Research Report on the Mechanism of Glycyrrhizic Acid in Alleviating Neurotoxicity Induced by Semen Strychni

Background Introduction

Semen Strychni, derived from the dried mature seeds of Strychnos Nux-vomica, has been found to possess various pharmacological activities, including anti-tumor, anti-inflammatory, immunosuppressive, and neuroexcitatory functions (Zheng et al., 2012; Agrawal et al., 2011a, b; Rao and Prasad, 2013). However, long-term or excessive use of Semen Strychni can lead to multi-organ damage, especially neurotoxicity, which limits its clinical application (Chen et al., 2014; Philippe et al., 2004). Research has shown that the main active components of Semen Strychni - strychnine and brucine - are also its primary toxic components (Wu et al., 2007). Among them, excessive strychnine can cause excessive inhibition of the cerebral cortex and involuntary contraction of respiratory muscles, ultimately leading to cardiac arrest or suffocation (Philippe et al., 2004).

Research Objectives

The purpose of this study is to investigate the role of High Mobility Group Box 1 (HMGB1) in neurotoxicity induced by Semen Strychni and to evaluate the potential mitigating effects of Glycyrrhizic Acid (GA). GA, as a natural anti-inflammatory and antiviral component, is widely used clinically, mainly for treating acute and chronic hepatitis (Liu et al., 2012). We hypothesize that GA can alleviate the neurotoxicity of Semen Strychni by inhibiting the phosphorylation and release of HMGB1.

Paper Source and Authors

This paper was written by Changwei Yu, Yalan Xiang, et al., from institutions including the Second Xiangya Hospital of Central South University and Hunan Provincial Maternal and Child Health Hospital, and published in the Journal of Neuroimmune Pharmacology in 2024.

Research Process

Experimental Materials

Semen Strychni (batch number 201,022,891) was provided by Sanxiang Co., Ltd. in Changsha, Hunan Province. The experimental animals were SPF grade SD rats provided by Hunan SJA Laboratory Animal Co., Ltd. This study was approved by the Experimental Animal Management Committee of Central South University.

Methods

Herbal Extraction

Semen Strychni was crushed and soaked in 75% acidic ethanol solution (pH 5, 1:12, w/v) for 12 hours, then refluxed three times with heat, each time for 1 hour. The filtrate was concentrated using a rotary evaporator, and finally, the composition of the extract was detected using High-Performance Liquid Chromatography-Ultraviolet Detection (HPLC-UV-MS).

Animal Experiment Design

48 rats were randomly divided into the following four groups (n = 12): 1. Control group: Intraperitoneal injection of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, followed by gavage with 0.5% CMC-Na solution. 2. Semen Strychni poisoning group (SS): Intraperitoneal injection of Semen Strychni extract (175 mg/kg), followed by gavage with 0.5% CMC-Na solution. 3. Low-dose GA detoxification group (SS + LGA): Intraperitoneal injection of Semen Strychni extract, followed by gavage with 75 mg/kg of GA. 4. High-dose GA detoxification group (SS + HGA): Intraperitoneal injection of Semen Strychni extract, followed by gavage with 150 mg/kg of GA.

Samples were collected and various experiments were conducted four days after administration.

Sample Collection and Processing

Cardiac blood was collected during dissection and centrifuged to obtain serum. Completely separated brain tissues were washed multiple times with saline and then immersed in paraformaldehyde.

Pathological Examination

Nissl staining, TUNEL staining, and FJB staining were performed. Images were observed and recorded through a microscope.

ELISA and Protein Analysis

ELISA was used to detect levels of ACHE, MAO, HMGB1, TNF-α, and IL-1β in serum. Western blotting was used to detect proteins such as HMGB1, p-HMGB1, and NF-κB in brain tissues.

Research Results

Glycyrrhizic Acid Alleviates Neuronal Damage Induced by Semen Strychni

• Nissl staining results showed a significant reduction in the number of neurons in the CA1 region of the hippocampus in SS group rats, with loss of cellular integrity. After GA treatment, neuronal damage was alleviated.
• FJB staining results showed a significant increase in FJB-positive cells in the CA1 and CA3 regions of SS group rats. After GA treatment, the number of positive cells decreased.
• TUNEL staining results showed a significant increase in TUNEL-positive cells in the cortex of SS group rats. GA treatment reduced the number of positive cells.

Glycyrrhizic Acid Alleviates Neurometabolic Enzyme Disorders Caused by Semen Strychni

• Serum levels of ACHE and MAO were significantly reduced in SS group rats. High-dose GA significantly upregulated ACHE and MAO levels, indicating that GA can effectively regulate neurotransmitter metabolic disorders caused by Semen Strychni.

Glycyrrhizic Acid Inhibits HMGB1 Release and Nucleocytoplasmic Translocation

• Serum HMGB1 levels were significantly increased in the SS group. GA treatment restored HMGB1 levels to normal.
• Immunofluorescence staining showed a large distribution of HMGB1 outside the nucleus in the SS group, while GA treatment reduced the translocation of HMGB1 to the cytoplasm.

Glycyrrhizic Acid Regulates Interactions Between HMGB1 and Phosphatases and Protein Kinases

• Immunoprecipitation results showed decreased binding of HMGB1 to PP2Ac and increased binding to PKCα in the SS group. GA treatment enhanced the binding of HMGB1 to PP2Ac and reduced binding to PKCα.

Glycyrrhizic Acid Inhibits Semen Strychni-Induced Inflammatory Factor Release and Inflammatory Pathway Activation

• Serum levels of TNF-α and IL-1β were significantly elevated in SS group rats. GA treatment significantly reduced inflammatory factor levels.
• Phosphorylation levels of JNK and p38 proteins in the MAPK pathway were significantly increased in the SS group, while GA treatment downregulated phosphorylation levels.

Research Conclusions and Significance

This study demonstrates that neurotoxicity induced by Semen Strychni is associated with the phosphorylation and release of HMGB1. GA alleviates neurotoxicity and inflammatory responses caused by Semen Strychni by inhibiting HMGB1 phosphorylation and reducing its cytoplasmic translocation. This finding provides new theoretical basis for the clinical use of GA in alleviating Semen Strychni toxicity, with important scientific value and application prospects.

Research Highlights

• This study is the first to systematically elucidate the key role of HMGB1 in neurotoxicity induced by Semen Strychni.
• It proposes and verifies the mechanism by which GA reduces neurotoxicity by regulating HMGB1 phosphorylation and inflammatory pathways.
• It provides the possibility of using a new natural drug, GA, in clinical detoxification applications.

Through this research, we have deepened our understanding of Semen Strychni toxicity and found a potentially effective detoxification method, providing scientific basis for improving its clinical safety.