摘要
为探究不同饮食模式对小鼠焦虑抑郁样行为的影响,采用慢性不可预见性温和应激(chronic unpredictability mild stress,CUMS)模型,将C57BL/6小鼠随机分为正常饮食组(normal diet,ND)、正常饮食应激组(normal diet stress,NDS)、高脂饮食组(high fat diet,HFD)和高脂饮食应激组(high fat diet stress,HFDS),对小鼠的行为、体质量、脂质和神经递质等指标进行测定。结果显示,与ND组相比,HFD组小鼠糖水偏好显著降低,旷场试验中总运动距离、进入中心区域次数和跨格次数降低,HFDS组部分行为学指标较HFD组更低,且HFDS组部分行为学指标低于NDS组;HFD组体质量、体脂比、总胆固醇(TC)和甘油三酯(TG)水平最高,CUMS显著降低高脂饮食和正常饮食小鼠的体脂比、TC和TG水平,且与NDS组相比,这些脂质指标水平在HFDS组更低。结果表明,高脂饮食易诱导焦虑抑郁样行为,且高脂饮食会加重CUMS诱导的焦虑抑郁样行为,其机制可能与脂质代谢、中枢神经递质代谢途径有关。
慢性压力是一个普遍存在的社会健康问
雄性C57BL/6小鼠购于湖北省疾控中心,共40只,体质量为(20±2) g。饲养环境为12 h的明暗循环,温度为24~26 ℃,湿度为60% 相对湿度,通风良好,小鼠自由进食饲料和水。适应性饲养1周后,随机分为对照组的正常饮食组(normal diet,ND)、高脂饮食组(high fat diet,HFD)和应激组的正常饮食应激组(normal diet stress,NDS)、高脂饮食应激组(high fat diet stress,HFDS),每组各10只,饲养12周。ND、NDS喂食基础饲料,HFD、HFDS喂食高脂饲料(D12492,睿迪生物科技(深圳)有限公司)。NDS和HFDS给予慢性不可预测温和应激,共12周。
参照Carter
测试之前,小鼠自由饮食24 h,保持小鼠能自由饮用2瓶不同的水,其中一瓶为蒸馏水,另一瓶为1%的蔗糖水。为了防止小鼠对糖水位置有偏爱,在此期间,每隔12 h对调蒸馏水瓶和蔗糖水瓶的位置。此训练结束后,小鼠禁食禁水12 h,蔗糖水瓶和蒸馏水瓶称质量后放到鼠笼上,给予小鼠自由饮水6 h,然后称量2个水瓶质量。根据以下公式计算糖水偏好率:糖水偏好率=(糖水消耗量/总液体消耗量)×100%。
小鼠单独放入敞箱(50 cm×50 cm×40 cm)的中央格中,自由探索5 min。用视频跟踪系统记录小鼠的运动距离、进入中心区域次数、支撑性站立次数等指标。每次试验结束后,用75%的乙醇清洗试验区域。
该测试装置为1个(42 cm×21 cm×25 cm)盒子,盒内分为小(1/3)暗室和大(2/3)亮室,1个3 cm高、4 cm宽的受限开口连接2个腔室。小鼠单个放置在有照明的隔间里,并可在2个隔间之间自由移动。用视频跟踪系统记录5 min内小鼠在光室和暗室的停留时间及穿梭次数。试验结束时,及时清理小鼠的排泄物。
高架迷宫装置的主体部分由大小相同的2个张开的臂和2个闭合的臂(5 cm×15 cm×35 cm)组成。1个中央开放区域(5 cm × 5 cm)连接4个手臂,让小鼠自由地进入每个臂。小鼠被单独地放在中央区域,面对张开的臂,自由探索5 min,视频跟踪系统将小鼠在迷宫中的活动记录下来。为避免前一只小鼠的气味干扰,后一只小鼠测试前,用75%的乙醇清洗除掉小鼠的尿液和粪便。
小鼠处死前,对其禁食但不禁水12 h。采用摘除眼球取血的方式取血后,立即颈椎脱臼处死小鼠,打开头颅取脑,并在冰上剥离海马体。打开腹腔,取出肾周脂肪组织、附睾脂肪组织、腹股沟脂肪组织等,称脂肪组织的质量。全血室温静置30 min后,1 300 r/min 4 ℃离心15 min,用移液枪小心吸取上层血清于干净EP管中备用,用于后续脂质指标及神经递质含量的测定。血清和海马体样本及时放在-80 ℃冰箱中保存。
使用试剂盒(A111-1-1,A110-1-1,南京建成生物工程研究所)测定血清中总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)含量;按照相应ELISA试剂盒(DB1296-Mu,DB459-Mu,上海沪鼎生物科技有限公司)操作手册,测定血清和海马体中的5-羟色胺(serotonin,5-HT)和去甲肾上腺素(norepinephrine,NE)含量。组织样本预处理方法如下:称取海马体组织,每10 mg组织加入100 μⅬ的RIPA(RIPA∶PMSF=100∶1),在冰上裂解30~40 min,然后于12 000 r/min 4 ℃离心10 min,小心吸取上清,进行测定。
如

