CN115607532A - Application of rhein in the preparation of drugs for improving the immune function of the aged body - Google Patents

Abstract

The invention belongs to the technical field of biological medical treatment, relates to a new application of Rhein (Rhein), and particularly relates to Rhein for improving the CD8 of an elderly organism ⁺ Novel use of T cell in virus-specific immune response function. The rhein comprises rhein, pharmaceutically acceptable salts or esters thereof, hydrates thereof or mixtures thereof, can be used as a medicament for clinically improving the antiviral infection specific immunity of the elderly, and provides help for improving the prognosis and outcome of patients; simultaneously strengthen the specificity CD8 of the old to the viral vaccine ⁺ The T cell immunoreactivity has wide clinical application value.

Description

Application of rhein in preparation of medicine for improving immune function of aged body

technical field

The invention belongs to the technical field of biomedicine. The invention relates to the application of rhein in the preparation of medicines for improving the immune function of aged organisms.

Background technique

The global population is aging rapidly. According to the World Health Organization, in almost every country, the population over the age of 60 is growing faster than any other population. It is estimated that by 2050, the number of people over the age of 60 will exceed 2 billion. Over the next 40 years, the proportion of people aged 60 and over is expected to rise to 22% of the total population. With age, the immune system response function of the elderly host declines, which we call "immunological aging", which includes functional changes in the innate immune system and acquired immune response. Innate immunity is relatively conservative, but healthy elderly people are still often accompanied by a high level of basic inflammatory state. Alterations in T cells in the adaptive immune system are particularly prominent. These changes included a global transition from naive T cells to memory phenotype T cells, resulting in a decrease in naive T cell numbers and an increase in memory phenotype T cells. Antigen-specific T cells themselves also change in nature with age.

Studies have shown that the specific immune response of CD8 ⁺ T cells in the elderly body is weakened, and there is evidence that the decrease in the diversity of CD8 ⁺ T cells is related to the decline in the immune response of the elderly host to new infections, including influenza viruses. The diversity of CD8 ⁺ T cells in aged mice (over 18 months old) is significantly reduced, the shift of dominant epitopes, the limitation of TCR diversity, and even the loss of some dominant epitopes of influenza, such as the MHC class of nuclear protein NP Absence of T-cell responses to the I-restricted epitope NP _366–374 /D ^b This loss of primary immune response to NP results in decreased resistance to viral infection

Overall, immune aging leads to the weakening of the body's acquired immune protection function, the reduction of vaccine response ability, the increase of basic inflammatory state, and the enhancement of immune inflammation pathology, so various infectious diseases (including influenza), autoimmune diseases and tumors occur. rate increased. Given that CD8 ⁺ T cells play a key role in clearing virus-infected cells in the specific immune response, improving the specific immune response function of CD8 ⁺ T cells in the elderly body will help the elderly host resist various viral infectious diseases.

Rhein (4, 5-dihydroxyanthraquinone) belongs to the monoanthracene nucleus 1, 8-dihydroxyanthraquinone derivative. Anti-inflammatory, anti-oxidation, anti-aging, anti-cancer and anti-pathogenic microorganisms and other pharmacological effects. Based on the current state of the art, the inventor of the present application intends to provide new medicinal uses of rhein, specifically related to the new use of rhein in improving the function of CD8 ⁺ T cells in the elderly body to specific immune responses to viruses .

Contents of the invention

The purpose of the present invention is to provide a new medicinal application of rhein based on the current state of the art, and specifically relate to the new application of rhein in improving the specific immune response function of CD8 ⁺ T cells in the elderly body to viruses.

The present invention has been proved by experiments that rhein has significant effects in regulating and improving the immune system of aging, especially T cell immune function. application, the rhein described in the present invention includes rhein or its pharmaceutically acceptable salt or ester, its hydrate or their mixture.

Specifically, the present invention provides an application of rhein in the preparation of a drug for improving the immune function of an aged body.

