黄芩汤对炎症相关性结直肠癌小鼠炎症及短链脂肪酸相关肠道菌群的影响

Effect of Huangqintang on Inflammation and Short-chain Fatty Acid-related Gut Microbiota in Mouse Model of Inflammation-associated Colorectal Cancer

摘要:

目的 构建氧化偶氮甲烷/葡聚糖硫酸钠（AOM/DSS）炎症相关性结直肠癌（CAC）小鼠模型，采用16S rRNA高通量测序方法，探讨CAC模型小鼠发生发展进程中黄芩汤对小鼠肠道菌群结构的影响。方法 将225只C57BL/6J小鼠随机分为5组，分别为正常组、模型组、美沙拉嗪组、黄芩汤高、低（18、9 g·kg^-1）剂量组。每组45只，除正常组外，每只小鼠在1周内分别于第1天和第5天进行10 mg·kg^-1 AOM注射，然后给予1.5% DSS溶液7 d，之后更换为无菌水14 d，此为1周期，共计3周期。在给予DSS第1天同时给予美沙拉嗪和黄芩汤高、低剂量组药物灌胃，正常组和模型组给予纯水进行灌胃，每日1次，直至3个周期结束。根据CAC发病进程，分为炎症阶段、增殖阶段及肿瘤生成阶段。每个周期结束后，分别测量各组小鼠体质量、结肠长度、记录小鼠结肠肿瘤数目，并进行疾病活动指数（DAI）评分。酶联免疫吸附测定（ELISA）检测每个周期小鼠血清中白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）及胃肠道肿瘤标志物糖类抗原199（CA199）含量；苏木素-伊红（HE）染色观察结肠病变。同时，采集正常组、模型组和黄芩汤高剂量组小鼠新鲜粪便3~5粒，按照DNA提取试剂盒说明书步骤提取小鼠粪便 DNA，进行16S rRNA高通量测序并分析结果。结果 与正常组比较，模型组小鼠3个阶段体质量显著下降（P<0.01），DAI评分逐渐上升，结肠长度明显缩短（P<0.05）。与正常组比较，增殖阶段模型组小鼠，IL-1β表达上升（P<0.05），IL-6、TNF-α表达明显上升（P<0.05）；CA199在炎症和增殖阶段升高，肿瘤阶段显著升高（P<0.01）。HE病理结果显示，与正常组比较，模型组小鼠，炎症阶段炎症细胞浸润明显，随着疾病进展到增殖和肿瘤阶段，肌层增厚，黏膜层细胞异常增生，最终形成晚期高级别上皮内肿瘤病变。黄芩汤给药后，与模型组比较，在炎症、增殖和肿瘤生成阶段各组小鼠体质量未见明显改善，DAI评分降低，结肠长度增加；肿瘤生成阶段结肠长度显著增加（P<0.01）。在肿瘤生成阶段，黄芩汤给药组显著抑制了肿瘤生成和生长，CAC小鼠肿瘤数目显著减少（P<0.01）。黄芩汤高、低剂量组IL-6、TNF-α、IL-1β在炎症、增殖和肿瘤阶段降低，IL-6、TNF-α 3个阶段均明显降低（P<0.05，P<0.01）；CA199在炎症阶段降低，增殖和肿瘤阶段明显降低（P<0.05，P<0.01）。HE结果显示，与模型组比较，给药组有效减轻了炎症反应、异常细胞增殖，延缓了肿瘤发生。肠道菌群结果显示，与正常组比较，在炎症、增殖和肿瘤生成阶段，模型组小鼠Alpha、Beta多样性降低，肠道菌群结构变化明显。与模型组比较，黄芩汤给药后，在肿瘤生成阶段明显调节模型小鼠的物种数量及Alpha、Beta多样性。黄芩汤给药后，在炎症、增殖和肿瘤生成阶段纠正肠道菌群回归正常丰度与多样性；在炎症阶段，门水平上，正向调节了小鼠的2种差异菌门（厚壁菌门、拟杆菌门）与3种差异菌属（Muribaculaceae、Rikenellaceae_RC9_gut_group、Flavonifractor）；在增殖阶段，门水平上，正向调节了小鼠的2种差异菌门（拟杆菌门、髌骨菌门）与5种差异菌属（Muribaculaceae、Rikenellaceae_RC9_gut_group、Candidatus_Saccharimonas、norank_f__UCG-010、Allobaculum）；肿瘤生成阶段，正向调节了小鼠的2种差异菌门（变形菌门、髌骨菌门）与8种差异菌属（Muribaculaceae、Candidatus_Saccharimonas、norank_f__UCG-010、Lachnospiraceae_UCG-006、Allobaculum、Bacteroides、Lachnospiraceae_NK4A136_group、Flavonifractor）。结论 黄芩汤能够较好地干预AOM/DSS 诱发的小鼠结肠炎癌转化进程，其作用机制可能与其纠正CAC模型小鼠炎症和短链脂肪酸相关菌群紊乱，直接或间接延缓结肠炎癌转化相关。

