data supporting research results Effect of <i>Centella asiatica</i> ethanol extract on zebrafish larvae (<i>Danio rerio</i>) insomnia model through inhibition of Orexin, ERK, Akt and p38

<b>Abstract</b><i>Background:</i> Insomnia is difficulty initiating or maintaining sleep for at least three nights a week or more and lasting for at least 3 months. One of the molecules that play a role in the circadian rhythm of arousal system is <i>hypocretin/orexin</i>. Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. <i>Centella asiatica</i> (CA) has a role in the signal work of the MAPK/ERK, Akt, and p38 path in many various diseases.<i>Methods:</i> The research method used is true laboratory experimental. The research approach used was randomized control group post-test only. Zebrafish embryos aged 0-7 dpf were used in this study. The treatment group consisted of 5 groups: normal, insomnia, insomnia + 2.5 μg/mL CA, insomnia + 5 μg/mL CA, and insomnia + 10 μg/mL CA. The locomotor motion of zebrafish larvae was observed using Basler cameras on days five-, six- and seven-day post fertilization (dpf), then analyzed by using Western Blot method.<i>Results:</i> The results proved that exposure to CA extract was able to reduce the expression of orexin (91963 ± 9129) and p38 (117425 ± 6398) as an arousal trigger in the sleep-wake cycle, with the most optimal concentration of CA 5 μg/mL. Exposure to CA extract was also able to reduce the expression of ERK (94795 ± 30830) and Akt (60113.5 ± 27833.5) with an optimum concentration of CA 2.5 μg/mL.<i>Conclusion:</i> Exposure to CA extract was able to improve the sleep activity of zebrafish larvae insomnia model by extending the total inactivity time (<i>cumulative duration</i>) and shortening the duration of first sleep (<i>latency to first</i>) in light and dark phases through inhibition of orexin, ERK, p38, and Akt.**摘要** *背景：* 失眠指每周至少3晚出现入睡困难或睡眠维持障碍，且病程持续至少3个月。参与觉醒系统昼夜节律调控的分子之一为下丘脑泌素/食欲素（hypocretin/orexin）。食欲素可激活p38丝裂原活化蛋白激酶（p38-MAPK）信号通路，并提升磷酸化ERK1/2的表达水平。积雪草（Centella asiatica, CA）可在多种疾病中调控MAPK/ERK、Akt及p38信号通路。 *方法：* 本研究采用纯正实验室实验法，研究设计为仅设后测的随机对照试验。实验对象为受精后0~7天（days post fertilization, dpf）的斑马鱼胚胎。实验组共设5组：正常对照组、失眠模型组、失眠+2.5 μg/mL积雪草组、失眠+5 μg/mL积雪草组及失眠+10 μg/mL积雪草组。分别于受精后第5、6、7天（dpf）采用巴斯勒（Basler）相机记录斑马鱼幼鱼的运动行为，随后通过蛋白质印迹法（Western Blot）进行分析。 *结果：* 结果显示，积雪草提取物干预可降低觉醒周期中作为觉醒触发因子的食欲素（91963 ± 9129）及p38丝裂原活化蛋白激酶（117425 ± 6398）的表达水平，最优干预浓度为5 μg/mL。此外，积雪草提取物还可降低ERK（94795 ± 30830）及Akt（60113.5 ± 27833.5）的表达水平，最优干预浓度为2.5 μg/mL。 *结论：* 本研究证实，积雪草提取物可通过抑制食欲素、ERK、p38及Akt的表达，延长斑马鱼失眠模型幼鱼在明、暗周期中的总静息时长（cumulative duration），缩短首次睡眠潜伏期（latency to first），从而改善其睡眠活动。