丁香酸调控胆汁酸代谢和肠道屏障改善胆汁淤积肝病的研究

罗欣; 成鹏; 陆茵; 韦忠红

doi:10.14148/j.issn.1672-0482.2024.0350

丁香酸调控胆汁酸代谢和肠道屏障改善胆汁淤积肝病的研究

doi: 10.14148/j.issn.1672-0482.2024.0350

罗欣^1,,
成鹏¹,
陆茵^{1, 2, 3},
韦忠红^{1, 3, ,}

1.
南京中医药大学药学院, 江苏省中药药效与安全性评价重点实验室, 江苏南京 210023;2.;3
2.
江苏省中医药防治肿瘤协同创新中心, 江苏南京 210023
3.
江苏省中医药与再生医学研究国际合作联合实验室, 江苏南京 210023

基金项目:

国家自然科学基金青年科学基金项目 82004124

江苏省研究生创新项目 KYCX22_2040

详细信息

作者简介:
罗欣, 女, 硕士研究生, E-mail: 20210761@njucm.edu.cn

通讯作者:
韦忠红, 女, 讲师, 主要从事基于肠道微生物开展中药调控消化系统疾病发生发展的研究, E-mail: wzh1225@njucm.edu.cn

中图分类号: R285.5
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- 文章访问数: 38
- HTML全文浏览量: 8
- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2023-11-13
- 网络出版日期: 2024-04-24
- 发布日期: 2024-04-10

Syringic Acid Improves Cholestatic Liver Disease by Regulating Bile Acid Metabolism and Intestinal Barrier

LUO Xin^1
,,
CHENG Peng¹,
LU Yin^{1, 2, 3},
WEI Zhonghong^{1, 3
, ,}

1.
Jiangsu Key Laboratory of Efficacy and Safety Evaluation of Traditional Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Jiangsu Collaborative Innovation Center for Cancer Prevention and Treatment of Traditional Chinese Medicine, Nanjing 210023, China
3.
Jiangsu Joint Laboratory for International Cooperation in Chinese Medicine and Regenerative Medicine Research, Nanjing 210023, China

摘要

摘要: 目的基于胆汁酸代谢和肠道屏障探讨丁香酸对胆汁淤积小鼠的调控作用。方法 20只小鼠随机分为对照组、模型组和丁香酸低、高剂量(70、140 mg·kg^-1)组。连续7 d灌胃给予相应药物, 于第5天给药2 h后腹腔注射α-萘异硫氰酸酯(ANIT)诱导肝内胆汁淤积小鼠模型。末次给药后, 记录小鼠体质量及肝质量变化; 检测小鼠血清中肝功能指标、组织病理学变化, qPCR验证小鼠结肠组织中紧密连接蛋白闭锁小带蛋白-1(ZO-1)、闭合蛋白(Occludin)、紧密连接蛋白-5(Claudin-5) mRNA的表达, 采用非靶向代谢组学分析血清中代谢产物的变化, 检测肝脏和粪便中总胆汁酸变化。结果丁香酸可以显著降低模型组小鼠血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)活性和总胆红素(TBIL)、直接胆红素(DBIL)水平(P＜0.05，P＜0.01), 减轻肝脏损伤和坏死; 降低模型组小鼠结肠的淋巴细胞浸润, 恢复隐窝形态。丁香酸高剂量组能显著升高结肠中ZO-1、Occludin、Claudin-5的mRNA表达水平(P＜0.05);显著上调11个代谢物, 下调29个代谢物, 代谢产物主要涉及次生代谢物的生物合成、次级胆汁酸生物合成和胆汁分泌通路。丁香酸降低肝脏中总胆汁酸含量及增加粪便总胆汁酸的排出(P＜0.05，P＜0.01)。结论丁香酸可以显著改善胆汁淤积小鼠肠道屏障的受损, 并且促进胆汁酸的代谢, 这可能是丁香酸改善胆汁淤积的关键调控环节。
- 丁香酸 /
- 胆汁淤积 /
- 肝损伤 /
- 肠道屏障 /
- 胆汁酸代谢
Abstract: OBJECTIVE To explore the regulatory effect of syringic acid in cholestatic mice based on bile acid metabolism and intestinal barrier. METHODS Twenty mice were randomly divided into control group, model group and low and high dose of syringic acid (70, 140 mg·kg^-1) groups. Intrahepatic cholestasis was induced by intraperitoneal injection of α-naphthalene isothiocyanate after 2 h of administration on the fifth day. After the last dose, the changes of body weight and liver mass of mice were recorded. Liver function indexes in serum and histopathology were detected, qPCR verified the expression of tight junction proteins Zonula Occludens Protein 1 (ZO-1), Occludin and Claudin-5 in mouse colon tissues, the changes of metabolites in serum were analyzed by using nontargeted metabolomics, and the changes of total bile acids in liver and feces were detected. RESULTS Syringic acid could significantly reduce the serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activity, total bilirubin (TBIL) and direct bilirubin (DBIL) levels (P < 0.05, P < 0.01) in the model group mice, and reduce liver damage and necrosis. Syringic acid reduced lymphocyte infiltration in the colon of mice in the model group and restored crypt morphology, while the high-dose syringic acid group significantly increased the mRNA expression levels of ZO-1, Occludin, and Claudin-5 in the colon (P < 0.05). The high-dose intervention of syringic acid significantly upregulated 11 metabolites and 29 metabolites, and the metabolites mainly involved the biosynthesis of secondary metabolites, secondary bile acid biosynthesis and bile secretion pathways. Syringic acid reduced the content of total bile acids in the liver and increased the excretion of total bile acids in feces (P < 0.05, P < 0.01). CONCLUSION Syringic acid can significantly improve the phenotype of cholestasis in cholestatic mice, improve the damage of intestinal barrier, and promote the metabolism of bile acids in cholestatic mice, which may be the key pathway for syringic acid to improve cholestasis.
- syringic acid /
- cholestasis /
- liver damage /
- intestinal barrier /
- metabolism of bile acids

