The effect and mechanism of Huangshui extract from Shengdi on sleep in rats
Insomnia has various names in the field of traditional Chinese medicine, such as “insomnia” and “unfocused eyes”. It is generally believed to be a restless phenomenon caused by various external and internal factors that stimulate the body, resulting in inability to fall asleep, morning drowsiness, and other restlessness. Insomnia is accompanied by other symptoms, such as difficulty in initiating and maintaining sleep, waking up early in the morning, and difficulty falling back asleep. The current global prevalence of insomnia is estimated to be between 10% and 40%. Different degrees of insomnia can have an impact on the body. For example, short-term insomnia can make people look haggard and mentally exhausted, while long-term insomnia can easily lead to negative emotions such as anxiety and depression, and may even cause psychological disorders and mental illnesses. Therefore, improving sleep quality and ensuring normal physiological routines are urgent social and health issues that need to be addressed.
At present, sedative and hypnotic drugs include benzodiazepines (such as triazolam, estazolam, nitrodiazepam, etc.), antidepressants, antihistamines, etc. However, continuous use may cause adverse reactions such as hangovers, delirium, and ataxia. The National 14th Five Year Plan and the 2035 Long Range Objectives outline the need to promote the inheritance and innovation of traditional Chinese medicine, and to leverage the important advantages of Chinese medicinal materials in disease prevention. Therefore, exploring the role of traditional Chinese medicinal materials in improving sleep and other aspects has become a new research hotspot. Rehmannia glutinosa Libosch., a perennial herbaceous plant in the family Scrophulariaceae, is a dried root of Rehmannia glutinosa Libosch. It is widely used as a traditional Chinese medicine for clearing heat, cooling blood, nourishing yin, and generating fluids. Modern pharmacological research has shown that Rehmannia glutinosa plays an important role in cardiovascular prevention and treatment, immune system regulation, lowering blood sugar and lipid levels, anti-aging, liver protection and bile retention, anti-tumor, antibacterial, and hemostatic effects. In addition, the combination of Rehmannia glutinosa and other traditional Chinese medicinal herbs, such as the Lily Rehmannia Decoction made by boiling Rehmannia glutinosa and Lily, the Ginseng Ling Granules composed of Suanzaoren, Xuanshen, Poria cocos, etc., and the Tianwang Buxin Pill composed of Ginseng, Yunling, Angelica sinensis, etc., also have the effect of calming and calming the mind. However, there is currently limited research on the efficacy and safety of Rehmannia glutinosa in improving sleep, and even fewer studies on its related mechanisms. Therefore, this study takes the “Methods for Functional Testing and Evaluation of Health Food” (2022 edition) as a reference to preliminarily determine the efficacy of Shengdi Huang Shui extract in improving sleep. Furthermore, by detecting changes in neurotransmitter content and corresponding receptor expression in the hypothalamus of rats, as well as changes in the content of cytokines in rat serum, the effect and possible mechanism of Shengdi Huang Shui extract in improving sleep are explored.

 

At present, experimental research on improving sleep mostly focuses on animal behavior and neurotransmitters. This experiment explored the effects of Rehmannia glutinosa on direct sleep, pentobarbital sodium and subthreshold dose induced sleep incidence and duration in rats, as well as sleep latency induced by barbiturates, through pharmacological studies of animal behavior. Due to the fact that the excitation or inhibition of the central system by drugs can be responded to through the behavior of rats, it has been demonstrated through the autonomous activity of rats that Rehmannia glutinosa can significantly reduce rat activity and has a clear effect on improving sleep. In addition, the high-dose group of Huangshui extract from Shengdi showed a significant increase in the incidence of sleep in rats induced by the subthreshold dose of pentobarbital sodium, indicating that Shengdi is not related to liver enzyme metabolism in improving sleep. The acute toxicity test on mice showed that when the water extract of Rehmannia glutinosa was 30000mg/kg · BW (equivalent to a human drug dose of 150g/d), there was no significant change in various characteristic indicators of mice. According to the drug toxicity grading standards reported in the literature, when the LD50>15000mg/kg dose, the substance was considered non-toxic. The maximum dose group in this experiment has exceeded 15000mg/kg, so it can be determined that the water extract of Rehmannia glutinosa is non-toxic and has good safety. The body can take it for a long time.

