Immune modulatory effects of food supplement products Zeng Jian Heath Tonic and Zhen Yuan Capsule in elderly mice

ABSTRACT Immunosenescence is the degeneration of immunological organs and cells due to aging. Some plants and fungi may have anti-aging benefits through regulating immunosenescence. In this study, mice were fed with two dietary supplement products: Zeng Jian Health Tonic (ZJ) contains extracts from Lentinula edodes, Poria cocos, Tremella fuciformis, cassia seed, Flammulina velutipes, Fructus lycii, chrysanthemum, and mulberry. Zhen Yuan Capsule (ZY) contains extracts from Ganoderma lucidum, Semen cuscutae, Poria cocos, Schisandra chinensis, Codonopsis pilosula, and Atractylodes macrocephala. In addition, the immune organ index, immune cell functions, inflammatory cytokines, and antioxidant enzyme activities were evaluated. The combination of ZJ and ZY could restore the function of lymphocytes and NK cells; decrease the level of pro-inflammatory cytokines including IL-1α, IL-1β, and GM-CSF; increase the level of anti-inflammatory cytokine IL-5; and enhance superoxide dismutase and glutathione peroxidase activities. Therefore, combining ZJ with ZY has anti-aging effects on elderly mice.


Introduction
The rate of global population aging is increasing as medical technology advances.The population aged 65 and older is predicted to rise from 10% in 2022 to 16% in 2050, roughly equaling the population under the age of 12 (United Nations Department of Economic and Social Affairs, Population Division, 2022).Therefore, aging will continue to be an economic and social burden.Throughout the course of aging, the immune system undergoes continual remodeling in response to internal and external stresses.This process is known as immunosenescence.Consequently, it increases the inflammatory response and causes oxidative damage, which promotes chronic diseases associated with aging, such as cancer, diabetes, Alzheimer's disease, cardiovascular disease, osteoporosis, and infectious and autoimmune diseases (Alexander et al., 2018;Bauer & Fuente, 2016;Santoro et al., 2021).As demonstrated by the COVID-19 pandemic, the vast majority of inpatient deaths caused by viral infection occur among the elderly, particularly those with chronic diseases (Y.Chen et al., 2021).Hence, using a food supplement with few side effects to delay immunosenescence may be a readily acceptable and effective way of reducing the social and economic burden of aging.
Immunosenescence is characterized by a dysfunction in the innate and adaptive immune systems.NK cells, as a representative of innate immune cells, play a crucial role in combating cancer, illness, aging, and infection.As more researchers examine the relationship between aging and changes in NK cell function, they discover that NK cell cytotoxicity decreases with age, thereby increasing the risk of viral infection and the accumulation of senescent and cancer cells in older people (Brauning et al., 2022).Thymus atrophy is a major contributor to adaptive immunological failure because it reduces naïve T-cell generation, T-cell receptor diversity, T-cell proliferative capability, etc. (Alves & Bueno, 2019).A decline in innate or adaptive immunity leads to an increase in the pro-inflammatory cytokine level and a decrease in the anti-inflammatory cytokine level.This disorder is known as "inflammaging" (Franceschi et al., 2000).Apart from inflammaging, oxidative damage to proteins, lipids, and carbohydrates during aging is also related to immune system failure (X.Li et al., 2020).Therefore, immunosenescence can be delayed by managing the aforementioned essential mechanism, such as preserving immune cell activity, balancing the release of pro-inflammatory and antiinflammatory cytokines, and reducing oxidative damage.
Although Western medicine with an anti-aging effect has made significant progress, its adverse effects and multidrug resistance remain to be an issue.By contrast, food supplement contains extracts from plants or fungi that have a multi-target mechanism, a low rate of side reactions, and other qualities that could lead to anti-aging advancements (Shen et al., 2017).Ingredients from plants or fungi can combat aging via four primary mechanisms (1) The antioxidant effect is accomplished by augmenting the concentration and activity of many antioxidant enzymes.Polysaccharides from Tricholoma lobayense and Astragalus could increase the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in mice liver to limit oxidative damage to tissues and organs (Ding et al., 2016;X. T. Li et al., 2012).(2) Reducing proinflammatory cytokine levels or increasing anti-inflammatory cytokine levels could improve inflammaging.For example, ginsenoside Rg1 reduced the levels of proinflammatory cytokines, such as IL-6, IL-1β, and tumor necrosis factor (TNFα), to promote healthy aging (Y.Yang et al., 2017).In addition, extracts of Apocynum venetum tea containing multiple compounds, such as neochlorogenic acid, rutin, and isoquercitrin among many others, enhanced the equilibrium between pro-and anti-inflammatory responses in mice (C.Li et al., 2019).(3) Immune aging could be regulated by restoring immune cell function.Evidence shows that Korean red ginseng, which includes ingredients such as ginsenosides Rg1, Rb1, and Rg3, and carbohydrate, increases the percentage of regulatory T-cells and cytotoxic natural killer (NK) cells in the spleen of elderly mice (Shin et al., 2020).Moreover, Dendrobium polysaccharides promotes lymphocyte proliferation in aged mice (Wang et al., 2018).(4) The balance of intestinal flora could be regulated to delay the aging of the immune system by taking particular herbal extracts, such as Paeonia lactiflora petal flavonoid extract (L.Liu et al., 2021) and bilberry anthocyanin extract (J.Li et al., 2019).

