肝脏Adora1受体介导非酒精性脂肪肝形成与黄芪多糖的干预机制研究

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, which is part of metabolic syndromes. NAFLD is also characterized as Spleen-Qi deficiency in Traditional Chinese Medicine (TCM). There are very few drugs in clinic that are of good safety and efficacy so far. According to the TCM theory, patients with metabolic syndrome could be treated by enhancing Spleen-Qi with some traditional formulas. We previously confirmed the anti-obesity effect of a traditional formula Fangji Huangqi decoction, as well as the extraction of Hangqi ( Astragali Radix). Moreover, we found the main component of Hangqi, Astraglus polysaccharides (APS) also inhibited the fatty liver formation in high-fat diet fed mice, restored the gut dysbiosis, increased the relative abundance of short-chain fatty acids (SCFAs)-producing bacteria, and contents of SCFAs in feces. We also screened the hepatic gene expression by using gene microarray and identified that hepatic Adora1 gene was significantly up-regulated in high-fat diet fed mice, but was down-regulated by APS. In addition, our results indicated that the up-regulation of hepatic Adora1 was prior to the formation of NAFLD in mice. Then, our preliminary data indicated that the DPCPX, a specific antagonist of Adora1, could inhibited the formation of NAFLD induced by high-fat diet in mice. As a result, we proposed our hypothesis that “hepatic Adora1 may be involved in modulating the formation of NALFD, and APS could be metabolized into SCFAs by gut microbiota to suppress hepatic Adora1 resulting to attenuation of NALFD”. In this project, we will first test the impacts on NAFLD formation by specifically knocking down or overexpressing the hepatic Adora1 expression by using a liver-specific Adora1 knockout mice, as well as injection with adenovirus-mediated Adora1 plasmid in mice. Then, we will elucidate the underlying mechanisms of hepatic Adora1 in modulating fatty liver development both in vivo and in vitro. This study will uncover a potential therapeutic target for NALFD----hepatic Adora1, and elucidate the mechanisms of APS in treating NAFLD. The results will promote the R&D of APS for NAFLD therapy by strengthening its scientific basis.

非酒精性脂肪肝（NAFLD）是最常见的慢性肝病，至今仍缺乏安全、有效的药物。根据肥胖的“从脾论治”思想，我们证实了黄芪多糖(APS)的减肥作用，并发现APS显著抑制小鼠NAFLD形成、恢复小鼠肠道菌结构、增加肠道产短链脂肪酸（SCFAs）细菌丰度和SCFAs的含量；利用基因芯片筛选发现APS抑制NAFLD小鼠肝脏腺苷A1受体（Adora1）表达，且发现高脂喂养小鼠肝脏Adora1上调早于脂肪肝形成；证实Adora1拮抗剂抑制小鼠NAFLD形成。因此提出假说“肝脏Adora1很可能调节NAFLD形成，APS可能是经细菌代谢成SCFAs调控肝脏Adora1防治脂肪肝”。本项目将通过构建肝脏特异性Adora1敲除小鼠和过表达等方式，在动物和细胞水平验证该假说。该研究有望发现NAFLD的新靶标----肝脏Adora1，揭示APS调控肝脏Adora1抑制脂肪肝的机理，为APS研究开发提供新的科学证据

本项目利用LoxP-Cre系统构建了肝脏特异性敲除Adora1小鼠，并利用腺相关病毒（AAV）为载体构建了肝脏特异性过表达Adora1小鼠，通过正反两个方面明确了肝脏Adora1激活对于NAFLD的保护作用。利用一系列的分子生物学方法，进一步揭示了肝脏 Adora1 激活减少 SREBP1c 蛋白成熟和核定位，抑制肝细胞的脂肪酸从头合成，从而抑制脂肪肝形成。因此，肝脏 Adora1不是黄芪多糖（APS）改善NAFLD的靶标。为了解释APS防治NAFLD的机理，我们利用靶向代谢组学方法发现并证实乙酸是APS发挥防治NAFLD作用的关键分子。进一步我们利用16S rDNA测序结合宏基因组学技术，发现APS显著增加了肠道Desulfovibrio vulgaris (D. vulgaris)菌的丰度，且意外发现D. vulgaris菌在体外具有直接产生乙酸的能力。我们还发现D. vulgaris菌干预可以增加动物肠道中乙酸的含量，并具有改善脂肪肝的作用，提示APS通过调节肠道菌群，增加肠道D. vulgaris菌的含量，促进肠道乙酸的生成可能是其发挥防治NAFLD的关键机理。综上，本项目的研究证实了APS防治NAFLD的作用依赖于其对动物肠道菌群的调节，尤其是增加肠道D. vulgaris菌含量和乙酸水平，而D. vulgaris菌可能是APS干预下产生乙酸的关键功能菌之一。另外，通过本项目的研究，我们首次证实肝脏Adora1的激活对于脂肪肝的形成具有保护作用，因此，我们推测高脂饮食喂养动物中肝脏Adora1的激活可能是机体一种适应性反应。肝脏Adora1可能是NAFLD治疗的关键新靶标，值得进一步的深入研究。

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