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基于胆汁酸代谢组学探讨复方大黄煎剂保留灌肠对轻微型肝性脑病大鼠模型的治疗作用
DOI: 10.3969/j.issn.1001-5256.2023.10.012
伦理学声明：本研究方案于2022年8月28日经由广西中医药大学实验动物伦理委员会审批，批号：DW20220826－162，符合实验室动物管理与使用准则。所有实验操作严格按照《关于善待实验动物的指导性意见》进行。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：王萌、毛德文负责课题设计；杜沅沁负责资料分析，撰写论文并最后定稿；钟瑞熙、徐健、黄国初、黄良江负责实验实施，分析数据；黄晶晶、谭钦文负责数据整理；姚春负责实验指导。
Therapeutic effect of retention enema with compound rhubarb decoction on a rat model of minimal hepatic encephalopathy based on bile acid metabolomics
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摘要:
目的 观察大黄煎剂保留灌肠对轻微型肝性脑病（MHE）大鼠模型的治疗作用，并基于胆汁酸（BA）代谢组学探讨其作用机制。 方法 将55只雄性SD大鼠随机分为空白组（NC组，n＝10）、肝性脑病组（HE组，n＝15）、轻微型肝性脑病组（MHE组，n＝15）和MHE大黄煎剂治疗组（MHEY组，n＝15）。腹腔注射CCl4和硫代乙酰胺（TAA）诱导MHE、HE大鼠模型，给药2周后处死。检测血清AST、ALT、ALP、TBil、总胆汁酸（TBA）和血氨的含量；取结肠内容物检测pH值；取肝组织和脑组织进行HE染色观察大鼠肝组织病理形态学改变；取胆汁用LC－MS进行BA靶向代谢组学分析。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD－t检验。 结果 与NC组比较，HE组、MHE组寻台潜伏期（造模后、用药后）显著增加，穿台次数显著减少（P值均<0.05）；与MHE组相比，MHEY组寻台潜伏期（用药后）显著降低而穿台次数显著增加，HE组寻台潜伏期显著增加而穿台次数显著减少（P值均<0.05）。与NC组比较，HE组和MHE组AST、ALT、ALP、TBil、TBA、血氨及结肠pH值显著增加（P值均<0.05）；与MHE组相比，MHEY组AST、ALT、ALP、TBil、TBA及血氨及结肠pH值减少（P值均<0.05），HE组AST、ALT、ALP、TBil、TBA、血氨及结肠pH值增加（P值均<0.05）。MHE组TBA、初级BA和次级BA均低于NC组（P值均<0.05）；HE组TBA和初级BA低于MHE组（P值均<0.05）；MHEY组TBA、初级BA高于MHE组（P值均<0.05）。MHE组与NC组对比，GCDCA、GUDCA、GHDCA、TCDCA、TUDCA、GLCA和TLCA减少（P值均<0.05），γ－MCA、THCA、7－KDCA、AlloLCA、α－MCA增加（P值均<0.05）。MHEY组与MHE组对比，THDCA、TMCA、TCDCA、TUDCA和TLCA增加（P值均<0.05）。 结论 大黄煎剂保留灌肠可通过调节BA肠－肝循环改善CCl4和TAA诱导的MHE大鼠模型肝损伤和认知功能，其作用机制可能与牛磺酸结合BA合成增加有关。 Abstract:Objective To investigate the therapeutic effect of rhubarb decoction （RD） retention enema on a rat model of minimal hepatic encephalopathy （MHE） and its mechanism of action based on bile acid （BA） metabolomics. Methods A total of 55 male Sprague－Dawley rats were randomly divided into blank group （NC group with 10 rats）， hepatic encephalopathy group （HE group with 15 rats）， MHE group with 15 rats， and MHE+rhubarb decoction treatment group （MHEY group with 15 rats）. Intraperitoneal injection of carbon tetrachloride （CCl4） and thioacetamide （TAA） was performed to establish a rat model of MHE or HE， and the rats were sacrificed after 2 weeks of administration. The serum levels of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， alkaline phosphatase （ALP）， total bilirubin （TBil）， and total bile acid （TBA） and the concentration of blood ammonia were measured； the colonic contents were collected to measure pH value； liver and brain tissue samples were collected， and HE staining was used to observe