The rhein described in the present invention includes, its pharmaceutically acceptable salt or ester, its hydrate or their mixture.

In particular, the medicine of the present invention uses rhein as the only active ingredient.

The immune function of the present invention is CD8 ⁺ T cell anti-virus specific immune response function, especially the number of CD8 ⁺ T cells induced increases and the functional effect is enhanced.

The elderly body in the present invention refers to a human body older than 60 years old, or a rodent body older than 18 months old.

In one embodiment, the present invention collects from aged organisms [for example, spleens are taken from aged C57BL/6 mice over 18 months old, or peripheral blood mononuclear cells (PBMCs) are taken from elderly people over 60 years old] to sort CD8+ T cells;

In one embodiment, the above-mentioned sorted CD8 ⁺ T cells of the elderly body are placed in an in vitro cell culture environment, a certain concentration of rhein is added to the culture medium, and incubation and culture treatment is carried out for a certain period of time to obtain rhein treated in vitro CD8 ⁺ T cells of aged organisms (mouse or human);

In one embodiment, cell activity detection is performed on the above-mentioned aged body (mouse or human) CD8 ⁺ T cells treated with rhein in vitro to ensure that the activity is above 90%.

In one embodiment, the CD8 ⁺ T cells of the aged body (mice or human body) treated with rhein in vitro are injected into the old body (mice or human body) by intravenous injection after centrifugation and washing; The CD8 ⁺ T cells of aged organisms (mice or human) treated with rhein in vitro can be used as control cells for intravenous infusion.

In one embodiment, after virus infection or virus vaccination, the number of virus-specific CD8 ⁺ T cells and the secretion of cell effectors in different treatment groups are detected.

In one embodiment, the aged body (mice or human) is directly orally or intravenously infused with rhein; the aged body (mice or human) without oral or intravenous infusion of rhein is used as a control;

In one embodiment, after virus infection or virus vaccination, the number of virus-specific CD8 ⁺ T cells and the secretion of cell effectors in the above-mentioned different treatment groups are detected.

The present invention confirms that the specific immune response function of CD8 ⁺ T cells in the elderly body of the rhein drug treatment group is significantly enhanced through the above implementation. The experimental results strongly suggest that rhein or its pharmaceutically acceptable salt or ester, its hydrate or their mixture has the function of enhancing the anti-virus specific immune response of CD8 ⁺ T cells in the aged body.

The meanings of all technical and scientific terms used in this application are intended to be the same as commonly understood by those skilled in the art, including those technical changes or equivalent technical replacements obvious to those skilled in the art. As used herein, the terms "comprises," "comprising," "has," "containing," or other variations thereof herein are inclusive or open-ended and do not exclude other Unenumerated elements or method steps.

Description of drawings

Figure 1: Detection of cytotoxicity after rhein drug treatment of cells, wherein,

At different concentrations (0, 15, 30, 60, 120uM) the cytotoxicity of rhein treated cells for 24h, 36h and 48h was detected, and the cytotoxicity of 60uM rhein treated cells for 24h was lower than 10% (9.6%), Treatment of aged CD8 ⁺ T cells under this condition had little effect on cell viability.

表示，*P<0.05,**P<0.01,***P<0.001)注：设CD45.2⁺CD8⁺CD44^high T细胞群。Figure 2: Adoptive transfer of rhein-treated and untreated control CD8 ⁺ T cells from CD45.2 ⁺ C57BL/6 aged mice into CD45.1 ⁺ C57BL/6 mice, and infection of recipient CD45. 1 ⁺ C57BL/6 mice, on day 8 and day 32 after the first infection (i.e. 4 days after the second infection), compared the Rhein-treated and untreated mice transferred to different tissues in the recipient mice Proportion and number of influenza virus-specific tetramer-positive cells in aged CD45.2 ⁺ CD8 ⁺ T cells in the control, AD. Represents spleen (Spleen), mediastinal lymph node (MLN), peripheral blood (Blood), lung (Lung) Proportion of virus-specific CD8 ⁺ T cells produced by activated CD8 ⁺ T cells in C57BL/6 aged mice (CD45.2) treated with Rhein and without intervention ); the number of mice in each group was 4. results in

Indicates, *P<0.05, **P<0.01, ***P<0.001) Note: CD45.2 ⁺ CD8 ⁺ CD44 ^high T cell population is assumed.

