Study on the protective effect of total flavonoids from Platycodon grandiflorus on acute liver injury rats based on SIRT6/NF – κ B signaling pathway
Acute liver injury (ALI) refers to a disease characterized by sudden abnormal liver function caused by various reasons in the short term. It is the initial stage of many liver diseases and its pathogenesis is complex; Acute liver injury caused by drug poisoning, immune response, viral infection, ischemia-reperfusion, etc. is closely related to oxidative free radicals, inflammatory factors, and cell apoptosis. Based on the current research status of treating acute liver injury both domestically and internationally, Western medicine often diagnoses liver disease through clinical manifestations and pathological changes. However, there is still a certain deviation from clinical practice, and there is still a lack of efficient multi-target hepatoprotective drugs for liver injury caused by various reasons in clinical practice. With the continuous deepening of traditional Chinese medicine’s basic theory and clinical research on liver disease, it has gradually shown advantages in the prevention and treatment of liver disease. Research on acute liver injury in traditional Chinese medicine theory has found that its pathogenesis is mostly due to the inability of positive deficiency to resist external pathogens, leading to the entry of damp heat and toxic pathogens into the bloodstream, blood stasis, and liver depression and qi stagnation. Therefore, exploring drugs for treating acute liver injury and studying their mechanisms of action are of great significance.

Inflammatory response has received widespread attention in the treatment of acute liver injury and other diseases, among which nuclear transcription factor – κ B (NF – κ B) is an important inflammatory transcription factor. When the liver undergoes an inflammatory response, it stimulates the acetylation of NF – κ B, initiates the transcription of downstream inflammatory factors such as TNF – α, IL-1 β, IL-6, and binds to the VCAM-1 and MCP-1 gene promoter regions for regulation, increasing the release of inflammatory factors and the synthesis of inflammatory mediators. Silencing information regulatory factor 2 (SIR2) is a widely present nicotinamide adenine dinucleotide (NAD+) – dependent deacetylase in living organisms. There are seven proteins in humans that are homologous to SIRT2: SIRT1~SIRT7. SIRT6 is located at the 13.3 end of chromosome 19, encoding proteins of 355 and 328 amino acids in length by 8 and 7 exons, respectively. It exerts an inhibitory effect on gene transcription by deacetylating and modifying the H3K9 site of many genomic proteins, including NF – κ B, on chromatin, thereby downregulating the expression of inflammatory factors and exerting anti-inflammatory effects.

Polygonum perfoliatum L., a plant belonging to the Polygonaceae family and the Polygonum genus, is a whole plant used to treat conditions such as jaundice, nephritis edema, mastitis, pertussis, chronic eczema, venomous snake bites, and damp heat jaundice. It has antibacterial, antiviral, anti mutagenic, blood pressure lowering, anti-tumor, and hemostatic effects. Guangxi Zhuang people commonly use it for the prevention and treatment of liver diseases. In recent years, our research group has conducted systematic pharmacological, pathological, and chemical component extraction, separation, and content determination studies on the active ingredient total flavonoids, which have hepatoprotective and anti liver injury properties, in order to determine the pharmacological substance basis for the prevention and treatment of liver disease by using the medicinal herb “Gangbangui”. The experiments have shown that TFP can reduce the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), enhance the activity of antioxidant enzymes such as total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH Px), and its mechanism may be related to its antioxidant free radical, lipid peroxidation inhibition, immune enhancement, and reduction of inflammatory mediator levels.

This experiment aims to explore the protective effect of TFP on acute liver injury induced by carbon tetrachloride (CCl4) in rats based on the above pathogenesis and characteristics, and to explore its hepatoprotective mechanism from the aspects of oxidative stress and inflammation, providing experimental basis for the development and application of TFP.

Seventy female SD rats were randomly divided into seven groups based on body weight: normal group, TFP control group (200 mg/kg), CCl4 model group, CCl4+silymarin group (120 mg/kg), and CCl4+TFP high, medium, and low dose groups (200, 100, 50 mg/kg), with 10 rats in each group. The normal group and CCl4 model group were given distilled water (8 mL/kg) by gavage, while the other groups were given corresponding doses of the drug by gavage once a day for 10 consecutive days. After the last administration, except for the normal group and TFP control group, all other groups were intraperitoneally injected with 12% CCl4 olive oil solution (5 mL/kg) to establish an acute liver injury model. Fasting without water for 16 hours, and collecting liver tissue after taking blood from the eyeball. Biochemical methods are used to detect liver function and oxidative stress indicators in serum; Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in liver tissue; Western blot was used to detect the expression levels of SIRT6/NF – κ B pathway related proteins in liver tissue; HE staining was used to examine the degree of liver tissue lesions, and immunohistochemistry was used to observe the expression level of p-NF – κ B p65 in liver tissue.

