The effect of gentiopicroside on TLR-4/NF – κ B and AMPK/Nrf2 pathways in non-alcoholic fatty liver disease
Non alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by diffuse hepatic steatosis, excluding alcohol and other clear liver damaging factors. It is closely related to insulin resistance (IR) and oxidative stress. With the improvement of people’s living standards and the change of diet structure, the incidence rate of NAFLD in China is on the rise. Internal and external factors such as genetics, diabetes, high-fat diet, lack of exercise and so on can induce NAFLD. Modern medicine believes that NAFLD is caused by secondary or multiple attacks of oxidative stress and insulin resistance, and this concept is widely recognized by scholars at home and abroad. Therefore, classic strategies such as anti-inflammatory and antioxidant can serve as the basic treatment principles for this disease. The TLR-4/NF – κ B signaling pathway is a classic inflammatory pathway, which is the most common pathway for liver changes caused by various stimuli. The stimulation of TLR-4 activates NF – κ B through a series of signaling pathways, which in turn promotes the release of inflammatory factors, ultimately inducing hepatocyte necrosis and activating the inflammatory cascade. Inhibiting the activation of the TLR-4/NF – κ B signaling pathway is a key pathway for reducing liver tissue inflammation and a marker for downregulating inflammatory expression related to oxidative stress. Meanwhile, the AMPK/Nrf2 signaling pathway plays an important role in resisting oxidative stress and improving lipid deposition processes in vivo. Nrf2 is the main regulatory factor in the cellular defense system’s resistance to oxidative stress, and there is evidence to suggest that genetic loss of Nrf2 is associated with more severe NAFLD. AMPK is a central regulator of cellular energy homeostasis and inflammation, capable of regulating fatty acid biosynthesis and inhibiting oxidative stress and inflammatory responses. Due to the critical roles of Nrf2 and AMPK in oxidative stress and lipid metabolism, this pathway can serve as a potential therapeutic target for treating liver lipid infiltration and inflammation. Meanwhile, previous studies have shown that the AMPK/Nrf2 pathway may be a key pathway regulating the progression of non-alcoholic fatty liver disease.

Gentiopicroside (GPS) is a terpenoid glycoside extracted from the Gentiana plant in the Gentianaceae family. Pharmacological studies have shown that GPS has clinical effects such as anti-inflammatory, analgesic, antioxidant, and free radical scavenging. Its hepatoprotective and choleretic effects are significant, but its hepatoprotective mechanism is not yet clear. Many studies have shown that GPS has great potential for applications in combating pathogenic microorganisms, gastrointestinal, pancreatic, cardiovascular diseases, respiratory system diseases, and neurological and psychiatric disorders. Meanwhile, GPS has significant antioxidant and liver protective effects, and can promote bile secretion and other biological activities. There are reports indicating that GPS has a protective effect on chemical substances and D-galactosamine/lipopolysaccharide induced liver injury in mice. In addition, GPS can improve symptoms of alcoholic fatty liver disease through the AMPK-PPAR α signaling pathway, but there are currently no reports on the association between GPS and TLR-4/NF – κ B, AMPK/Nrf2 signaling pathways in NAFLD models. Therefore, this experiment established a rat model of NAFLD and used metformin as a control to preliminarily study the protective effect of GPS on the liver and its regulatory effect on the TLR-4/NF – κ B and AMPK/Nrf2 signaling pathways, providing a basis for the clinical treatment of NAFLD with GPS.

Sixty SD rats were randomly divided into a normal group, a model group, a metformin group (200 mg/kg), and GPS high, medium, and low dose groups (120, 60, 30 mg/kg). The normal group was given standard feed, while the other groups were fed high-fat feed for 14 weeks to establish a rat NAFLD model. Biochemical methods were used to detect liver function, oxidative stress, and lipid accumulation. Enzyme linked immunosorbent assay (ELISA) was used to detect insulin resistance and inflammatory factor levels. Western blot was used to detect the expression of TLR-4/NF – κ B and AMPK/Nrf2 pathway proteins in rat liver tissue; Observation of Pathological Changes in Liver Tissue by Oil Red O Staining

 

In recent years, the incidence rate of NAFLD has shown an obvious upward trend and has become one of the main causes of chronic liver disease and abnormal liver function. At present, most of the drugs used to treat NAFLD need to be metabolized through the liver, which increases the burden on the liver while treating NAFLD. Moreover, the therapeutic effect of drugs on NAFLD is limited, and no specific drugs have been found yet. Therefore, finding drugs with definite efficacy and minimal side effects to treat NAFLD has great practical significance.

