补中益气汤体内外对脾虚证两种特征菌数量的影响

Effects of Buzhong Yiqi Decoction on the Number of Two Characteristic Bacteria in Spleen Deficiency Syndrome in Vitro and in Vivo

目的前期研究发现脾虚证患者的肠道菌群发生显著变化,针对其中丰度明显改变的艾克曼菌ATCC BAA-835(Akkermansia muciniphila ATCC BAA-835,AKK)和单形拟杆菌(Bacteroides uniformis,BU)建立实时荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,QPCR)检测方法,通过体外孵育和体内动物实验考察补中益气汤对其的影响。方法采用模拟人体胃肠道代谢方法,将补中益气汤水煎剂与人工胃/肠液孵育后,与健康人群/脾虚证患者粪便共孵育,将粪便样品、24 h孵育样品进行微生物多样性测序;筛选AKK和BU特异性引物并建立其QPCR分析方法,检测补中益气汤与健康人群/脾虚证患者粪便共孵育后AKK和BU菌不同时间点的动态变化;采用番泻叶灌胃塑造脾虚证小鼠模型并给予补中益气汤治疗,考察补中益气汤对脾虚证小鼠体内AKK和BU菌含量的影响。结果测序结果表明,健康人群和脾虚证患者肠道菌群结构存在明显差异,补中益气汤对粪便孵育液中肠道微生物具有一定的调节作用。方法学考察结果表明,建立的AKK和BU的QPCR定量检测方法重复性、稳定性良好,定量结果可靠。补中益气汤体外与健康人群/脾虚证患者粪便共孵育,其中AKK和BU呈现动态变化过程,其对健康人群/脾虚证患者粪便中AKK和BU均具有一定影响。孵育24 h,补中益气汤对脾虚证患者粪便样本中的AKK菌无显著影响,但可显著降低脾虚证患者粪便中BU菌含量(P<0.01)并恢复至健康人的水平。体内动物实验结果表明,补中益气汤体内可显著增加脾虚证小鼠粪便样本中AKK菌含量(P<0.01)并降低BU菌含量(P<0.01)。结论补中益气汤体内外可直接调节BU菌含量,降低BU菌含量可能是其治疗脾虚证的分子作用机制之一。

Objective Previous studies found significant changes in the gut microbiota of patients with spleen deficiency, and a real-time quantitative real-time polymerase chain reaction(QPCR) assay was established for Akkermansia muciniphila ATCC BAA-835(AKK) and Bacteroides uniformis(BU) in which the abundance was significantly altered. To explore the effects of Buzhong Yiqi Decoction the intestinal flora of patients with spleen deficiency through in vitro incubation and in vivo animal experiments.Methods Buzhong Yiqi Decoction was incubated in artificial gastric juice, artificial intestinal juice, and intestinal bacteria sequentially, then Illumina high-throughput sequencing technology was used to detect bacterial structure of fecal and 24 h incubation samples. After screening the specific primers of AKK and BU, we attempted to establish quantitative real-time polymerase chain reaction(QPCR)analysis methods to detect their quantitative changes in different time points samples. Mice were orally administered Sennae Folium decoction to establish a spleen-deficiency model, then treated with Buzhong Yiqi Decoction. Fresh feces were collected to detect AKK and BU amount.Results The sequencing results showed that the structure of intestinal microflora between healthy people and spleen-deficiency patients was different, and Buzhong Yiqi Decoction had modulation effects on the intestinal microflora in incubation samples. The established QPCR methods had good repeatability, stability and reliability. In vitro, Buzhong Yiqi decoction had a certain effect on the AKK and BU in feces of healthy population/spleen deficiency patients, in which AKK and BU showed a dynamic process in 0-24 h incubation. After 24 h incubation, Buzhong Yiqi Decoction had no significant effects on AKK amount in spleendeficiency patients, but could significantly reduce BU amount(P<0.01) and made it no difference with healthy people.What’s more, in vivo, Buzhong Yiqi Decoction could increase AKK amount and reduce BU amount in spleen-deficiency mice significantly(P<0.01).Conclusion Buzhong Yiqi Decoction can directly adjust the amount of BU, and decreasing the amount of BU may be one of the molecular mechanisms of its treatment on spleen-deficiency syndrome.