Figure 3: After PR8 infection of recipient C57BL/6 mice (CD45.1) on day 8 and day 32 (i.e., 4 days after the second infection), aged cells were compared in spleen cells after Rhein intervention and untreated controls CD45.2 ⁺ CD8 ⁺ T cells secreted effector factors Granzyme A, Granzyme B, TNF-α, IFN-γ after stimulation and activation by influenza virus-specific peptides,

表示，*P<0.05,**P<0.01,***P<0.001)注：设CD45.2⁺CD8⁺CD44^high T细胞群。A. Ratio of activated CD8 ⁺ T cells secreting cell effectors Granzyme A, Granzyme B, TNF-α, IFN-γ in the spleen of C57BL/6 aged mice (CD45.2) treated with Rhein and non-intervention (A, Dot pattern), the proportion statistical diagram of cell effectors (B), the absolute number of cells secreting cell effectors statistical graph (C), the MFI value statistical graph of cell effectors (D), the number of mice in each experiment group is 4 , resulting in

Indicates, *P<0.05, **P<0.01, ***P<0.001) Note: CD45.2 ⁺ CD8 ⁺ CD44 ^high T cell population is assumed.

Figure 4: PR8 infection of recipient C57BL/6 mice (CD45.1) on days 8 and 32 (that is, 4 days after the second infection), comparing aged CD45 cells after Rhein intervention and untreated controls in mediastinal lymph nodes .2 ⁺ CD8 ⁺ T cells secreted effectors Granzyme A, Granzyme B, TNF-α, IFN-γ after stimulation and activation by influenza virus-specific polypeptides,

表示，*P<0.05,**P<0.01,***P<0.001)注：设CD45.2⁺CD8⁺CD44^high T细胞群。A. Ratio of activated CD8 ⁺ T cells secreting cell effectors Granzyme A, Granzyme B, TNF-α, IFN-γ in mediastinal lymph nodes of C57BL/6 aged mice (CD45.2) treated with Rhein (A , dot plot), the ratio statistics of cell effectors (B), the absolute number of cells secreting cell effectors (C), the MFI value of cell effectors (D), and the number of mice in each group was 4 Only. results in

Indicates, *P<0.05, **P<0.01, ***P<0.001) Note: CD45.2 ⁺ CD8 ⁺ CD44 ^high T cell population is assumed.

Figure 5: PR8 infection of recipient C57BL/6 mice (CD45.1) on days 8 and 32 (that is, 4 days after the second infection), comparing aged CD45 cells after Rhein intervention and untreated controls in peripheral blood .2 ⁺ CD8 ⁺ T cells secreted effectors Granzyme A, Granzyme B, TNF-α, IFN-γ after stimulation and activation by influenza virus-specific polypeptides,

表示，*P<0.05,**P<0.01,***P<0.001)注：设CD45.2⁺CD8⁺CD44^high T细胞群。A. Ratio of activated CD8 ⁺ T cells secreting cell effectors Granzyme A, Granzyme B, TNF-α, IFN-γ in the peripheral blood of C57BL/6 aged mice (CD45.2) treated with Rhein (A , dot plot), the ratio statistics of cell effectors (B), the absolute number of cells secreting cell effectors (C), the MFI value of cell effectors (D), and the number of mice in each group was 4 Only. results in

Indicates, *P<0.05, **P<0.01, ***P<0.001) Note: CD45.2 ⁺ CD8 ⁺ CD44 ^high T cell population is assumed.

