The Regulatory Effects Of Chinese Traditional Medicine Yin-Yang-Tonic Herbs On Hormone-Induced Inhibition Of Th1/Th2 Cytokine Production

Objective The immunoneuroendocrine is an integrated network that included immune, nerve and hormonal systems, and plays an important role in sustaining the homeostasis of health through their corresponding mediators of cytokines, neuromediators and hormones, respectively. It initiates and regulates the coordinated systematic responses to against pathological events, such as inflammation and tissue damages. The two major neuroendocrine signaling pathways that include Hypothalamus- Pituitary-Adrenal axis (HPA) and Hypothalamus-Pituitary-Thyroid axis (HPT) have been found influencing immune system through the end effectors of glucocorticoid (GC) and thyroxine (T4) for HTA and HPT respectively. A number of studies has shown that both GC and T4 could directly involve in a broad range of immune response through interacting with the correspongding nuclear receptors in the immune cells.T lymphocytes are a high hyperheterogeneity cells and taking over about 60% of total lymphocytes in immune system. They are very sensitivity to the changes of HPA or HPT. These characteristics of diversity and sensitivity of T cells are necessary for its function that against pathogen invasions and tissue damages, as well as for maintaining the balance within immunoneuroendocrine network. T cells can be dissected into CD4~+ and CD8~+ subsets according to their cell surface markers. T helper cells (Th) that contain CD4 marker and serve as the central modulator inimmune response can be further clustered as Thl and Th2 cells mostly according to their cytokine-producing profiles. While cytotoxic T cells (Tc) contain CD8 marker are classified into Tel and Tc2 relying on the profile of cytokine secretion similar to Thl and Th2. Thl cells produce IFN- Y , IL-2 that are mainly responsible for cell-mediated immune response to antitumor, antivirus and antiparasites. Excessive Thl-type cytokines have been found to be associated with the tissue destruction in autoimmune diseases, such as leprosy, tubercle, viral hepatitis sarcoidosis. In contrast with Thl cells, Th2 cells produce IL-4, IL-5, IL-6, and IL-13 ususally functional promote humoral immune response and antibody production, which are essential for antibiosis and antitoxin to elimination of extracellular organisms. The over-accumulated Th2-cype cytokines have been implicated in allergic asthma and parasitic diseases. It has been found that the ratio of Thl and Th2 (Thl/Th2) type cytokines is consistent under the healthy condition to maintain the immune system in a quite staus, however in response to a pathologic condition, the ratio of Thl/Th2 type cytokine will shifts that is when one type cytokine is excessive expressed, another type will responsibly reduced, so that to keep the balance of Thl and Th2 dynamically.Regarding to the relationship between immune and neuroendocrine systems, the observations of that GC and T4 induced T cells differentiation and cytokine productions have shown the direction influences of GC and T4 to immune system. Nevertheless, the molecular mechanisms of such influences, particularly the effects to Thl and Th2 cytokine productions, are still unknown. On the other hands, hormone therapy for severe inflammation and autoimmune diseases have demonstrated a limited effects because the serious adverse events. Additionally, the elevated endogen hormones that induced by overreacting to pathogens have been observed in many diseases and have became a considerable problem for patients. Therefore, elucidating the basic mechanisms of hormone induced Thl and Th2 cytokine productions will shed the light for developing the effective drugs for clinical therapy.Yin-yang theory is an essential part of Traditional Chinese Medicine (TCM) in diagnosis and treatment based on overall analysis of symptoms and signs, whichcaused by the interplay, interdependence, transformation and suppression relationships between the two opposite factors of yin-yang. With the great development of modern TCM, Shen ziyi and his colleagues have demonstrated that yang-deTicieni is related to the changes of HPA axis, while j/n-deficient is related to the changes of HPT axis. The GC-induced serials symptoms, including cold, tremble, ache and knee, reflect yang-deficient and has been treated with YGW, a standard prescription of yang-tonic herbs for altering HPA axis. In contrast, the T4-induced symptoms of irritability, dysphoria and insomnia, reflect y/n-deficient and has been treated with ZGW for altering HPT axis. However, the immunopharmocological mechanisms of both YGW and ZGW remain vague. In this study, we employed GC-induced jwjg-deficient mice and T4-induced ym-deficient mice as models to systematically study the effects of YGW and ZGW, respectively, on Thl and Th2 type cytokine expressions.This study attempts to explore the effects of yin-yang tonic herbs on the abnormal