Use of hemoglobin-based oxygen carriers in the preparation of antiviral drugs

Abstract

The present application relates to the technical field of biological medicine, and in particular to application of hemoglobin oxygen carrier (HBOCs) in preparation of antiviral drugs, and provides a novel mechanism of action of HBOCs, and reveals that HBOCs change the distribution of immune cell subgroups in mouse bone marrow, up-regulate the expression level of antiviral genes, thereby enhancing the resistance of the body to viruses, and provides a new idea for development and preparation of antiviral drugs. Isg15 ​

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of hemoglobin oxygen carriers in the preparation of antiviral drugs. Background Technology

[0002] Antiviral immunomodulators enhance the body's antiviral capacity by activating the host's immune response (such as enhancing the function of T cells and dendritic cells) or inhibiting excessive inflammatory responses (such as regulating cytokine storms). Immune cells proliferate rapidly during antiviral activity, often in an oxygen-deficient microenvironment.

[0003] Oxygen is essential for maintaining physiological processes such as cell growth, differentiation, metabolism, and tissue homeostasis. Red blood cells, as natural oxygen carriers, bind oxygen in the lungs and transport it throughout the body. Hemoglobin (Hb) is the primary oxygen carrier in red blood cells, facilitating oxygen delivery through its well-known oxygen-carrying function. Many adverse factors, such as trauma, blood loss, and pernicious anemia, can lead to a decrease in red blood cell and hemoglobin levels in the blood. Timely transfusion of red blood cells to restore blood volume and ensure an adequate oxygen supply is crucial for saving lives. Hemoglobin oxygen carriers (HBOCs) are a class of blood substitutes obtained from natural hemoglobin through polymerization, cross-linking, and polymer modification. Due to their excellent oxygen binding and release properties, they are widely used clinically for patients with tissue ischemia or hypoxia. Currently, glutaraldehyde-crosslinked bovine hemoglobin (HBOC-201) has been approved for the treatment of acute pernicious anemia in South Africa in 2001 and in Russia in 2010.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of hemoglobin oxygen carriers in the preparation of antiviral drugs. This invention mainly utilizes scRNA-seq technology to explore a novel mechanism of action of HBOCs, providing an effective strategy for preparing drugs that enhance the body's resistance to viruses. It reveals that HBOCs significantly enhance the antiviral capacity of immune cells in mouse bone marrow, providing a novel approach for the development and preparation of new antiviral drugs.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: One aspect of the present invention relates to the use of hemoglobin oxygen carriers in the preparation of antiviral drugs.

[0007] This invention utilizes single-cell RNA sequencing (scRNA-seq) technology to reveal that HBOCs alter the distribution of immune cell subsets in mouse bone marrow and upregulate antiviral genes. Isg15This invention enhances the body's resistance to viruses by increasing the expression level of these substances. It provides a new approach for the development and preparation of antiviral drugs.

[0008] Hemoglobin oxygen carriers are red blood cell substitutes with oxygen-carrying capacity, prepared from hemoglobin through chemical modifications such as cross-linking, polymerization, and coupling, or by encapsulating it in a synthetic membrane. This invention does not specifically limit the type or preparation method of the hemoglobin oxygen carrier; existing hemoglobin oxygen carriers in the art can be used to implement the technical solutions of this invention. In some specific embodiments, the hemoglobin oxygen carrier includes, but is not limited to, glutaraldehyde-crosslinked bovine hemoglobin.

[0009] Furthermore, the hemoglobin oxygen carrier exerts its antiviral effect by activating signaling pathways related to immune cell subsets and the body's viral defense and immune response.

[0010] Furthermore, mouse bone marrow cells were grouped using classic immune cell markers to analyze the distribution changes of immune cell subsets.

[0011] Furthermore, differential gene expression (DEGs) analysis and signaling pathway enrichment analysis were used to screen for differentially expressed genes among mice in different groups and to identify the important signaling pathways enriched in these genes.

[0012] Furthermore, the immune cell subsets include, but are not limited to, at least one of neutrophils, dendritic cells, monocytes, T cells, and B cells.

