| ObjectiveImmunosenescence-related diseases like autoimmune diseases and bone metabolic diseases have become the key factors threatening health of the aged man as the society is aging rapidly. Bone destruction caused by the abnormalities of bone metabolism is one of the important characteristics of immunosenescence-related diseases. To reduce the harm of this kind of disease, improving the state of immunosenescence should be put in an important place.Imbalance of T cell subsets is the key of immunosenescence. Aging and long-term inflammation stimulation destroy the diversity, plasticity and balance of T cell phenotype and function, which directly threaten the internal environment stabilization of osteoimmune, then abnormal activation of osteoclast and bone mass loss appear as a consequence, finally lead to bone metabolic diseases, such as osteoporosis and inflammatory bone loss. Therefore, keeping the diversity and plasticity of T cell repertoire, reconstructing T cell subsets balance can help restoring immune internal environment stabilization, delaying immunosenescence and preventing bone metabolic diseases.We have learned in former study that You-Gui-Wan and partial effective ingredients can regulate differentiation and development of T cell subsets,protect against T cell immunosenescence induced by Dexamethasone, and we found that some of its effective ingredients contributed to increase na?ve T cell quantity, which was helpful to enhance the body immunity against new antigens. Whether the abnormal differentiation of osteoclasts can also be restrained by these medicines as T cells, this is our focus. We will further explore the correlation between T cell immunosenescence and bone metabolism abnormalities, and optimized the prescription based on the effector mechanism of You-Gui-Wan and its ingredients so as to find an effective medicine resisting T cell immunosenescence and bone metabolism abnormalities, which will lay a scientific foundation for resisting and preventing diseases caused by immunosenescence and bone metabolism abnormalities. Methods 1. The preparation of Chinese medicine You-Gui-Wan compound: according to the classical recipe, this compound was prepared by using traditional water extraction and alcohol precipitation method.The YGW partial effective ingredients were aconitine(AC), catapol(CA), cinnamaldehyde(CI). 2. The establishment of T cell immunosenescence group and intervention of Chinese medicine compound: establish T cell immunosenescence model in mice by hormone-inducing.The model were divided into 4 groups which are CA group(6mg·kg-), CI group(0.126mg·kg-), AC group(10μg·kg-) and YGW group(7.5g·kg-). 3. Thymic T cell apoptosis of each group was observed by Using Annexin V/PI double staining. 4. The detection of peripheral blood,naive T cells(CD3+CD45RA+CD62L+),central memory T cell,effector memory T cell,and the change of CD4+/CD8+ by Flow cytometry. 5. Th1(CD3+CD4+IFN-γ+), Th2(CD3+CD4+IL-4+), Th17(CD3+CD4+IL-17+) and Treg(CD4+CD25+foxp3+) were detected by internal and external factors intracellular flow cytometry staining. 6. The mouse bone morphology’s changes were determined by HE staining. 7. The expression levels of TRACP5 b in serum were measured by ELISA. 8. The expression levels of osteoclast differentiation-related signaling molecules such as TRAF6, P38, MEK before and after the experiment were measured by Western blot. 9. The proliferation and differentiation of osteoclasts under the influence of YGW and some active ingredients were analyzed by MTT in vitro.Optimized the best concentration of medicines. 10. The expression levels of RANK, NFATc1 m RNA of osteoclasts were measured by RT-PCR.Then detected the direct effects of YGW and partial effective ingredients. Results 1. Hormones can induce T cell immunosenescence in mice.Compared with the control group,there was a substantial reduction of aging model group mice’s thymocyte cells(P<0.05), thymocyte cells apopotosis increased by Annexin-FITC /PI double staining(P<0.01).FACS tests showed that the proportion of Tn cells decreased in peripheral-blood and spleen(P<0.05),the proportion of TCM decreased(P<0.05) and TEM increased(P<0.05); the ratio of CD4/CD8 in the spleen decreased(P<0.05);T cell subsets Th1’s IFN-γ production decreased;Th2 had no significant change;Th17 increased;Treg cells’ decreased;Th1/Th2 decreased;Th17/Treg increased;it’s a classical pattern of hermone-induced T cell immunosenescence that T cell subsets is in an unbalanced state. 2. Effects on bone metabolism of hermone-induced T cell immunosenescence in miceWith the changing of T cell in bone microenvironment, bone marrow cells decreased significantly,HE staining showed that hormone can magnify the bone lacuna,induce femurs trabe cular of mice in model group(P<0.05).It is showed that the expression of TRACP5 b in serum of model group increased.T cell subsets imbalance accompanied with bone metabolic abnorm alities. 3. YGW and partial effective ingredients can reverse hermone-induced T cell immunosenescence significantlyCompared with hermone-induced immunosenescence model mice, YGW and partial effective ingredients can significantly decrease thymocyte apopotosis, increase the initial ratio of T cells(p<0.05), increase the proportion of TCM, decrease TEM relatively(P<0.05), improve the ratio of CD4/CD8 cells, enhance the Th1 cell’s ability to secrete IFN-γ in spleen, up-regulate the ratio of Th1/Th2(P<0.05), decrease Th17 with rising Treg a slightly and the ratio of Th17/Treg recovers from imbalance.(P<0.05). 4. YGW and part of the active ingredients can significantly reverse imbalance of T cell subsets in bone marrow microenvironment significantlyCompared with the hormone-induced aging mice, YGW and some active ingredients can increase the proportion of Tn in bone marrow(P<0.05); enhance the Th1 cell’s ability to secrete IFN-γ in bone marrow, reduce the proportion of Th17 cell subsets. 5. Protection of YGW and part of the active ingredient on bone metabolism in hermone-induced miceHE staining showed that YGW and part of the active ingredient can inhibit area of trabecular’s loss, protect the bone loss which induced by hormone, reduced the serum level of expression of TRACP5 b. Western blot analysis showed: YGW and some active ingredients can reduce the expression of the molecule TRAF6 in bone marrow cells(P <0.05), decreased activation of MAPK signaling pathway, thereby inhibit osteoclast maturation. 6. YGW and some active ingredients can affect the differentiation of abnormal hormone-induced osteoclast in vitro directlyMTT tests showed:YGW and some active ingredients can inhibit the proliferation of osteoclasts significantly, the efficacy in a time-and dose-dependent manner; at the same time, high concentrations of YGW and part of the effective ingredients can reduce the expression of specific genes RANK and NFATc1 m RNA about osteoclast differentiation. Conclusions 1. The distinguishing features of hermone-induced T cell immunosenescence are that ratios of Tn、TCM decrease,TEM increases relatively and ratios of CD4/CD8,Th1/Th2 and Th17/Treg become unbalanced. 2. YGW and partial effective ingredients have an obvious protection against hermone-induced T cell immunosenescence.They can increase the ratio of Tn、TCM,decrease TEM ratio and put each subsets such as CD4/CD8,Th1/Th2,Th17/Treg back in balance. 3. YGW and partial effective ingredients can reduce the expression of TRACP5 b in serum, restrain osteoclasts from enhancing bone resorption activity and improve the bone quality. 4. YGW and partial effective ingredients reverse the T cell immunosenescence-induced prosoplasia of OC maybe by restrain the activation of MEK and P38 in MAPK signal pathway,so that restrain the differentiation and maturation of OC 5. YGW and partial effective ingredients can directly restrain expressions of osteoclast’s RANK、NFATc1 m RNA in vitro.Within these effective ingredients, CI is obviously effective. |
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