脂肪组织巨噬细胞M1/M2型转化改善慢性炎症的机制研究

The chronic inflammatory state accompanied by obesity is closely related to the occurrence of insulin resistance (IR) and diabetes. The accumulation of macrophages in adipose tissue is considered as an important cause for the chronic inflammatory. Studies have found that the macrophages exist as the alternative activated macrophage phenotype (M2 phenotype) in lean subjects. The M2 phenotype macrophages could secrete various anti-inflammatory factors such as interleukin-10 (IL-10) and TGF-β (transforming growth factor-β). And moreover this M2 phenotype could synthesize the Arginase -1, which inhibit the pro-inflammatory effect of the inducible nitric oxide synthase (iNOS). However, the macrophages accumulation increases in adipose tissue of obesity, and are polarized toward the classical activated macrophage subtype (M1 phenotype). The M1 macrophages can release many pro-inflammatory factors including tumor necrosis factor-α(TNF-α) and IL-6, and additionally the expression and activities of iNOS also increases. With the development of obesity, the M1 macrophages are dominant and secrete abundant of pro-inflammatory factors leading to the IR. If the pro-inflammatory M1 macrophages can be transformed into anti-inflammatory M2 macrophages, the chronic inflammatory state will be improved in obesity, and then the insulin sensitivity in adipose tissue and other peripheral tissues will be enhanced. Activation of peroxisome proliferator-activated receptor-γ ((PPARγ) is a key step in activating the M2 macrophages. Due to the strong plasticity of macrophage, we speculate that the M1 macrophages can be transformed into M2 in the obese state by activating the PPARγ pathway, and amend the inflammatory and IR. Thus,in vitro, we'll construct the co-culture system of adipocytes and macrophages, and induce the macrophages into M1 with free fatty acids, and observe whether the M1 macrophages can be transformed into M2 by activating the PPARγ pathway.In vivo, we'll establish the obesity mice model by high-fat diet, and interfere with PPARγ agonist rosiglitazone. The switch of polarization state of macrophages in adipose tissue will be observed. Flow cytometry is used to identify the specific markers of different macrophages subsets (M1-type: CD11c and M2: CD206, etc). The expression of pro-inflammation factors (TNF-α, IL-6, IL-1, MCP1, etc) and anti-inflammation factors (IL-10, Arg1, etc) will be tested by RT-PCR and Western blot, and further distinguish the macrophages subsets. From the respect of immunology, this study is conducted to prevent the chronic inflammatory state in adipose tissue, and will provide an important theoretical basis to reveal the pathogenesis of obesity and related metabolic disorders and will find out new therapeutic targets.

肥胖伴随的慢性炎症状态与胰岛素抵抗、糖尿病等的发生密切相关。脂肪组织巨噬细胞的聚集是慢性炎症发生的重要原因。在正常个体，脂肪组织巨噬细胞为M2型，分泌抗炎因子；而在肥胖时，外周血中的巨噬细胞趋化聚集至脂肪组织增多，并被活化为M1型，M1细胞分泌多种炎症因子，具有促炎活性。激活PPARγ是介导M2型活化的关键步骤。但脂肪组织中已活化为M1型的细胞能否再转化为M2型并不清楚。巨噬细胞的可塑性高，我们推测激活PPARγ通路可能使肥胖时已活化为M1型的促炎的巨噬细胞转化为抗炎的M2型细胞，从而缓解炎症状态，减轻胰岛素抵抗。为此，我们拟建立脂肪细胞与巨噬细胞共培养体系，并构建高脂喂养的肥胖小鼠模型，予PPARγ激动剂罗格列酮干预，观察脂肪组织巨噬细胞能否由活化的M1型转化为M2型。本课题从免疫学角度干预脂肪组织慢性炎症状态，将为揭示肥胖及相关代谢疾病的发病机理提供新的理论基础，寻找到新的治疗靶点。

背景.肥胖伴随的慢性炎症状态与胰岛素抵抗、糖尿病的发生密切相关。正常个体脂肪组织巨噬细胞为M2型，分泌抗炎因子；而肥胖个体巨噬细胞被活化为M1型，分泌促炎因子。肥胖时脂肪组织慢性低度炎症的发生可能与巨噬细胞浸润及极化有关。如能减少M1型，增加M2型可能改善慢性炎症。研究报道激活AMPK具有抗炎作用，二甲双胍为AMPK激动剂，能否改善巨噬细胞极化从而减轻炎症并不清楚。我们予二甲双胍在体内外干预，观察巨噬细胞极化状态及炎症指标的变化。PPARγ通路是调节M2型细胞极化的另一重要环节，我们用PPARγ激动剂干预观察巨噬细胞极化状态的改变。.主要研究内容 .体外试验.（1）用棕榈酸刺激骨髓来源巨噬细胞建立炎症模型，流式细胞仪鉴定细胞亚型、检测炎症因子表达。.（2）二甲双胍、AMPK激动剂（AICAR）及二甲双胍联合AMPK抑制剂分别干预棕榈酸诱导的巨噬细胞，观察细胞亚型及炎症因子的变化。.（3）PPARγ激动剂罗格列酮、姜黄素及姜黄素联合PPARγ抑制剂分别干预棕榈酸诱导的巨噬细胞，观察细胞亚型及炎症因子的变化。.体内试验.（1）构建高脂喂养肥胖小鼠模型。观察二甲双胍对小鼠体重、脂肪含量、血糖、胰岛素抵抗、炎症因子等指标的影响。.（2）提取小鼠附睾脂肪组织，观察巨噬细胞表面标志物及炎症因子表达，及二甲双胍的干预作用。.（3）观察二甲双胍干预后小鼠肠道菌群的变化。.结果.(1)成功诱导巨噬细胞炎症模型，棕榈酸刺激后MI型细胞比例及炎症因子表达升高，M2型细胞比例下降。.(2)二甲双胍及AICAR干预棕榈酸诱导巨噬细胞炎症模型后，M1型细胞比例及促炎因子表达下降，M2型细胞比例升高，加入AMPK抑制剂后，二甲双胍作用被阻断。.(3) 成功建立高脂喂养肥胖小鼠模型。肥胖组血循环中TNF-a、IL-6明显升高，加用二甲双胍后明显降低， M1相关标志物表达减少。.(4)二甲双胍可改变高脂喂养肥胖小鼠肠道菌群结构。 .(5)姜黄素干预棕榈酸诱导炎症模型，可改善巨噬细胞极化状态，减少M1型细胞比例及促炎因子表达，增加M2型细胞比例。加入PPARγ抑制剂后姜黄素作用被阻断。.科学意义.二甲双胍通过改善巨噬细胞极化状态而减轻炎症，从而改善胰岛素抵抗，其机制依赖于AMPK的激活。姜黄素也可通过激活PPARγ通路改善巨噬细胞极化状态。为以后治疗慢性炎症状态相关的代谢性疾病提供新的靶点。