expression of Thl/Th2 type cytokine productions that were induced by GC or T4 and the possible immunopharmacological mechanisms of the yin-yang-tomc herbs using molecular biology techniques. This study is expected to evaluate the uses of yin-yang-toviic herbs in clinical for immune disorder, particularly in the use of immunesuppression related hormone exposures.Methods1. Animal model: Inbred BALB/cA mice were injected (i.m.) with Hydrocortisone (HO and treated with Thyroxin (i.g.) respectively to establish the animal models of hormone induced Thl/Th2 type cytokine abnormal expression. Meanwhile, model mice were treated with You-Gui-Wan (YGW) and Zuo-Gui-Wan (ZGW) separately compared with control mice (normal mice treated with PBS) and matched control mice (model mice treated with PBS). 7days later, the mcie were injected with ConA (v.s. PBS) lh after end treatment. RT-PCR, ELISA and intracellular cytokine stain by flowcytomytre were used to analyze the changes of IFN- Y, IL-2 (Thl), IL-4, IL-10 (Th2) mRNA expression and protein secretion as well as the cytokine-producing T cells with or without the treatment of YGW andZGW in hormone-treated mice. 2. Cell cultureSplenocytes were extracted from model or treated mice and cultured in ConA condition, RT-PCR was used to exam the epression of IFN-y ^ IL-2, IL-4> IL-10 mRNA of cultured splenocytes. Moreover, the splenocytes from normal mice were respectively cultured in the following condition: The culture system contained T4, T4+ZGW, HC or HC+YGW. 4h later, IFN- y , IL-2, IL-4> IL-10 were detected by RT-PCR. This operation was to further insure the effects of hormone on Thl/Th2 type cytokines and the protection of TCM in vitro.All the experiments were designed to explore the mechanism of regulation of yin-yang tonic TCM on hormone-inducce inhibition of Thl/Th2 type cytokine whether due to the protection of cytokine transcription or to the restoretion of cytokine-producing T cells?Results1. YGW prevented the GC induced inhibition of Thl/Th2 typecytokine production and cell proliferation in mice(1). GC induces the inhibition of Thl/Th2 cytokine production in vivo and in vitroFollowing the treatment of GC, the mice had the typical "yawg-deficient" symptoms of depression, accidie and the changes of splanic coefficient. IFN- Y, IL-10, IL-2, IL-4 transcription by RT-PCR were significantly decreased in a time-and dose-dependent manner. The splenocytes from GC treated mice have the decreased reactivity to ConA stimulation in vivo. Moreover, GC showed the same inhibition on cytokine transcription in vitro and the splenocytes cultured with GC also exhibited a weakened activity in responds to ConA stimulation in vitro. IFN- Y production in murine serum was suppressed from 210. 8±26 to 145. 7±47(pg/ml) by GC detected by ELISA. The results showed that GC inhibited the cytokine expression both in yivo and in vitro.(2). The inhibition effects of Thl and Th2 cytokine production by GC arecoupled with the specific subtype of T cell proliferationIn order to further examine the states of cytokine-secreting T lymphocyte under GC condition, we applied flow cytometry to label cell surface and intracellular cytokines to determine the cytokine production by CD4+ and CD8+ T cell. The percentage IFN-y+ CD4+ T cells from HC-treated mice were markedly decreased from 16.5% to 7.3% while the percentage IFN-y+ CD8+ T cells from HC treated mice were decreased from 18.6% to 6.7% compared with control group. Both of IL-10-and IL-4-producing CD4+ and CD8+ T cells only slightly decreased without statistical significance. The result suggests GC severely suppress the IFN-y-secreting T cell population.Consequently, GC can inhibites the expression of Thl/Th2 type cytokine through suppressing gene expression and attenuating the population of cytokine-producing T cell.(3). YWGcan induce both Thl and Th2 cytokine productionsThe splenocytes from treated mice with oral administration of YGW show increased transcription of IFN- y , IL-10, IL-2 fd IL-4, and the changes of IFN- Y , IL-10 had the statistical significance. As a result, the production of IFN- Y in serum was markedly enhaced at large dose of YGW(0.4ml/mouse) from 227.8 ±23 to 256.8+27 (pg/ml) .(4). YGW prevents the inhibitory effects of GC induced Thl/Th2 cytokine productionWith oral administration of YGW during GC injection, the inhibitory effects of GC on transcriptions of IFN-y, IL-2, IL-4, and IL-10 by freshly isolated splenocytes were restored by YGW treatment associated with conrespondint alteration of the "yang-deficient" symptoms. Meanwhile, YGW improved the impaired reactivity to ConA stimulation induced by GC. In addition, ELISA analysis showed that YGW administration protected the HC-treated mice from inhibition of IFN-y production in the serum level (181.4 + 25 vs 145.7 + 47). The protective effects of YGW on GC-induce inhibition of Thl/Th2 cytokine transcription were further confirmed by in vitro study. During pharmacological concentration, YGW showed adose-dependent protection on cytokine expression under GC condition. The results suggested that YGW has profound restoration on GC-induced inhibition of Thl/Th2 tyoe cytokine and effectively protect the mice free from "yang-deficient" symptoms.