图 1 糖水偏好试验
Fig.1 Sucrose preference test
A:液体总消耗量;B:糖水偏好率。柱上不同字母表示组间具有显著性差异,P<0.05,n=6。A:Total liquid consumption; B:Sucrose preference.Different letters in the same graph indicate significant differences,P<0.05,n = 6.
如

图 2 旷场试验
Fig.2 Open field test
A:运动轨迹;B:运动总距离;C:进入中心区域次数;D:跨格次数。柱上不同字母表示组间具有显著性差异,P<0.05,n=6~8。A:Trajectory diagram; B:Total distance; C:Number of entries to the center; D:Number of crossed squares.Different letters in the same graph indicate significant differences,P<0.05,n=6-8.
如

图 3 明暗箱试验
Fig.3 Light/dark test
A:待在暗箱时间;B:待在明箱时间。图中不同字母表示组间具有显著性差异(n=6~8),P<0.05。A:Time in dark box; B:Time in light box.Different letters in the same graph indicate significant differences,P<0.05.
如

图 4 高架十字迷宫试验
Fig.4 Elevated plus maze test
A:进入开放臂次数的百分比;B:待在开放臂时间的百分比。图中不同字母表示组间具有显著性差异(n=6~8),P<0.05。A:Percentage of the entries to the open arms; B:Percentage of the time in the open arms.Different letters in the same graph indicate significant differences,P<0.05.
小鼠的体质量变化如

图 5 小鼠体质量和血脂指标
Fig.5 Body weight and blood lipids
A:1~12周体质量;B:第12周的体质量;C:体脂比;D:血清总胆固醇;E:血清甘油三酯。图中不同字母表示组间具有显著性差异(n=6~8),P<0.05。A:Body weight; B:Weight at week 12; C:Adipose/Weight; D:TC; E:TG.Different letters in the same graph indicate significant differences,P<0.05.
如

图 6 血清(A、B)和海马体(C、D)中的5-羟色胺及去甲肾上腺素含量
Fig.6 5-HT and NE levels in serum (A,B) and hippocampus (C,D)
图中不同字母表示组间具有显著性差异(n=6~8),P<0.05。Different letters in the same graph indicate significant differences,P<0.05.
本研究利用CUMS诱导焦虑抑郁模型,探究了不同饮食模式对小鼠行为、体质量、血脂、单胺类神经递质的影响,揭示了高脂饮食在CUMS诱导的焦虑抑郁样行为的形成过程中的作用。结合行为学试验结果表明,长期高脂饮食易诱导焦虑抑郁样行为的产生,且高脂饮食联合CUMS会加重抑郁样行为。与正常饮食组(ND)相比,高脂饮食组(HFD)小鼠的糖水偏好率、旷场中的运动总距离、进入旷场中心区域次数、跨越格子次数、待在明箱时间和进入开放臂次数显著降低,表明长期高脂饮食会诱导小鼠愉悦感、自发活动和探索能力降低。同样地,黄志
脂代谢异常与肥胖、神经精神障碍等代谢紊乱疾病的发展密切相
单胺类神经递质,例如5-羟色胺、多巴胺、去甲肾上腺素等,直接或间接参与调节人的情绪,其功能障碍与焦虑抑郁等精神障碍疾病有
综上所述,高脂饮食更易诱导焦虑抑郁样行为的形成,且高脂肪饮食会加重CUMS诱导的焦虑抑郁样行为。不同饮食模式在CUMS诱导的小鼠焦虑抑郁样行为形成中的作用可能与脂质代谢、单胺类神经递质代谢途径有关,但是其具体机制仍待进一步研究。
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