Pharmaceutical grade saponins Rb₁ and Re were purchased from the Natural Medicine Chemistry Laboratory of Jilin University and stored at 4°C according to the drug instructions. Aging model mice (C57BL/6.18 months old) were purchased from Changchun Yishi Experimental Animal Technology Co., LTD., and raised in Jilin Provincial Health Inspection Center {approved by the Experimental Animal Management Committee of Jilin Provincial Center for Disease Control and Prevention [JCDC (2023) No. 1]}. Mouse T lymphocyte population (CD3, CD4, CD8) ELISA kit, Shanghai Varan Biological Company. Mouse interleukin INF ELISA kit, Mouse interleukin TNF ELISA kit, Mouse interleukin 27 (IL-27) ELISA kit, Mouse interleukin 17 (IL-17) ELISA kit, Mouse interleukin 10 (IL-10) ELISA kit, Mouse interleukin 6 (IL-6) ELISA kit, Mouse interleukin-4 (IL-4) ELISA kit, Mouse interleukin 2 (IL-2) ELISA kit, All purchased from Shanghai Enzyme Research Biology. Mouse immunoglobulin G (IgG) test kit, Mouse immunoglobulin M (IgM) test kit, Mouse immunoglobulin A (IgA) test kit, All purchased from BioLegend.

Forty healthy male C57BL/6 mice (18 months old, weight 28–32 g) were selected. The aging mouse models were put into the mouse house for 7 days, then they were divided into 4 groups on average, with 10 mice in each group. They were divided into Aged model group, Rb₁ administration group (Rb₁), Re administration group (Re) and Rb₁, Re administration group (Rb₁ + Re). The dosage was formulated according to the optimal concentration of the team’s previous trial, that is, the dosage of Rb₁ was 30 mg/kg, and the dosage of Re was 15 mg/kg. In addition, 10 6-month-old male healthy C57BL/6 mice (weight 20–25 g) were used as a control group (Col).

The mice were raised in Jilin Provincial Health Inspection Center in a constant temperature (21°C ± 2°C) and 50% humidity environment, and the light was set to 12 h dark light cycle, and they were free to drink and eat. The saponin monomer drug was dissolved in distilled water according to the experimental design, and the mice were given the drug through drinking water every day. Control and aging groups drank distilled water. Before feeding, each mouse was starved for 12 h and then numbered and weighed. After 8 weeks of continuous feeding, each mouse was starved for 12 h, and then weighed. The mice were killed 20 h after the last administration and the relevant indexes were detected.

The mouse model was anesthetized with 0.5% pentobarbital sodium, measured the body length, removed the eyeball and collected blood. The blood was left for 15 min, centrifuged at 3,000 r/min at 4°C for 10 min, and the upper serum was collected and placed in the EP tube at −80°C for freezing. At the same time, the heart, liver, spleen, lung, kidney and thymus tissues were quickly washed in pre-cooled normal saline, dried by filter paper and weighed, part of them were fixed in 4% paraformaldehyde solution to prepare paraffin sections, and part of them were placed in frozen tubes and put into liquid nitrogen, and frozen at −80°C to be measured. The remaining biological tissues after dissection were treated harmlessly.

Electronic balance is used to weigh the thymus and spleen to the exact milligram. The thymus index and spleen index are calculated according to the Eqs 1, 2. Thymus index  % = Thymus   weight   mg Weight   g × 1000 × 100 % (1) Spleen index  % = Spleen   weight   mg Weight   g × 1000 × 100 % (2)

The weight growth rate of each mouse was calculated by the Eq. 3: Weight growth rate  % = W 2 g − W 1 g W 1 g × 100 % (3)

Where W₁ was the weight before treatment and W₂ was the weight after 8 weeks of administration.

After the mice were anesthetized and killed, the thymus tissue was dissected, and cell suspension was prepared using a tissue fragmentation apparatus, and the cell concentration was adjusted to 5 × 10⁶/mL. The cells were washed with PBS for 3 times and cultured with RPMI 1640 medium. After overnight culture, the culture solution was removed, cleaned with PBS for 3 times, and then centrifuged at 1,500 r/min for 5 min. Then RNase was added and incubated in a metal bath at 37°C for 30 min. Then PBS was used to clean it three times, TUNEL dye solution and tritonX-1000 were added and stored at 4°C for 30 min away from light. Then, all cells were stained with H33342, and after adding H33342 dye solution, they were stored at 4°C for 30 min away from light. They were then observed under a fluorescence microscope. In the visual field, green is apoptotic cells and blue is all cells. The overall state of thymus was judged according to the proportion of apoptotic cells.

