Sanwei DouKou Decoction ameliorate Alzheimer disease by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway

Abstract

Ethnopharmacological relevance

Sanwei DouKou decoction (SDKD) is a traditional Chinese medicine (TCM) prescription derived from the Tibetan medical book “Si Bu Yi Dian” and is clinically used for the treatment of Alzheimer's disease (AD). However, the potential mechanism of SDKD treatment for AD remains elusive.

Aim of the study

This study aims to explore the potential mechanism by which SDKD alleviates AD.

Materials and methods

Extracts of SDKD were identified with Gas chromatograph-mass spectrometer (GC-MS). 5 × FAD mice were treated with SDKD for 8 weeks. The efficacy of SDKD against AD was evaluated by in-vivo experiments. Morris water maze and contextual fear conditioning tests were used to detect the learning and memory ability of mice. Hematoxylin-eosin staining (H&E) staining was used to observe the pathological changes of brain tissue. Immunohistochemistry was used to detect the positive expression of Nestin in hippocampus. In in-vitro experiments, the Cell Counting Kit 8 (CCK-8) technique was used to detect cell viability, the proliferation of neural stem cells was detected by immunofluorescence staining, the intracellular protein expression was detected by Western Blot.

Results

The results of this study suggested that SDKD may ameliorate AD. SDKD significantly shortened the escape latency of mice in the Morris water maze experiment, increased the number of times the mice crossed the target quadrant, and prolonged freezing time in the contextual fear memory experiment. SDKD also improved neuronal pathology in the hippocampus, decreased neuronal loss, and increased Nestin protein levels. Furthermore, in in-vitro experiments, SDKD could significantly increase Neural stem cells (NSCs) viability, promoted NSCs proliferation, and also effectively activated the Wnt/β-catenin signalling pathway, increased Wnt family member 3A (Wnt3a), β-catenin and CyclinD1 protein levels, activated the NSCs proliferation pathways in AD model mouse brain tissue.

Conclusions

The present study demonstrated that sanwei doukou decoction can ameliorate AD by increasing endogenous neural stem cells proliferation through the Wnt/β-catenin signalling pathway. Our observations justify the traditional use of SDKD for a treatment of AD in nervous system.

Introduction

Alzheimer's Disease (AD) is a chronic neurodegenerative disease characterized by cognitive and motor impairment, and the ability to live independently may be lost in the later stages of the disease, which seriously affects the quality of life of the patients (Abubakar et al., 2022; McDade, 2022). Besides the deteriorating effects on human health and the quality of life, there is also a tremendous economic impact associated with the disease (Maccioni et al., 2022). At present, economical cost of AD in the world has reached to one billion dollars a year. At present, there are around 50 million AD patients worldwide and this number will increase to reach 152 million by 2050 (Breijyeh and Karaman, 2020). AD has become a global health problem. Neuronal loss is the major cause and fundamental pathological changes of Alzheimer's disease. Stem cell therapy has been proven to alleviate Alzheimer's disease progression (Si and Wang., 2021). It is characterized by neural stem cells through proliferation, migration and differentiation into neurons, to supplement the lost neurons, repair the neural network, to restore the function of brain tissue. In fact, current therapy tends to focus on the exogenous neural stem cells therapy, that is, the surgical injection of neural stem cells into the brain tissue. However, the difficult source of stem cells and easy tumor development should not be ignored (Liu et al., 2020). Studies have confirmed that large numbers of neural stem cells also exist in adult brain tissue. Compared with exogenous neural stem cells therapy, there are no ethical problems and tumor formation problems in promoting endogenous neural stem cells proliferation, which is of important research significance (Wang et al., 2021). Therefore, accelerating drug development that promotes the proliferation of endogenous neural stem cells deserves wide attention.

