Mechanism of Sancao Anshen Prescription in Regulating cAMP/PKA Signaling Pathway to Improve Sleep and Depressive States in Zebrafish with SD Models
10.13422/j.cnki.syfjx.20251302
- VernacularTitle:三草安神方调控cAMP/PKA信号通路改善SD模型斑马鱼睡眠及抑郁状态的作用机制
- Author:
Mengying HUANG
1
;
Ping WANG
2
Author Information
1. Institute of Gerontology，Hubei University of Chinese Medicine，Wuhan 430065，China
2. Engineering Research of TCM Protection Technology and New Product Development for the Elderly Brain Health，Ministry of Education，Hubei University of Chinese Medicine，Wuhan 430065，China
- Publication Type:Journal Article
- Keywords:
Sancao Anshen prescription;
sleep deprivation;
cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA） signaling pathway;
depression;
zebrafish
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2026;32(1):180-190
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveBy establishing a light sleep deprivation （SD） model and using the traditional Chinese medicine （TCM） compound Sancao Anshen prescription for intervention， this study aims to observe one of the effects of Sancao Anshen prescription on the sleep and depressive states of zebrafish in the SD model and explore the mechanism of action of this drug in regulating the cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA） signaling pathway. MethodsA total of 240 six-month-old wild-type AB zebrafish were randomly divided into a blank group， a model group， low-dose， medium-dose， and high-dose groups of Sancao Anshen prescription （0.28， 0.83， 2.48 g·L-1）， and a melatonin group （0.2 g·L-1）， with 40 fish in each group. Except for the blank group， all others were exposed to LED lights （150 lux） for three days to construct the sleep deprivation model， and were treated with the corresponding doses of Sancao Anshen decoction and melatonin solution for three days. 24 h movement behavior was used to detect diurnal movement trajectories. A T-shaped maze was employed to detect learning and memory functions， and a new tank experiment was conducted to detect depression-like behaviors in zebrafish. Hematoxylin-eosin （HE） staining was used to observe the morphology of hypothalamic neurons， and transmission electron microscopy was utilized to observe the ultrastructure of hypothalamic cells. Immunohistochemical （IHC） was used to measure the positive expression of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in the hypothalamus， and enzyme-linked immunosorbent assay （ELISA） was employed to determine the content of cAMP， 5-hydroxytryptamine （5-HT）， gamma-aminobutyric acid （GABA）， and glutamic acid （Glu） in brain tissue. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to measure the expression of PKA， cAMP response element-binding （CREB）， and brain-derived neurotrophic factor （BDNF） mRNAs and proteins and their phosphorylation levels （p-PKA， p-CREB） in brain tissue. ResultsCompared with those in the blank group， the resting time and resting count in the model group decreased significantly （P<0.01）， while the gross motor time and gross motor count increased significantly （P<0.01）. The latency time to enter the EC region in each administration group increased significantly （P<0.01）. The exploration time towards the top and the number of times entering the top decreased significantly （P<0.01）， and the incubation period of the first ascent increased significantly （P<0.01）. The number of hypothalamic neurons decreased significantly， and the neurons exhibited irregular shapes， sparse arrangement， and nuclear condensation. Nuclear collapse， nuclear membrane rupture and dissolution， chromatin condensation， mitochondrial swelling and deformity， and plate-like cristae rupture or disappearance were observed. The positive expression of TNF-α and IL-1β in the hypothalamus was significantly decreased （P<0.01）. The content of cAMP， GABA， and 5-HT in brain tissue was significantly downregulated， while the content of Glu was significantly upregulated （P<0.01）. The mRNA of PKA， CREB， and BDNF was significantly downregulated （P<0.01）， and the protein expression of p-PKA， p-PKA/PKA， p-CREB， p-CREB/CREB， and BDNF was significantly decreased （P<0.05， P<0.01）. Compared with those in the model group， the resting time in the medium-dose and high-dose groups of Sancao Anshen prescription and the melatonin group increased （P<0.05， P<0.01）， and the resting count in each administration group significantly increased （P<0.05， P<0.01）. The gross motor time and gross motor count significantly decreased （P<0.05， P<0.01）. The latency time to enter the EC region decreased （P<0.01）， and the exploration time towards the top increased （P<0.01）. The time for the first ascent in the high-dose group of Sancao Anshen prescription and the melatonin group was shortened （P<0.05， P<0.01）， and the number of times entering the top increased （P<0.01）. The morphology of neurons in each administration group improved， with the gap decreased， the nuclear membrane relatively intact， and mitochondrial swelling improved. The positive expression of IL-1β in each administration group significantly decreased （P<0.01）. The positive expression of TNF-α in the medium-dose and high-dose groups of Sancao Anshen prescription and the melatonin group significantly decreased （P<0.01）. The Glu content in the low-dose group of Sancao Anshen prescription decreased， while cAMP and GABA levels increased （P<0.05， P<0.01）. There was no statistically significant difference in 5-HT content. The medium-dose and high-dose groups of Sancao Anshen prescription and the melatonin group all showed significant increases in cAMP， 5-HT， and GABA levels （P<0.01）， and a significant decrease in Glu content （P<0.01）. The mRNA of CREB in the low-dose group of Sancao Anshen prescription was significantly upregulated （P<0.01）， while there was no statistically significant difference in the mRNA of PKA and BDNF. The mRNA of PKA， CREB， and BDNF in the medium-dose and high-dose groups of Sancao Anshen prescription and the melatonin group were all significantly upregulated （P<0.01）. The protein expression of p-PKA， p-PKA/PKA， p-CREB， p-CREB/CREB， and BDNF in the high-dose group of Sancao Anshen prescription and the melatonin group increased significantly （P<0.05， P<0.01）， and the protein expression of p-PKA， p-CREB， and BDNF in the medium-dose group of Sancao Anshen prescription increased （P<0.05）. ConclusionThe improvement of sleep and depressive states in zebrafish with the SD model by Sancao Anshen prescription may be related to the inhibition of inflammatory factors such as TNF-α and IL-1β， the reduction in Glu， and the elevation in the content of neurotransmitters such as GABA and 5-HT via the cAMP/PKA signaling pathway.