Figure 6: PR8 infection of recipient C57BL/6 mice (CD45.1) on day 8 and day 32 (that is, 4 days after the second infection), comparison in lung tissue between aged mice treated with Rhein and untreated controls CD45.2 ⁺ CD8 ⁺ T cells secreted effector factors Granzyme A, Granzyme B, TNF-α, IFN-γ after stimulation and activation by influenza virus-specific peptides,

表示，*P<0.05,**P<0.01,***P<0.001)注：设CD45.2⁺CD8⁺CD44^high T细胞群。A. Ratio of activated CD8 ⁺ T cells secreting cell effectors Granzyme A, Granzyme B, TNF-α, IFN-γ in the lungs of C57BL/6 aged mice (CD45.2) treated with Rhein (A, dot pattern), the ratio statistics of cell effectors (B), the absolute number of cells secreting cell effectors (C), the MFI value statistics of cell effectors (D), (the number of mice in each group was 4 only. Results end with

Indicates, *P<0.05, **P<0.01, ***P<0.001) Note: CD45.2 ⁺ CD8 ⁺ CD44 ^high T cell population is assumed.

detailed description

Example 1 Preparation of C57BL/6 aged mouse (CD45.2 ⁺ ) spleen cells

Twelve female C57BL/6 mice (CD45.2 ⁺ ) aged 18-24 months in SPF grade were taken, and the mice were first killed by decapitation, fixed on the dissecting board, and sprayed with 75% disinfectant alcohol on the surface of the mice. Spleen was dissected with sterilized dissecting instruments.

(1) Take a 10cm cell culture dish, put in a 200-mesh filter screen of appropriate size, and then add 3ml of R10 culture medium to the dish. Place the spleen on a strainer in a Petri dish, 5 ml syringe for trituration

(2) After grinding, take a 15ml centrifuge tube and mark it on the tube wall. Use a 1ml pipette gun to transfer the cell suspension to a centrifuge tube. After the transfer, insert the centrifuge tube into an ice box to ensure cell viability.

(3) Centrifuge at 500g at 4°C for 5 minutes and discard the supernatant. Add 1ml 1× Cracking Red Solution, mix gently and place on ice for 5 minutes.

(4) Add 4ml of R10 culture medium and mix well to stop cracking. Centrifuge at 500 g at 4°C for 5 minutes and discard the supernatant. (5) Add 1ml R10 culture medium to resuspend the cells, use a 1ml pipette gun to absorb the cell suspension filter, and transfer to a new centrifuge tube.

(6) Spleen cell counting: absorb 10 ul of cell suspension and dilute the cells 200 times for counting.

Example 2 Sorting of CD8 ⁺ T cells in the spleen of C57BL/6 aged mice (CD45.2 ⁺ )

(1) Centrifuge the spleen cells obtained in the experimental technical scheme 1 at 500 g for 5 minutes, discard the supernatant, and resuspend the spleen cells in MojoSort ^TM Buffer (buffer is prepared well in advance).

(2) Filter the cells with a 70 μm cell strainer, centrifuge at 300 g for 5 min, and add an appropriate volume of MojoSort ^TM Buffer to make the final concentration 1×10 ⁸ /ml.

(3) Add 10ul Biotin-Antibody Cocktail to every 100ul (10 ⁷ cells), mix well, and incubate on ice for 15min.

(4) Add 10ul Streptavidin Nanobeads to each 10 ⁷ cells, mix well, and incubate on ice for 15min

(5) Add 2.5ml of MojoSort ^TM Buffer to the cells, and gently pipette to mix.

(6) Put the column into the magnetic separator, add 3mL of cell separation buffer to the column to wash the column.

(7) Add the cell suspension into the column, and collect the unlabeled target cells after 5 minutes.

(8) Transfer the cells to a 15ml centrifuge tube, centrifuge at 300g for 5min, discard the supernatant, resuspend in 1ml R10, and count the cells.