After blood coagulation, mouse serum was obtained by centrifugation at 3,000 r/min for 10 min and stored at −20°C until use. According to the kit instructions, the mouse immunoglobulin G (IgG) test kit was used to determine the total IgG concentration in mouse serum. The brief steps are as follows: (Szczuka et al., 2019): Take a sufficient number of enzyme-labeled coated plates, set standard product holes, sample holes to be measured and blank control holes, and add standard product 50 μL into the standard product holes; Add 10 μL of the sample to be measured in the sample hole, and then add 40 μL of the sample diluent (that is, the sample dilution is 5 times); Blank control hole is not added; (Liu et al., 2022); Incubate in a 37°C incubator for 30 min, Discard the liquid, pat dry on the absorbent paper, fill each hole with washing liquid, leave for 1 min, shake off the washing liquid, pat dry on the absorbent paper, repeat washing board 4 times; (Qiang et al., 2020); Add 50 μL of enzyme-labeled working liquid, and do not add the blank control hole; (Punja, 2011); Incubate in a 37°C incubator for 30 min, discard the liquid, pat dry on absorbent paper, fill each hole with washing liquid, leave for 1 min, shake off the washing liquid, pat dry on absorbent paper, repeat washing board 4 times; (Bell et al., 2022); Each well was first added with developing agent A 50 μL, then with developing agent B 50 μL, and developed for 15 min at 37°C away from light; (Vuksan et al., 2019); Remove the enzyme label plate, add 50 μL termination solution to each hole, terminate the reaction, adjust the zero with blank holes, and measure the light absorption value of each hole with 450 nm wavelength within 15 min after termination; (Liu et al., 2020); According to the concentration of the standard product and the corresponding OD value, the linear regression equation of the standard curve is calculated, and then the corresponding sample concentration is calculated on the regression equation according to the OD value of the sample. The final concentration is the actual measured concentration multiplied by dilution.

Serum was isolated according to the method described in 2.6. The content of interleukin was determined according to the kit instructions. The brief steps are as follows: (Szczuka et al., 2019): The number of slats required was calculated in advance, and 30 min before the experiment, the kit was taken out, restored to room temperature, 100 μL standard working liquid and test sample were added to each reaction hole, the standard product was repeated three times, the plate was sealed and incubated at 37°C for 90 min, the liquid was discarded and dried. 100 μL biotin-labeled interleukin antibody was added to each reaction hole, and the plates were sealed and incubated at 37°C for 60 min (Liu et al., 2022). Discard the liquid, shake dry, add 350 μL washing solution to each reaction hole, soak for 1–2 min, shake dry the washing solution, repeat 3 times; (Qiang et al., 2020); Each reaction hole was added with 100 μL HRP labeled streptavidin and incubated at 37°C for 30 min after plate sealing; (Punja, 2011); Add 300 μL washing liquid to each reaction hole, and shake the washing liquid dry at an interval of 30 s. Repeat 4 times; (Bell et al., 2022); Add 90 μL color developer (dark) to each reaction hole, and hide color from 37°C for about 15 min after sealing the plate; (Zhang YT. et al., 2023); Add 50 μL termination solution into each reaction hole, and immediately measure OD value with enzyme-labeled instrument at 450 nm wavelength (Within 5 min), calculate the average OD value of the standard and sample: the OD value of each standard and sample should be subtracted from the OD value of the blank hole. The content of interleukin in serum samples was calculated according to the standard curve.