[0013] Furthermore, the hemoglobin oxygen carrier regulates genes Isg15 The expression plays an antiviral role.

[0014] Another aspect of the invention relates to a composition comprising a hemoglobin oxygen carrier and excipients.

[0015] The composition obtained by combining hemoglobin oxygen carriers and excipients required for antiviral drugs can be used to enhance the body's antiviral capabilities.

[0016] This invention does not specifically limit the type of excipients, but the excipients include, but are not limited to, conventional excipients in the field of antiviral drugs.

[0017] Furthermore, the composition can also be used in combination with other conventional antiviral drugs in the art to synergistically exert antiviral capabilities.

[0018] Another aspect of the invention relates to the use of the said composition in the preparation of antiviral drugs.

[0019] This invention verifies through relevant experiments that hemoglobin oxygen carriers can enhance the body's antiviral ability and reveals the antiviral mechanism of hemoglobin oxygen carriers. Compositions using hemoglobin oxygen carriers as the main active ingredient have good antiviral effects.

[0020] Another aspect of the present invention relates to a method for analyzing the antiviral mechanism of hemoglobin oxygen carriers, comprising the following steps: (a) Hemoglobin oxygen carriers were injected intravenously into mice, and the distribution changes of immune cell subsets in the bone marrow of mice were analyzed, and significantly differentially expressed genes and signal enrichment pathways were screened. (b) Analyze the expression levels of the significantly differentially expressed genes in different viral models to verify the role of the significantly differentially expressed genes; (c) Pathological analysis and RT-qPCR experiments were performed on the spleen tissue of mice injected with hemoglobin oxygen carrier to verify the application potential of hemoglobin oxygen carrier in the preparation of antiviral drugs.

[0021] The method described above for analyzing the antiviral mechanism of hemoglobin oxygen carriers has the advantages of being simple, easy to operate, and not relying on complex processes. It reveals and verifies the antiviral mechanism of hemoglobin oxygen carriers from multiple perspectives, providing a solid theoretical basis for the antiviral activity of hemoglobin oxygen carriers.

[0022] Furthermore, the distribution changes of immune cell subsets in mouse bone marrow were analyzed using single-cell RNA sequencing analysis (scRNA-seq).

[0023] scRNA-seq is a technique for studying gene expression differences at the single-cell level. This technique reveals the unique gene expression patterns of each cell by sequencing RNA transcripts in a single cell, providing a powerful tool for studying cellular heterogeneity. Its basic principle is to convert mRNA transcripts in a single cell into cDNA, and then perform high-throughput sequencing.

[0024] This invention does not specifically limit the type of virus model; any virus model with typical characteristics in the art can be used to implement the technical solutions of this invention. In some specific embodiments, the virus model includes, but is not limited to, at least one of: Severe Acute Respiratory Syndrome Coronavirus 2, Sendai virus, hepatitis C virus, or hepatitis B virus.