Sanwei DouKou decoction (SDKD) is a traditional Chinese medicine formula, which has been used in Tibetan medicine for more than 2000 years. SDKD is a classic prescription recorded in “Si Bu Yi Dian”which is a classic medical book of Tibetan medicine for improving sleep and memory (Yundangongbu, 1982). Numerous studies have confirmed the significant effect of SDKD (An et al., 2021; Li et al., 2021). SDKD is made from cardamom, cumin and long pepper boiled in yak's milk. Cardamom is a plant in the ginger family, and the dried ripe fruit is a medicine. Numerous studies have shown that cardamom has antibacterial, anti-asthmatic, and antioxidant effects and has the potential to treat neurological diseases (Alam et al., 2021; Paul et al., 2020). Cumin is a plant in the cumin genus, and the seed is a medicine. Numerous studies have shown that cumin has antibacterial, anti-inflammatory and nerve-stimulating effects (Ghasemi et al., 2019; Kang et al., 2019). Long pepper is a plant in the Piperaceae, and the dried ripe fruit is a medicine. Numerous studies have shown that long pepper has antibacterial and anti-inflammatory effects, and also has an apparent neurological protective effect, making it a potential drug for treating neurological diseases (Wang et al., 2020). Cardamom, cumin and long pepper belong to the “aromatic drugs” in traditional Chinese medicine and has the potential to treat neurological diseases. A book in the Yuan Dynasty named ⟪Ao shi shang han jin jing lu⟫records “if serious, treating with niuhuangwan or zhibaodan, the aromatic drugs can enlighten”, which is the first time to clearly put forward the use of aromatic medicine to treat diseases with abnormal consciousness (Liu, 2021). ⟪Bencao gangmu⟫ records that “musk can open the orifices, Open the meridians … to treat stroke … " (Li, 1982), which point out that aromatic medicine can treat mental diseases such as stroke. According to the position and structure, the “orifice” in traditional Chinese Medicine can be divided into “the outer orifice” and “the inside orifice”, and “the inside orifice” includes meridians, qichongmen, blood channels, mind orifice, heart orifice and brain orifice. Brain orifice disease is a disease with the opening and closing disorders of brain orifice, with unconsciousness, ravings, syncope, convulsion and fainting as clinical symptoms (Zhou and Tan, 2020), which is similar to neurological diseases such as stroke and Alzheimer's disease in modern medicine. “Aromatic enlightenment” in Chinese medicine is to treat Alzheimer's disease, depression, stroke and other mental diseases with aromatic drugs. Modern pharmacological studies believe that aromatic drugs have great potential in regulating the permeability of the blood-brain barrier (BBB) because they contain a large number of volatile oils, so that they have great potential in the treatment of neurological diseases (Zhang et al., 2022). Modern pharmacological studies have found that eucalyptol in cardamom can inhibit the production of Oxygen radicals and prevent the accumulation of Aβ42 (Paul et al., 2020), Cuminaldehyde in cumin can inhibit the fibrosis process of synapsin (Iranshahy and Javadi, 2019), and piperine in long pepper can reduce acetylcholinesterase activity, reduce inflammation and reduce oxidative stress (Wang et al., 2019). Moreover, piperine has been confirmed to ameliorate neurological diseases such as epilepsy, insomnia, dementia (Azam et al., 2022). SDKD is a Traditional Chinese Medicine can enhance nerve impulse transmission, improve memory and ameliorate cognition, which can be used to ameliorate neurological diseases such as AD (An et al., 2021; Li et al., 2021).

Accumulative evidence showed that the amelioration of Alzheimer's disease is associated with the proliferation, migration, and differentiation of neural stem cells, which is important for nervous system repair (Ma et al., 2021). Based on the current achievements, endogenous neural stem cells are considered to be a potential therapeutic direction (Zhang et al., 2022). The Wnt/β-catenin signalling pathway plays a key role in regulating neural regeneration in the adult mouse brain and can promote NSCs proliferation (Austin et al., 2021). When the Wnt/β-catenin signalling pathway is activated, the β-catenin expression increases. When β-catenin accumulation in the cytoplasm reaches a certain level, it enters the nucleus and activates downstream regulated genes. CyclinD1 is a downstream gene of the Wnt/β-catenin signalling pathway that can regulate the cell cycle (Yuan et al., 2022). CyclinD1 expression increases, and it binds to cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), enabling cells to enter S phase and promoting cellular proliferation and division (Wang et al., 2021). Therefore, activation of the Wnt/β-catenin signalling pathway induces high β-catenin and CyclinD1 expression and promotes NSCs proliferation. However, whether SDKD could prevent AD via the activation of Wnt/β-catenin signalling pathway is still unclear. The aim of this study was to explore the effect of SDKD alleviating AD and understand its underlying mechanism.

In this study, 5 × FAD mice were used to investigate the behavioural and brain histopathological changes. We also use the neural stem cells model to examine NSCs proliferation and the levels of Wnt family member 3A (Wnt3a), β-catenin, and CyclinD1 related with Wnt/β-catenin signalling pathway. Altogether, our results determine the role and mechanism of SDKD in preventing and treating AD.