The effect of myricetin on the immune function of cyclophosphamide induced immunosuppressed mice
The immune system can protect organisms from infections caused by pathogens to maintain balance and overall health. Immunosuppression is a long-term or temporary immune dysfunction. Due to immune system damage, organisms become more sensitive to pathogens and have lower resistance. Even minor pathogens that are not usually taken seriously can easily cause infections. Currently, many immunomodulators used in clinical practice have some side effects, such as levamisole and Bacillus subtilis. Therefore, it is still necessary to research and develop new immunomodulators to provide assistance in the treatment of immune suppression related diseases.

In the late 18th century, a light yellow crystal called myricetin (MYR) was extracted from the bark of Yangmei in the Myriaceae family (see Figure 1). MYR is a flavonoid compound found in fruits, vegetables, tea, and wine, and is an important active ingredient and additive in many foods. Modern pharmacological research shows that MYR has therapeutic effects on a variety of diseases, including different types of cancer, inflammatory diseases, atherosclerosis, Alzheimer’s disease and pathogenic microbial infection. However, the function of these flavonoids is still not fully understood, especially in regulating the immune system.

Cyclophosphamide (CTX) is an immunosuppressive inducer that has been widely used as a chemotherapy and disease modifier to combat certain solid tumors, lymphomas, and some autoimmune diseases. CTX can damage the DNA of normal cells, which is one of the reasons why it can perform immunosuppression. Currently, it is also used as an immunosuppressant in clinical settings. MYR is also a commonly used modeling drug for various laboratories to construct ideal animal models of immune deficiency.

Therefore, we used CTX to intraperitoneally inject mice to reduce their immunity and establish an immunosuppressive model. We treated the model mice with MYR and evaluated the spleen and thymus indices, spleen lymphocyte proliferation ability, serum hemolysin levels, antibody forming cell ability, as well as serum cytokine interleukin-2 (IL-2), interferon – γ (IFN – γ), interleukin-4 (IL-4), interleukin-6 (IL-6) secretion levels, in order to develop MYR as a new immunomodulatory agent.

 