Animal experiment and sample collection
Male and female C57BL/6JNifdc adult (aged four months; n = 5) and aged mice (aged 24 months; n = 5) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. and housed in a specific pathogen-free animal facility on a 12 h light/dark cycle with free access to food and water.As shown in Table 1, mice were divided into five groups and fed with ZJ(1055 mg/kg), ZY(369 mg/kg), ZJ(1055 mg/kg)+ZY(369 mg/kg) or phosphate-buffered saline (PBS) intragastrically once per day (200 µL/mouse) for 35 days (Table 2).Following a course of intragastric treatment lasting 35 days, the blood from the orbital venous plexus, spleen, thymus, and lung tissue was collected from euthanized mice.Furthermore, the immune organ index, the proliferative capacity of spleen lymphocytes, the cytotoxicity of spleen NK cells, the cytokines secreted in serum, and the SOD and GSH-Px enzyme activities in lung tissues were detected.The study protocol was approved by the Institutional Animal Ethics Committee of Foshan Huamio Biotechnology Company Ltd (Approval number: No.00295716).

Immune organ index
Mice were weighed before euthanasia.After rinsing with PBS, the spleen and thymus were dried and weighed.Thymus and spleen indices of mice were calculated in accordance with the ratio of thymus weight (milligrams)/body weight (kilograms) and spleen weight (milligrams)/body weight (kilograms), respectively.

Lymphocyte proliferation assay
The freshly isolated spleen was placed in a Petri dish containing 4 mL of RPMI-1640 and was gently grated with the piston of a syringe.The cell suspension was mixed in the dish and transferred to a centrifuge tube containing 4 mL of Ficoll solution.After centrifugation at 400×g for 35 min at 18°C, the milky white layer cells were aspirated into another centrifuge tube.The cell was washed two times with RPMI-1640, and the supernatant was removed after centrifugation.The proliferative capacity of T-cells was measured by the MTT assay.In brief, the cell concentration was adjusted to 1 × 10 6 cells/mL.Afterward, 100 µL of cell suspension per well was added into a 96-well plate.Then, 20 µL of ConA with a final concentration of 5 μg/mL was added to each well except for the control well (20 μL of medium was added to the control well), and each well was assayed in duplicate.
After incubation at 37°C for 48 h, the absorbance value for each well was measured at 570 nm.The proliferative capacity of splenocytes was expressed by using the stimulus index (SI), which was calculated as follows: SI = Absorbance of sample well/Absorbance of control well.

NK cell cytotoxicity assay
Target cells (YAC-1) were cultured for 24 h and washed three times with RPMI-1640.Afterward, the cell concentration was adjusted to 1 × 10 5 cells/mL for target cells and 5 × 10 6 cells/mL for effector cells (splenic lymphocytes).
Then, 100 μL of effector cells and 100 μL of target cells (effector cells:target cells = 50:1) were added into each well of a 96-well plate.Two control wells for the natural release of target cells (100 μL of target cells +100 μL of RPMI-1640) and for maximum release (100 μL of target cells +80 μL of RPMI-1640) were set.The plate was incubated at 37°C for 4 h.One hour before the end of incubation, the plate was removed from the incubator, and 20 μL of LDH release reagent was added.Cytotoxicity was measured and calculated as follows: Cytotoxicity (%) = (Absorbance of sample well − Absorbance of natural release well/(Absorbance of maximum release well − Absorbance of natural release well).

Determination of SOD and GSH-Px activities in lung tissues
For lung homogenate preparation, mouse lungs were washed with PBS to remove blood and then centrifuged at 3000 rpm for 10 min.The supernatant was used to determine the content of SOD and GSH-Px following the instruction of the SOD and GSH-Px kit from Nanjing Jiancheng Bioengineering Institute.

