目的采用Illumina高通量测序技术和实时荧光定量PCR（Real-Time PCR,RT-PCR）法研究何首乌（PM）致肝损伤与肠道微生物组间的关系,并验证两种定量方法的一致性。方法雄性SD大鼠随机分为5组：对照组、脂多糖（LPS）组、LPS+对乙酰氨基酚（APAP）组、PM组和LPS+PM组；大鼠尾iv给予4.0 mg/kg LPS建立肝损伤模型,各组相应每天1次ig给予0.625 g/kg APAP和12 g生药/kg PM,记录大鼠体质量；分别于造模后2、14 h、5和8 d,对大鼠粪便中细菌16SrRNA基因的V4高变区进行Illumina高通量测序,根据测序结果得出的差异物种,采用RT-PCR进行验证；取造模后8 d大鼠肝脏组织,HE染色,光学显微镜观察。结果大鼠肝脏病理学检查结果显示,与对照组比较,LPS组大鼠存在肉芽肿,PM组无异常病变；与LPS组比较,LPS+PM大鼠肝细胞出现轻度变性和微小肉芽肿增多,LPS+APAP组可见微小肉芽肿和淋巴细胞浸润。Illumina高通量测序结果提示,与对照组比较,随着PM给药次数增加,单独给予PM的大鼠肠道微生物无显著变化；LPS+PM组表现为肠球菌科和毛螺旋菌科细菌逐渐增加,乳杆菌属细菌减少,且与LPS组有差异；RT-PCR结果显示,与对照组比较,随着PM给药次数的增加,单独给予PM的大鼠肠道微生物无显著变化；LPS+PM组肠球菌科、毛螺旋菌科细菌数显著增加（P<0.05）,乳杆菌属细菌数显著减少（P<0.05）,且与LPS组比较有显著差异。结论 PM肝损伤大鼠存在不同程度的菌群失衡,且Illumina高通量测序和RT-PCR检测结果具有良好的一致性,但Illumina高通量测序技术可获得更多的微生物信息,更具优势。

Objective To study the relationship between Polygonum multiflorum (PM) induced liver injury and gut flora by Illumina high-throughput sequencing and Real-time PCR (RT-PCR). Methods Male SD rats were randomly divided into control group, LPS group, LPS + acetaminophen (APAP) group, PM group and LPS + PM group. Rats were iv administered with 0.004 g/kg LPS in tail to make liver injury model. Rats in corresponding group were ig administered with 0.625 g/kg APAP and 12 g/kg (crude drug) PM once a day, record the body weight of rats at the same time. Illumina high-throughput sequencing was used to sequence the V4 hypervariable region of 16S rRNA gene of rat fecal bacteria. According to the results of the sequencing of different species, taken the RT-PCR to test and verify the biomarker, observed the relationship between PM induced liver injury and gut flora, and compared the consistency of the two methods. The liver tissue of rats was stained with HE and observed by optical microscope 8 d after modeling. Results Histopathological analysis revealed that, compared to control group, LPS group existed some microgranuloma, PM group have no significances; Compared to LPS group, LPS + PM group induced mild degeneration of liver cells and increased the number of microgranuloma, LPS + APAP group existed some micro granuloma and lymphocytes infiltration. Illumina high-throughput sequencing showed that, compared to control group, as the times of taking PM, the bacterium number of Enterococcaceae and Lachnospiraceae increased, while the Lactobacillus decreased in LPS + PM group, and had a difference LPS with group. The RT-PCR showed the bacterium number of Enterococcaceae and Lachnospiraceae increased, while the Lactobacillus decreased in LPS + PM group, compared to control group, and had an obvious difference with LPS group. Conclusion The liver injury rats' gut flora induced by PM show imbalance to some extent, and the two methods apply in quantitative analysis shows a good consistency, while Illumina high-throughput sequencing obtains more information about microbiota and has a greater advantage than RT-PCR.

重大新药创制科技重大专项（2013ZX09302303；2012ZX09301003-001-008）；北京市科委基金项目（Z131100006513010）；广东省科学院创新药物安全性评价研究团队（2016GDASRC-0104）