Example 3 Exploration of working conditions of rhein-treated cells

Firstly, rhein (storage concentration: 1000uM) was gradiently diluted with R1640 medium to 0, 15uM, 30uM, 60uM, and 120uM, and three replicate wells were set up for each gradient. At the same time, three times for treating cells (mouse splenocytes) were set: 24h, 36h, and 48h. The LDH-Cytox ^TM Assay Kit, a kit for detecting cytotoxicity, was used to detect the cytotoxicity of drugs at different concentrations and for different time periods. The conditions of use of the drug are determined with a cytotoxicity of less than 10%.

(1) Medium preparation: RPMI 1640+5%FBS+1% double antibody+2mmol/l l-glutamine+50μmol/lβ-mercaptoethanol

(2) Set up three 96-well plates, and mark them according to the time of treating cells respectively. Pipette 100ul of the cell suspension diluted with culture medium to the experimental wells of the 96-well plate (2x10 ⁶ /well). Set high control, low control, and background control at the same time, and set three duplicate holes for each control. Add 100ul of cell suspension to the high control well and low control well, and add 100ul of cell-free medium to the background control

(3) Add 100ul of drug-containing medium to each well of the experimental well, and add 100ul of drug-free medium to the control well.

(4) The 96-well plates corresponding to different times were cultured in a 5% CO ₂ incubator at 37°C for 24h, 36h, and 48h respectively.

(5) After adding 10ul Lysis Buffer to the high control well, add 10ul medium to the low control well and the background control well. Incubate in a 5% CO ₂ incubator at 37°C for 30 min.

(6) Centrifuge the 96-well plate for 5 minutes (500×g) to pellet the suspended cells.

(7) Pipette 100ul supernatant from each well to a new 96-well plate.

(8) After adding 100ul Working Solution to each well, incubate at room temperature in the dark for 15min.

(9) After adding 50ul Stop Solution to each well, immediately detect the absorbance at 490nm with a microplate reader.

(10) Calculation of cytotoxicity

A: absorbance of sample; B: absorbance of high control; C: absorbance of low control

Example 4 Treatment of rhein on CD8 ⁺ T cells in the spleen of C57BL/6 aged mice (CD45.2 ⁺ )

(1) Medium preparation: RPMI 1640+5%FBS+1% double antibody+2mmol/l l-glutamine+50μmol/lβ-mercaptoethanol

(2) The CD8 ⁺ T cells sorted by magnetic beads were resuspended in medium, and the cell concentration was adjusted to 2×10 ⁷ /ml.

(3) Pipette 100 μul of cell suspension into a 96-well plate (2×10 ⁶ /well), and plate 24 wells (12 wells for each of the experimental group and the control group).

(4) Add 100 μl of medium containing Rhein (final concentration 60 μM) to each well of the experimental group, and add 100 μl of medium without drugs in the control group, and gently blow and mix with a pipette gun.

(5) Place the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours.

(6) After the treatment, centrifuge at 500 g for 5 minutes, discard the medium, wash twice with PBS, resuspend in PBS, collect cells, and perform cell counting.

Example 5 Adoption of aged mouse CD8 ⁺ T cells (CD45.2 ⁺ ) to congenic C57BL/6 mice (CD45.1 ⁺ )

(1) Take 24 SPF grade female C57BL/6 mice aged 8-12 weeks, and divide them into drug experiment group and control group, with 12 mice in each group. NOTE: Three days before the start of the experiment we transferred the mice to a biosafety level 2 laboratory of the Department of Experimental Animals. The mice were grouped, divided into four cages per cage, and marked by piercing their ears.

(2) Use a 1.5ml insulin needle to draw the drug-treated cells and untreated control cells (cell volume: 2×10 ⁶ /120ul PBS) and inject them into CD45.1 ⁺ mice respectively by way of retro-orbital venous plexus injection.

