Liuwei Dihuang Formula Ameliorates Aberrant Cholesterol Metabolism in Postmenopausal APOE-/- Mice by Regulating Intestinal Microbiota and Bile Acid Metabolism
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摘要:
目的 探究六味地黄方调节肠道菌群和胆汁酸代谢改善小鼠绝经后胆固醇代谢异常及可能的作用机制。 方法 利用双侧卵巢摘除的APOE-/-小鼠给予高脂饮食, 复制绝经后胆固醇代谢异常模型, 并给予0.13 mg·kg-1的雌二醇和9.0 g·kg-1的六味地黄方进行治疗。90 d治疗结束后, 生化法检测小鼠血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-c)、高密度脂蛋白胆固醇(HDL-c)和肝脏、粪便中TC水平; ELISA法检测小鼠血清肿瘤坏死因子α(TNF-α)、白介素6(IL-6)、白介素1β(IL-1β)水平; 肝脏HE和油红O染色观察小鼠肝脏脂质损伤; 生化法检测小鼠肝脏、血清、胆囊和粪便中总胆汁酸(TBA)含量; 液相质谱联用技术(UPLC-MS)对小鼠肝脏和粪便胆汁酸组分定量分析; 16S rRNA测序分析小鼠结肠菌群, 并分析粪便胆汁酸组分和肠道菌群之间的相关性。 结果 六味地黄方可以降低模型小鼠血清TC、TG、LDL-c以及TNF-α、IL-1β、IL-6水平(P < 0.01), 提高HDL-c水平(P < 0.01);并减少肝脏脂质堆积, 减轻肝脏损伤; 还可降低血清、胆囊和肝脏TBA水平(P < 0.01), 增加粪便中TBA排出(P < 0.01);减少粪便TαMCA、TβMCA、TCA、TCDCA、TDCA和CA结合型胆汁酸的含量(P < 0.05, P < 0.01), 增加次级胆汁酸LCA的含量(P < 0.01)。六味地黄方恢复了模型小鼠结肠菌群的平衡, 上调粪异杆菌属(Allobaculum)的相对丰度(P < 0.01), 并下调颤螺菌属(Oscillospira)和普氏菌(Prevotella)的相对丰度(P < 0.05)。 结论 六味地黄方通过重塑结肠菌群的结构恢复菌群的平衡, 增加粪便中次级胆汁酸的排出以降低血清和肝脏中胆固醇沉积, 从而改善模型小鼠胆固醇代谢异常。 Abstract:OBJECTIVE To investigate the regulation of intestinal microbiota and bile acid metabolism by Liuwei Dihuang formula (LW) to ameliorate postmenopausal cholesterol metabolism abnormalities and its possible mechanism of action. METHODS APOE-/- mice with bilateral ovariectomy were given a high-fat diet to replicate the postmenopausal cholesterol abnormality model, 0.13 mg·kg-1 estrogen and 9.0 g·kg-1 LW were used for treatment. The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) in serum, TC in liver and feces were measured by biochemical methods. The levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in serum were measured by enzyme-linked immunoassay. The levels of total bile acids (TBA) in the liver, serum, gallbladder and feces were measured by biochemical methods. Hepatic lipid damage was observed by HE and oil red staining. Fecal bile acids were quantified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). 16S rRNA sequencing was used to analyze the colonic microbiota of mice. The correlation between postmenopausal fecal bile acid fractions and intestinal microbiota was analyzed. RESULTS LW reduced serum TC, TG, LDL-c as well as TNF-α, IL-1β, IL-6 levels (P < 0.01) and increased HDL-c level in models (P < 0.01). LW ameliorated liver injury and reduced liver lipid accumulation. LW also reduced serum, gallbladder and hepatic TBA levels (P < 0.01) and increased fecal TBA excretion (P < 0.01); it also reduced fecal levels of conjugated bile acids such as TαMCA, TβMCA, TCA, TCDCA, TDCA and CA(P < 0.05, P < 0.01) and increased the level of secondary bile acid LCA (P < 0.01). LW restored the balance of colonic microbiota in model mice, upregulated the relative abundance of Allobaculum (P < 0.01), and downregulated the relative abundance of Oscillospira and Prevotella (P < 0.05). CONCLUSION LW ameliorates the cholesterol abnormalities in model mice by reshaping the composition of the colonic microbiota to restore the balance of the microbiota and increasing the excretion of fecal secondary bile acids to reduce cholesterol deposition in the serum and liver. -
Key words:
- Liuwei Dihuang formula /
- postmenopausal /
- APOE-/- mice /
- cholesterol metabolism /
- bile acids /
- intestinal microbiota
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图 1 六味地黄方对绝经后APOE-/-小鼠的血清脂质和炎症因子的影响
注: C57+ND.正常对照组; APOE-/-+OVX+HFD.模型组; APOE-/-+OVX+HFD+E2.雌二醇治疗组; APOE-/-+OVX+HFD+LW.六味地黄方治疗组; 与正常对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=5~6。
Figure 1. Effect of LW on serum lipids and inflammatory factors in postmenopausal APOE-/- mice
图 2 六味地黄方对绝经后APOE-/-小鼠肝脏胆固醇和胆汁酸堆积的影响
注: A.肝脏HE和油红O染色图，比例尺=50 μm；B.HE染色评价病理损伤面积百分比的定量分析图；C.油红O染色阳性面积百分比的定量分析图; D~E.肝脏TC和TBA含量图；F.肝脏差异胆汁酸含量图; C57+ND.正常对照组; APOE-/-+OVX+HFD.
模型组; APOE-/-+OVX+HFD+E2.雌二醇治疗组; APOE-/-+OVX+HFD+LW.六味地黄方治疗组; 与正常对照组比较, #P < 0.05, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=5~6。Figure 2. Effect of LW on hepatic cholesterol and bile acid accumulation in postmenopausal APOE-/- mice
图 3 六味地黄方对绝经后APOE-/-小鼠的粪便胆固醇和胆汁酸排泄的影响
注: C57+ND.正常对照组; APOE-/-+OVX+HFD.模型组; APOE-/-+OVX+HFD+E2.雌二醇治疗组; APOE-/-+OVX+HFD+LW.六味地黄方治疗组; 与正常对照组比较, #P < 0.05, ##P < 0.01;与模型组比较, * *P < 0.05, * *P < 0.01。x±s, n=5~6。
Figure 3. Effect of LW on fecal cholesterol and bile acid excretion in postmenopausal APOE-/- mice
图 4 六味地黄方对绝经后APOE-/-小鼠肠道菌群组成的影响
注: A.物种多样性韦恩图；B.物种多样性PCoA聚类图；C.门水平差异物种丰度图; D.属水平益生菌、致病菌与其他菌组成图；E~H.
属水平差异物种丰度图。C57+ND.正常对照组; APOE-/-+OVX+HFD.模型组; APOE-/-+OVX+HFD+E2.雌二醇治疗组; APOE-/-+OVX+HFD+LW.六味地黄方治疗组; 与正常对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=5。Figure 4. Effects of LW on intestinal microbial composition in postmenopausal APOE-/- mice
图 5 粪便胆汁酸组分与肠道菌群相关性分析
注: 红色表示2类变量之间呈正相关关系; 灰色表示2类变量之间呈负相关关系。
*表示在0.05级别(双尾)相关性显著; * *表示在0.01级别(双尾)相关性显著。Figure 5. Correlation analysis of fecal bile acids and intestinal microbiota
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