Aconitine inhibits GSK-3 β and counteracts β – amyloid protein induced neuronal damage
Alzheimer’s disease (AD) is one of the most significant central neurodegenerative diseases, characterized by progressive cognitive impairment and behavioral damage. With the increasing aging of society, it has become a global public health issue. According to the World Alzheimer’s Report released by the International Alzheimer’s Association, there are approximately 50 million AD patients worldwide, with an estimated 82 million by 2030 and 152 million by 2050. The abnormal increase of beta amyloid protein (A β) in neural tissue is not only considered one of the main pathological changes in AD, but also an important cause of neuronal damage in AD. The research evidence on the neurotoxicity of A β continues to accumulate. However, several clinical trials of drugs that specifically clear A β have ended in failure, prompting drug developers to refocus their attention on enhancing the tolerance of nerve cells to the cytotoxic effects of A β. However, there is currently no commercially available new drug.

Traditional Chinese medicine has a good effect on improving patients’ cognitive ability in the treatment of AD. Regarding the understanding of the etiology and pathogenesis of AD, the Traditional Chinese Medicine Fu Yang School believes that “excessive Yin transformation, insufficient Yang transformation, and endogenous turbid evil” are the main causes and pathogenesis of AD. “Yuan Yang deficiency and” Yang deficiency and Yin excess “play a leading role in the occurrence and development of AD. Fuzi is known as the first product of traditional Chinese medicine for “returning yang and rescuing reverse”, with significant effects of tonifying fire and assisting yang. It is widely used in the clinical treatment of AD by the traditional Chinese medicine school of tonifying yang, and has created a number of clinically effective compound preparations such as Wu Zang Wen Yang Hua Yu Tang. Aconitine is an important active ingredient in Aconitum carmichaelii. Previous studies have found that Aconitine has pharmacological effects such as cardiotonic, analgesic, anti-tumor, and immunomodulatory effects. Its pharmacological mechanism is related to the activation of phosphatidylinositol 3-kinase (PI3K). Glycogen synthase kinase 3 β (GSK-3 β) is an important downstream target kinase that negatively regulates the PI3K signaling pathway. GSK-3 β phosphorylated at the Tyr216 site is its activated form, and A β – induced overactivation of GSK-3 β is considered one of the important causes of neuronal damage in the AD process. So far, there is no evidence to suggest that aconitine can alleviate neuronal damage caused by A β – mediated GSK-3 β hyperphosphorylation.

This study used a cell model induced by A β 1-40 in vitro to investigate whether aconitine exerts neuroprotective effects on SH-SY5Y neurons by regulating the phosphorylation level of the Tyr216 site of GSK-3 β, which is helpful for understanding the molecular mechanism of using traditional Chinese medicine Aconitum for AD clinical treatment at the molecular level.

As early as 15-20 years or even earlier before typical cognitive impairment and behavioral impairment symptoms of AD appear, A β, which has cytotoxic effects on nerve cells, has already undergone pathological aggregation in the brains of AD patients, but there are no obvious symptoms of memory loss at this time. A β has significant cytotoxicity towards nerve cells, not only inducing cellular oxidative stress and neuroinflammation, but also interfering with the opening of cell membrane ion channels, leading to an imbalance of ion concentration gradients inside and outside the cell, abnormal transmembrane potential of the cell membrane, and directly damaging cholinergic nerve conduction function, resulting in cholinergic nerve signal transmission disorders, as well as directly inducing neuronal apoptosis. When patients exhibit typical cognitive and behavioral impairments, their brain’s nerve cells have been extensively lost, and significant brain atrophy can be observed on brain imaging. At this point, it is difficult to achieve ideal clinical efficacy by using drugs to clear A β, which is also one of the reasons why AD has been clinically difficult to treat since its formal definition in 1907. Finding effective drugs that can enhance the tolerance of nerve cells to the cytotoxic effects of A β cells and intervening with drugs in the early stages of AD pathology is one of the important components of a comprehensive drug therapy plan to effectively prevent the sustained progression of AD. Starting treatment early usually means that patients require a prolonged period of medication, and the benefits of medication treatment must be carefully balanced against the risks of medication safety. Considering that traditional Chinese medicine and its active ingredients generally have good safety, and their multi-target pharmacological characteristics are suitable for complex chronic diseases such as AD, early intervention with traditional Chinese medicine and its active ingredients may be a reasonable potential choice.

The Traditional Chinese Medicine Fu Yang School highly values the warming yang and removing blood stasis treatment principles of Fuzi, and has achieved good results in improving cognitive and behavioral symptoms in the clinical treatment of AD. Aconitine is not only an important pharmacological substance of Aconitum, but also an important cause of Aconitum poisoning. Therefore, the balance of medication in Aconitum decoction is complex, and it is also the dominant reason for selecting Aconitine as the research object. Aconitine, as an important bioactive component in Aconitum, has been found to activate the pharmacological activity of PI3K in previous studies. The activation of PI3K can negatively regulate the activation of GSK-3 β caused by phosphorylation of Tyr216 site. GSK-3 β is involved in the generation, aggregation, and subsequent neuronal damage of A β, while A β – mediated neuroinflammation and oxidative stress can also induce phosphorylation activation of GSK-3 β. In addition, overactivated GSK-3 β can also disrupt the activity of the important neurotransmitter acetylcholine, accelerate neuronal axonal degeneration, hinder axonal transport, and further exacerbate cognitive dysfunction. Based on the above research findings, it can be reasonably inferred that aconitine helps enhance the resistance of nerve cells to the cytotoxic effect of A β, and this beneficial pharmacological effect is related to its inhibition of GSK-3 β phosphorylation at the Tyr216 site and activation. However, this speculation requires experimental evidence to confirm.

In this study, a cell injury model was successfully established by incubating SH-SY5Y cells with A β 1-40, which was characterized by an increase in LDH levels in the cell culture supernatant, accompanied by a significant increase in apoptosis and necrosis rates. 5 nmol/L aconitine did not show significant cytotoxicity, but pretreatment of SH-SY5Y cells with aconitine significantly reduced cell damage caused by A β 1-40. The release of LDH from cell damage was significantly reduced, and both apoptosis and necrosis rates were significantly decreased. At the same time, the phosphorylation level of GSK-3 β at the Tyr216 site was significantly reduced, indicating that the cellular safety of aconitine at this concentration is acceptable and enhancing the ability of cells to resist A β cytotoxicity. This effect may be related to its inhibition of GSK-3 β phosphorylation at the Tyr216 site and activation.

It is generally believed that aconitine has cardiotoxicity. Therefore, methods such as soaking in clean water, processing with gall bladder, and repeated steaming are used to reduce aconitine in aconite. The hydrolysis of aconitine is promoted through long-term frying. However, trace amounts of aconitine still enter the human body and produce corresponding pharmacological effects. There are research reports on the mechanism by which aconitine enters the central nervous system through the blood-brain barrier. A proton coupled organic cation reverse carrier is involved in the blood-brain barrier transport of aconitine alkaloids. The results of this study indicate that even at an extremely low concentration level of 5 nmol/L, aconitine can still exert a neuroprotective effect on nerve cells, significantly enhancing their resistance to A β cytotoxic damage. This suggests that the efficacy of the traditional Chinese medicine formula for warming yang and removing blood stasis in treating AD is closely related to aconitine’s enhancement of nerve cell resistance to A β cytotoxicity. Given the inherent cardiotoxicity of aconitine, it is clearly necessary to design rigorous in vivo experimental studies to verify its in vivo activity in enhancing neuronal resistance to A β cytotoxic damage, while ensuring safety without causing cardiotoxicity.