Study on the hepatotoxicity mechanism of different extracts of Sophora alopecuroides based on oxidative stress
Sophora alopecuroides L. belongs to the family Fabaceae and genus Sophora. In 1977, it was included in the Pharmacopoeia of the People’s Republic of China and is an important component of traditional Chinese medicine plant resources and ecological vegetation in provinces such as Gansu, Inner Mongolia, and Ningxia. Kudou beans have the functions of clearing heat, detoxifying, and dehumidifying. Alkaloids, flavonoids, and sugars are the main active ingredients, which have good therapeutic effects on gastrointestinal diseases. Wang et al. found that the alkaloids in Sophora alopecuroides are mainly quinolones, which play important roles in antioxidant, antiparasitic, and antibacterial activities. The study found that the flavonoids of Sophora alopecuroides have a certain antagonistic effect on hepatitis B virus. At present, a bitter bean plant industry chain has been formed in the domestic market, focusing on ecological environment protection, characteristic product development, and regional development.

In traditional applications, bitter beans have never attracted attention due to their toxicity. In recent years, drug-induced liver injury has become the second largest non infectious liver disease in China and the leading cause of acute liver failure in European and American countries, accounting for up to 60% of cases. There are increasingly more clinical case reports on the hepatotoxicity caused by traditional Chinese medicine, and its potential toxicity has become a hot research topic both domestically and internationally. Early acute toxicity experiments found that Sophora alopecuroides extract has certain toxicity to rat liver. The study of the toxicity of the liver, as the main organ for metabolism and detoxification in the body, is of great significance. At present, the specific mechanism of liver toxicity caused by Sophora alopecuroides is still unclear. Therefore, this study conducted subacute toxicity experiments to observe the effects of four extracts, namely water decoction (WD), water ultrasound (WU), ethanol reflux (ER), and ethanol ultrasound (EU), on the general behavior, tissue morphology, and oxidative stress indicators of rats after administration. The content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of rats after administration, as well as the expression and distribution of reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and nuclear factor erythroid 2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1), SOD1, and SOD2 proteins in the liver, were measured to explore the effects of Sophora alopecuroides on rats. The mechanism of hepatotoxicity in mice provides scientific data and reference for clinical basic research on Sophora alopecuroides.

The change in liver coefficient can be an important indicator of liver lesions. After administration of different extracts of Sophora alopecuroides, the liver coefficient of rats significantly increased. The increase in liver coefficient suggests that the liver may undergo reactions such as edema and inflammation. Pathological observation of liver tissue revealed necrosis of liver cells and an increase in inflammatory cells. Through the detection of liver injury sensitivity indicators for ALT and AST, it was found that the levels of ALT and AST in the serum of rats in each group increased to varying degrees after administration of different extracts. This indicates that long-term administration of Sophora alopecuroides extract can cause liver function damage, which is consistent with literature reports. In order to further investigate the liver toxicity of different extracts of Sophora alopecuroides, this paper conducted microscopic morphological observations of the liver and found that rat liver mitochondria were swollen, even with disappearance of cristae, and exhibited obvious autophagy. Among them, autophagy is a response of the body to eliminate foreign substances and respond to unfavorable external factors. After administration of Sophora alopecuroides extract, significant autophagy occurred in the liver of rats. Basal levels of autophagy have a protective effect on cells, but excessive autophagy can cause autophagic cell death and even accelerate disease progression. Meanwhile, Ravanan et al. demonstrated that inhibiting autophagy can reduce the inflammatory response caused by the NF κ B pathway. After administration of different extracts of Sophora alopecuroides, the degree of autophagy in rat liver increased, which may be due to Sophora alopecuroides accelerating liver toxicity by increasing autophagy.

Mitochondria are the energy metabolism centers of cells, playing important roles in cell proliferation, genetic information transmission, immune regulation, cell growth cycle regulation, apoptosis, and other processes. It is also a key site for cells to produce ROS like substances, and mitochondrial damage and dysfunction can cause metabolic abnormalities and organ dysfunction in the body. Therefore, mitochondrial damage may also be an important cause of liver toxicity. Through morphological observation, this study found that after administration of different extracts of Sophora alopecuroides, rat liver mitochondria swelled and even disappeared cristae, especially in the WD group. The reason may be mitochondrial damage, which leads to an increase in ROS free radical generation and ultimately causes organ dysfunction in the body. This study found a significant correlation between rat liver toxicity and oxidative stress by measuring related oxidative stress indicators such as MDA, GSH, SOD, etc. Different extracts of Sophora alopecuroides can cause significant changes in oxidative stress indicators to varying degrees in female and male rats after administration, with a significant increase in MDA and a significant decrease in SOD compared to the blank group. MDA is the final product of lipid peroxidation, which can reflect the degree of lipid peroxidation and thus the degree of liver cell damage. Under normal circumstances, SOD can convert superoxide into H2O2, CAT can convert H2O2 into H2O, and GSH can directly counteract the toxicity of oxygen free radicals by supplying H+, clearing superoxide ions and other free radicals in the body, thereby preventing liver cell damage. In fact, oxidative stress plays a crucial role in the pathogenesis of many diseases, such as colitis, atherosclerosis and Alzheimer’s disease. This study found that the main mechanism by which bitter beans cause liver damage is oxidative stress.
The Nrf2 signaling pathway is the main pathway for oxidative stress and antioxidant activity in the body. Under normal circumstances, Nrf2 mainly binds to Keap1 to form a complex that exists in the cytoplasm and maintains a low level to maintain cellular homeostasis. When the body is exposed to external stimuli, such as drug stimulation and the presence of oxygen free radicals, the complex formed by Nrf2 and Keap1 separates, and Nrf2 protein accumulates and moves to the nucleus. In the nucleus, Nrf2 binds to the antioxidant response element ARE, inducing the expression of downstream antioxidant proteins HO-1 and SOD1. SOD2 is mainly distributed in mitochondria and is a key protein that clears oxygen free radicals to protect the stability of the mitochondrial environment. Its activity changes with changes in ROS in the body. HO-1 and SOD1 can catalyze the degradation of hemoglobin to produce bilirubin, CO, and Fe2+, all of which can exert positive antioxidant effects. SOD2, also known as Zn SOD, can clear superoxide anions. The Western blotting results showed that there was a significant difference in Nrf2 protein expression in the liver of male rats after administration of WD, WU, and EU extracts compared to the blank group. HO-1 protein expression was significantly reduced in all four extract groups after administration (P<0.05, see Figure 6). SOD1 protein expression was significantly reduced in the liver of female rats, while it also showed a decreasing trend in the liver of male rats, with the WD and ER groups being the most obvious (P<0.05). SOD2 protein expression was significantly reduced in the liver of male rats, while it showed an increasing trend in each group of female rats, with significant differences compared to the blank group in WD, WU, and ER. Although there are differences in the changes of indicators between male and female rats, both can cause liver toxicity by affecting the liver oxidative stress indicators of rats. Female rats may restore antioxidant function through compensatory mechanisms to maintain internal balance under oxidative stress. In the early stage of acute toxicity experiments, it was found that male mice were more sensitive to the toxicity of different extracts, had a higher mortality rate, and the physiological structures of male and female rats were different, which may also lead to certain differences in various indicators between male and female rats. Chen et al. also found significant differences in indicators between male and female rats when conducting acute and subacute toxicity experiments on the extract of golden iron lock in mice and rats.
In summary, Sophora alopecuroides extract can cause liver toxicity, mainly by regulating the relevant proteins in the Nrf2/HO-1 pathway, leading to oxidative stress in the body.