柴胡疏肝散加减可改善胆囊胆固醇结石肝郁证小鼠肝郁和结石程度

doi:10.12122/j.issn.1673-4254.2026.03.15

摘要/Abstract

摘要：

目的探讨柴胡疏肝散加减对胆囊胆固醇结石（CS）肝郁证模型小鼠的治疗作用及其可能通过调节肠道菌群与胆汁酸代谢发挥作用的机制。方法将60只6周龄C57BL/6雄性小鼠按体质量分区间法随机分为空白组（n=9）、模型组（n=51），模型组小鼠予2%高脂致石饲料喂养8周，建立CS小鼠模型。8周CS造模后随机取3只模型组小鼠查看胆囊结石情况，将剩余48只小鼠分为CS组、CS肝郁组、柴胡疏肝散组及熊去氧胆酸组，12只/组。除CS组外，其余3组采用“慢性不可预见性温和应激（CUMS）+孤养”法建立CS肝郁证小鼠模型，柴胡疏肝散组、熊去氧胆酸组分别予柴胡疏肝散加减和熊去氧胆酸浓缩液（0.02 mL/g）灌胃，其余组灌胃等量生理盐水，连续干预3周。观察肝郁证造模期间小鼠一般情况及体质量变化；造模前后对各组小鼠进行行为学实验；HE染色观察回肠组织病理学变化；ELISA检测胆汁中总胆固醇、总胆汁酸含量；q-PCR检测回肠组织中G蛋白偶联胆汁酸受体GPbar1 （TGR5）、胰高血糖素样肽-1/2的mRNA表达；16SrDNA测序与代谢组学检测小鼠粪便与肠内容物的微生物多样性与胆汁酸。结果与空白组及CS组比较，CS肝郁组小鼠体质量下降（P<0.01），渐趋淡漠懒动、反应迟钝；行为学指标显示旷场移动总距离、中心停留时间、糖水消耗减少（P<0.05），游泳、悬尾不动时间增加（P<0.05）；胆囊体积胀大、结石晶体明显，胆汁浑浊；肠绒毛变短、肠道形态破坏；胆汁总胆固醇含量增高（P<0.05）、总胆汁酸含量降低（P<0.05）；肠GLP1/2、TGR5的mRNA表达均升高（P<0.01）。与CS肝郁组相比，柴胡疏肝散加减组小鼠体质量升高（P<0.05）；行为学指标显示旷场移动总距离、中心停留时间、糖水消耗增加（P<0.05），游泳、悬尾不动时间减少（P<0.05）；胆囊体积缩小、结石晶体减少、胆汁清透；上述肠、胆囊病理改变恢复；胆汁总胆固醇降低（P<0.01）、总胆汁酸增高（P<0.05）；肠GLP1/2、TGR5的mRNA表达降低（P<0.01）。肠道菌群检测显示，结石组和结石肝郁组的ace指数、Shannon指数降低（P<0.01）；疣微菌门及其主要菌属阿克曼氏菌属在CS组、CS肝郁组相对丰度上调（P<0.01），拟杆菌门及其主要菌属Muribaculaceae在CS组下调（P<0.01）；胆汁酸谱分析显示，CS组牛磺脱氧胆酸、脱氧胆酸、甘脱氧胆酸上调（P<0.05），CS肝郁组胆酸、3-酮脱氧胆酸上调；相关性分析显示，促石胆汁酸与有害菌正相关，脱氧胆酸与扭瘤胃球菌群、毛螺菌科丰度呈正相关（P<0.05）；甘脱氧胆酸、GCA、牛磺脱氧胆酸与土里肠杆菌属、扭瘤胃球菌群丰度呈正相关（P<0.01）。结论柴胡疏肝散加减可以改善胆囊胆固醇结石肝郁证模型小鼠肝郁程度和结石程度，其机制可能与改善肠道菌群紊乱及胆汁酸代谢有关。

关键词: 胆囊胆固醇结石, 肝郁证, 柴胡疏肝散, 肠道菌群

Abstract:

