datasheet1_Excessive Intake of Longan Arillus Alters gut Homeostasis and Aggravates Colitis in Mice.docx

Background: Longan is the fruit of Dimocarpus longan Lour. and the longan arillus has long been used in traditional Chinese medicine possessing various health benefits. However, the excessive intake of longan is found in daily life to cause “shanghuo” syndrome. “Shanghuo” has been linked to increased disease susceptibility. The present study thus aimed to investigate the toxicological outcomes after excessive longan treatment.
Methods: Longan extract at a normal dosage of 4 g/kg and two excess dosages of 8 and 16 g/kg was orally administered to normal C57BL/6J mice for two weeks or to C57BL/6J mice with DSS-induced colitis. Mouse gut microbiome were analyzed by 16S rRNA sequencing. Short chain fatty acid (SCFA) contents in colonic contents were measured by GC-MS. Colon tissue was used for histopathological observation after H and E staining, detection of protein expression by western blot, analysis of gene expression by qPCR, and detection of apoptotic cells by TUNEL assay. ELISA was used for biochemical analysis in serum.
Results: In normal mice, repeated longan intake at excess doses, but not the normal dose, increased infiltration of inflammatory cells, elevated serum levels of TNF-α and IL-6 and reduced production of SCFAs. In DSS-induced colitic mice, longan intake at 4 g/kg did not promote colitis in mice, while excessive longan (8 or 16 g/kg) aggravated colitis in mice, showing increased inflammation, more serious histological abnormalities, increased gut permeability, and increased epithelia injury when compared to DSS alone. Excessive longan induced a significant reduction of microbial diversity in colitic mice, accompanied with aggravated alterations of DSS-associated bacteria including the increase of Proteobacteria phylum and genera of Bacteroides, Akkermansia, Turicibacter and Escherchia-Shigella, and the decrease of norank_f__Muribaculaceae. The changed microbial compositions were accompanied with decreased SCFAs when longan was supplemented with DSS. The aggravated colon injury by excessive intake of longan in colitic mice was tightly correlated with the altered microbial communities and decreased SCFAs production.
Conclusion: Excessive longan intake disturbs gut homeostasis and aggravates colitis via promoting inflammation and altering gut microbe compositions and associated metabolism in mice. Our findings warrant rational longan arillus consumption as a dietary supplement or herbal medicine.

### 背景
龙眼（Longan）为龙眼树（Dimocarpus longan Lour.）的成熟果实，其假种皮（longan arillus）在传统中医药领域应用历史悠久，具备多种保健益处。然而日常生活中发现，过量食用龙眼可引发“上火（shanghuo）”综合征，而“上火”与疾病易感性升高存在关联。因此本研究旨在探究过量摄入龙眼后产生的毒理学效应。

### 方法
本研究采用灌胃方式，给正常C57BL/6J小鼠连续两周灌胃给予正常剂量（4 g/kg）及两种高剂量（8 g/kg、16 g/kg）的龙眼提取物，同时对右旋糖酐硫酸钠（Dextran Sulfate Sodium, DSS）诱导的结肠炎C57BL/6J小鼠开展相同处理。通过16S rRNA测序（16S rRNA sequencing）技术分析小鼠肠道微生物组（gut microbiome）；采用气相色谱-质谱联用仪（Gas Chromatography-Mass Spectrometry, GC-MS）测定结肠内容物中的短链脂肪酸（Short Chain Fatty Acid, SCFA）含量；对结肠组织进行苏木精-伊红（Hematoxylin-Eosin, H&E）染色以开展组织病理学观察，通过蛋白质印迹（Western Blot）检测蛋白表达水平，采用实时荧光定量聚合酶链式反应（quantitative Polymerase Chain Reaction, qPCR）分析基因表达，并用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL assay）检测凋亡细胞；采用酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）对血清进行生化指标分析。

### 结果
在正常小鼠中，过量摄入龙眼（而非正常剂量）可增加炎性细胞浸润，升高血清肿瘤坏死因子-α（Tumor Necrosis Factor-α, TNF-α）与白细胞介素-6（Interleukin-6, IL-6）水平，并降低短链脂肪酸（SCFA）的生成量。在DSS诱导的结肠炎小鼠模型中，4 g/kg剂量的龙眼摄入并未加重结肠炎，而8 g/kg或16 g/kg的过量龙眼摄入则会加剧结肠炎：与单纯DSS处理组相比，过量组小鼠炎症反应更强、组织病理学异常更为严重、肠道通透性更高且上皮损伤更显著。过量摄入龙眼可显著降低结肠炎小鼠的微生物组多样性，同时加剧DSS相关菌群的改变：包括变形菌门（Proteobacteria）、拟杆菌属（Bacteroides）、阿克曼菌属（Akkermansia）、图里西杆菌属（Turicibacter）以及埃希菌-志贺菌属（Escherchia-Shigella）的丰度升高，而未分类Muribaculaceae科（norank_f__Muribaculaceae）的丰度降低。在DSS联合龙眼处理组中，微生物组组成的改变与短链脂肪酸生成量减少相伴发生。结肠炎小鼠中过量摄入龙眼所导致的结肠损伤加剧，与微生物群落改变及短链脂肪酸生成减少密切相关。

### 结论
本研究结果表明，过量摄入龙眼可通过促进炎症反应、改变肠道微生物组成及相关代谢途径，扰乱小鼠肠道稳态并加剧结肠炎。本研究结果提示，将龙眼假种皮（longan arillus）作为膳食补充剂或中药材使用时，需合理控制摄入量。