[0025] This invention analyzes the antiviral mechanism of hemoglobin oxygen carriers from three aspects, as detailed below: In the first aspect, this invention performs scRNA-seq analysis on mouse bone marrow cells to analyze the distribution changes of immune cell subsets in the bone marrow of mice injected with hemoglobin oxygen carriers and control mice, and screens for significantly differentially expressed genes and signal enrichment pathways to reveal the antiviral mechanism of HBOCs. Bone marrow cells from mice in different treatment groups were grouped and subgrouped based on classic immune cell markers. The changes in the composition ratio of important immune cell groups (such as neutrophils, dendritic cells, monocytes, T cells, and B cells) were analyzed. The results showed that the number of neutrophils and monocytes decreased, while the number of dendritic cells (DCs), B cells, and T cells increased, indicating the effect of HBOCs on the immune function of mice. DEGs and GO analyses of these immune cell populations revealed that HBOCs significantly activated signaling pathways in the immune cell's response to the virus. Specifically, the expression level of Isg15 mRNA was significantly upregulated in all five immune cell types, suggesting... Isg15 Genes play a role in antiviral responses; Secondly, this invention analyzes bulk RNA-seq data from the public GEO database to detect the expression level of Isg15 mRNA in antiviral responses of different models, thereby verifying... Isg15 The crucial role of genes in the body's defense against viruses; Analysis of bulk RNA-seq data from mouse models or humans infected with different viruses, such as SARS-CoV-2, Sendai virus (SeV), hepatitis C virus (HCV), and hepatitis B virus (HBV), showed that the expression level of Isg15 mRNA was significantly increased in the virus-infected group compared with the normal group, indicating that... Isg15 It plays an important role in the body's defense against viruses; Thirdly, this invention verifies the potential of HBOCs as antiviral drugs through hematoxylin-eosin (H&E) staining of tissue sections and RT-qPCR experiments. Pathological analysis of H&E-stained sections of spleen tissue from mice in different treatment groups revealed extensive neutrophil infiltration and blurred boundaries between red and white pulp in the HBOC group, suggesting the antiviral effect of the spleen tissue. RT-qPCR analysis of Isg15 mRNA expression in mouse spleen tissue showed significantly elevated Isg15 mRNA levels compared to the normal group, indicating that HBOCs enhance the antiviral activity of the spleen. Isg15 The expression of this substance enhances the body's antiviral ability, suggesting its potential for development in the preparation of antiviral drugs.

[0026] Isg15 mRNA encodes... Isg15 Messenger RNA of proteins. Isg15 It is a ubiquitin-like protein induced by interferon and is widely found in various organisms such as mammals, birds, and insects. It has a variety of biological functions, including antiviral, antitumor, immune regulation, and participation in autophagy and metabolic processes.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses single-cell RNA sequencing technology to reveal that HBOCs alter the distribution of immune cell subsets in mouse bone marrow and upregulate antiviral genes. Isg15 The expression level of this substance, thereby enhancing the body's ability to resist viruses, provides a new research idea for the development and preparation of antiviral drugs.

[0028] This invention provides a composition obtained by combining hemoglobin oxygen carriers and excipients required for antiviral drugs. This composition is used to enhance the body's antiviral capabilities. It can also be used in combination with other conventional antiviral drugs in the field to synergistically enhance antiviral capabilities and broaden the dosage regimens for antiviral drugs.

[0029] This invention verifies through relevant experiments that hemoglobin oxygen carriers can enhance the body's antiviral ability and reveals the antiviral mechanism of hemoglobin oxygen carriers. Compositions using hemoglobin oxygen carriers as the main active ingredient have good antiviral effects.

[0030] The method for analyzing the antiviral mechanism of hemoglobin oxygen carriers provided by this invention has the advantages of being simple, easy to operate, and not relying on complex processes. It reveals and verifies the antiviral mechanism of hemoglobin oxygen carriers from multiple perspectives, providing a solid theoretical basis for the antiviral effect of hemoglobin oxygen carriers. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1This diagram illustrates the antiviral mechanism of HBOCs. (A) UMAP of bone marrow cell populations in the control / HBOC group mice (n=42414); (B) Bubble chart showing characteristic markers of different bone marrow cell populations; (C) Composition ratio of different cell populations; (D) GO analysis of significantly upregulated genes among the five immune cell groups, showing 10 representative signal enrichment pathways. The p-value was corrected using the Benjamini-Hochberg method, and the corrected p-value was <0.05; (E) Venn diagram showing the genes involved in viral defense responses in the five immune cell groups. Isg15 The expression of [a substance] was significantly upregulated in all five immune cell types.

[0033] Figure 2 The enrichment of oxidation-related pathways in gene expression in HBOC compared to the control group (A) and the enrichment score of the antiviral gene set (B).

[0034] Figure 3 for Isg15 The role of Isg15 in the body's antiviral response is validated in the diagram. (AE) shows a box plot (n=3) of Isg15 mRNA expression levels in different viral infection models from the GEO database. (A) shows bulk RNA-seq data from lung tissue of normal or SARS-CoV-2 infected mice (GSE264189). (B) shows bulk RNA-seq data from lung tissue of normal or SeV infected mice (GSE267631). (C) shows bulk RNA-seq data from liver tissue of normal or HCV infected mice (GSE200811). (D) shows bulk RNA-seq data from liver tissue of normal or HBV infected mice (GSE163285). (E) shows bulk RNA-seq data from liver tissue of healthy or SARS-CoV-2 infected patients (GSE206635). Statistical differences were analyzed using the two-tail Student's t-test. p<0.05, p<0.001, p<0.0001.