HTML全文

图 1 丁香酸对ANIT小鼠肝功能的影响

注：与对照组比较, ^#P＜0.05，^##P＜0.01，^###P＜0.001;与模型组比较，^*P＜0.05，^{* *}P＜0.01。x±s，n=5。

Figure 1. Effect of syringic acid on liver function in ANIT mice

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图 2 丁香酸对ANIT小鼠肝脏组织病理学的影响

注：箭头指向炎性浸润。标尺=100 μm。

Figure 2. Effect of syringic acid on pathological changes of liver tissue in ANIT mice

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图 3 丁香酸对ANIT小鼠结肠组织病理学的影响(标尺=100 μm)

Figure 3. Effect of syringic acid on pathological changes of colon tissue in ANIT mice(Scale bar=100 μm)

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图 4 丁香酸对ANIT小鼠结肠紧密连接蛋白表达的影响

注：与对照组比较，^#P＜0.05;与模型组比较，^*P＜0.05。x±s，n=5。

Figure 4. Effect of syringic acid on colonic tight junction protein expression in ANIT mice

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图 5 模型组和丁香酸高剂量给药组PLS-DA图(A)及置换检验图(B)

Figure 5. PLS-DA plots (A) and permutation plots (B) in model group and syringic acid high-dose dosing group

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图 6 小鼠血清代谢产物火山图

Figure 6. The volcano map of metabolites in mice serum

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图 7 小鼠血清代谢产物KEGG通路富集分析

Figure 7. The KEGG pathway of metabolites in mice serum

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图 8 丁香酸对ANIT小鼠肝脏中总胆汁酸含量的影响

注：与对照组比较，^###P＜0.001;与模型组比较，^{* * *}P＜0.001。x±s, n=3。

Figure 8. Effect of syringic acid on total bile acid concentration in liver in ANIT mice

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图 9 丁香酸对ANIT小鼠粪便中总胆汁酸含量的影响

注：与模型组比较，^*P＜0.05，^{* *}P＜0.01。x±s, n=3。

Figure 9. Effect of syringic acid on total bile acid concentration in feces in ANIT mice

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表 1 引物序列

Table 1. Primer sequence

引物	正向(5’→3’)	反向(3’→5’)
Gapdh	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
ZO-1	AACCCGAAACTGATGCTGTGGATAG	CGCCCTTGGAATGTATGTGGAGAG
Occludin	TGGCTATGGAGGCGGCTATGG	GCGATGAAGCAGAAGG
Claudin5	CTGCCTTCCTGGACCACAACATC	CACCACGCACGACATCCACAG

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表 2 丁香酸对ANIT小鼠肝脏指数的影响(x±s, n=5)

Table 2. Effect of syringic acid on liver indexin ANIT mice (x±s, n=5)

组别	体质量/g	肝脏质量/g	肝脏指数/%
对照组	22.92±0.77	1.16±0.07	5.08±0.22
模型组	20.82±0.40	1.32±0.05	6.36±0.31^##
SA低剂量组	22.30±0.53	1.11±0.04	4.96±0.15^**
SA高剂量组	23.22±0.47	1.17±0.06	5.02±0.21^**
注: 与对照组比较, ^##P＜0.01;与模型组比较, ^{* *}P＜0.01。

