健康与遗传心理学研究室知识库

抑郁症发病机制复杂，细胞因子是诱发抑郁症的重要原因之一。TLR4(toll-like receptor 4)是参与免疫反应的跨膜受体蛋白，应激通过TLR4介导来调控细胞因子表达，它在应激引起的抑郁样行为中具有潜在作用。但是TLR4对社会挫败应激引起的抑郁样行为是否有直接影响目前并不知道。在炎性免疫反应中，TLR4启动子区的DNA甲基化可能在TLR4表达的调控中发挥重要作用，但是TLR4的表观遗传修饰是否参与抑郁症的发生却不清楚。

为考察TLR4对社会挫败应激引起的抑郁样行为的直接影响，以及TLR4启动子区DNA甲基化是否参与抑郁样行为的发生，本研究利用社会挫败应激造模抑郁行为，采用强迫游泳测试、社会交往测试、明暗箱测试观察行为指标，利用western blotting技术检测抑郁症核心脑区海马内TLR4和TNF-a (tumor necrosisfactor-a)蛋白表达变化，使用亚硫酸氢盐测序法检测海马内TLR4基因启动子区的DNA甲基化水平变化。

研究分三部分。第一部分研究考察TLR4是否与社会挫败应激引起的抑郁样行为有关。该部分研究包括实验一和实验二。实验一考察社会挫败应激对抑郁样行为和海马TLR4表达的影响。结果显示社会挫败应激增加了小鼠强迫游泳测试中不动时间，降低了小鼠社会交往测试中社交比例，减少了小鼠明暗箱测试中明箱活动时间，同时引起小鼠海马内TLR4蛋白表达水平升高。实验二考察抗抑郁药氟西汀对社会挫败应激所致抑郁样行为和海马TLR4, TNF-a表达变化的影响，进一步探索TLR4与社会挫败应激引起的抑郁样行为是否有关。结果显示氟西汀能够抑制社会挫败应激引起的海马内TLR4和TNF-a蛋白表达水平的升高，并显著改善社会挫败应激引起的小鼠强迫游泳测试中不动时间增加，改善小鼠社会交往测试中社交比例降低，改善小鼠明暗箱测试中明箱活动时间减少。实验一和实验二的结果从抑郁样行为的发生和逆转两方面表明TLR4与社会挫败应激引起的抑郁样行为有关。

第二部分研究考察TLR4对社会挫败应激引起的抑郁样行为的直接影响。该部分研究包括实验三和实验四。实验三考察TLR4抑制剂对社会挫败应激所致抑郁样行为和海马TLR4, TNF-a表达变化的影响。结果显示急性注射TLR4抑制齐TAK-242能够抑制社会挫败应激引起的小鼠海马内TLR4和TNF-a蛋白表达水平升高，并显著改善社会挫败应激引起的小鼠强迫游泳测试中不动时间增加，但不影响小鼠社会交往测试中社交比例降低和明暗箱测试中明箱活动时间减少。实验四考察TLR4敲除对社会挫败应激所致抑郁样行为和海马TNF-a表达变化的影响。结果显示TLR4基因敲除能够抑制社会挫败应激引起的海马内TNF-a蛋白表达水平升高，显著改善社会挫败应激引起的小鼠强迫游泳测试中不动时间增加，但不影响小鼠社会交往测试中社交比例降低和明暗箱测试中明箱活动时间减少。TLR4基因敲除也可以改善腹腔注射TLR4激动剂LPS (fipopolysaccharide)引起的强迫游泳测试中不动时间的增加。实验三和实验四的结果共同证明TLR4可以直接影响社会挫败应激引起的绝望行为，但是不影响社会挫败应激引起的社交逃避行为和焦虑样行为。

第三部分研究考察海马内TLR4启动子区DNA甲基化是否参与抑郁样行为的发生。该部分研究包括实验五和实验六。实验五考察社会挫败应激对小鼠海马内TLR4基因启动子区DNA甲基化水平的影响。结果显示社会挫败应激可以引起绝望行为的小鼠海马内TLR4基因启动子区一486 CpG位点DNA甲基化水平显著降低。实验六考察甲基供体S-腺普甲硫氨酸对社会挫败应激所致绝望行为、海马内TLR4基因启动子区DNA甲基化水平和TLR4表达变化的影响。结果显示甲基供体S-腺普甲硫氨酸可以逆转社会挫败应激引起的小鼠强迫游泳测试中不动时间增加，改善了应激所致的绝望行为，逆转了社会挫败应激引起的TLR4基因启动子区一486 CpG位点DNA甲基化水平降低，同时抑制了社会挫败应激引起的TLR4蛋白表达水平的升高。实验五和实验六的结果表明海马TLR4基因启动子区一486 CpG位点DNA甲基化水平降低参与调控社会挫败应激引起的TLR4蛋白表达水平的升高和绝望行为的发生。

