抑郁模型小鼠海马中胆汁酸受体变化的研究

doi:10.3969/j.issn.1674-8115.2021.12.012

摘要/Abstract

摘要：

目的·探索胆汁酸受体在抑郁模型小鼠海马中的表达变化以及可能的机制。方法·将30只4周龄C57BL/6雌性小鼠随机分为对照（control，CON）组10只、地塞米松（dexamethasone，DEX）组10只、慢性不可知性应激（chronic unexpected mild stress，CUMS）组10只。CUMS组每日给予不同的CUMS；DEX组每日2次给予0.2 mg/kg的DEX灌胃给药，CON组和CUMS组每日给予等体积的羧甲基纤维素钠（溶剂）灌胃。造模持续5周，每周称重。造模结束后，各组小鼠分别进行强迫游泳实验、悬尾实验和糖水偏好实验，观察小鼠的行为学变化。实验结束后处死小鼠，分离并称取小鼠海马及各脏器质量，采用Western blotting检测小鼠海马中脑源性神经营养因子（brain derived neurotrophic factor，BDNF）、法尼酯X受体（farnesoid X receptor，FXR）和G蛋白偶联胆汁酸受体1（G protein-coupled bile acid receptor 1，GPBAR1，又称为TGR5）的改变；小鼠血清胆囊收缩素（cholecystokinin，CCK）和成纤维细胞生长因子15（fibroblast growth factor 15，FGF15）含量使用ELISA试剂盒检测。同时使用不同浓度DEX处理大鼠胶质瘤细胞（C6）株，采用Western blotting检测细胞中FXR和TGR5蛋白水平，使用ELISA试剂盒检测C6细胞培养基中BDNF和胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor，GDNF）含量，活性氧试剂盒检测细胞内氧化应激水平。用5 mmol/L N-乙酰-L-半胱氨酸（NAC）提前预处理C6细胞2 h，观察DEX对胆汁酸受体影响的变化。结果·与CON组相比，DEX组和CUMS组糖水偏好度降低，强迫游泳实验和悬尾实验的不动时间延长（均P<0.05），胆囊系数显著升高（均P<0.05）。DEX组血清FGF15水平较CON组显著升高，CUMS组则显著降低（均P<0.05）；CUMS组CCK水平较CON组显著增高（P<0.05）。此外，2种抑郁模型小鼠海马中TGR5蛋白表达水平显著下降，FXR显著增加（均P<0.05）。C6细胞使用200 μmol/L和400 μmol/L DEX处理3 d后，细胞内TGR5表达量显著减少，FXR表达量显著增加，培养基中GDNF和BDNF（BDNF仅在400 μmol/L组）水平显著降低（均P<0.05）。200 μmol/L DEX可显著提高C6细胞中的活性氧水平。NAC预处理可部分逆转DEX诱导的C6细胞胆汁酸受体改变。结论·DEX和CUMS抑郁模型小鼠海马内存在相似的胆汁酸受体改变，即TGR5减少，FXR增加；该变化可能与氧化应激水平升高有关。

关键词: 抑郁症, 胆汁酸代谢, 胆汁酸受体, 海马, 氧化应激

Abstract:

Objective·To explore the changes of bile acid receptors in hippocampi of depression mouse models.

Methods·Thirty female C57BL/6 mice with the age of 4 weeks were randomly divided into control (CON, n=10) group, chronic unexpected mild stress (CUMS, n=10) group and dexamethasone (DEX, n=10) group. CUMS group mice were treated with different CUMS every day, DEX group mice were administered with DEX (0.2 mg/kg) by oral gavage twice a day, and CON group and CUMS group were given equal volumes of CMC-Na (solvent) by oral gavage every day. The treatments lasted for 5 weeks, and the mice were weighed once a week. After modeling, the forced swimming test, the tail suspension test and the sucrose preference test were applied to detect the depression-like behavior in mice. After the mice being sacrificed, the hippocampus and the other organs were separated and weighed. The expressions of brain-derived neurotrophic factor (BDNF), farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1, also TGR5) in the hippocampus were evaluated by Western blotting. The serum levels of fibroblast growth factor 15 (FGF15) and cholecystokinin (CCK) were detected by ELISA kits. Meanwhile, in the rat glioma cell line (C6) incubated with different concentrations of DEX, the expressions of FXR and TGR5 were detected by Western blotting, and the levels of BDNF and glial cell line-derived neurotrophic factor (GDNF) in the cell culture medium were detected by ELISA kits. The ROS assay kit was used to detect intracellular oxidative stress levels. C6 cells were pretreated with 5 mmol/L N-acetyl-L-cysteine (NAC) for 2 h, and the effects of DEX on bile acid receptors were observed.