Immunosuppression is a manifestation of immune dysfunction in the human body. Under the combined influence of one or more factors, immune damage in the human body can make it more susceptible to infection and result in immune related lesions. MYR is a flavonoid substance found in many foods, which has effects such as liver protection, anti-tumor, anti-inflammatory, antithrombotic, antioxidant, etc. There have also been many reports on its preclinical immune regulation. MYR can improve skin damage symptoms induced by calcitriol in atopic dermatitis mice, regulate local Th1/Th2 balance, and repair damaged skin barriers. In vitro experiments, Hou et al. found that MYR has an inhibitory effect on inflammatory factors in human immortalized keratinocytes induced by tumor necrosis factor – α (TNF – α)/IFN – γ, and can block the STAT1 and NF – κ B signaling pathways. MYR significantly increased the proportion of Treg cells and promoted the restoration of immune balance in colitis mice.
CTX is a type of alkylating agent with broad anti-tumor properties. It was first referred to as a “latent” broad-spectrum anti-cancer drug, which has selectivity towards T cells and can induce DNA damage and repair in vivo. Due to its ability to treat autoimmune diseases and regulate excessive immunity, as well as its high convenience in modeling and simple material requirements, CTX is currently a commonly used inducer for immunocompromised animal models in laboratories. This experiment uses CTX modeling to evaluate the immune level of MYR. Compared with the normal control group, the immune response related indicators such as spleen, thymus index, and cytokine secretion level were significantly reduced in the Mod treatment group, indicating the successful establishment of the immune deficient mouse model. The immune function of the body is closely related and mutually influenced by the functional status of immune organs such as the spleen and thymus. When the immune ability of the body is suppressed, the spleen and thymus will shrink. In this study, MYR was found to alleviate the atrophy of the spleen and thymus immune organs in mice caused by MYR, leading to an increase in the weight and index of the immune organs. This indicates that MYR can enhance the immune level of mice to some extent. Compared with the Mod group data, all three groups of MYR treatment significantly increased spleen and thymus indices, and the degree of increase in this trend indicates a certain dependence of treatment efficacy on drug dosage. It is worth noting that after applying high doses of MYR, both values were close to the control group level. The results indicate that MYR can alleviate immune organ damage caused by CTX and has an immune enhancing effect on immune organs.
The adaptive immune system, including cellular and humoral immune responses, can protect the body from the invasion of specific pathogens. Lymphocyte proliferation plays a crucial role in the activation of adaptive immunity and is often used to evaluate the immune response ability of animals. ConA and LPS can stimulate the proliferation of T and B lymphocytes, respectively. To further confirm the immune effect of MYR, we detected the secretion levels of lymphocytes under the action of ConA and LPS. The experimental results indicate that the application of MYR can significantly alleviate the inhibition of CTX on lymphocytes, and the changes observed from the application of different concentrations of MYR are somewhat dependent on the dosage of the drug. Research has shown that an imbalance in the ratio of CD4+T lymphocytes to CD8+T lymphocytes has an impact on immune function. Through flow cytometry analysis, it was found that the CD4+and CD8+subgroups of mice treated with CTX were significantly reduced, and the CD4+/CD8+subgroup ratio decreased (P<0.05 or P<0.01), indicating that the mice were in an immunosuppressive state. Compared with the Mod model group, each dose group of MYR can increase the number of CD4+and CD8+T cell subsets, as well as the CD4+/CD8+subset ratio (P<0.05 or P<0.01), with the high-dose group showing a significant dose-dependent effect. MYR may be involved in affecting the levels of CD4+and CD8+cells, regulating their immune imbalance and restoring them to normal levels. Therefore, it indicates that MYR can enhance both cellular and humoral immunity, which is consistent with the conclusion drawn from its effects on spleen and thymus indices.
The changes in serum hemolysin can reflect the amount of antibodies produced in the body at the molecular level, and an increase in serum hemolysin can enhance the specific antibody response to thymus dependent cell antigens. Based on the data results of the control experiment, we found that compared with the blank group, the hemolysin content in the CTX induced immunosuppressive mouse group decreased, and the number of antibody producing cells decreased. Different doses of MYR significantly increased the formation of serum hemolysin in CTX induced immunosuppressive mice, and also had a certain enhancing effect on the number of cells producing specific antibodies, indicating that MYR can enhance the cellular immune function of mice. In order to investigate the mechanisms associated with this enhancement, we conducted a more in-depth in vivo study on the effects of MYR on lymphocytes and inflammatory factors.
Cytokines, as one of the elements in the immune system, serve as bridges and links between cells in immune responses, playing a crucial role in coordinating immune and inflammatory responses. For example, increasing the secretion of anti-inflammatory cytokine IL-4 by TH2 cell subsets can control inflammation and improve symptoms of allergic rhinitis. IFN – γ plays an important role in regulating immune responses, inducing T cell generation, activating macrophages, and cross regulating TH1 and TH2 cells. IL-2 can regulate Treg to exert immunosuppressive or immunostimulatory effects, playing a crucial role during immune homeostasis. IL-4 has multiple immunomodulatory effects, both stimulating and inhibiting, and can stimulate B cell proliferation. It is an important factor in promoting hematopoietic cell development and differentiation, as well as the formation of IgG and IgE immunoglobulin. The cell specific expression of IL-6 can lead to various biological reactions, playing an important mediating role in acute inflammatory responses, and can inhibit effector CD8+T cell responses by reducing the production of IFN – γ. The experimental results showed that the expression levels of cytokines IL-2 and IL-4, IL-6, and IFN – γ in mice injected with CTX showed a significant downward trend compared to the control group, and the differences in the data of the other three groups, except for IL-4, were extremely significant. MYR can reverse this phenomenon by dose dependently bringing the secretion levels of cytokines IL-2 and IL-4, IL-6, and IFN – γ close to the normal range of the control group, and has a good regulatory effect on CTX induced immune deficiency in mice. Among them, the concentrations of IL-6 and TNF – γ in the high-dose drug group were restored to the levels of the control group. This result indicates that MYR restores the balance of the body’s immune system and improves symptoms of immune deficiency by regulating the interactions between immune cells and cytokines. Overall, MYR has a greater effect on the production of cytokine IFN – γ secreted by Th1 cell subsets than on cytokines IL-4 and IL-6 secreted by Th2 cell subsets, indicating that MYR mainly enhances the immune function of immunocompromised model mice by strengthening cellular immune function.
In summary, the impact of MYR on the immune system is complex, as it can induce changes in the expression of various lymphocytes and inflammatory factors, and regulate the interactions between these factors. Based on the results of this study, we conclude that MYR plays a positive role in immune function, especially under high-dose conditions. We have demonstrated that MYR can reverse immune suppression induced by CTX. Therefore, our experimental results provide evidence for the immune enhancing effect of MYR and scientific basis for its further development and potential clinical applications. In future research, we hope to continue exploring the impact of MYR on some diseases caused by low immunity or immune disorders.