Establishment and evaluation of lactose intolerance model with liver depression and spleen deficiency syndrome
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摘要: 目的 建立和评价乳糖不耐受并符合肝郁脾虚证证侯特点的病证结合动物模型。方法 实验1：大鼠随机分为对照(Con)组、高FODMAPs(FOD)组、高乳糖(Lac)组、避水应激(WAS)组、高FODMAPs+避水应激(FOD+WAS)组、高乳糖+避水应激(Lac+WAS)组。避水应激每天2 h，连续10 d；高FODMAPs同期给予10% FODMAPs饲料13 d；高乳糖同期给予高剂量乳糖(5.25 g/kg)灌胃13 d。对行为学、体重、进食等指标进行一般情况观察，并对粪便颗粒数、蔗糖水偏好、AWR评分等进行检测，苏木精-伊红染色结肠组织，Elisa测定结肠5-HT、SERT、TPH1水平。实验2：应激因素改为束缚加夹尾应激，大鼠随机分为对照组、高FODMAPs组、束缚夹尾组、高FODMAPs+束缚夹尾组。束缚每日2 h，夹尾20 min，休息10 min后，再次夹20 min，连续10 d，余同实验1。观察一般情况、粪便颗粒数、蔗糖水偏好、AWR评分。结果 与Con组相比，FOD组及WAS组粪便颗粒数升高(P< 0.05)，疼痛阈值下降(P< 0.05)；FOD+WAS组相较于FOD、WAS组粪便颗粒数升高(P< 0.05)，疼痛阈值下降(P< 0.05)；Lac组相较于Con组，Lac+WAS组相较于WAS组差异无统计学意义。糖水偏好率：WAS、FOD+WAS、Lac+WAS组相较于Con组显著降低，余组间差异无统计学意义。Elisa：FOD+WAS组相较于Con组，5-HT、TPH1显著升高，SERT显著降低(P< 0.01)。FOD+WAS组相较于FOD组，5-HT显著升高(P< 0.05)；相较于WAS组，5-HT、TPH1显著升高，SERT显著降低(P< 0.01)。实验2结果与实验1趋势一致。结论 高FODMAPs饮食叠加避水应激可以成功建立乳糖不耐受肝郁脾虚证模型，叠加组存在结肠5-HT合成升高及再摄取障碍。
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关键词:
- 乳糖不耐受 /
- 肝郁脾虚证 /
- 高FODMAPs饮食 /
- 避水应激 /
- 病证结合模型
Abstract: Objective To establish and evaluate lactose intolerance model with liver depression and spleen deficiency syndrome.Methods Experiment 1: Rats were randomly divided into control group, high FODMAPs group(FOD group), high lactose group(Lac group), water avoidance stress group(WAS group), high FODMAPs+water avoidance stress group(FOD+WAS group), High lactose+water avoidance stress group(Lac+WAS group). Water avoidance stress was performed 2 hours a day for 10 consecutive days. High FODMAPs were given 10% FODMAPs feed for 13 days, and high lactose was given high dose of lactose(5.25 g/kg) by gavage for 13 days. Behavior, body weight, eating and other indicators were observed in general, and the number of fecal particles, sucrose water preference, and AWR score were detected, colon tissue was stained with HE, and the levels of 5-HT, SERT, and TPH1 in colon were determined by Elisa. Experiment 2: The stress factor was changed to restraint and tail clipping stress, and the rats were randomly divided into control group, high FODMAPs group, restraint-tail clip group, high FODMAPs+restraint-tail clip group. Restraining stress was performed for 2 hours; Tail clipping stress was performed for 20 minutes, and after a 10-minute rest, it was clipped again for 20 minutes for 10 consecutive days. The rest was the same as experiment one. The general condition, the number of fecal particles, the preference for sucrose water, and the AWR score were observed.Results Compared with the control group, the number of fecal particles in the FOD group and the WAS group increased(P< 0.05), the pain threshold decreased(P< 0.05); Lac was not significantly different from the control group, and Lac+WAS group was not significantly different from the WAS group. Sucrose water preference rate: WAS, FOD+WAS, Lac+WAS groups were significantly lower than those of the control group, and there was no significant difference between the remaining groups. ELisa: Compared with the control group, FOD+WAS group significantly increased 5-HT and TPH1 and significantly decreased SERT (P< 0.01). Compared with the FOD group, the FOD+WAS group had a significant increase in 5-HT(P< 0.05). Compared with the WAS group, 5-HT and TPH1 were significantly increased, and SERT was significantly decreased(P< 0.01). The results of experiment 2 were consistent with the trend of experiment 1.Conclusion The model of lactose intolerance with liver depression and spleen deficiency syndrome could be successfully established by high FODMAPs diet combined with water avoidance stress, and the superimposed factors induce colonic 5-HT synthesis and reuptake disorder. -
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表 1 对照及高FODMAPs饲料配方
g 成分 AIN-93G标准配方 高FODMAPs配方 酪蛋白 4.000 0 4.000 0 胱氨酸 0.060 0 0.060 0 乳糖 0 0.695 6(3.5%) 果糖 0 0.695 6(3.5%) 低聚果糖 0 0.596 2(3.0%) 玉米淀粉 7.950 0(40.0%) 5.962 5(30.0%) 麦芽糖糊精 3.040 0 3.040 0 蔗糖 2.000 0 2.000 0 纤维素 1.000 0 1.000 0 豆油 1.000 0 1.000 0 酒石酸氢胆碱 0.050 0 0.050 0 AIN矿物质混合物 0.700 0 0.700 0 AIN维生素混合物 0.200 0 0.200 0 BHT 0.002 8 0.002 8 合计 20.002 8 20.002 8 注：AIN为美国营养学会(American Institute of Nutrition)；BHT为二丁基羟基甲苯；()内数字为构成比例。 
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表 2 疼痛阈值比较
X±S 组别 只数 疼痛阈值/mL Con组 6 0.73±0.08 Lac组 6 0.72±0.07 FOD组 6 0.62±0.061) WAS组 6 0.38±0.011) Lac+WAS组 6 0.39±0.16 FOD+WAS组 6 0.22±0.011)2)3) 与Con组比较，1)P < 0.05；与FOD组比较，2)P < 0.05；与WAS组比较，3)P < 0.05。
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表 3 400倍视野下嗜酸性粒细胞个数
X±S 组别 只数 数量/个 Con组 9 3.78±0.79 FOD组 9 4.56±0.96 WAS组 9 8.44±1.071) FOD+WAS组 9 10.78±1.471)2)3) 与Con组比较，1)P < 0.01；与FOD组比较，2)P < 0.01；与WAS组比较，3)P < 0.01。
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表 4 实验1其余4组大鼠5-HT、SERT、TPH1组间比较
ng/g，X±S 组别 5-HT SERT TPH1 Con组 22.97±5.28 2439.23±200.09 1.26±0.11 FOD组 32.20±2.892) 1881.29±211.382) 1.40±0.13 WAS组 30.77±3.511) 2217.89±257.37 1.21±0.08 FOD+WAS组 39.41±5.222)3)4) 1677.75±341.762)5) 1.50±0.122)5) 与Con组比较，1)P < 0.05，2)P < 0.01；与FOD组比较，3)P < 0.05；与WAS组比较，4)P < 0.05，5)P < 0.01。
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