综上所述，本研究使用氟西汀、TLR4抑制剂TAK-242及TLR4基因敲除鼠三种方法证明TLR4可以通过调控海马内TNF-a表达从而影响社会挫败应激引起的绝望行为，但是不影响社会挫败应激引起的社交逃避行为和焦虑样行为。海马TLR4基因启动子区一486 CpG位点DNA甲基化水平的降低参与调控社会挫败应激引起的TLR4蛋白表达水平升高和绝望行为。这些发现首次证明TLR4在社会挫败应激诱导的绝望行为中发挥着独特作用，TLR4基因启动子区DNA甲基化水平降低参与社会挫败应激诱导的TLR4蛋白表达水平升高和绝望行为的发生。

The pathogenesis of depression is complex. Proinflammatory cytokines play an important role in the pathogenesis of depression. And TLR4 (toll-fike receptor 4), a kind of transmembrane receptor, is involved in immune response. Stress increases the expression of cytokine, depending on TLR4. Therefore, TLR4 could be associated with depression induced by stress. But the exact effects of TLR4 on depressive-like behaviors induced by chronic social defeat stress (CSDS) are not known. DNA methylation at promoter may play a potential role in TLR4 expression regulation during inflammation. But whether the epigenetic regulation of TLR4 gene is involved in depression is not known.

Thus, the aim of the present study was to investigate the effects of TLR4 on depressive-like behaviors induced by CSDS, and whether DNA methylation at TLR4 promoter is involved in depressive-like behaviors. The behavioral paradigm used was the model of depressive-like behaviors induced by CSDS. The animal behaviors were assessed by forced swimming test (FST), social interaction test (STT) and light-dark box test (LDT). The expressions TLR4 and TNF-a (tumor necrosis factor-a) protein in the hippocampus were measured using western blotting. DNA methylation level at TLR4 promoter in the hippocampus was measured using bisulfate sequencing PCR.

In the first group of Experiment 1 and Experiment 2, whether TLR4 was involved in depressive-like behaviors induced by CSDS was examined. The results of Experiment 1 showed that CSDS induced behavioral despair in FST, social avoidance in SIT, anxiety-like behavior in LDT, and increased expression of TLR4 in the hippocampus. The results of Experiment 2 showed that antidepressant fluoxetine treatment significantly reversed behavioral despair in FST, social avoidance in SIT， anxiety-like behavior in LDT, and prevented the increased expression of TLR4 and TNF-a in the hippocampus induced by CSDS. The results from Experiments 1 and 2 indicated that TLR4 was involved in the occurrence and recovery of depressive-like behaviors induced by CSDS.

In the second group of Experiments 3 and 4, the direct effects of TLR4 on depressive-like behaviors induced by CSDS were examined by using TLR4 inhibitor (TAK-242) and TLR4 knockout mice. The results of Experiment 3 showed that acute injection of TLR4 inhibitor (TAK-242) could block behavioral despair, but not social avoidance and anxiety-like behavior induced by CSDS. TLR4 inhibitor could reverse the increased expression of TLR4 and TNF-a in the hippocampus induced by CSDS. The results of Experiment 4 further showed that knockout of TLR4 could block behavioral despair, but not social avoidance and anxiety-like behavior induced by CSDS. Knockout of TLR4 could reverse the increased expression of TNF-a in the hippocampus induced by CSDS. In addition, knockout of TLR4 could also block behavioral despair induced by lipopolysaccharide. The results from Experiments 3 and 4 indicated that TLR4 could modulate CSDS-induced behavioral despair, but not social avoidance and anxiety-like behavior.

In the third group of Experiments _5 and 6, whether DNA methylation at TLR4 promoter participated in the modulation of depressive-like behaviors was examined. The results of Experiment 5 showed that CSDS induced specific CpG site(一486 CpG) demethylation at TLR4 promoter in hippocampus from mice which showed behavioral despair induced by CSDS. The results of Experiment 6 showed that S一adenosyl methionine treatment reversed behavioral despair in FST, normalized specific CpG site(一486 CpG) demethylation at TLR4 promoter and the increased expression of TLR4 in hippocampus induced by CSDS. The results from Experiments 5 and 6 showed that DNA methylation at TLR4 promoter participated in the modulation of depressive-like behaviors induced by CSDS.

In conclusion, these findings demonstrate for the first time that TLR4 plays a distinct role in despair behavior induced by CSDS, and that specific CpG site demethylation at TLR4 promoter participates in the modulation of expression of TLR4 in hippocampus and despair behavior induced by CSDS.