[0035] Figure 4This is a diagram validating the antiviral mechanism of HBOCs. (A) is an H&E staining image of spleen tissue from control / HBOC group mice (left scale bar is 50μm, right scale bar is 5μm). White arrows indicate the boundary between red and white pulp, and black arrows indicate neutrophils. (B) is an expression diagram of Isg15 mRNA in mouse spleen tissue. The error bar represents the Mean ± SD (n=3) of three replicate experiments. Statistical differences were analyzed using a two-tail Student's t-test. p<0.05.

[0036] Figure 5 For the adeno-associated virus (AAV) titer detection experiment, the uptake of AAV by macrophages treated with HBOC was analyzed. Data are expressed as mean ± standard deviation, and statistical analysis was performed using a two-tailed Student's t test.

[0037] Figure 6 Flowcharts and survival curves for HBOC treatment of C57BL / 6J mice infected with SARS-CoV-2 (A) and HSV-1 (B). Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0039] Aofide is a product developed by Runfang (Beijing) Biomedical Research Institute Co., Ltd., containing 6.0~7.0 g / dL bovine polymerized hemoglobin. The solution is a modified lactated Ringer's solution containing 100 g / dL water for injection, 113 mmol / L sodium chloride, 4 mmol / L potassium chloride, 1.4 mmol / L calcium chloride dihydrate, 10 mmol / L sodium hydroxide, 27 mmol / L sodium lactate, and 200 mg / dL N-acetyl-L-cysteine.

[0040] Example 1: Investigation into the antiviral mechanism of HBOCs

[0041] This study performed scRNA-seq analysis on bone marrow cells from control and HBOC groups to investigate the distribution changes of important immune cell populations in mouse bone marrow, and screened for significantly differentially expressed genes and signaling pathways between the groups, revealing a novel antiviral mechanism of HBOCs. Related data can be found in [link to relevant data]. Figure 1 .

[0042] The scRNA-seq analysis experiment was performed according to the following steps: (1) Sample processing: Healthy and suitable male C57BL / 6J mice were randomly divided into two groups (n=3): control group and HBOC group. Mice in the HBOC group were intravenously injected with Ofrode (600mg / kg) once a day for 3 consecutive days; mice in the control group were given an equal volume of PBS in the same way. On the fourth day, the femur and tibia of the mice were taken for subsequent bone marrow cell isolation, and the spleen tissue was used for subsequent analysis.

[0043] (2) Bone marrow cell isolation: Under sterile conditions, the femur and tibia, with their ends trimmed, were removed, and the surrounding muscle and fibrous tissue were removed. The bone marrow cells were thoroughly washed with PBS. The cell suspension was filtered through a cell filter and centrifuged. The supernatant was discarded, and the cell clumps were washed twice with PBS and centrifuged. After removing red blood cells with red blood cell lysis buffer, the cells were resuspended in PBS containing 0.04% BSA, and the viability of the single-cell suspension was assessed using trypan blue staining.

[0044] (3) scRNA-seq: Mouse bone marrow cell scRNA-seq was performed on the 10×Genomics Chromium Platform, with all experimental steps following the manufacturer's instructions. Single-cell suspensions were encapsulated into single-cell gel beads (GEMs) using the Chromium Next GEM Chip G Single Cell Kit (10×Genomics) on the Chromium Controller. Barcode cDNA was synthesized on a C1000 Touch Thermal cycler (Bio-Rad) and amplified using the Amplification Master Mix Kit (10×Genomics). The final library was constructed using the Chromium Next GEMSingle Cell 3' Reagent Kit v3.1 (10×Genomics) and quantified using an Agilent BioAnalyzer 2100. Sequencing was performed on an Illumina NovaSeq X Plus, with paired-end reads of 150 bp.