(3) 24 hours after adoption, recipient mice were infected with 0.5LD ₅₀ PR8 influenza virus intranasally, and on the 28th day after infection, 3LD ₅₀ nasal drops combined with 7LD ₅₀ tail vein injection were used for the second infection.

Example 6 Comparison of CD8 ⁺ T cell response function analysis of aged mice treated with rhein and CD8 ⁺ T cells of aged mice in the control group

In this embodiment, the CD8 ⁺ T cells of the experimental group and the control group were detected by flow cytometry on the 8th day after infection and the 32nd day after infection (the 4th day after the second infection). The level of virus-specific CD8 ⁺ T cells (Tetramer ⁺ CD8 ⁺ T) induced by infection, and the cellular effectors secreted by the two groups of CD8 ⁺ T cells after stimulation with influenza-specific peptides were also detected, including Granzyme A, Granzyme B, IFN - Levels of gamma, TNF-alpha.

1. Surface Staining: Proportion of Virus-Specific CD8 ⁺ T Cells

(1) Cell plating: add 5×10 ⁵ -1×10 ⁶ cells to each well of a 96-well U-shaped plate, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

(2) Shake the cell pellet on a shaker, use a pipette gun to absorb 200 μL of pre-cooled PBS to wash the cells, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

(3) The flow staining system is 20 μL, and the staining scheme and antibody concentration are as follows:

(4) Shake the cell pellet on a shaker, add 20 μL of the prepared staining mix antibody to each well, and pipette slowly with a pipette gun to make the antibody fully contact with the cells.

(5) Take a piece of tinfoil paper of appropriate size to wrap the 96-well plate, avoid light, incubate at 4°C for 30 minutes, take it out in the middle and shake and mix once.

(6) After staining, add 150 μL of staining buffer to each well to wash the cells twice, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

(7) Finally, add 200 μL of pre-cooled staining buffer to each well to resuspend the cells, transfer them into flow tubes and mark them, and then test them on the machine.

2. Cytokine staining

(1) Cell plating: add 2×10 ⁶ cells per well

(2) Influenza dominant epitope peptide NP _366-374 peptide (ASNENMETM) and PA _224-233 peptide (SSLENFRAYV) stimulate different tissue cells. The working concentration is 2.5μg/mL. In addition, a Golgi blocker was added at 1 ul/ml, mixed evenly, and cultured in a cell culture incubator at 37° C. for 5 hours.

(3) After the incubation, centrifuge at 500g for 5 minutes at 4°C and discard the supernatant. Wash the cells once with pre-cooled PBS.

(3) Cell surface staining, the operation steps are the same as above

(4) Add 150ul of permeabilization solution to each well, gently blow and mix with a pipette gun, wrap the 96-well plate with tin foil, and keep it away from light for 15 minutes at room temperature.

(5) After the cells are perforated, centrifuge at 1000g for 8 minutes and discard the supernatant.

(6) Vortex to suspend the cell pellet, add 150ul 1×wash buffer to each well to wash the cells twice, remove residual permeation solution, centrifuge at 1000g for 8 minutes, discard the supernatant, and vortex to suspend the cells rise.

(7) Intracellular cytokine staining

(8) Add 20ul of staining mix to each well, mix gently, protect from light, and incubate at 4°C for 30 minutes. Vortex to mix every 10 minutes.

(9) Add 200ul staining buffer to each well and wash twice, centrifuge at 1000g at 4°C for 8 minutes.

(10) Shake the cells, add 200ul staining buffer to each well, transfer the resuspended cells to flow tubes, and mark them.

(11) Streaming on the machine, followed by Flowjo software to analyze the data.

Example 7 Detection of cytotoxicity after rhein drug treatment of cells

When the cells were treated with rhein for 36 hours and 48 hours, the damage of the drugs to the cells was greater than 10%. When the cells were treated at different concentrations for 24 hours, the cytotoxicity increased with the increase of the drug concentration, and when the cells were treated with 120uM rhein, the cytotoxicity exceeded 20%. When the cells were treated with a concentration of 60uM, the cytotoxicity was less than 10% (9.6%). Therefore, treatment of aged CD8 ⁺ T cells under this condition had less effect on cell viability (Fig. 1).