Statistical analysis
All data are presented in mean±SD and were analyzed using Prism 9 (GraphPad Software Inc., San Diego, CA).Data were analyzed by one-way ANOVA or Brown -Forsythe and Welch ANOVA tests, followed by Dunnett's post-hoc test for comparisons among multiple groups.Differences were considered significant when the value of P < .05.

No major changes in immune organs
The spleen and thymus undergo structural changes as a result of aging, which affect the activity of immune cells and eventually lead to a decline in immunological response and function (Pangrazzi & Weinberger, 2020;Sun et al., 2018).As shown in Figure 1(a), the spleen index of elderly control mice was lower than that of young control mice.The spleen index was higher in the three product-treated groups than in the age-controlled group.However, no statistically significant difference was found.Similarly, the elderly control group had a lower thymus index than the young control group.
Compared with the elderly control group, the ZJ group exhibited a slightly greater thymus index, but the difference was not statistically significant (Figure 1(b)).These results indicated that a single product or a combination of products can mitigate the structural alteration of the spleen associated with aging to a certain extent, but this treatment had almost little effect on the thymus.Note: compared with the aged group.**: 0.001 < P < .01.

Combination of ZJ and ZY effectively facilitated T-cell proliferation in aged mice
Aging reduces the proliferative activity of splenic lymphocytes, which reflects the regenerative and rejuvenated function of immune cells (Yousefzadeh et al., 2021).As shown in Figure 2, the ability of aged control mice to proliferate lymphocytes in the spleen was less than that of their younger counterparts.The ZJ+ZY treatment had a substantial influence on the proliferation of splenic lymphocytes, whereas other treatments had no significant effect.Based on these findings, the combination of ZJ and ZY can effectively restore the decrease in lymphocyte activity.

Combination of ZJ and ZY significantly enhanced the lytic function of NK cells in aged mice
NK cells play a crucial role in the innate immune response as they directly release lysis granzymes or induce apoptosis by expressing Fas ligand (Prager & Watzl, 2019;Ritter et al., 2022;Terrén et al., 2019).Yac-1 cell is a mouse lymphoma cell line that is targeted by NK cells; thus, it is used to investigate the activity of NK cells (Elsner & Dressel, 2020).As shown in Figure 3, elderly animals exhibited impaired NK cell cytotoxicity in the absence of any treatment, which is consistent with previous findings (Chiu et al., 2013).The ZJ +ZY treatment considerably increased the activity of NK cells within the spleen compared with the elderly control group.By contrast, a few mice in the two groups treated with a single product displayed NK activation.Based on these results, ZJ+ZY treatment greatly restores the activity of NK cells in aged mice.

Combination of ZJ and ZY modulated inflammaging in aged mice
Inflammaging is associated with an increase in the level of proinflammatory cytokines and a decrease in the level of antiinflammatory cytokines during the aging process.With regard to pro-inflammatory cytokines, the ZJ+ZY treatment successfully decreased the levels of IL-1α, IL-1β, and GM-CSF.Meanwhile, ZJ alone suppressed the secretion of IL-1α and IL-1β in the serum of elderly mice.ZY alone showed no effect on other cytokines, except for IL-1α (Figure 4(a-g)).With regard to anti-inflammatory cytokines, ZY and ZJ+ZY played a crucial role in boosting IL-5, whereas ZJ alone showed no effect in any cytokine (Figure 4(h-j)).The cytokine profiles elicited by single products or product combinations are summarized in Table 3.Therefore, ZJ+ZY could observably regulate the equilibrium of inflammatory responses in elderly mice.

Combination products significantly improved the antioxidant enzyme activity in elderly mice
Oxidative damage of proteins, lipids, and carbohydrates, as well as apoptosis caused by high quantities of accumulated oxidative free radicals, are among the causes of immunological failure throughout aging (X.Li et al., 2020).SOD is an important antioxidant enzyme that enables the elimination of free radicals during biological oxidation (Sharma et al., 2018).GSH-Px is a crucial peroxidase in the body, which can prevent oxidative damage (Mu et al., 2021).The results reveal that SOD and GSH-Px enzyme activities of elderly control mice were considerably lower than those of young control mice.In addition, ZJ+ZY treatment remarkably improved the activity of two antioxidant enzymes in elderly mice, whereas the administration of ZJ or ZY alone had little effect on enzyme activity.(Figure 5).Therefore, a powerful antioxidant effect can be achieved by mixing ZJ with ZY.