[0045] (4) Data preprocessing: Cell Ranger software was used to convert the raw base call files (BCL) of the sequencing into FASTQ files and compare them with the mouse reference genome (mm10 / GRCm38) to generate a count matrix for each sample.

[0046] (5) Data Analysis: R software was used to normalize the matrix, screen for highly variable genes, perform principal component analysis (PCA), remove batch processing, and perform unbiased clustering to obtain two-dimensional visualization maps (UMAPs) of different cell subpopulations. Cell subpopulations were annotated based on classic immune cell markers. Significantly differentially expressed genes among five important immune cell groups (neutrophils, dendritic cells, monocytes, T cells, and B cells) were analyzed, and GO analysis was performed on these genes. Signaling pathways with p-values ​​less than 0.05 after adjustment using the Benjamini-Hochberg method were selected. Genes co-regulated by HBOCs in relevant signaling pathways were identified using the JVEEEN tool.

[0047] In this example, scRNA-seq was performed on bone marrow cells from C57BL / 6J mice under different treatments, and 10 cell subsets were identified by unbiased clustering based on previously reported characteristic markers of bone marrow cells: neutrophils, B cells, monocytes, T cells, dendritic cells, erythrocytes, macrophages, hematopoietic stem cells, mast cells, and mesenchymal stem cells (MSCs). Figure 1 AB). Compared with the control group, the HBOC group had the largest proportion of neutrophils among all immune cells, followed by B cells (AB). Figure 1 C). Notably, in the HBOC group, the number of B cells, T cells, and dendritic cells increased, while the number of neutrophils and monocytes decreased (C). Figure 1 (A, C). These results indicate that HBOCs have a certain impact on the composition and functional status of various immune cells in mouse bone marrow.

[0048] This case study focuses on neutrophils, dendritic cells, monocytes, T cells, and B cells, and performs GO analysis on DEGs among these five immune cell subsets. The analysis revealed that signaling pathways related to viral defense and immune responses were significantly activated. Figure 1 (D) This suggests that HBOCs may enhance the antiviral activity of these immune cells.

[0049] Furthermore, among the genes related to viral resistance, Isg15 The gene was found to be upregulated by HBOCs in all five types of immune cells. Figure 1 E), indicating that the antiviral mechanism of HBOCs is related to Isg15 The correlation between them. Therefore, this example uses scRNA-seq technology to reveal a novel antiviral mechanism of HBOCs.

[0050] Example 2: HBOC enhances the body's oxidation response and promotes antiviral response.

[0051] This example further analyzes processed mouse bone marrow single-cell sequencing data to determine the different RNA expression patterns in the control and HBOC groups, and explores the antiviral response of tissue cells after increased oxygen content. Related analysis results are shown below. Figure 2 .

[0052] (1) GO enrichment analysis: To identify biological processes and signaling pathways associated with the two groups of differentially expressed genes, GO analysis was performed using the clusterProfiler package (version 4.12.0) in R. Descriptive terms with p-values ​​less than 0.05, adjusted according to the Benjamini-Hochberg method, were considered statistically significant.

[0053] (2) Enrichment analysis of the antiviral gene set: Gene expression data of the control group and HBOC group were normalized, and genes were ranked according to the fold change in expression. An antiviral gene set was selected from the MSigDB database, and the enrichment score and significance of the gene set were calculated using GSEA software. Statistical significance was assessed through tests, and box plots and heatmaps were plotted to display the results. Figure 2 B.

[0054] In this example, HBOCs-treated mouse bone marrow immune cells showed a stronger oxidative stress response and enrichment of antiviral genes, indicating that HBOCs provide a local hyperoxygen environment for tissues and enhance the body's antiviral function.

[0055] Example 3 Gene Isg15 Verification of its role in the body's antiviral response

[0056] This example analyzes multiple relevant bulk RNA-seq data from the GEO database to detect the expression level of Isg15 mRNA in different viral infection models and validate the gene. Isg15 Its crucial role in the body's antiviral response. (See relevant data...) Figure 3 .