Compared with the control group, the levels of virus-specific CD8 ⁺ T cells induced by influenza infection were higher in aged CD8 ⁺ T cells in the rhein drug-treated group. On the 8th day and 32th day after the initial infection (that is, 4 days after the second infection), the proportion of virus-specific CD8 ⁺ T cells induced by CD8 ⁺ T cells in the drug treatment group in the spleen, mediastinal lymph nodes, and lung tissues, The number was higher than that of the control group. In peripheral blood, the ratio and number of virus-specific CD8 ⁺ T cells induced by CD8 ⁺ T cells in the drug treatment group were also higher than those in the non-intervention group on the 8th day after infection. Statistical difference, but the intervention group still has an increasing trend (Figure 2).

Compared with the control group, the levels of cellular effectors secreted by aged CD8 ⁺ T cells stimulated with influenza-specific peptides were higher in the drug-treated group.

In the spleen, the proportion, number and mean fluorescence intensity (MFI) of Granzyme A secreted by the activated CD8 ⁺ T cells in the drug treatment group were significantly increased, and the proportion, number and MFI of Granzyme B were significantly increased on the 8th day after infection, and were significantly increased on the 8th day after infection. There was a tendency to increase on day 32; the secretion of TNF-α and IFN-γ increased to varying degrees in the drug group (Figure 3). In the mediastinal lymph nodes, Granzyme A, TNF-α, and Granzyme B secreted by activated CD8 ⁺ T in the drug group were all significantly increased, and IFN-γ was significantly increased on the 8th day after infection, and there was a tendency to increase on the 32nd day. The absolute numbers and MFI values of the cytodynamic factors all increased to varying degrees (Fig. 4). The proportions of Granzyme A, TNF-α, and Granzyme B secreted by activated CD8 ⁺ T in peripheral blood were all significantly increased, and IFN-γ had a tendency to increase. Similarly, the absolute number of cells and MFI values of each cell efficacy factor also changed to different degrees. increased (Figure 5). In lung tissue, each cell effector had a tendency to increase to varying degrees (Fig. 6).

进行表示，使用统计作图软件GraphPadPrism7进行数据统计分析，两组数据统计比较采用非配对t检验统计分析。以P≤0.05为差异有统计学意义。All data are mean ± standard deviation

To express, use the statistical mapping software GraphPadPrism7 for statistical analysis of the data, statistical comparison of two groups of data using unpaired t-test statistical analysis. P≤0.05 was considered statistically significant.

In summary, after treating CD8 ⁺ T cells in aged mice with rhein, their specific immune response to influenza infection was improved. The main manifestations are as follows: 1) After rhein intervention, the number of virus-specific CD8 ⁺ T cells produced by CD8 ⁺ T cells in aged mice increased after influenza infection. 2) The levels of cellular effectors secreted by rhein-treated CD8 ⁺ T cells in aged mice stimulated by influenza-specific peptides increased.

Claims (6)

1. Application of rhein in preparing medicine for improving immunity of senile organism is provided.

2. Use according to claim 1, characterized in that: the rhein comprises rhein, pharmaceutically acceptable salt or ester thereof, a hydrate thereof or a mixture of the rhein and the salt or the ester.

3. Use according to claim 1 or 2, characterized in that: the rhein in the medicine is used as the only active component.

4. Use according to claim 3, characterized in that: the immune function is CD8 ⁺ T cell antiviral specific immune response function.

5. Use according to claim 4, characterized in that: the immune response function refers to CD8 ⁺ The number of T cell induction increases and the functional effect increases.

6. Use according to claim 1, characterized in that: an elderly organism refers to a human organism older than 60 years of age, or a rodent organism older than 18 months of age.

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