The collected fresh blood was added with heparin sodium for anticoagulation, and then centrifuged at 1,700 rpm for 10 min to separate the serum. After discarding the supernatant, 200 μL normal saline was added to reinsert the cells. 1 mL of Ficoll lymphocyte separation fluid was added to the new centrifuge tube, and then the resuspended whole blood cells were slowly added so that the cells were on top of the lymphocyte separation fluid, while the fluid level was kept stratified. After centrifugation at low temperature (1,200 rpm, 30 min), the centrifugation end page was divided into 4 layers, in which peripheral blood mononuclear cells (PBMC) were in the second layer, and the PBMC layer was completely absorbed by pipetting gun to obtain relatively pure PBMC. 2 mL normal saline was added to the extracted PBMC, then centrifuged at 1,700 rpm for 10 min, and the supernatant was discarded to obtain PBMC precipitation. PBMC precipitation was re-suspended in RPMI l640 medium with 50 μL. Appropriate amount of cell suspension was added to the cell incubation board, leukocyte combination stimulant was added to each hole, and the mixture was blown and cultured in the incubator for 1 h, and then protein transport inhibitor containing Monensin was added to the incubation hole, and the culture was continued for 3 h. After taking out the cell incubation plate, blow and mix all the liquid in the hole, transfer it to the flow tube as much as possible, add 1 mL PBS to mix, centrifuge at 1,200 rpm for 5 min, and discard the supernatant. 2 μL cell surface cytokine fluorescently labeled antibody was added to the detection tube, and isotype control antibody was added to the care, and incubated at 4°C for 30 min in the dark. Then 1 mL PBS solution was added into the flow tube, blown and mixed well, centrifuged at 1,200 rpm for 5 min, and the supernatant was discarded. Each tube was fixed with 4% paraformaldehyde, and then cleaned with 1 mL PBS solution. During the cleaning process, blow and mix well, then centrifuge at 1,200 rpm for 5 min, and discard the supernatant. 200 μL of film breaking liquid was added into each flow tube and placed at 4°C away from light for 20 min. Next, add 1 mL washing TM buffer to wash, blow and mix well during washing, then centrifuge at 1,200 rpm for 5 min, discard the supernatant, and repeat cleaning twice. Finally, the cells were suspended with 100 μL TM buffer and added cytokine antibodies, incubated at 4°C for 30 min in the dark, then centrifuged at 1,200 rpm for 5 min, the supernatant was discarded, and 400 μL PB solution was used for re-suspension precipitation, and flow detection was performed in flow cytometry.

SPSS 19.0 was used for statistical analysis. The two sets of data were directly compared with the practical one-way ANOVA or T-test. Three or more sets of data were compared for analysis using ANOVA. Data are expressed as mean ± standard deviation of no less than 3 biological replicates. The significance level was significant with p < 0.05.

After 8 weeks of drug administration, the mice were dissected and their internal organs were observed. The results showed that saponin monomer administration had a positive effect on the aging model. Administration of Rb₁ and Re alone produced different effects than simultaneous administration of Rb₁ + Re. Saponins had no significant effects on the heart, liver, lung and kidney. But it has positive effects on the spleen, thymus and liver. After analysis, the spleen index and thymus index were significantly increased in the saponin administration group.

The results of spleen index showed that the spleen index of Col group was the highest and significantly higher than that of aging model group. Rb₁ and Re administration alone can significantly increase the spleen index in aging models, ranging from 7% to 12%. Combined administration of Rb₁ + Re showed a higher spleen index than administration of Rb₁ or Re alone, with Rb₁ + Re leading to a 25.5% increase in spleen index. However, the thymus index in all treatment groups was significantly lower than that in Col group (Figure 1A). Accordingly, the combination of Rb₁ and Re has a more significant effect on spleen index, which may be caused by the superimposed effect of Rb₁ and Re.

The thymus index of Col group was the highest and significantly higher than that of Aged group. After Rb₁ and Re monomer treatment, the thymus index was significantly higher than that of Aged group. After analysis, compared with Aged group, the thymus index in Rb₁ group was 18.67% higher, that in Re group was 12.12% higher, and that in Rb₁ + Re combined therapy was 23.40% higher. However, there was no significant difference between all groups. In addition, the thymus index in the Re group was significantly higher than that in the Aged group, but significantly lower than that in the Col group. However, the Rb₁ + Re group showed no significant difference from the Rb₁ administration group and Col group (Figure 1B). Therefore, both Rb₁ and Re can have a positive effect on thymus index, and Rb₁ has a greater effect than Re.

By analyzing the effects of Rb₁, Re and Rb₁ + Re on the weight growth rate of aging models, this study found that Rb₁ and Re alone can significantly increase the weight growth rate, but the growth rate is far less than that of the control group. However, combined administration of Rb₁ + Re significantly increased the growth rate of body weight compared with administration of Rb₁ or Re alone, by 5.96 times and 1.08 times (Figure 1C). In addition, the weight growth rate of all groups was significantly higher than that of Aged group.

