy for infections.

2. The application according to claim 1, characterized in that, The drug enhances long-term immune protection of antibodies by protecting the mitochondrial function of plasma cells, promoting plasma cell production, and / or prolonging the survival time of plasma cells.

3. The application according to claim 1, characterized in that, The drug was administered to the subjects.

4. The application according to claim 3, characterized in that, The subjects included humans or animals.

5. The application according to claim 3, characterized in that, The routes of administration include oral or injectable administration.

6. The application according to claim 3, characterized in that, The drug is administered to the subject concurrently with and / or after the subject is vaccinated and / or exposed to the pathogen.

7. A pharmaceutical composition for enhancing the immune efficacy of a vaccine and / or for adjuvant treatment of infection, characterized in that, This includes N-acetylcysteine ​​or its pharmaceutically acceptable salts.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage forms of the pharmaceutical composition include oral preparations and injections.

10. A method for enhancing the protective effect of antibodies, characterized in that, This includes administering N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof during and / or after vaccination of subjects and / or exposure to pathogens.

Images