the histopathological changes of the liver； the bile was collected， and liquid chromatography－mass spectrometry was used to perform BA－targeted metabolomics analysis. Continuous data were expressed as mean±standard deviation； a one－way analysis of variance was used for comparison between multiple groups， and the least significant difference t－test was used for further comparison between two groups. Results Compared with the NC group， the HE group and the MHE group had a significant increase in searching platform latency （after modelling and after administration） and a significant reduction in the number of platform crossings （all P<0.05）； compared with the MHE group， the MHEY group had a significant reduction in searching platform latency （after administration） and a significant increase in the number of platform crossings， and the HE group had a significant increase in searching platform latency and a significant reduction in the number of platform crossings （all P<0.05）. Compared with the NC group， the HE group and the MHE group had significant increases in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）； compared with the MHE group， the MHEY group had significant reductions in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）， and the HE group had significant increases in AST， ALT， ALP， TBil， TBA， blood ammonia， and colon pH value （all P<0.05）. The MHE group had significantly lower TBA， primary BA， and secondary BA than the NC group （all P<0.05）； compared with the MHE group， the HE group had significantly lower TBA and primary BA （all P<0.05）， and the MHEY group had significantly higher TBA and primary BA （all P<0.05）. Compared with the NC group， the MHE group had significant reductions in GCDCA， GUDCA， GHDCA， TCDCA， TUDCA， GLCA， and TLCA （all P<0.05） and significant increases in γ－MCA， THCA， 7－KDCA， AlloLCA， and α－MCA （all P<0.05）， and compared with the MHE group， the MHEY group had significant increases in THDCA， TMCA， TCDCA， TUDCA， and TLCA （all P<0.05）. Conclusion RD retention enema can improve liver injury and cognitive function in a rat model of MHE induced by CCl4 and TAA by regulating the enterohepatic circulation of BA， possibly by increasing the synthesis of taurine－binding BA. -
Key words:
- Hepatic Encephalopathy /
- Rhubarb Decoction /
- Bile Acid
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图 1 肝组织HE染色（×200）
注： a，NC组；b，MHE组；c，MHEY组；d，HE组。中央静脉（黑色箭头），假小叶形成（红色箭头），肝细胞水肿相关变性（黄色箭头），炎症细胞浸润（蓝色箭头），胆管数量（绿色箭头）。
Figure 1. Liver tissue HE staining image（×200）
图 2 脑组织HE染色（×200）
