内源性大麻素系统对哈萨克族肥胖儿童异常空腹血糖受损状态影响的机制研究

Obesity is known to be associated with an increased risk of insulin resistance.The underlining mechanism is related with overactive of endocannabinoids system（ECs）and inflammation pathway.The prevalence of diabetes in Kazakh is lower than other nationalities whereas the prevalence of obesity is higher than others in local area.The results of research showed that human gut flora changing influence the mucosa expressing of ECs so as to interfere the metabolic pathway including glycometabolism and inflammation pathway.Our hypothesis is that Kazakh children intestinal flora may influnce on glucose metabolism by mucosa ECs based on the results of our former study about the decreased number of Bacteroidetes and lower frequence of fasting glucose impaire among obese Kazakh group. .High performance liquid chromatogram is used to test peripheral AEA and 2-AG. Real-time fluorescence quantitative PCR (Q-PCR) is used to evaluate the levels of MyD88，TRAF6 expression of peripheral blood mononuclear cells and intestinal flora while the concentration of plasma cytokines such as LPS, IFN-αare measured by enzyme-linked immunosorbent assay(ELISA)．Blood glucose, insulin concentration and other index will be determined after overnight fasting using a modified hexokinase enzymatic method. The aims of this study are explore the relationship among ECs, plasma cytokines, LPS and index of glycometabolism after intestinal flora changing.Therefore,the results of our research may be helpful for understanding of machanism underlining glycometabolism result from obesity and new thinking of therapeutic aspects.

肥胖增加胰岛素抵抗（IR）风险，其发生与内源性大麻素系统（ECs）及炎性通路过度激活等相关。而肥胖患病率较高的哈萨克族较少发生糖尿病，机制不明。已知人体肠道菌群构成改变可影响肠上皮ECs表达，可进一步作用于包括糖代谢途径、炎性通路在内的许多代谢途径。课题组已发现：哈族肥胖儿童肠道菌群构成异常、空腹血糖受损比例低，推测其特有肠道菌群构成可能通过ECs影响到该人群糖代谢途径，发挥保护作用。.本项目采用病例对照方法，高效液相色谱‐质谱联用技术检测外周血ECs 的AEA和2-AG，RT-PCR检测炎性信号途径分子（MyD88、TRAF6等）mRNA表达及肠道菌群构成；酶联免疫检测LPS、IFN-α等水平；生化方法测血糖、胰岛素浓度等。通过探索哈族肥胖儿童肠道菌群改变后ECs与炎性因子、内毒素（肠道菌群相关）、糖代谢指标等相关性，为肥胖相关糖代谢异常机制提供参考，有助于为新的治疗方向提供依据。

肥胖后部分人群可导致胰岛素抵抗（IR），其发生与炎性通路过度激活等相关。内源性大麻素系统（ECs）则被发现与肠道炎症、能量代谢相关。肥胖患病率较高的哈萨克族较少发生糖尿病，为研究肥胖后胰岛素敏感性差异的原因，本项目采用病例对照方法，在学龄儿童群体中探究ECs、肠道菌群及炎症通路之间的相关性，具体采用高效液相色谱‐质谱联用技术检测外周血ECs的AEA和2-AG，RT-PCR检测炎性信号途径分子（MyD88、TRAF6等）mRNA表达及肠道菌群构成；酶联免疫检测LPS、IFN-α等水平；生化方法测血糖、胰岛素浓度等。关键数据如下：①肥胖组的LPS、INFɑ、INFβ、VCAM-1、IL-1β、TNFɑ、IL-6的表达水平均明显升高(P＜0.05)，CXCL2、TLR4、MyD88、RANTES、GAPDH、P65、TRAM的基因相对表达量在观察组水平均明显升高(P＜0.05)；②TLR4（r=0.522，P＜0.05）及其下游MyD88（r=0.689，P＜0.05）、TRAM（r=0.562，P＜0.05）与BMI呈正相关，与HOMR-IR、FIN（P>0.05）均无相关性；③观察组较对照组相比，其乳酸杆菌属（7.88±0.14，7.48±0.33）、梭菌属（5.98±0.26，5.48±0.77）、肠球菌（6.88±0.27，6.58±0.56）拷贝数均增高，而普氏菌属则低于对照组（1.48±0.63，2.33±0.42）；拟杆菌属、双歧杆菌属两组间差异无统计学意义；④观察组AEA浓度[(7.89±1.46)pmol/mL]较对照组升高[(2.99±0.97)pmol/mL]（P＜0.05）；2-AG浓度在两组间差异[(1.88±0.42)pmol/mL,(1.69±0.56)pmol/mL]无统计学意义。⑤关联分析发现LPS与AEA之间存在一定相关性（r=0.529，P＜0.05）；梭菌属与AEA存在一定关联（r=0.589，P＜0.05），与血糖无明显关联（r=0.089，P>0.05）；提示了人体肠道菌群构成改变影响了ECs水平，可能通过LPS激活炎性通路影响到糖代谢通路的表达。本项目通过探索哈族肥胖儿童肠道菌群改变后ECs与炎性因子、内毒素（肠道菌群相关）、糖代谢指标等相关性，为肥胖相关糖代谢异常机制提供参考，有助于为新的治疗方向提供依据。