A high-fat diet can cause an increase in free fatty acids (FFA) in the serum. FFA accumulates continuously in the body, causing liver damage and increased cell membrane permeability, leading to the overflow of ALT and AST from cells into the bloodstream. The activity of ALT and AST in the serum of the model group increased, accompanied by an increase in TC, TG, LDL-C content and a decrease in HDL-C content, indicating the successful establishment of the NAFLD model in this experiment. The research results show that GPS effectively improves NAFLD, regulates transaminase and blood lipids, and has a protective effect on the liver.

The specific pathogenesis of NAFLD is still unclear, and the “multiple parallel strikes” pathogenesis has been widely accepted. Various factors such as inflammation, oxidative stress, and insulin resistance collectively contribute to the occurrence and progression of NAFLD. The liver fat infiltration caused by IR as the center produces a large amount of FFA. If the body’s oxidative pathway cannot process excessive FFA, it will lead to an increase in TG, which in turn can cause fat accumulation and induce fat degeneration. At the same time, an increase in FFA stimulates the secretion of inflammatory response regulatory factors TNF – α, IL-1 β, and IL-6 by liver Kupffer cells, promoting inflammation and leading to inflammatory infiltration, necrosis, and fibrosis of liver cells. When the liver undergoes an inflammatory response, TLR-4 binds to its ligand to activate NF – κ B, leading to the synthesis and release of a series of inflammatory factors and triggering an inflammatory response in the body. The results of this experiment showed that the high, medium, and low dose groups of GPS could reduce insulin resistance index, TG, TC, LDL-C, and levels of inflammatory factors TNF – α, IL-1 β, and IL-6, increase HDL-C levels, and effectively inhibit TLR-4 and NF – κ B signaling pathways. Among them, the high dose group of GPS showed the most significant improvement, suggesting that GPS may protect the liver by inhibiting inflammation, regulating lipid metabolism, and regulating the TLR-4/NF – κ B pathway.

Oxidative stress is widely recognized as one of the damaging factors in non-alcoholic fatty liver disease. The abundance of mitochondria in liver cells facilitates the oxidation of FFA, providing energy to cells while also producing a large amount of reactive oxygen species. The body’s antioxidant defense system can exert protective effects by inhibiting the production of free radicals, clearing free radicals, repairing damage, and inducing antioxidant enzymes. SOD and GSH Px are important antioxidant enzymes that can effectively clear free radicals, inhibit the formation of lipid peroxidation reactions, and protect the body. MDA is the final product of free radicals involved in lipid peroxidation reactions, and its abnormal expression can severely damage the structure of cell membranes, causing cell swelling and necrosis. Activation of AMPK can promote nuclear translocation of downstream molecule Nrf2 and upregulate Nrf2 expression. Nrf2 exerts anti-inflammatory and antioxidant effects by regulating factors, mediating downstream antioxidant proteins and enzymes such as HO-1 to alleviate liver oxidative stress. The results of this study indicate that compared with the model group, the GPS dose groups can significantly enhance SOD and GSH Px activity and reduce MDA content, while significantly increasing p-AMPK and Nrf2 protein expression levels. This suggests that GPS may activate the AMPK/Nrf2 signaling pathway, thereby inhibiting oxidative stress and achieving hepatoprotective effects.

In summary, GPS can effectively improve the injury status of NAFLD rats, regulate insulin resistance, inhibit oxidative stress levels and inflammatory responses, regulate TLR-4/NF – κ B and AMPK/Nrf2 signaling pathways, and provide new strategies for clinical applications.