二仙汤下调更年期大鼠内脏脂肪MCP-1抑制巨噬细胞聚集及极化的实验研究

Inner Canon of Huangdi wrote when forty-nine the beauties of women disappear. This notion is consistent with the modern discovery in women that after menopause women usually have central obesity. The accumulated visceral fat could secrete MCP-1 which results in macrophage accumulation and polarization in visceral fat. This ultimately leads to insulin resistance. Traditional Chinese medicine considered that central obesity in menopausal women is associated with the loss of shen jing. Therefore, bu shen is supposed to inhibit central obesity. On the basis of our previous results, this study investigates the internal mechanism of the inhibitory effects of er xian decotion on secretion of MCP-1 and macrophage polarization in visceral fat. Firstly, by using Real-time PCR, immunofluorescence and Western-blot we observe the role of NFκB pathway in the inhibitory effects of er xian decotion on visceral adipose MCP-1 secretion and the accumulation and polarization of macrophages in menopausal rat model. By using siRNA, luciferase reporter,Elisa and Transwell assays we then investigate the inhibitory effects of serum containing er xian decotion on macrophage migration through down regulation of MCP-1 and the inactivation of NFκB in adipocyte model. This study helps to describe the scientific connotation of Inner Canon of Huangdi and annotates the internal mechanism of bushen treatment on central obesity in menopausal women.

"女子七七…故形坏而无子也"。"七七形坏"观点涵盖现代医学对绝经后女性体态呈向心性肥胖的认识。增生的内脏脂肪分泌MCP-1促进巨噬细胞聚集与极化，最终诱发胰岛素抵抗。中医认为绝经后向心性肥胖与肾精渐亏有关，应从肾论治。本课题在前期研究基础上，从整体、细胞分子水平探讨补肾方二仙汤下调内脏脂肪MCP-1抑制巨噬细胞聚集及极化的内在机制。首先在更年期大鼠模型中，应用Real-time PCR、免疫荧光、Western-blot等技术观察NFκB通路在二仙汤下调内脏脂肪MCP-1抑制巨噬细胞聚集与极化中的作用；其次，应用siRNA、报告基因、Elisa、Transwell等技术在细胞模型中进一步探索二仙汤含药血清下调脂肪细胞MCP-1并抑制巨噬细胞迁移的效应及NFκB通路在其中的作用。本研究将丰富女子"七七形坏"的科学内涵，并揭示 "从肾论治" 绝经后女性向心性肥胖的内在机制。

研究显示女性在更年期时体重较绝经前显著升高，且体态呈“向心性”肥胖。中医认为“肾气”下降是更年期相关疾病产生的重要原因。我们采用补肾方二仙汤干预更年期动物模型，观察二仙汤对更年期动物模型胰岛素敏感性的影响以及对内脏脂肪内分泌功能及内脏脂肪中巨噬细胞极化的影响。在卵巢切除大鼠及卵巢切除高脂饲料喂养小鼠模型中，我们发现二仙汤可降低卵巢切除的更年期动物模型体重及内脏脂肪的重量并可提高模型组胰岛素敏感性，表现为糖耐量曲线下面积下降且胰岛素抵抗指数下降；应用qPCR和免疫荧光技术我们发现二仙汤可抑制内脏脂肪中巨噬细胞细胞的聚集及M1型极化并改善其内分泌功能；应用Western-blot技术我们发现卵巢切除大鼠内脏脂肪中细胞核p65蛋白表达显著升高，二仙汤可降低内脏脂肪细胞核p65蛋白的表达水平。在体外缺氧诱导的脂肪细胞模型中，应用Elisa及qPCR技术我们发现缺氧可诱导3T3-L1脂肪细胞分泌MCP-1，应用不同信号通路阻断剂我们发现这一效应与激活NFκB信号通路有关。二仙汤含药血清预干预后缺氧诱导3T3-L1脂肪细胞分泌的MCP-1水平与对照组血清干预后缺氧诱导3T3-L1脂肪细胞分泌的MCP-1水平相比较无统计学差异；应用Tranwell小室细胞迁移实验我们发现脂肪细胞缺氧诱导的条件培养液可促进RAW264.7巨噬细胞的迁移，二仙汤含药血清与对照血清干预缺氧的脂肪细胞的条件培养液促RAW264.7巨噬细胞迁移效应相比无统计学差异。这提示在体外二仙汤含药血清不能直接降低缺氧诱导脂肪细胞MCP-1的分泌及由此引起的巨噬细胞迁移。我们的研究提示二仙汤在整体水平可改善更年期动物模型胰岛素敏感性，这可能与其调节内脏脂肪内分泌功能并抑制内脏脂肪巨噬细胞聚集及M1型极化有关。