[0057] Bulk RNA-seq data analysis was performed using the following steps: (1) Data Acquisition: Bulk RNA-seq data of mouse or human tissues infected with different viruses were obtained from the GEO database, and the relevant counting matrices were downloaded. These included lung tissue data of normal or infected SARS-CoV-2 mice (GSE264189), lung tissue data of normal or infected SeV mice (GSE267631), liver tissue data of normal and infected HCV mice (GSE200811), liver tissue data of normal or infected HBV mice (GSE163285), and data of healthy or infected SARS-CoV-2 patients (GSE206635).

[0058] (2) Data analysis: R software was used to normalize and transform the matrix, and the expression of Isg15 mRNA in the normal group / virus-infected group was visualized. The two-tail Student's t-test was used to test the statistical differences, and the p value should be less than 0.05.

[0059] This example selects bulk RNA-seq data from mouse or human models infected with different viruses from a public database and analyzes the expression level of Isg15 mRNA. Figure 3 The images show the expression levels of Isg15 mRNA in these viral infection models. The results indicate that, compared to the normal group, the expression levels of Isg15 mRNA were significantly increased in the viral infection groups, suggesting gene... Isg15 It plays a role in the body's response to viruses. Therefore, this example verifies... Isg15 Its key role in antiviral responses, suggesting that HBOCs regulate... Isg15 The mechanism by which it exerts its antiviral effect is expressed.

[0060] Example 4: Verification of the antiviral mechanism of HBOCs

[0061] This study investigated the pathological changes in spleen tissue and the difference in Isg15 mRNA expression levels in mice from the control group and HBOC group using H&E staining and RT-qPCR experiments, thus verifying the antiviral mechanism of HBOCs. Relevant data can be found in... Figure 4 .

[0062] I. The HE staining experiment shall be performed according to the following steps: Mouse spleen tissue was fixed in 10% neutral formalin buffer, dehydrated, and embedded in paraffin blocks. The paraffin blocks were uniformly cut into 5 μm thick sections, dewaxed, rehydrated, and stained with hematoxylin and eosin, respectively. The sections were observed under a microscope, and histological images were obtained for subsequent pathological analysis.

[0063] II. The RT-qPCR experiment shall be performed according to the following steps: (1) RNA extraction: After homogenizing the tissue by sonication, the tissue was lysed with TRNzol (Invitrogen) reagent and the supernatant was collected by centrifugation. The RNA precipitate was collected using chloroform / isopropanol, washed twice with 75% ethanol, and centrifuged. The ethanol was discarded, and the extracted RNA was dissolved in RNase-free ddH2O.

[0064] (2) Reverse transcription and RT-qPCR detection: Genomic DNA removal and reverse transcription were performed using the PrimeScript™ RT reagent Kit (Takara Bio) according to the manufacturer's instructions to synthesize cDNA. The relative RNA expression level of the samples was detected using Universal BlueqPCR SYBR Green Master Mix (Yeasen). The primer sequences are as follows: Isg15 forward 5'-GGAACGAAAGGGGCCACAGCA-3' (SEQ ID No.1), reverse 5'-CCTCCATGGGCCTTCCCTCGA-3' (SEQ ID No.2); GAPDH forward: 5'-CTCAGGAGAGTGTTTCCTCGTC-3' (SEQ ID No.3), reverse 5'-ATGGGCTTCCCGTTGATGAC-3' (SEQ ID No.4). The experiment was repeated three times, with GAPDH as the quantitative internal control, and the mRNA expression level was calculated using the -ΔΔCt standard method.

[0065] In this case, the spleen tissue of mice in the control group / HBOC group was subjected to HE staining for pathological analysis. The results showed that compared with the normal group, the spleen of mice in the HBOC group had a large number of neutrophil infiltrations and blurred boundaries between the red pulp and white pulp, suggesting that HBOCs enhance the antiviral effect of the mouse spleen. Figure 4 A).

[0066] This study used RT-qPCR to detect the expression level of Isg15 mRNA in the spleen of mice. The results showed that the Isg15 mRNA level in the HBOC group was significantly higher than that in the normal group. Figure 4 B), indicating that HBOCs can be regulated... Isg15 The expression of this enhances the body's antiviral ability.