Neurotransmitters are crucial for sleep regulation and can serve as endogenous indicators of insomnia. This experiment investigated the mRNA expression of amino acid neurotransmitters and monoamine neurotransmitters, as well as their receptor proteins, to explore the potential mechanism of action of Huangshui extract in improving sleep in rats. GABA and Glu are a class of amino acid neurotransmitters, with GABA being an inhibitory neurotransmitter in the human and mammalian brain and Glu being an excitatory neurotransmitter. The two types of amino acid neurotransmitters have a circulating pathway, with GABA mainly obtained by decarboxylation of Glu through glutamate decarboxylase catalysis, and Glu also being formed by catalysis of GABA through gamma aminobutyric transaminase. Therefore, the steady-state balance between GABA and Glu is crucial for the body’s sleep. Gao et al. found that the dual extract of sour jujube kernel and schisandrin alcohol water can significantly increase the GABA content in PCPA induced insomnia rats, and reduce the Glu level and Glu/GABA ratio, which can improve the sleep condition of PCPA induced insomnia rats. Yu et al. found that the sedative, hypnotic, and anti anxiety effects of Schisandra vinegar are related to an increase in GABA content and a decrease in Glu content in brain tissue. In this experiment, all groups of Huangshui extract from Shengdi significantly increased the GABA content in the hypothalamus of insomnia rats, and the Glu content and Glu/GABA ratio were also reduced compared to the control group. Further research has found that GABAA receptors can specifically bind to GABA and exert inhibitory effects between synapses, with GABAA α 1 and GABAA α 2 subtypes being the two common receptor subtypes. Diazolidine drugs can bind to GABAA α 1 receptors to exert sedative effects, and bind to GABAA α 2 receptors to exert anti anxiety effects. Metabolic glutamate receptors belong to G protein coupled receptors, among which mGluR1 and mGluR2 can significantly affect the disorder of glutamate regulation in the nervous system. Under pathological conditions, glutamate can accumulate in large quantities in the central nervous system, leading to the opening of Ca2+channels and Ca2+overload, damaging important components of neurons and causing insomnia. By measuring the gene expression levels of GABA and Glu related receptors in the treatment group, it was found that the extract of Huangshui can affect the expression levels of GABAA receptor genes, significantly upregulate the mRNA expression levels of GABAA α 1 and GABAA α 2 receptors, and reduce the mRNA expression levels of mGluR1 and mGluR2 receptors. This regulatory effect will also enhance the transmission of GABA neural signals, inhibit central nervous system disorders caused by Glu accumulation, and induce sleep in rats.

5-HT, NA, and DA are a class of monoamine neurotransmitters, among which 5-HT can regulate central nervous system homeostasis and participate in the occurrence of sleep processes. The widely used PCPA induced animal insomnia model internationally is based on the method of PCPA inhibiting 5-HT synthesis to induce corresponding insomnia symptoms in animals. NA promotes neuronal excitation to maintain wakefulness, while DA plays an important role in sleep arousal. In this study, the changes in the content of these three neurotransmitters in the hypothalamus of rats treated with the extract of Huangshui from Shengdi are shown in Table 4. Compared with the model group, the NA content in the treated group slightly decreased, but it was not statistically significant and may be influenced by multiple factors. The results need further analysis. The content of 5-HT and DA both changed, especially in the middle and high dose groups of Huangshui extract, which showed significant changes. Due to the fact that 5-HT must bind to corresponding receptors to exert its effects, among the known receptors, the 5-HT1 α and 5-HT2 α receptors are most relevant to anti insomnia research. They mainly participate in the awakening activity of sleep, and their content is closely related to the length of their awakening cycle. This study found that the mRNA expression levels of 5-HT1 α and 5-HT2 α receptors in the hypothalamus of the treatment group were significantly increased. Combined with changes in 5-HT content, it suggests that the pathway of improving sleep and the increase in 5-HT content of Huangshui extract are related to changes in its receptor mediated physiological effects. Due to the important role of dopamine receptor D2 in maintaining wakefulness, the mRNA expression level of dopamine receptor D2 in rats significantly decreased after administration, indicating that the extract of Huangshui can improve sleep in rats by regulating dopamine and its receptors.

The regulation of inflammatory factors can also affect the occurrence of sleep wake processes. The secretion of inflammatory cytokines during normal sleep follows a regular pattern, while sleep disorders can lead to abnormal immune responses. For example, long-term chronic sleep restriction leads to an increase in the levels of pro-inflammatory cytokines TNF – α and IL-1 β in the brain, but excessive elevation can also impair sleep. The anti-inflammatory cytokine IL-10 levels are significantly reduced, and a decrease in total nighttime sleep time can also lead to changes in systemic inflammatory status. Porkka et al. also revealed the relationship between sleep rhythm and immune activity, as sleep deprivation can lead to changes in the expression of immune related genes and increase the production and release of pro-inflammatory cytokines. Therefore, the disorder of inflammatory factors is also an important influencing factor of insomnia. In this study, the extract of Rehmannia glutinosa improved sleep in rats while also causing changes in the cytokines TNF – α, IL-1 β, and IL-10. It is speculated that the improvement of sleep by Rehmannia glutinosa extract is closely related to the regulation of cytokines. When Sun et al. studied the sedative and hypnotic effects of Shuangxia Tang, they also found that the levels of inflammatory factors TNF – α and IL-1 were significantly reduced in insomnia rats. In addition, the levels of 5-HT and its metabolites HIAA, receptors 5-HT1A and 5-HT2A were increased in insomnia rats, indicating the involvement of the 5-HT system and immune system. They also believed that PCPA induced excitation of the hypothalamic pituitary adrenal cortex (HPA) axis in insomnia rats may be due to 5-HT blockers reducing 5-HT levels, leading to a significant increase in TNF – α and promoting 5-HT synthesis. Based on the changes in neurotransmitter content and receptors in the central nervous system of this experiment, it is demonstrated that the extract of Huangshui can improve sleep in rats by regulating neurotransmitters and cytokines.

In summary, based on the preliminary judgment that the extract of Huangdi water can improve sleep and safety in rats, this study further explored the mechanism of Huangdi water extract in improving sleep using the PCPA insomnia rat model. The results showed that the extract of Rehmannia glutinosa can significantly improve the levels of GABA, 5-HT, Glu, and DA neurotransmitters and the expression of corresponding receptor mRNA in the hypothalamus of insomnia rats. In addition, the levels of cytokines TNF – α, IL-1 β, and IL-10 also changed accordingly, indicating that the extract of Rehmannia glutinosa can improve sleep in rats by regulating neurotransmitters and cytokines. Further in-depth research is needed in the future on the physiological functions of the main components in the extract of Huangshui, as well as how to further improve sleep pathways and mechanisms of action.