This experiment established a rat model of acute liver injury induced by CCl4 and intervened with total flavonoids (TFP) from Scutellaria baicalensis. From the aspects of liver function indicators, pathological sections, and expression of related inflammatory factors, the alleviating effect of TFP on acute liver injury in rats was found. In research, establishing a successful animal model of liver injury is one of the key steps in screening hepatoprotective drugs. CCl4 mainly induces free radical metabolism, leading to oxidative stress that destroys the cell membrane structure of liver tissue, increases cell membrane permeability, and causes inflammatory infiltration of neutrophils. The levels of ALT, AST, ALP, TBIL, and γ – glutamyl transpeptidase (γ – GT) in the cytoplasm increase, and the production of lipid peroxide malondialdehyde (MDA) continues to damage the cell membrane structure, creating a vicious cycle. In addition, CCl4 can also cause a decrease in the activity of antioxidant enzymes total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH Px), leading to an imbalance of the enzyme antioxidant system, which may form a vicious cycle of oxidative stress and exacerbate inflammatory reactions. Therefore, the increase in relevant indicators such as ALT, AST, ALP, TBIL, γ – GT, MDA content, and the decrease in T-SOD and GSH Px activity can be considered as important criteria for determining the severity of acute liver injury. In this experiment, after CCl4 modeling treatment, compared with the normal group, the serum levels of ALT, AST, ALP, TBIL, γ – GT, and MDA in the model group rats were significantly increased, while the activities of T-SOD and GSH Px were significantly decreased. Observing pathological sections, HE staining showed severe damage to the liver tissue structure, with liver cells exhibiting severe vacuolar deformation and being infiltrated by a large number of inflammatory cells. The liver lobular structure was severely damaged, indicating the successful establishment of the acute liver injury model.

According to reports, SIRT6 regulates the expression of telomere chromatin and downstream genes in the NF – κ B signaling pathway. The transcription factor NF – κ B plays a key role in environmental stress response by regulating genes related to proliferation, apoptosis, differentiation, inflammation, and immune system regulation. Under non stress conditions, NF – κ B is isolated in the cytoplasm by its inhibitor I κ B α. Under stress conditions, I κ B α is degraded, allowing NF – κ B transcription factors to migrate to the nucleus and activate transcription of key genes. Given the importance and tissue specificity of these target genes, the activation of the NF – κ B pathway is strictly regulated by several mechanisms, one of which involves NAD+- dependent deacetylase and ADP ribosyltransferase SIRT6. After the pathway is activated, SIRT6 binds to the promoter region of the NF – κ B target gene, promoting gene silencing by deacetylating lysine 9 of histone H3. TNF – α secreted by mononuclear macrophages has the effect of promoting inflammatory response and activating macrophages, controlling the production and secretion of inflammatory mediators. IL-1 β is mainly secreted by mononuclear macrophages and lymphocytes. In addition to triggering pathogenic inflammatory reactions, it also promotes the expression of various inflammatory factors and participates in acute phase inflammatory responses. IL-6 is an inflammatory factor secreted by mononuclear macrophages with various biological activities. When the liver is damaged, mononuclear macrophages release excessive IL-6, which leads to massive necrosis of liver cells and can develop into liver fibrosis or even cirrhosis in the long run. At the same time, a large amount of IL-6 aggregates accelerate the contact between white blood cells and endothelial cells through VCAM-1, and more monocytes reach the damaged liver site through MCP-1, exacerbating the inflammatory response.

Modern research has shown that Kangban Gui has rich pharmacological effects such as cough and phlegm relief, antioxidant and anti-inflammatory, and antiviral. The results of this experiment show that it is also commonly used in clinical practice to treat various diseases such as burns, cough, herpes zoster, acute nephritis, and bacterial dysentery, all of which have good therapeutic effects. At the same time, Kangban Gui has also been developed into various dosage forms and widely used in clinical practice. This experiment tested various indicators commonly used in clinical practice to determine liver function. The results showed that after TFP administration, compared with the normal group, the main transaminase and liver function indicators in the serum of TFP control group rats had no statistically significant effect, indicating that TFP does not have adverse effects on liver function; Compared with the model group, the levels of transaminase in the serum of rats in each dose group of CCl4+TFP decreased, while the levels of antioxidant factors increased, indicating that TFP can effectively improve liver function indicators and enhance the body’s antioxidant capacity. It is expected to be developed into an efficient and low toxicity hepatoprotective preparation. Microscopic observation revealed that after treatment, the liver tissue structure, vacuolar size, and degree of deformation in rats were significantly improved compared to the model group. Inflammatory cell infiltration was significantly reduced, and the expression of p-NF – κ B p65 was weakened. Western blot was used to detect the expression of SIRT6, p-NF – κ B p65, MCP-1, and VCAM-1 proteins. The results showed that the expression of SIRT6 increased and the expression of p-NF – κ B p65 decreased in each dose group of TFP. The expression of key pro-inflammatory factors MCP-1, VCAM-1, TNF – α, IL-6, and IL-1 β decreased, suggesting that TFP may effectively improve acute liver injury in rats by reducing liver inflammation and enhancing antioxidant capacity. This result suggests that TFP may exert anti acute liver injury effects by promoting SIRT6 expression and inhibiting NF – κ B transcription to reduce the release of downstream inflammatory factors.

In summary, TFP can effectively alleviate liver injury, and its mechanism may be related to the inflammatory response and antioxidant mediated by the SIRT6/NF – κ B pathway. The research results of this article may provide new methods and strategies for the clinical use of TFP to alleviate liver injury.