Protective effect of Astragaloside IV on high-dose S-ketamine induced PC12 cell injury
Millions of infants and young children worldwide receive general anesthesia every year. Numerous studies have shown that single and short-term anesthesia does not have adverse effects on the development of the nervous system, but high doses or repeated exposure to anesthetic drugs can cause cognitive dysfunction and neurological developmental disorders. The impact of anesthetic drugs on the developing brain has attracted widespread attention from many parents.

S-ketamine (SK) is the S enantiomer of ketamine and was officially approved by the FDA as an adjuvant drug for the treatment of severe depression in 2019. It is also a commonly used anesthetic in pediatric and obstetric surgeries. It is worrying that people seem to be only interested in studying its antidepressant effects, without paying too much attention to the potential risks it may cause. A recent study analyzed 962 adverse events related to SK and found that female patients, patients receiving high-dose SK, and patients receiving multiple drug treatments had a higher probability of experiencing adverse reactions. Therefore, it is of great significance to explore the mechanism of SK induced nerve damage and find corresponding coping measures for both infant and adult anesthesia.
Astragaloside IV (ASIV) is one of the main active ingredients isolated from the traditional Chinese medicine Astragalus membranaceus. Studies have shown that it has a wide range of pharmacological effects, including anti-tumor, anti-inflammatory, antioxidant, anti fibrotic, and anti apoptotic effects. In addition, it has been proven to be a natural neuroprotective agent. However, there are few reports on whether ASIV can alleviate nerve damage caused by anesthetic drugs. Therefore, this study selected commonly used neural cell line rat adrenal pheochromocytoma cells, namely PC12 cells, to construct an in vitro injury model of SK, explore the neuroprotective effect of ASIV, and provide reference for the rational use of SK and the development of new drugs for ASIV.

The neurotoxicity of anesthetic drugs has always been a concern of the international community. The US Food and Drug Administration and the International Association for the Study of Anesthesiology held a joint meeting to discuss the importance of standardizing preclinical research and establishing reporting standards for anesthesia induced perinatal neurotoxicity, and suggested the use of novel methods to address some of the major issues in this field. As for ketamine, unlike most anesthetics, it is an NMDA receptor antagonist. Drug blockade of NMDA receptors can alter the excitatory inhibitory balance necessary for brain development, leading to neurotoxicity. Studies have shown that ketamine can induce neurotoxicity in humans through the mitochondrial apoptosis pathway in a time-dependent and dose-dependent manner, while its enantiomer S-ketamine has not been reported to produce similar side effects. Therefore, it is speculated that when a certain concentration threshold is reached, SK can also activate the mitochondrial apoptosis pathway and cause cell apoptosis.
Mitochondria, as the center of cellular metabolism and the main regulator of redox balance, play a crucial role in the occurrence and development of diseases. Among the many physiological reactions mediated by mitochondria, oxidative stress is one of the key factors in activating the mitochondrial apoptosis pathway, and excessive ROS is the material basis for the occurrence of oxidative stress. Commonly used anesthetics in clinical practice have been proven to promote or reduce the degree of oxidative stress. For example, inhalation anesthesia sevoflurane can induce neuronal apoptosis by increasing intracellular Ca2+concentration, raising ROS levels, and increasing the expression of mitochondrial apoptosis pathway related proteins Cleaved-Caspase-9, Cytochrome C, and Bax/Bcl-2. The local anesthetic bupivacaine activates ROS mediated autophagy, leading to impaired autophagy flux and decreased cell viability. However, the use of reactive oxygen species scavengers can significantly reverse this autophagic damage. In the mitochondrial pathway, the increase in ROS content leads to changes in the relative expression levels of Bax and Bcl-2. Bax is activated and oligomerized at the outer membrane of mitochondria, and subsequently binds to specific molecules to alter mitochondrial membrane permeability. Cytochrome C is therefore released from mitochondria into the cytoplasm, binding with factors such as Apaf-1 to form apoptotic bodies, and activating Pro-Caspase-9 for cleavage. The cleaved Caspase-9 further activates downstream related apoptotic factors, promoting cell apoptosis.

Astragaloside IV is a natural active substance with strong antioxidant properties, and research has shown that some of its effects are closely related to mitochondria. For example, ASIV can promote mitochondrial autophagy, reduce the accumulation of damaged mitochondria and the generation of mitochondrial reactive oxygen species. Liu et al. also found that ASIVs can exert neuroprotective effects by targeting alpha synuclein, a protein located in the inner membrane of mitochondria and highly expressed in the human brain. It can be seen that ASIV has the potential to treat nerve damage caused by anesthetic drugs.
Based on the above research results and analysis, this study conducted an in vitro experiment using the mitochondrial apoptosis pathway as a clue to treat high-dose SK induced nerve damage with ASIV. The final experimental concentrations of SK and ASIV were determined to be 450 μ g/mL and 25 μ mol/L, respectively, using CCK-8 assay. In addition, the experiment found that PC12 cells exposed to high doses of SK not only showed a significant increase in apoptosis rate and ROS content, but also activated the main upstream molecules Bax, Bcl-2, Caspase-9, and Cytochrome C in the mitochondrial apoptosis pathway. This indicates that high-dose SK induces apoptosis in PC12 cells by activating the ROS mediated mitochondrial apoptosis pathway. After treatment with ASIV, these changes induced by SK were significantly inhibited, demonstrating that ASIV has a certain protective effect on PC12 cells during this process.
In summary, Astragaloside IV can alleviate high-dose S-ketamine induced damage in PC12 cells, and its mechanism may be related to the inhibition of mitochondrial apoptosis pathway.