目的采用Illumina高通量測序技術(shù)和實時熒光定量PCR（Real-Time PCR,RT-PCR）法研究何首烏（PM）致肝損傷與腸道微生物組間的關(guān)系,并驗證兩種定量方法的一致性。方法雄性SD大鼠隨機分為5組：對照組、脂多糖（LPS）組、LPS+對乙酰氨基酚（APAP）組、PM組和LPS+PM組；大鼠尾iv給予4.0 mg/kg LPS建立肝損傷模型,各組相應(yīng)每天1次ig給予0.625 g/kg APAP和12 g生藥/kg PM,記錄大鼠體質(zhì)量；分別于造模后2、14 h、5和8 d,對大鼠糞便中細菌16SrRNA基因的V4高變區(qū)進行Illumina高通量測序,根據(jù)測序結(jié)果得出的差異物種,采用RT-PCR進行驗證；取造模后8 d大鼠肝臟組織,HE染色,光學(xué)顯微鏡觀察。結(jié)果大鼠肝臟病理學(xué)檢查結(jié)果顯示,與對照組比較,LPS組大鼠存在肉芽腫,PM組無異常病變；與LPS組比較,LPS+PM大鼠肝細胞出現(xiàn)輕度變性和微小肉芽腫增多,LPS+APAP組可見微小肉芽腫和淋巴細胞浸潤。Illumina高通量測序結(jié)果提示,與對照組比較,隨著PM給藥次數(shù)增加,單獨給予PM的大鼠腸道微生物無顯著變化；LPS+PM組表現(xiàn)為腸球菌科和毛螺旋菌科細菌逐漸增加,乳桿菌屬細菌減少,且與LPS組有差異；RT-PCR結(jié)果顯示,與對照組比較,隨著PM給藥次數(shù)的增加,單獨給予PM的大鼠腸道微生物無顯著變化；LPS+PM組腸球菌科、毛螺旋菌科細菌數(shù)顯著增加（P<0.05）,乳桿菌屬細菌數(shù)顯著減少（P<0.05）,且與LPS組比較有顯著差異。結(jié)論 PM肝損傷大鼠存在不同程度的菌群失衡,且Illumina高通量測序和RT-PCR檢測結(jié)果具有良好的一致性,但Illumina高通量測序技術(shù)可獲得更多的微生物信息,更具優(yōu)勢。

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.

重大新藥創(chuàng)制科技重大專項（2013ZX09302303；2012ZX09301003-001-008）；北京市科委基金項目（Z131100006513010）；廣東省科學(xué)院創(chuàng)新藥物安全性評價研究團隊（2016GDASRC-0104）