The macroscopic condition of thymus can be evaluated by detecting apoptotic cells of thymus. The results showed that there were a large number of apoptotic cells in the aging mouse model, but fewer apoptotic cells in the Col group. However, after the intervention of Rb₁ or Re alone, the apoptotic cells of thymus decreased slightly, and the proportion of apoptotic cells decreased. It is worth noting that apoptotic cells in the thymus were significantly reduced after the combined intervention of Rb₁ and Re, which indicates that the combined intervention of Rb₁ and Re has a alleviating effect on thymocytes in the senescent state (Figure 2). This is in good agreement with the thymus index. However, after the joint intervention of Rb₁ and Re, the thymocytes still could not restore to the Col group state. This may be due to the fact that saponin monomers can remove oxidative free radicals in aging body, maintain REDOX balance, and promote the growth of thymus cells (Rais et al., 2017). In addition, the results of this study also suggest that Rb₁ and Re combined use has a stronger immune-enhancing effect than the single use.

It is well known that immune antibodies come from T cells and B cells. They recognize antigens produced by external microorganisms or toxins (Nelsen et al., 2014). Once the antibody finds the intruder, the cell quickly defends itself against the threat. There are five main immunoglobulin (Ig) antibodies in the body: IgA, IgG, IgM, IgD, and IgE (Cavazzana et al., 2017). These antibodies are all produced by B cells. Among them, IgA, IgM, and IgG are commonly used for diagnosis. When our body is infected by an organism, our immune system rapidly produces IgA and IgM and acts as the earliest antibodies to defend against or delay the infection (Milner, 2018). Within a few weeks, our immune system produces IgG, while IgA and IgM gradually break down. Thus, IgA and IgM work in the short term, while IgG is involved in the long term response. The results of this study showed that the levels of IgA, IgM, and IgG in aging model mice were significantly higher than those in control group. However, when saponin monomer was administered, IgA and IgG contents increased significantly, but IgM contents did not show significant changes. There was no significant difference between Rb₁ administration alone and Re administration alone. However, when Rb₁ and Re were administered simultaneously, IgG levels in aging mouse models were significantly increased (Figure 3). Different from the aging model mice, the levels of various immunoglobulins in the Col group were lower, which also indicated that the inflammation of the Col group mice was lower.

At the same time, the results of this study also showed that after 8 weeks of administration, American ginseng monomer Rb₁ and Re produced a long-term response to the aging model, so the level of IgA and IgM increased limited, while IgG increased significantly. In particular, IgA content increased significantly after administration, but the increase was small, while IgM did not change significantly, so the effect of American ginseng monomer Rb₁ and Re on the aging model is long-term. In addition, simultaneous administration of Rb₁ and Re produced significantly better effects than single administration.

The results of this study showed that the contents of IL-2, IL-4, and IL-10 in the Col group were significantly higher than those in the Aged group, but the corresponding levels of IL-2, IL-4, and IL-10 in the Aged group were significantly increased after Rb₁ and Re treatment. In particular, IL-4 levels in the aging model were not significantly different from those in the Col group after Rb₁ and Re administration. When Rb₁ and Re were administered at the same time, the content of IL-10 increased to a level close to Col. In addition, IL-6, IL-17, and IL-27 concentrations were found to be highest in the aging model, but lowest in Col. However, after administration of Rb₁, Re or Rb₁ + Re at the same time, its content decreased significantly, and the effect of simultaneous administration was the most obvious. At the same time, interferon TNF and INF were measured in this study, and the results showed that the aging model had higher levels of TNF and INF than the control group. After administration, INF levels returned to levels comparable to Col, but TNF was not affected. Simultaneous administration of Rb₁ and Re significantly reduced TNF levels in the aging model mice, but they were still much higher than those in the Col group (Figure 4).

The thymus and spleen are the main places of immune function and the natural barrier of defense against invasion. Thymus index and spleen index can reflect the immune level of the body to a certain extent (Leite-De-Moraes et al., 2001). Studies have shown that aging can inhibit the proliferation of immune cells and impair the development of immune organs, thus affecting the immune response ability of the body (Onodera et al., 2017). However, drug intervention can have positive effects on thymus index and spleen index. The results of this study show that the thymus index and spleen index of the Aged group model are significantly lower than those of the Col group, which indicates that the immune organs of the aged model have certain atrophy, which also indicates that their immunity is declining. After 8 weeks of Rb₁ and Re intervention, the thymus index and spleen index of the aging model were significantly higher than those of the Aged group, but lower than those of the Col group, indicating that saponin Rb₁ and Re had a positive effect on the immune organs of aging mice, but the thymus index and spleen index could not be restored to the level of Col group by administration of Rb₁ and Re. In addition, the combination of Rb₁ and Re produced a better effect than the administration alone. In addition, by measuring the body weight growth rate, it was found that the combined administration of Rb₁ + Re could effectively increase the body weight growth rate, which indicated that the body capacity of the aging model was well restored.