(5). YGW prevents the inhibitory effects of GC induced Thl/Th2 cell proliferationFollowing treatment with YGW+HC, the protective activity of YGW on HC-inducing inhibition of Thl/Th2 lymphacyte proliferation was observed, IFN-y+-CD4+ T cells were recovered from 7.3% to 13.8% and IFN-y+- CD8+ T cells were elevated from 6.7% to 14.5%, IL-4+-CD4+ and IL-4+ -CD8+ T lymphacytes were also enhanced above normal level (p<0.01). Moreover, along with the monocyte counts in spleen, thymus and bone marrow being highly exceeded the levels of HC-treated mice, the population of cytokine-producing T cell were expanded.All the results suggested YGW could effectively protect the Thl/Th2 lymphocytes against damnification of GC.2. ZGW prevented the T4 induced shift of Thl/Th2 typecytokine production and cell proliferation in mice(1). T4 induces the inhibition of Thl/Th2 cytokine production in vivo and in vitroAfter treatment with T4, the mice showed typical "yin-deficient" symptoms of excited, agitate, high temperature and changes of splanic coefficient, the transcriptions of IFN- Y , IL-10, IL-2, IL-4 were significantly decreased in a time-dependent manner, and IFN- v production in T4-murine serum by ELISA was suppressed from 210. 8 ±26 to 158 + 67 (pg/ml) .The splenocytes from T4 treated mice have the decreased reactivity to ConA stimulation in vivo. Moreover, T4 showed the same inhibition on cytokine transcription in vitro in a dose-dependent manner. The splenocytes cultured with T4 also exhibited a weakened activity in responds to ConA stimulation in vitro. The result suggested T4 inhibited the expression of Thl/Th2 type cytokine.(2). The inhibition effects ofThl/Th2 cytokine production by T4 are coupled with the specific subtype of T cell proliferationTo insure the reduction of Thl/Th2 type cytokine expression due to T lymphocytes alteration, we detected the changes of cytokine-producing T cell, the results showed the proportion of IFN- y +> IL~10+T cell were significantly decreased. It means the inhibition of T4 on Thl/Th2 type cytokine mainly by inhibiting transcrioption of cytokine and by attenuating the population of Thl/Thl lymphocyte subsets.(3). ZWGcan induce both Thl and Thl cytokine productions The splenocytes from ZGW-treated mice show decreased expression of IFN- Y , IL-10, IL-2 #] IL-4 by RT-PCR, and the changes of IFN- Y , IL-4 had the statistical significance. Meanwhile, ZGW impaired the cytokine expression under ConA stimulation. The inhibition of ZGW on cytokine production was approved in vitro study. In addition, The splenocytes from ZGW teated mice showed decreased proportion of IFN- Y +CD4+ from 12.5% to 3.6%, IFN- y +CD8+ T cell from 11.7% to 3.1%, and IL-4+,IL-10+ T cells were slightly decreased without significance. ELISA showed the fallen production of IFN- Y from 210.8+26 to 167.6 + 67 (pg/ml). All the results showed ZGW inhibited Thl/Th2 cytokine expression (4). ZGW prevents the inhibitory effects ofT4 induced Thl/Thl cytokine shift The typical "yin-deficient" symptoms derived from T4 treatment were associated with the shift state of Thl/Th2 type cytokine. Compared with control mice IFN- y, IL-2, IL-4 and IL-10 were decreased 18%, 33.6%, 42.5% and 48.3%. T4 showed more inhibition on Th2 type cytokine than on Thl. The balance state of Thl/Th2 in normal circumstance was break down by T4 treatment. In T4+ZGW treated mice IFN- y, IL-2, IL-4 and IL-10 were decreased 43.5%, 44.5%, 46.5% and 46.4% in contrast to control mice, IFN- y production (83.2 + 49.1) in T4+ZGW murine serum was decreaed, and the typical "yin-deficient" symptoms was hold back. The results suggested that ZGW could withstand the "yin-deficient" symptoms induced by T4 may be related to reestablishment of Thl/Th2 balance.ConclusionsThe results have suggested that the inhibitory effects of Thl/Th2 cytokine productions by hormones is through negative regulation of the cytokine gene'stranscription and downregulation of cytokine secretion, as well as inhibiting the proliferations of corresponding cells. On one side, such biological functions can be used for immunotherapy, and on another side, it could causes the immunotoxins too. ZGW and YGW, the yin-yang-enrichment prescriptions of TCM, can recover the shifts of Thl/Th2 that induced by hormones to the normal balance. Our results have shown that YGW can prevent GC induced inhibition on both cytokine production and Thl/Th2 subsets proliferation; ZGW can regulate the shift state of Thl/Th2 by acting as cytokine suppression. The results of present study have provided the molecular evidences for the prevention effects of YGW and ZGW on hormones induced Thl/Th2 shifting, and that will greatly improve the understanding and appropriate utilizing of yin-yang tonic in TCM.