[0067] Example 5: Investigation into the antiviral effects of HBOCs

[0068] This study analyzed the antiviral activity of bone marrow cells from control and HBOC groups at both the cellular and mouse levels to explore the antiviral effects of HBOCs at both the cellular and animal levels, revealing a novel antiviral mechanism of HBOCs. Relevant data can be found in [link to relevant data]. Figure 5 and Figure 6 .

[0069] The cell antiviral experiment was performed according to the following steps: RAW246.7 macrophages were cultured in vitro for 24 hours, and HBOC (300 mg / L) was added as a preservative. The control group was cultured in the same manner with an equal volume of PBS for 24 hours, and then infected with adeno-associated virus (AAV). Forty-eight hours after infection, the culture supernatant was concentrated, and the viral titer was determined according to the specified ratio. Figure 5 ).

[0070] The antiviral experiment in mice was conducted according to the following steps: Healthy, 6-week-old male C57BL / 6J mice were randomly divided into two groups (n=10): a control group and an HBOC group. Mice in the HBOC group (yellow line) received intravenous injections of Eppendorf (600 mg / kg) once daily for 2 consecutive days; mice in the control group (black line) received an equal volume of PBS using the same method. Initially, each mouse received 1250 PFU of SARS-CoV-2 virus via oral-nasal administration, and each mouse received 1 × 10⁻⁶ PFU of HSV-1 virus. 6 Each mouse was observed daily for a total of fourteen days, and the survival curve was recorded and statistically analyzed. Figure 6 ).

[0071] This example demonstrates the antiviral effects of HBOCs at both the cellular and animal levels, increasing the uptake of viruses by macrophages and prolonging the survival period of mice after viral infection.

[0072] In summary, this example demonstrates that HBOCs enhance the antiviral effect of mouse spleen, suggesting that HBOCs have certain prospects and development potential in the preparation of novel antiviral drugs.

[0073] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. Application of hemoglobin oxygen carriers in the preparation of antiviral drugs.

2. The application according to claim 1, characterized in that, The hemoglobin oxygen carrier includes: glutaraldehyde cross-linked bovine hemoglobin.

3. The application according to claim 1, characterized in that, The hemoglobin oxygen carrier exerts its antiviral effect by activating signaling pathways related to immune cell subsets and the body's viral defense and immune response.

4. The application according to claim 3, characterized in that, The immune cell subsets include at least one of neutrophils, dendritic cells, monocytes, T cells, and B cells.

5. The application according to claim 1, characterized in that, The hemoglobin oxygen carrier exerts its antiviral effect by regulating the expression of the Isg15 gene.

6. A composition, characterized in that, Includes hemoglobin oxygen carriers and excipients.

7. The use of the composition according to claim 6 in the preparation of an antiviral drug.

8. A method for analyzing the antiviral mechanism of hemoglobin oxygen carriers, characterized in that, Includes the following steps: (a) Hemoglobin oxygen carriers were injected intravenously into mice, and the distribution changes of immune cell subsets in the bone marrow of mice were analyzed, and significantly differentially expressed genes and signal enrichment pathways were screened. (b) Analyze the expression levels of the significantly differentially expressed genes in different viral models to verify the role of the significantly differentially expressed genes; (c) Pathological analysis and RT-qPCR experiments were performed on the spleen tissue of mice injected with hemoglobin oxygen carrier to verify the application potential of hemoglobin oxygen carrier in the preparation of antiviral drugs.

9. The method for analyzing the antiviral mechanism of hemoglobin oxygen carriers according to claim 8, characterized in that, The distribution changes of immune cell subsets in mouse bone marrow were analyzed using single-cell RNA sequencing.

10. The method for analyzing the antiviral mechanism of hemoglobin oxygen carriers according to claim 8, characterized in that, The virus models include at least one of: Severe Acute Respiratory Syndrome Coronavirus 2, Sendai virus, hepatitis C virus, or hepatitis B virus.

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