Abstract:

Objective To construct a mouse model of inflammation-associated colorectal cancer （CAC） by using azoxymethane （AOM）/dextran sulfate sodium （DSS） and investigate the effect of Huangqintang on the gut microbiota structure of mice during the occurrence and development of CAC by 16S rRNA gene high-throughput sequencing.Method A total of 225 C57BL/6J mice were randomized into 5 groups （n=45）： Normal， model， positive drug （mesalazine）， and high （18 g·kg^-1） and low （9 g·kg^-1）-dose Huangqintang. Except those in the normal group， each mouse was injected with 10 mg·kg^-1 AOM on day 1 and day 5 within 1 week and then given 1.5% DSS solution for 7 days， which was then changed to sterile water for 14 days. This process referred to as one cycle， and mice were treated for a total of 3 cycles. On the first day of DSS treatment， mice were administrated with corresponding drugs by gavage， and the normal group and the model group were administrated with pure water by gavage， once a day until the end of the third cycle. The progression of CAC was divided into inflammation， proliferation， and tumorigenesis stages. At the end of each cycle， the body weight and colon length were measured for mice in each group， and the number of colon tumors in mice was recorded. Meanwhile， the disease activity index （DAI） was determined. The serum levels of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and carbohydrate antigen-199 （CA199）， a tumor marker in the gastrointestinal tract of mice， were measured by ELISA. Hematoxylin-eosin staining was employed to observe colon lesions. At the same time， 3-5 pellets of fresh feces of mice in the normal group， model group， and high-dose Huangqintang group were collected， from which the fecal DNA of mice was extracted for 16S rRNA gene high-throughput sequencing.Result Compared with the normal group， the model group showed decreased body weight （P<0.01）， increased DAI， and shortened colon length （P<0.05） at the three stages. Compared with the normal group， the model group showed elevated levels of IL-1β， IL-6， and TNF-α （P<0.05） at the proliferation stage and elevated levels of CA199 at the inflammation， proliferation， and tumorigenesis （P<0.01） stages. Compared with the normal group， the model group presented obvious infiltration of inflammatory cells at the inflammation stage， thickening of the muscle layer and abnormal proliferation of mucosal layer cells at the proliferation and tumorigenesis stages， and final formation of advanced intraepithelial tumor lesions. Compared with the model group， the Huangqintang groups showed no significant improvement in the body weight， decreased DAI score， and increased colon length at the three stages， and the increase of colon length in the tumorigenesis stage was significant （P<0.01）. At the tumorigenesis stage， the administration of Huangqintang inhibited tumor formation and growth， reduced the number of tumors （P<0.01）， lowered the levels of IL-6 （P<0.05， P<0.01）， TNF-α （P<0.05， P<0.01）， and IL-1β at the three stages， and decreased CA199 at the inflammation stage as well as at the proliferation and tumorigenesis stages （P<0.01， P<0.05）. Compared with the model group， the administration of Huangqintang reduced inflammation and abnormal cell proliferation， delaying the occurrence of tumors. Compared with the normal group， the model group showcased decreased alpha and beta diversity and altered structure of gut microbiota at the inflammation， proliferation， and tumorigenesis stages. The administration of Huangqintang adjusted the abundance and diversity of gut microbiota to the normal levels. At the inflammation stage， Huangqintang positively regulated two differential phyla （Firmicutes and Bacteroidetes） and three differential genera （Muribaculaceae， Rikenellaceae_RC9_gut_group， and Flavonifractor） in mice. At the proliferation stage， Huangqintang positively regulated two differential phyla （Bacteroidetes and Patescibacteria） and five differential genera （Muribaculaceae， Rikenellaceae_RC9_gut_group， Candidatus_Saccharimonas， norank_f__UCG-010， and Allobaculum）. At the tumorigenesis stage， Huangqintang positively regulated two differential phyla （Proteobacteria and Patescibacteria） and eight differential genera （Muribaculaceae， Candidatus_Saccharimonas， norank_f_UCG-010， Lachnospiraceae_UCG-006， Allobaculum， Bacteroides， Lachnospiraceae_NK4A136_group， and Flavonifractor） in mice.Conclusion Huangqintang can intervene in the AOM/DSS-induced transformation of inflammation to CAC in mice by correcting inflammation and short-chain fatty acid-related microbiota disorders.