TNF-α-let-7-IKKε通路调控儿茶酚胺抵抗参与儿童肥胖发生的作用机制研究

Recently, the prevalence of childhood obesity is being increased significantly and it has been to the focus of public health and clinical problem all over the world. The pathogenesis of obesity has not yet been clarified, which leads to poor efficacy of weight loss in obese children. Catecholamine resistance as an upstream mechanism leads to childhood obesity. On the basis of previous research, we propose that the increased TNF-α promotes the catecholamine resistance through regulating of let-7- IKKε signaling pathway in obese children. First of all, we want to clarify the relativity of the expression level of TNF-α, let-7 and IKKε of adipose tissue with BMI of obese chirdren. Secondly, we will clarify the relativity of the expression level of TNF-α, let-7 and IKKε with obesity degree of mice, reveal the impact of changes of expression level TNF-α, let-7 and IKKε on the adipocyte sensitivity to catecholamine, and confirm the effect of TNF-α regulating let-7- IKKε signaling pathway, by using molecular and cellular biology technology, based on the establishment of obese mice and the primary cell culture of human preadipocyte. Finally, using several biological technology such as gene knock-in and gene knock-out, our study will elucidate the molecular mechanism of TNF-α promoting catecholamine resistance through regulating the let-7-IKKε signaling pathway. The aim of this project is to explore new ideas and approaches for the targeted drug to relieve catecholamine resistance and promote the energy consumption in childhood obesity.

儿童肥胖发病逐年增多，成为全球关注的公共卫生和临床问题，其发生机制尚未搞清是制约减重疗效的关键。儿茶酚胺抵抗是导致儿童肥胖发生的上游机制。本课题组在前期研究的基础上，提出肥胖儿童体内升高的TNF-α通过调控let-7-IKKε通路，促使肥胖儿童儿茶酚胺抵抗发生的假说。本研究将首先明确肥胖儿童体内TNF-α、let-7、IKKε的表达变化，及其与肥胖程度的相关性；进而，通过建立肥胖小鼠模型和原代培养人前体脂肪细胞，结合分子和细胞生物学技术，确证肥胖个体脂肪组织内TNF-α、let-7和IKKε的表达水平与脂肪组织对儿茶酚胺敏感性及肥胖程度的相关性，并明确TNF-α对let-7-IKKε通路的调控作用；最后，通过高表达和低表达等生物学技术，阐明TNF-α通过调控let-7-IKKε通路，介导儿茶酚胺抵抗发生的分子机制。这将为进一步研究改善儿茶酚胺抵抗、促进肥胖儿童能量消耗的靶向药物开辟新思路。

儿童肥胖的发生率逐年升高，已成为全球关注的公共卫生和临床问题。脂肪组织儿茶酚胺抵抗是儿童肥胖的特征，并且这一现象在肥胖早期阶段就已出现。因此阐明肥胖儿童脂肪组织功能异常的分子机制，是儿童肥胖的防治关键。肥胖儿童脂肪组织功能异常与其体内的慢性低度炎症有关，TNF-α通过多种机制参与调解脂肪细胞的功能。脂肪细胞IKKε的表达异常是促使肥胖儿童儿茶酚胺抵抗发生的关键分子，然而关于引起脂肪细胞中IKKε 表达增加的分子机制尚不清楚。本课题旨在通过研究肥胖儿童腹部皮下脂肪组织IKKε的表达水平，运用细胞和分子生物学技术，明确IKKε与肥胖患儿肥胖程度、TNF-α表达水平、以及脂肪细胞儿茶酚胺敏感性的相关性；运用过表达和低表达技术，阐明TNF-α上调脂肪细胞IKKε的表达，抑制脂肪细胞儿茶酚胺敏感性的具体分子机制。主要研究结果：肥胖儿童腹部皮下脂肪组织中IKKε的表达水平明显上调，且与脂肪组织中TNF-α的表达水平呈正相关。TNF-α上调了脂肪细胞内IKKε的表达，并且随着TNF-α的浓度增大作用逐渐增强；同时抑制了异丙肾上腺素诱导的脂肪细胞HSL和Perilipin的磷酸化水平。证实了IKKε为let-7a的靶基因。转染let-7a inhibitor后的应激状态脂解作用明显受到抑制，IKKɛ的表达水平明显增加，同时HSL的磷酸化水平明显受到抑制。转染Lin28B siRNA后明显改善了TNF-α对细胞的应激状态脂解作用的抑制，下调了脂肪细胞中IKKɛ的表达，但上调了let-7a的表达。let-7a mimic转染后，明显改善了TNF-α对脂肪细胞的应激状态脂解作用的抑制，同时抑制了脂肪细胞内IKKɛ的表达。肥胖儿童脂肪组织中let-7a的RNA表达水平明显下降，而Lin28B mRNA表达水平明显高于非肥胖儿童。IKKɛ的mRNA表达水平与let-7a的RNA表达水平呈负相关，与Lin28B mRNA表达水平呈正相关。最终阐明了TNF-α通过激活脂肪细胞Lin28B/let-7a信号轴，上调脂肪细胞中IKKε的表达，降低异丙肾上腺素诱导的HSL的磷酸化水平，使得脂肪细胞的应激状态脂解作用受到抑制，最终引起脂肪细胞儿茶酚胺抵抗的发生。这将为揭示儿童肥胖发病机制，进一步寻找治疗肥胖的有效药物分子靶点提供实验及理论依据。