注： a，NC组；b，MHE组；c，MHEY组；d，HE组。黑色箭头表示神经胶质细胞，红色箭头表示假定的神经元。
Figure 2. HE staining image of brain tissue （×200）
表 1 水迷宫数据
Table 1. Water maze data
组别 动物数 （只） 寻台潜伏期（s） 穿台次数 测试 造模前 造模后 用药后 NC组 10 15.38 ± 3.23 14.02 ± 2.44 17.41 ± 4.56 15.44 ± 3.95 10.1 ± 1.4 HE组 15 16.80 ± 4.33 17.69 ± 3.71 51.43 ± 6.111）2） 51.23 ± 5.501）2） 2.9 ± 1.01）2） MHE组 15 14.66 ± 3.68 15.75 ± 3.23 32.94 ± 6.941） 32.43 ± 7.311） 6.1 ± 1.21） MHEY组 15 16.15 ± 3.18 14.97 ± 2.67 31.31 ± 6.59 21.80 ± 5.312） 8.0 ± 0.82） F值 0.654 2.594 51.995 76.982 75.242 P值 0.586 0.068 <0.000 1 <0.000 1 <0.000 1 注：与NC组比较，1）P<0.05；与MHE组比较，2）P<0.05。 
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表 2 四组间各指标比较
Table 2. Comparison of indicators among four groups
指标 NC组 （n＝10） HE组 （n＝10） MHE组 （n＝10） MHEY组 （n＝10） F值 P值 ALT（U/L） 52.5 ± 18.4 1 403.9 ± 263.51）2） 787.0 ± 70.31） 400.4 ± 75.72） 166.823 <0.000 1 AST（U/L） 141.7 ± 13.8 1 445.6 ± 103.31）2） 785.9 ± 79.61） 408.1 ± 53.52） 638.414 <0.000 1 ALP（U/L） 152.7 ± 10.9 513.9 ± 37.81）2） 335.9 ± 22.41） 246.4 ± 16.72） 406.490 <0.000 1 TBiL（μmol/L） 0.15 ± 0.03 2.37 ± 0.061）2） 1.66 ± 0.081） 1.36 ± 0.052） 2 531.005 <0.000 1 TBA（μmol/L） 11.9 ± 1.0 106.9 ± 7.01）2） 77.4 ± 4.41） 53.4 ± 3.62） 789.005 <0.000 1 结肠pH 6.77 ± 0.04 7.41 ± 0.061）2） 6.99 ± 0.071） 6.64 ± 0.072） 305.991 <0.000 1 血氨（μmol/L） 103.3 ± 8.5 226.6 ± 12.91）2） 169.3 ± 9.41） 134.1 ± 7.82） 289.720 <0.000 1 注：与NC组比较，1）P<0.05；与MHE组比较，2）P<0.05。
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表 3 胆汁中BA代谢产物的统计分析
Table 3. Statistical analysis of bile acid metabolites in bile
BA代谢产物 MHE组 vs NC组 MHEY组 vs MHE组 HE组 vs MHE组 MHE组 含量 P值 VIP值 MHEY组 含量 P值 VIP值 HE组 含量 P值 VIP值 GCDCA ↓ <0.001 1.44 ↑ 0.105 0.99 ↓ 0.314 1.26 GUDCA ↓ <0.001 1.36 ↑ 0.991 0.11 ↓ 0.314 0.71 GHDCA ↓ 0.001 1.22 ↓ 0.879 0.11 ↑ 0.314 1.10 TCDCA ↓ 0.001 1.16 ↑ <0.001 1.66 ↑ 0.976 0.30 TUDCA ↓ <0.001 1.18 ↑ 0.001 1.47 ↑ 0.314 0.93 THDCA ↓ 0.026 0.80 ↑ <0.001 1.47 ↑ 0.011 2.06 GLCA ↓ 0.002 1.17 ↑ 0.067 1.23 ↓ 0.703 0.49 TLCA ↓ 0.000 1.24 ↑ <0.001 1.83 ↑ 0.314 0.74 TMCA ↓ 0.239 0.43 ↑ 0.003 1.30 ↑ 0.314 1.01 γ－MCA ↑ <0.001 1.50 ↓ 0.181 0.62 ↓ 0.236 1.46 THCA ↑ 0.001 1.19 ↑ 0.057 1.17 ↓ 0.976 0.41 7－KDCA ↑ <0.001 1.43 ↓ 0.156 0.66 ↓ 0.314 1.17 AlloLCA ↑ 0.013 1.00 ↓ 0.097 0.94 ↓ 0.314 1.29 α－MCA ↑ 0.001 1.17 ↓ 0.150 0.74 ↓ 0.314 1.00 注：GCDCA，甘氨鹅脱氧胆酸；GUDCA，甘氨熊脱氧胆酸；GHDCA，甘氨猪脱氧胆酸；TCDCA，牛磺鹅脱氧胆酸；TUDCA，牛磺熊脱氧胆酸；THDCA，牛磺猪脱氧胆酸；GLCA，甘氨石胆酸；TLCA，牛磺石胆酸；TMCA，牛磺鼠胆酸；γ－MCA，γ－鼠胆酸；THCA，牛磺猪胆酸；7－KDCA，7－酮脱氧胆酸；AlloLCA，别胆酸；α－MCA，α－鼠胆酸。↓表示MHE组比NC组、MHEY组比MHE组、HE组比MHE组代谢物的数值低；↑表示MHE组比NC组、MHEY组比MHE组、HE组比MHE组代谢物的数值高。
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