Effects of Four Commonly Used Chinese Medicines with Clearing Heat and Drying Dampness on Intestinal Flora Mediated Bile Acids and Short Chain Fatty Acids Metabolism
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摘要:
目的 探究在长期临床等效剂量下4种清热燥湿类中药对正常小鼠肠道菌群、胆汁酸及短链脂肪酸的影响。 方法 30只Balb/c雄性小鼠被随机分为对照组、苦参组、黄连组、黄柏组和黄芩组, 每组各6只。除对照组外, 苦参组、黄柏组、黄连组和黄芩组给药剂量分别为2.34、3.12、1.3 g·kg-1和2.6 g·kg-1, 连续灌胃2周, 采用液相色谱-串联质谱法检测粪便中短链脂肪酸和胆汁酸含量, 利用16S rRNA高通量基因测序技术分析肠道内容物中菌群结构变化。 结果 清热燥湿类中药对正常小鼠粪便中6种短链脂肪酸无明显影响。在常见的23种胆汁酸中, 与正常组相比, 黄芩组、苦参组、黄连组和黄柏组分别有4、3、2、1种胆汁酸显著变化。从肠道菌群丰度变化上看, 与对照组相比, 除苦参组外, 黄连组、黄芩组和黄柏组小鼠厚壁菌门均呈现下降趋势, 拟杆菌门均呈现上升趋势, 其中黄连组变化更显著。 结论 苦参、黄柏、黄连和黄芩对正常小鼠长期药效剂量下短链脂肪酸影响较小, 但可以通过改变肠道菌群结构影响胆汁酸含量的变化。 Abstract:OBJECTIVE To explore the effects of four kinds of heat-clearing and dampness-drying herbs on intestinal flora, bile acids and short chain fatty acids (SCFAs) in normal mice at long-term clinical equivalent doses. METHODS Thirty Balb/c male mice were randomly divided into control group, Sophora flavescens Ait. (KS) group, Phellodendron chinense Schneid. (HB) group, Coptis chinensis Franch. (HL) group and Scutellaria baicalensis Georgi (HQ) group, with 6 mice in each group. Except for the control group, KS group, HB group, HL group and HQ group were orally administrated at a dosage of 2.34 g·kg-1, 3.12 g·kg-1, 1.3 g·kg-1 and 2.6 g·kg-1, respectively, for two weeks. The contents of short chain fatty acids and bile acids in feces were detected by liquid chromatography tandem mass spectrometry, and the structural changes of microflora in intestinal contents were detected by 16S rRNA high-throughput gene sequencing technology. RESULTS There was no significant effect of heat-clearing and dampness-drying Chinese medicine on the contents of six short-chain fatty acids in the feces of normal mice. Among the 23 common bile acids, when compared with the control group, the numbers of the significantly changed bile acids in HQ group, KS group, HL group and HB group were 4, 3, 2 and 1, respectively. From the perspective of changes in the abundance of intestinal flora, compared with the control group, except for the KS group, Firmicutes in the HL group, HQ group and HB group all showed a downward trend, and the Bacteroides showed an upward trend, among which the changes in the HL group were the most significant. CONCLUSION Sophora flavescens Ait., Phellodendron chinense Schneid., Coptis chinensis Franch. and Scutellaria baicalensis Georgi have little effect on SCFAs in normal mice, but can affect bile acid content by changing the structure of intestinal flora. -
图 1 黄连、黄芩、黄柏和苦参组小鼠短链脂肪酸含量图
Figure 1. Content of short-chain fatty acids in mice of HL, HQ, HB and KS groups
图 2 黄连、黄芩、黄柏和苦参组小鼠胆汁酸PCA图(A)和热图(B)
Figure 2. PCA (A) and Heatmap (B) of bile acids in mice of HL, HQ, HB and KS groups
图 3 黄连、黄芩、黄柏和苦参组小鼠粪便中差异胆汁酸含量图
注：*P < 0.05, **P < 0.01。
Figure 3. Bile acid content differences in feces of mice in HL, HQ, HB and KS groups
图 4 黄连、黄芩、黄柏和苦参组小鼠肠道菌群PCA(A)和PCoA(B)图
Figure 4. PCA (A) and PCoA (B) of intestinal flora of mice in HL, HQ, HB and KS groups
图 5 黄连、黄芩、黄柏和苦参组小鼠物种组成与差异分析图
注：A.门水平物种组成图; B.属水平物种组成图; C~F.黄连、黄芩、黄柏和苦参组属水平物种差异图。*P < 0.05, **P < 0.01。
Figure 5. Species composition and difference analysis of mice in HL, HQ, HB and KS groups
图 6 黄连、黄芩、黄柏和苦参组小鼠属水平肠道菌群与短链脂肪酸(A)和胆汁酸(B)相关性热图
注：*P < 0.05, **P < 0.01, ***P < 0.001。
Figure 6. Heatmap of correlation between intestinal flora at genus level and short-chain fatty acids (A) or bile acids (B)
表 1 胆汁酸及内标的LC-MS/MS参数
Table 1. LC-MS/MS parameters for bile acids and internal standard
ID Q1 Q3 DP/V CE/eV CXP LCA 375.300 375.300 -210.000 -10.000 -10.000 DCA、HDCA、UDCA、CDCA 391.300 391.300 -230.000 -10.000 -10.000 Dehydro-CA 401.200 401.200 -200.000 -10.000 -10.000 CA、α-MCA、β-MCA、ω-MAC 407.200 407.200 -245.000 -10.000 -10.000 GUDCA、GCDCA、GDCA、HDCA 448.200 74.000 -210.000 -83.000 -9.000 GCA 464.200 74.000 -170.000 -88.000 -11.000 TDCA、THDCA、TUDCA、TCDCA 498.200 80.000 -280.000 -130.000 -9.000 TCA、T-α-MCA 514.200 80.000 -205.000 -130.000 -9.000 CDCA-d4 395.200 359.200 -210.000 -10.000 -10.000 GCA-d4 468.200 74.000 -170.000 -88.000 -11.000 
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表 2 黄连、黄芩、黄柏和苦参组小鼠肠道菌群α多样性指数比较(x±s)
Table 2. Comparison of intestinal flora α diversity indices of mice in HL, HQ, HB and KS groups(x±s)
组别 ACE Chao Shannon Simpson C 401.18±80.85 405.41±86.75 3.67±0.49 0.07±0.05 HB 353.61±18.4 360.17±15.31 3.52±0.32 0.08±0.04 HL 255.31±28.56**** 257.02±27.55**** 2.9±0.38** 0.13±0.05 HQ 376.42±54.55 379.69±53.43 3.81±0.23 0.04±0.01 KS 455.58±26.99 470.4±30.29 3.73±0.49 0.08±0.05 注: 与C组比较，**P < 0.01, ****P < 0.000 1。
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