Results·Compared with CON group, both DEX group and CUMS group had decreased sucrose preference, prolonged immobility time in the forced swimming test and the tail suspension test (P<0.05) and greater organ relative mass of gallbladder (P<0.05). The level of serum FGF15 in DEX group was significantly higher than that in CON group, but significantly lower in CUMS group (P<0.05); the level of CCK in CUMS group was significantly higher than that in CON group (P<0.05). Additionally, in the hippocampi of the two different depression models, the protein levels of TGR5 and FXR decreased and increased, respectively (P<0.05). The 200 μmol/L and 400 μmol/L DEX increased the expression of FXR, while significantly inhibited the expression of TGR5 in C6 cells; the levels of GDNF and BDNF (BDNF only in 400 μmol/L group) significantly decreased (P<0.05) in the culture medium. DEX at 200 μmol/L significantly increased the levels of ROS in the C6 cells. The cells pretreated with NAC partially reversed the overexpression of FXR and the low expression of TGR5 induced by DEX.

Conclusion·The similar changes of bile acid receptors, i.e. decreased TGR5 and increased FXR, are found in the hippocampi of depression mouse models induced by CUMS and DEX, respectively. These changes may be related to the elevated level of oxidative stress.

Key words: depression, bile acid metabolism, bile acid receptor, hippocampus, oxidative stress

中图分类号:

R964

吴静, 李学义, 陈京红, 王泽剑. 抑郁模型小鼠海马中胆汁酸受体变化的研究[J]. 上海交通大学学报(医学版), 2021, 41(12): 1628-1634.

Jing WU, Xue-yi LI, Jing-hong CHEN, Ze-jian WANG. Study on changes of hippocampal bile acid receptors in the depression mouse models[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(12): 1628-1634.

图/表 8

图1 小鼠建模过程中体质量动态变化趋势Note：①P=0.011, ②P=0.001, ③P=0.004, vs CON group at the same time.

Fig 1 Dynamic change trend of body mass in mice during modeling

图2 小鼠建模完成后行为学指标变化Note： A. FST. B. TST. C. SPT. ①P=0.000, ②P=0.007, ③P=0.030, vs CON group.

Fig 2 Behavioral changes after mice modeling

图3 各组小鼠胆囊系数及血清中FGF15和CCK的含量Note： A. Gallbladder organ coefficient. B. FGF15 content. C. CCK content. ①P=0.003, ②P=0.000, ③P=0.041, ④P=0.018, ⑤P=0.001, vs CON group.

Fig 3 Gallbladder organ coefficient and serum levels of FGF-15 and CCK in each group

图4 小鼠建模后海马内相关蛋白水平的变化Note：A. The expression of FXR, TGR5 and BDNF verified by Western blotting. B-D. Quantitation of FXR (B), TGR5 (C) and BDNF (D). ①P=0.012, ②P=0.005, ③P=0.001, ④P=0.003, vs CON group.

Fig4 Changes in related protein levels in mouse hippocampus after modeling

图5 C6细胞使用DEX处理后相关蛋白水平的变化Note： A. The expression of FXR and TGR5 verified by Western blotting. B/C. Quantitation of FXR (B) and TGR5 (C). ①P=0.000, vs 0 μmol·L－1 group.

Fig 5 Changes in related protein levels in C6 cells treated with DEX

图6 C6细胞使用DEX处理后培养基中神经营养因子的含量变化Note： A/B. The concentrations of BDNF (A) and GDNF (B) in the culture medium. ①P=0.025, ②P=0.018, ③P=0.000, vs 0 μmol·L－1 group.

Fig 6 Changes of BDNF and GDNF concentrations in the cell culture medium in C6 cells treated with DEX

图7 C6细胞加入DEX孵育3 d后细胞内ROS水平的变化Note： A. ROS assay in C6 cells after exposure to 200 μmol·L－1 DEX for 3 d. B. Quantitative analysis of the images obtained from florescence microscopy. ①P=0.000, vs control group.

Fig7 Change of ROS level in the C6 cells treated with DEX for 3 d

图8 NAC预处理2 h对DEX处理3 d的C6细胞内FEX和TGR5表达变化的影响Note： A. The expression of FXR and TGR5 verified by Western blotting. B/C. Quantitation of FXR (B) and TGR5 (C). ①P=0.038, ②P=0.009, ③P=0.003, vs Control group.

Fig 8 Effect of NAC pretreatment for 2 h on the expression of FEX and TGR5 in the C6 cells treated with DEX for 3 d

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