激活TRPM8调控巨噬细胞Cx43介导白色脂肪棕色化拮抗肥胖的机制研究

Adipocyte differentiation is closely related with phenotypic transformation of macrophage，the regulation of macrophage phenotypic transformation to M2 can promote white fat browning, of which Ca2+ plays an important role. Our recent research revealed that the expression of Cx43 was down-regulated in white adipose tissue during obesity,which caused visceral fat remodeling through decreasing Ca2+ influx induced by Cx43,whereas activation of TRPM8 enhanced the expression of UCP1 in adipocyte of brown fat,and regulated endoplasmic reticulum and mitochondrial Ca2+.Other research showed thermogenesis induced by UCP1 was associated with SERCA operated calcium ,Ca2+ is an important signal molecule of TRPM8, Cx43 and UCP1. It remains to be confirmed whether the activation of TRPM8 can promote browning of white fat through intercellular Cx43. For this purpose, we hypothesized that activation of TRPM8 promote phenotypic transformation of macrophage to M2 phenotype by regulating calcium homeostasis,which result in the interaction of macrophage with adipocyte by Cx43, thus promoting the browning of white fat by activating PKA/UCP1 pathway. To test the hypothesize, diet-induced obese mice model，TRPM8-/- and Cx43-/- Macrophage specific mice model were established to explore the effects of TRPM8 in regulating the phenotype of macrophage on browning of white fat and its mechanism. Our study will expand the understanding of obesity and shed light on new approaches for preventing obesity.

脂肪细胞分化与巨噬细胞表型密切相关，调控巨噬细胞表型转化为M2型可致白色脂肪棕色化，Ca2+在其中发挥重要作用。我们前期研究发现肥胖时白色脂肪组织Cx43下调，脂肪重构与Cx43介导的Ca2+减少有关；激活TRPM8增加棕色脂肪细胞UCP1表达，并可调控内质网及线粒体钙。他人研究发现UCP1介导的产热与SERCA调控钙有关，而Ca2+是TRPM8、Cx43和UCP1共同的信号分子，激活TRPM8通过细胞间Cx43调控白色脂肪棕色化的机制有待探索。为此，我们提出假说“激活TRPM8调控巨噬细胞内钙稳态致其转化为M2型，通过Cx43介导巨噬细胞-脂肪细胞间的对话，激活PKA/UCP1信号通路促进白色脂肪棕色化”。拟采用高脂诱导肥胖小鼠、巨噬细胞特异TRPM8-/-和Cx43-/-小鼠为模型，从整体、细胞和分子水平揭示TRPM8调控巨噬细胞表型转化在白色脂肪棕色化中的作用，为肥胖防治提供新靶点。

慢性低度炎症是肥胖及其相关糖尿病、心血管病发生的关键机制，其中，巨噬细胞发挥着重要作用。线粒体功能障碍可导致巨噬细胞极性转变，而线粒体钙超载是导致其功能障碍的重要原因，但缝隙连接蛋白43（Cx43）在其中的作用及机制仍未明确。为此，我们构建了巨噬细胞特异性Cx43敲除小鼠并进行高脂喂养（HFD），结果显示巨噬细胞Cx43敲除可显著改善HFD喂养引起的体重增加及糖耐量异常，同时伴有脂肪组织促炎型巨噬细胞浸润减少。体外试验中发现抑制或敲减Cx43可显著改善棕榈酸（PA）引起的线粒体相关内质网膜（MAMs）及线粒体Ca2+单向转运体（MCU）介导的线粒体钙超载，进而抑制琥珀酸脱氢酶（SDH）过度活化引起的线粒体功能障碍，从而促进抗炎型巨噬细胞的极化，提示Cx43可作为线粒体中调控Ca2+稳态的通道，参与调节巨噬细胞极化及肥胖的发生发展。SIRT3是线粒体上重要的去乙酰化酶，对于维持线粒体功能具有重要作用，但其在巨噬细胞介导的肥胖进展中的作用及机制仍不明了。为此，我们构建巨噬细胞特异性SIRT3敲除小鼠并进行HFD喂养，结果显示巨噬细胞SIRT3敲除加重HFD引起的体重增加及糖耐量异常，同时伴有整体代谢水平降低以及脂肪组织中促炎型巨噬细胞增加。体外实验表明，SIRT3缺乏使得琥珀酸脱氢酶亚基（SDHA）乙酰化水平增加，导致其活性降低，引起琥珀酸（Suc）的累积，进而引起Kruppel-like factor 4 （KLF4）启动子甲基化水平增加，导致其转录下降，从而促进促炎型巨噬细胞的极化及脂肪细胞功能障碍，因此SIRT3可作为肥胖防治的重要靶点。临床研究部分，我们进行了一项前瞻性GLP1受体激动剂-利拉鲁肽干预研究，显示其可通过直接激活背外侧前额叶皮层和前额叶眶面皮层脑区的功能，不依赖于减重及降糖的方式改善2型糖尿病患者的认知功能。此外，通过建立模型对糖尿病下肢血管病变进行评估，可早期发现下肢血运障碍，从而有助于预防糖尿病足的发生。其次，通过随访糖尿病下肢血管病变介入术后的患者，膝下血管病变再狭窄率较高，发现总胆固醇（TC）和术前糖化血红蛋白（HbA1c）是影响手术疗效及再狭窄的危险因素，为防治再狭窄提供依据。