Discussion
The immune system undergoes constant remodeling and adaptation as the body ages.Among the detrimental effects of aging on the immune system are as follows: (1) The gradual atrophy of immunological organs and the rapid degeneration of immune cells.(2)Low-grade, sterile chronic inflammation is generated by the overexpression of pro-inflammatory cytokines and the accumulation of free radicals resulting from the oxidative destruction of proteins, lipids, and carbohydrates (Alexander et al., 2018;Santoro et al., 2021).Thus, restoring  the normal function of immune organs and immune cells, reducing inflammaging, and enhancing the activity of antioxidant enzymes can lessen the harm caused by immunosenescence and promote healthy aging.Based on our study, the combination of ZJ and ZY can repair the function of lymphocytes and NK cells, regulate the balance between proinflammatory and anti-inflammatory cytokines, enhance the enzymatic activities of SOD and GSH-Px, and reverse spleen atrophy, thereby promoting healthy aging.
The quality and function of atrophied immunological organs steadily decline with aging.Thus, the immune organ index of the spleen and thymus in mice, which can indicate aging-related changes of immune organs, were measured.It is indicated that the spleen and thymus indices of old mice were lower than those of young mice.Based on previous studies, the spleen and thymus indices can be partially restored by anti-aging Chinese medicine in naturally and chemically induced aged mice models (Dong & Liu, 2021;Xi et al., 2021;Xiong et al., 2011).In this investigation, the spleen index of mice fed with food supplement products or their combination showed only an upward trend probably because the mice used in previous studies were younger than those used in this study, where immunological organ structure is harder to reverse.
In this study, ZJ+ZY had a significantly larger anti-aging effect than either ZJ or ZY alone on all the factors influencing aging.This result may be due to a multi-target mechanism generated by the superposition of herbal and fungal ingredients (Wei et al., 2019).Xiangsha Liujunzi could provide patients with functional dyspepsia, a significant symptomatic relief (Lv et al., 2017).In addition, the Xiaoyao pill increased the Hamilton Rating Scale score of digestion (Du et al., 2014).Therefore, the synergistic effect mechanism must be clarified in the future because of the intricate nature of superposition of herbal and fungal ingredients.
As the subject of research on aging, humans have become complex due to various limiting factors, such as ethical issues, long life spans, and various environmental influences (Mitchell et al., 2015).Accordingly, animal models have been established to study aging.Aquatic animals, rodents, and pigs are commonly used to evaluate the effects of herbal medicines.Aquatic animals exhibit the advantages of low breeding cost and high prolificity (Belo et al., 2021), and they are suitable for a wide range of studies (Cherr et al., 2017;Seto et al., 2015;Wang et al., 2021;Yu et al., 2022).As a representative of aquatic animal models, zebrafish has been used as animal models in the research on aging diseases.However, considering the differences in disease pathogenesis and drug treatment between zebrafish and humans (S.Liu, 2022), and the significant environmental effect on their experimental results (Keller & Keller, 2018), zebrafish is actually not a good model for human antiaging research.Pigs exhibit the advantage of having certain similarities in anatomy and physiology to humans (Lunney et al., 2021;Singh et al., 2016).However, given the long breeding cycle and high costs of pigs (Zhu et al., 2020), rodents are more commonly used in research on aging diseases than pigs.The genetic proximity of mice to humans, the easy manipulation of the mouse genome, and the availability of many mutants and inbred strains have made mice the most popular mammalian model.In addition, the short life span of mice and the fact that mouse tissues can be analyzed at all stages of aging (Vanhooren & Libert, 2013) make mice a potential animal model for studying diseases related to aging.

Figure 1 .
Figure 1.Immune organ index.Body weight, spleen weight, and thymus weight of mice were weighed.The spleen index (a) and thymus index (b) were calculated using the following formula: spleen index = spleen weight (milligrams)/body weight (kilograms); thymus index = thymus weight (milligrams)/body weight (kilograms).

Figure 2 .Figure 3 .
Figure2.Lymphocyte proliferation in the spleen.Lymphocyte proliferation was detected following the preparation of splenic lymphocytes using the MTT assay.The proliferation ability was expressed by using the stimulus index (Si=absorbance of sample well/Absorbance of control well).Note: compared with the aged group.*: P < 0.05, ***: 0.0001 < P < 0.001.

Table 2 .
Conversion dose in mice based on body surface area (The dose used in mice was calculated by multiplying human dose by12.3 (Food and Drug  Administration, 2005)).

Table 3 .
Summary of cytokine secretion in food supplement product -treated aged mice.