粗壮女贞提取物对肠道菌群的调节与减肥机制的研究

Obesity and its related metabolic disorders have become an urgent public health problem. Recent studies demonstrated that regulating the intestinal flora could be a new potential strategy for the obesity prevention and treatment. In the preliminary study, we have found that the extraction of Ligustrum robustum which is abound in the mountain area of Yunnan, Guizhou, and Sichuan etc. have effects on the regulation of the intestinal flora and prevention of obesity. In order to further confirm and explore the effect and its mechanism, in vitro antibacterial test will be used to evaluate the bacteriostatic effect of different Ligustrum robustum extracts, and in vivo animal models for weight losing evaluation. Tranditional culture methods, real-time RCR and T-RFLP will be employed to detect the changes of gut flora and NMR analysis on stool metabolomics will be carried out to detect its relationship with gut flora changes. In vitro enzymatic reaction and flow cytometry will be used to study the effect of extraction of Ligustrum robustum on fatty acid synthase, and then an epidemiological survey will be conducted on the population who have a drinking habit of the Ligustrum robustum. By combining the results of in vitro antibacterial test and fatty acid synthase enzyme assay, in vivo animal intervention experiments and population-based study, we could not only interpret the effect of Ligustrum robustum on prevention and treatment of the obesity of experimental animals and population, but also clarify its possible mechanisms. The results of this proposed project will provide direct evidences for the further use of Ligustrum robustum as a safe therapeutic way on obesity and its related metabolic disorders prevention and treatment.

肥胖及相关的代谢性疾病引起的危害已成为亟待解决的公众健康问题。国内外研究结果显示，调节肠道菌群可能成为防治肥胖的新策略。项目组前期研究结果提示，盛产于云、贵、川等山区的粗壮女贞可能具有调节肠道菌群和预防肥胖的效果。为了进一步证实和研究粗壮女贞对肠道菌群的调节与减肥机制，本课题将通过体外抑菌实验探讨抑菌和成分的关系；动物干预实验观察不同提取物的减肥效果；采用培养法和分子生物学方法检测肠道菌群的改变，探讨肠道菌群改变和减肥的关系；进行粪便代谢组研究，探讨肠道菌群改变与粪便代谢组的关系；采用体外酶促反应和流式细胞术，探讨粗壮女贞对脂肪酸合酶的影响；调查粗壮女贞饮用人群和非饮用人群的肥胖相关信息，初步探讨其人群抗肥情况。从体外实验、体内实验以及人群调查三方面，阐明粗壮女贞调节肠道菌群及其减肥的可能机制，为粗壮女贞预防和治疗肥胖提供直接依据，为寻找安全的防治肥胖及相关的代谢性疾病的方法提供参考。

本研究观察了粗壮女贞不同提取物的体外抑菌效果并分析其主要成分，探索了粗壮女贞提取物防治肥胖的效果及对肠道菌群的影响，研究了粗壮女贞水提物减少细胞内甘油三酯堆积及其机制，通过人群干预试验，探讨了饮用粗壮女贞苦丁茶后的抗肥降脂效果及机制。.研究发现：①粗壮女贞水提物具有选择性抑菌作用，对革兰阳性菌的抑制性总体强于革兰阴性菌，对金黄色葡萄球菌耐药菌株、产气荚膜梭菌等致病菌有较好抑制作用，对婴儿双歧杆菌、德氏乳杆菌无明显抑制；抑菌物质可能主要为极性较大物质，正丁醇萃取层、剩余水层黄酮含量和粗多糖含量较高可能与其抑菌活性有关。②动物实验发现粗壮女贞可有效预防高脂饮食动物宿主肥胖，帮助高脂饮食宿主胆固醇、甘油三酯和LDL-C/HDL-C等指标恢复正常（P<0.05）。此外粗壮女贞还可提高高脂饮食动物宿主肠道内乳杆菌、降低肠球菌，且随着粗壮女贞剂量的增高，实验组肠道菌群与肥胖组肠道菌群的相似性成反比（P<0.05）。③粗壮女贞可减少胞内甘油三酯堆积，或通过增加PPARα及p-AMPK的表达（P<0.05）调控甘油三酯及游离脂肪酸代谢过程关键酶活性实现降脂。粗壮女贞具有抑制细胞分化的效果，并可通过上调表达PPARγ、SREBP-1c和C/EBPα（P<0.05）等途径来抑制脂肪细胞的过度肥大。粗壮女贞苦丁茶的抗氧化能力在多个方面优于或相当于绿茶和普洱茶；并对氧化损伤的HUVEC细胞有保护效应（P<0.05）。④粗壮女贞饮用可降低受试者体重、血糖、谷丙转氨酶、谷草转氨酶、脂联素、瘦素等指标；提升高密度脂蛋白和GIP水平（P<0.05）。⑤此外，我们还发现粗壮女贞水提物具有选择性杀伤肿瘤细胞的能力，并可延长裸鼠的生存时间（P<0.05）。.综上，本研究发现粗壮女贞可能通过调节肠道菌群，调控甘油三酯及游离脂肪酸代谢过程关键酶活性、抑制脂肪细胞分化肥大等多途径来实现减肥降脂功效。本研究从体外实验、动物实验、人群实验三个层次证实了粗壮女贞的抗肥效果和可能机制，为人们寻找安全的预防和治疗肥胖及相关代谢性疾病的方法提供参考，达到了预期研究目的。