Objective To investigate the therapeutic effect of modified Chaihu Shugan San (CHSGS) in mice with cholesterol gallstones (CS) and liver depression syndrome and its potential mechanism. Methods Sixty male C57BL/6 mice were randomly divided into blank group (n=9) and model group (n=51) with CS induced by high-lithogenic diet feeding. After verification of successful modeling in 3 mice, the remaining 48 mouse models were divided into CS group, CS-liver depression (CS-LD) group, CHSGS group, and ursodeoxycholic acid (UDCA) group, and the latter 3 groups were subjected to CS-LD modeling by chronic unpredictable mild stress (CUMS) and solitary raising and treated with saline, CHSGS or UDCA gavage for 3 weeks. The mice were assessed for general state, body weight, depressive behavior, ileal pathologies, biliary total cholesterol (TC) and total bile acid (TBA) levels, and ileal mRNA expressions of TGR5, GLP-1, and GLP-2 using q-PCR. 16S rDNA sequencing and metabolomics studies were used to analyze fecal microbiota and bile acids of the mice. Results Compared to the blank control and CS mice, the CS-LD mouse models showed decreased body weight, depressive behaviors, enlarged gallbladder with crystals, intestinal villi damage, increased biliary TC, decreased TBA, and increased ileal GLP-1/2 and TGR5 mRNA expressions, which were obviously improved by CHSGS treatment. Microbiota analysis revealed decreased alpha diversity in CS and CS-LD groups, with altered abundances of Verrucomicrobiota, Akkermansia, Bacteroidota, and Muribaculaceae. Bile acid profiling revealed significant upregulation of TDCA, DCA, and GDCA in CS group and increased CA and 3-ketodeoxycholic acid levels in CS-LD group. Correlation analysis suggested positive correlations between pro-lithogenic bile acids and the harmful gut bacteria. Conclusion Modified CHSGS alleviates liver depression and cholesterol gallstones in mice likely by ameliorating gut microbiota dysbiosis and bile acid metabolism disorders.

Key words: cholesterol gallstone, liver depression syndrome, Chaihu Shugan Powder, intestinal flora

梁仁杰, 王荷颖, 李兆艳, 陈惠萍, 王奕淳, 闵莉. 柴胡疏肝散加减可改善胆囊胆固醇结石肝郁证小鼠肝郁和结石程度[J]. 南方医科大学学报, 2026, 46(3): 615-628.

Renjie LIANG, Heying WANG, Zhaoyan LI, Huiping CHEN, Yichun WANG, Li MIN. Modified Chaihu Shugan Powder alleviates cholesterol gallstones in mice with liver depression syndrome by regulating gut microbiota and bile acid metabolism[J]. Journal of Southern Medical University, 2026, 46(3): 615-628.

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参考文献 37

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Primer		Sequence (5'-3')	Length (bp)
GAPDH	F	5'-TGTGTCCGTCGTGGATCTGA-3'	151
GAPDH	R	5'-TTGCTGTTGAAGTCGCAGGAG-3'	151
GLP1/2	F	5'-CCAGAAGAAGTCGCCATTGCC-3'	118
GLP1/2	R	5'-TCAGCCAGTTGATGAAGTCCCT-3'	118
TGR5	F	5'-TTATGGCCTCCTGTTGCCTG-3'	135
TGR5	R	5'-GCCAGGGTTGAGGGTACATC-3'	135

Primer		Sequence (5'-3')	Length (bp)
GAPDH	F	5'-TGTGTCCGTCGTGGATCTGA-3'	151
GAPDH	R	5'-TTGCTGTTGAAGTCGCAGGAG-3'	151
GLP1/2	F	5'-CCAGAAGAAGTCGCCATTGCC-3'	118
GLP1/2	R	5'-TCAGCCAGTTGATGAAGTCCCT-3'	118
TGR5	F	5'-TTATGGCCTCCTGTTGCCTG-3'	135
TGR5	R	5'-GCCAGGGTTGAGGGTACATC-3'	135

Group	Initial body weight (g)	Final body weight (g)
Control	27.40±0.81	28.20±0.54
CS	30.30±1.24^∆∆	31.70±1.11^∆∆
CSD	30.30±1.04^∆∆	26.78±0.90**
ZY	29.82±0.27^∆	29.02±0.80^{# ▲}
XY	29.80±1.93^∆	26.98±1.57

Group	Initial body weight (g)	Final body weight (g)
Control	27.40±0.81	28.20±0.54
CS	30.30±1.24^∆∆	31.70±1.11^∆∆
CSD	30.30±1.04^∆∆	26.78±0.90**
ZY	29.82±0.27^∆	29.02±0.80^{# ▲}
XY	29.80±1.93^∆	26.98±1.57

Group	Time	Total distance (mm)	Center time (s)
Control	Pre-modeling	16036.73±1577.49	43.17±9.59
Control	Post-modeling	15749.01±2721.57	44.93±6.28
CS	Pre-modeling	15449.44±2032.69	37.62±5.28
CS	Post-modeling	15281.21±2350.29	31.71±6.41^∆∆
CSD	Pre-modeling	14860.68±2342.23	42.02±10.07
CSD	Post-modeling	8713.92±850.85**	18.57±5.04*
ZY	Pre-modeling	15541.03±1598.71	29.08±5.29
ZY	Post-modeling	13396.29±482.19^{# ▲}	29.44±3.16^#
XY	Pre-modeling	15811.60±1340.74	31.37±10.77
XY	Post-modeling	7785.30±2518.09	22.64±5.01