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表 3 SA高剂量组调控的血清代谢物

Table 3. Serum metabolites regulated by the SA high dose group

编号	代谢物	T_R/min	Calc. MW	碎片峰	ESI	Log₂ FC
1	9Z-Octadecen-12-ynoicacid	7.53	278.224 7	219.056 0, 201.046 7, 149.023 8, 95.086 1	ESI⁺	1.13
2	2-Oxindole	1.98	133.053 1	134.060 4, 106.065 8, 79.055 1	ESI⁺	-1.18
3	3'-hydroxyacetophenone	4.46	136.052 7	122.036 6, 94.042 0, 79.055 0	ESI⁺	5.17
4	4-(2-Aminophenyl)-2_4-dioxobutanoate	1.64	207.053 6	162.055 3, 134.096 9, 84.960 7	ESI⁺	-3.63
5	Arachidoyl Ethanolamide	9.10	355.345 2	338.341 7, 106.086 7, 88.976 5	ESI⁺	-4.23
6	Corticosterone	4.15	346.214 5	329.2120, 123.081 1, 97.065 5	ESI⁺	2.09
7	Hippuric acid	1.87	179.058 6	105.034 1, 95.0499 4, 77.039 4	ESI⁺	-1.61
8	Nonivamide	4.46	293.199 2	229.958 2, 137.060 0	ESI⁺	5.78
9	Palmitoylcarnitine	9.50	399.335 0	341.268 4, 144.102 1, 95.086 5, 85.029 1, 57.070 6	ESI⁺	-7.05
10	Muroctasin	21.43	886.559 4	675.478 1, 303.233 7, 283.264 3, 241.012 0, 152.995 3, 78.958 0	ESI^-	3.12
11	(3S, 5Z, 7E)-26, 26, 26, 27, 27, 27-Hexafluoro-9, 10-secocholesta-5, 7, 10-triene-3, 25-diol	10.83	508.280 0	279.233 0, 227.032 5, 152.995 0	ESI^-	-4.59
12	[STtrihydroxy(2∶0)]11beta_17_21-trihydroxypregn-4-ene-3_20-dione_21-sulfate	5.31	442.166 1	265.127 1, 222.107 7, 113.023 3	ESI^-	-2.83
13	{2-[2-(Isobutyryloxy)-4-methylphenyl]-2-oxiranyl}methyl 2-methylbutanoate	7.07	334.178 2	289.181 4, 245.191 5, 219.826 4	ESI^-	-2.42
14	1-(3, 4-Dimethoxyphenyl)-3, 5-decanediyl diacetate	3.34	394.235 9	242.919 2, 224.527 7, 153.091 4	ESI^-	1.72
15	Histamine	19.56	111.080 0	/	ESI⁺	2.31
16	Cumyluron	3.05	302.118 8	254.852 7, 228.046 1, 79.956 1	ESI^-	-3.60
17	Hexahydrocurcumin	9.12	374.172 5	329.176 3, 285.186 4, 270.162 9, 229.123 5	ESI^-	-1.65
18	2-Phenylethyl D-glucopyranoside	2.44	284.126 0	265.108 3, 221.118 0, 203.107 3, 177.127 8	ESI^-	-1.17
19	Protoporphyrin Ⅸ	18.35	580.398 2	403.358 5, 113.023 3, 85.028 3	ESI^-	-1.38
20	3-Chlorotyrosine	1.86	215.034 5	/	ESI^-	-2.07
21	4-(Heptyloxy)benzoic acid	4.07	236.140 9	191.143 7, 146.937 7, 103.919 2	ESI^-	-1.76
22	4-Allyl-2-methoxyphenyl 6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside	2.60	458.179 3	266.098 2, 193.034 9, 113.023 3	ESI^-	-1.04
23	4-Hydroxy-N, N, N-trimethyl-9-oxo-7-[(propiony-loxy)methyl]-3, 5, 8-trioxa-4-phosphatetracosan-1-aminium 4-oxide	16.61	552.366 8	375.326 7, 220.545 2, 175.023 9, 113.023 3	ESI^-	-1.32
24	8'-Hydroxyabscisate	4.07	280.131 1	235.134 0, 191.143 7, 162.838 2	ESI^-	-1.63
25	Aderbasib	3.53	416.204 8	239.164 7, 193.035 2, 113.023 4	ESI^-	1.01
26	Catalpol	3.93	362.122 3	313.111 6, 80.964 1	ESI^-	-2.76
27	Cholic acid	4.93	408.287 6	352.102 3, 220.830 4	ESI^-	-5.86
28	Tegobuvir	13.35	517.114 8	/	ESI^-	-1.38
29	Deoxycholic acid	8.09	392.292 9	345.279 9, 221.415 0	ESI^-	-1.98
30	Helenalin	6.91	262.1205	235.134 0, 217.122 7, 191.143 5	ESI^-	-2.03
31	Hippuric acid	1.86	179.057 6	134.987 0, 106.934 7	ESI^-	-1.40
32	Isodomedin	4.13	392.220 1	305.805 8, 221.422 2, 164.835 6, 123.081 9, 78.427 5	ESI^-	2.06
33	Lusitanicoside	3.90	442.184 0	193.035 0, 175.024 3, 113.023 4	ESI^-	-1.10
34	MFCD00273074	10.37	344.292 9	233.214 3, 136.908 9	ESI^-	-2.53
35	MFCD12546417	1.47	189.993 0	/	ESI^-	-1.14
36	NBenzyloxycarbonyl-L-leucine	5.77	265.131 6	220.134 1, 192.139 1, 163.112 1, 82.028 9	ESI^-	1.24
37	NP-005196	3.32	282.146 7	263.129 1, 237.149 5, 219.138 9	ESI^-	-1.41
38	NP-021701	6.02	266.151 9	247.134 0, 203.144 8, 191.144 0	ESI^-	-1.08
39	Taurodeoxycholic acid	5.90	499.296 8	397.434 5, 221.893 0	ESI^-	-2.10
40	Tetrofosmin	15.41	382.239 2	301.830 3, 96.959 1	ESI^-	3.15
注: FC(SA high dose vs Model)。

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