T细胞免疫衰老对破骨细胞增殖分化的影响及右归丸的逆转效应

Imbalance of T lymphocyte subsets is the major cause of immunosenescence. The increasing age and inflammation contribute to the deterioration of T-cell phenotype plasticity and functional diversity and equilibrium, while directly threating homeostasis in the osteoimmunology and resulting in unusual activated osteoclasts and bone destruction. This research project focuses on how the imbalance of T cell subsets affects the normal differentiation of osteoclasts. In our previous study, the experimental results indicated that You Gui Wan(YGW) and its active ingredients can adjust the polarization of the T-cell differentiation and development, at the same time, they have a significant protective effect on T cell immunosenescence induced by glucocorticoid. We have to solve the key problem is that whether these drugs can reverse the imbalance of T cell subsets when they improve the abnormal differentiation of osteoclasts. The purpose of this study is to establish a immune aging model in mice to observe T cell subsets phenotypic and functional changes, and these changes effect on the proliferation and differentiation of osteoclasts in model of murine, on this account to investigate the connection between T-cell immunosenescence and the abnormal bone metabolism. Then through observation the regulation of imbalance of T cell subsets and unusual activated osteoclasts by YGW and its part active ingredients, looking for the immunological basis on these prescriptions of tonifying kidney to strengthen the bone and clarifying the pharmacological mechanism and targets, on which to lay the foundation for modern science of traditional Chinese medicine for effective prevention and control bone metabolic diseases caused by T cell immunosenescence.

T细胞亚群失衡是免疫衰老的核心,增龄与炎症刺激的累积使T细胞表型与功能的多样性、可塑性和均衡性受到破坏，并直接威胁骨免疫内环境的稳定，造成破骨细胞的异常活化和骨破坏。T细胞亚群失衡如何影响破骨细胞的正常分化是本研究的关注焦点。我们以往研究右归丸及其有效成分调节T细胞分化发育和效应极化时发现，该方药对由激素诱导的T细胞免疫衰老有明显的保护作用。这些药物在逆转T细胞亚群失衡的同时是否能改善破骨细胞的异常分化?这是我们要解决的关键问题。本研究拟建立免疫衰老小鼠模型，观察模型小鼠T细胞亚群表型和功能变化、以及这种变化对破骨细胞增殖分化的影响，以此探讨T细胞免疫衰老与骨代谢异常的相关性；通过观察右归丸及部分有效成分对T细胞亚群及破骨细胞异常分化的调节作用，寻找该方药补肾壮骨的免疫学依据，阐明其药理机制和作用靶点，为中医药有效防治由T细胞免疫衰老导致的骨代谢疾病奠定科学基础。

T细胞的免疫衰老现象主要表现为初始T细胞（Naive T, Tn）减少和T细胞亚群失衡两方面。增龄与炎症刺激的日积月累导致Tn过度消耗和生成不足，并直接影响T细胞各亚群失衡，这一过程中包含了体内激素水平不断变化对T细胞免疫损伤的累积。RANKL与其诱骗配体—骨保护素(Osteoprotegerin，OPG)是近年来骨代谢研究领域中影响OC分化和功能、调节骨重建的核心调节因素，RANKL和OPG之间的平衡决定了骨吸收的程度。激素（GC）水平的不断变化不仅可以损伤T细胞免疫平衡，同时也是影响RANKL和OPG平衡的重要因素之一。因此，我们认为，由GC诱导的T细胞亚群的异常分化能够直接影响RANKL和OPG之间的平衡，破坏骨内环境稳定，T细胞的免疫衰老程度与OC增殖分化密切相关。我们的关注焦点是右归丸及有效成分对GC诱导的T细胞亚群失衡的纠偏效应是否可以改善破骨细胞的异常分化以及其作用靶点。本项目通过体外实验筛选出右归丸部分有效成分的优化组合配伍；通过体内实验建立免疫衰老小鼠模型，观察模型小鼠初始和效应性T细胞亚群表型和功能的变化规律，以及这种变化对OC增殖分化的影响，进一步证实了T细胞的免疫衰老与骨代谢异常的密切相关，并通过补肾阳经典方剂-右归丸及部分有效成分对T细胞免疫衰老及OC异常分化的保护效应，为中医药从“肾”论治免疫衰老导致的骨相关疾病提供免疫学依据和中药疗效证据。本研究结果表明：.1. T细胞的免疫衰老与骨代谢异常的密切相关Th17、TEM亚群比例与OC增殖分化呈显著负相关(P < 0.05)。.2. 右归丸及有效成分优化组合对GC诱导的T细胞亚群失衡有明显的保护作用，同时可以改善破骨细胞的异常分化，其作用靶点可能是通过抑制MAPK通路中的Ras/ ERK途径来抑制OC分化成熟。.3. 尽管右归丸方中的单体有效成分的作用特点各不相同，而且，与离体实验结果有所差异，不同的组合也表现出不同的作用。但根据我们对模型小鼠免疫衰老保护作用的研究中可以得出如下结论：右归丸单体有效成分的最佳组合是桂皮醛+梓醇+乌头碱。. 本研究明确了初始和效应T细胞亚群的表型和功能的变化规律对OC增殖分化的影响，系统地阐明了T细胞免疫衰老对OC增殖分化的相关性，深入探讨了中药单体有效成分的免疫药理基础，摆脱了复方中药多靶点调节作用的臆测性和模糊性。.