Study on the mechanism of action of polysaccharides from dog liver vegetables in alleviating liver fibrosis based on the LIN28A/NF – κ B signaling pathway
Hepatic fibrosis (HF) is caused by various harmful stimuli, including viral infections, drug toxicity, alcohol, and sustained wound healing responses to autoimmune liver disease, leading to liver damage. It is an important pathological marker of chronic liver disease, a key event in the occurrence of cirrhosis, and a major cause of end-stage hepatocellular carcinoma and advanced cirrhosis. As a clinical medication for liver fibrosis and cirrhosis, colchicine mainly inhibits the early activation and proliferation of hepatic stellate cells, promotes hepatic stellate cell apoptosis, interferes with collagen metabolism, and has varying therapeutic effects on HF caused by different etiologies. Colchicine can also cause many side effects, such as abdominal pain, nausea, loss of appetite, etc. High doses of colchicine can also lead to liver and kidney failure. Therefore, new safe and effective therapeutic drugs still need to be researched and developed. Dicliptera cinensis (L.) Juss, as a traditional Chinese medicinal herb, has a sweet taste and a cool nature. It has the effects of clearing heat, cooling blood, diuresis, detoxification, and calming the liver. It has a relieving effect on various types of liver injury models such as CCl4, D-galactosamine hydrochloride, and lipopolysaccharide. In previous studies, it was found that dog liver vegetable polysaccharides (DCP) have a good inhibitory effect on liver lipid metabolism disorders, mainly related to alleviating inflammatory reactions and inhibiting oxidative stress. LIN28A is a highly conserved RNA binding protein that plays an important role in inducing pluripotent stem cells, regulating development, cell growth, and metabolism. Overexpression of LIN28A has been found in various cancer cells such as esophageal cancer and ovarian cancer. Studies have shown that liver specific deletion of LIN28A can alleviate mouse hepatocellular carcinoma and effectively prolong the lifespan of cancer affected mice, suggesting that LIN28A may become a potential target for the treatment of hepatocellular carcinoma or liver diseases. However, there is currently no literature reporting its association with HF. This study aims to explore the association between LIN28A and HF and investigate whether DCP can protect the liver by inhibiting LIN28A.

Liver fibrosis is a pathological lesion characterized by the progressive accumulation of extracellular matrix (ECM) in the perihepatic space, and is the main problem of chronic liver disease. Due to its ultimate cause of cirrhosis, it has become the leading cause of morbidity and mortality worldwide, with an estimated 2% of the global population affected by cirrhosis. Therefore, studying the protective effect of DCP on the liver is of great significance.
The experiment used CCl4 to induce liver fibrosis, which caused pathological changes very similar to human clinical HF. HA, LN, PCIII, and IV-C are the main components of the extracellular matrix in liver cells. In liver fibrosis, due to increased synthesis and insufficient degradation of extracellular matrix, the degree of elevation in their indicator levels is positively correlated with the degree of liver fibrosis. In the study, the levels of HA, LN, PC Ⅲ, and Ⅳ – C in the model group rats significantly increased. The results of H&E staining and Masson staining showed that the liver tissue of the model group rats exhibited significant lipid degeneration and collagen deposition, indicating successful modeling. After administration of DCP, the levels of HA, LN, PC Ⅲ and Ⅳ in rat serum significantly decreased, and the H&E staining pathological changes were alleviated, indicating that DCP has a protective effect on the liver and liver cells.
NF kB is a nuclear transcription factor that regulates a large number of genes that play important roles in regulating cell apoptosis and inflammation. When the liver undergoes an inflammatory response, NF kB is activated and releases inflammatory factors such as TNF – α, IL-6, and IL-1 β, leading to an exacerbation of liver tissue inflammation. Inducing the expression of enzymes COX-2 and iNOS at the site of inflammation will also increase. Excessive inflammatory response also serves as a marker of inflammation. The experimental results showed that the mRNA relative content and protein expression levels of NF-kBp65/\ COX-2 and iN-OS in the liver tissue of the model group rats were significantly increased, and the mRNA relative content of TNF – α, IL-6, and IL-1 β was also significantly increased; Compared with the model group, the mRNA relative content and protein expression levels of NF-2kBp65, COX-2, and iNOS in the liver tissue of rats in the low, medium, and high dose groups of DCP were significantly reduced. The mRNA relative content of inflammatory factors TNF – α, IL-6, and IL-1 β also decreased to varying degrees. This result indicates that DCP can exert anti-inflammatory effects by inhibiting the expression of NF-kBp65 protein and reducing the release of downstream inflammatory factors.
TGF – β 1 is a multifunctional cytokine (pro fibrotic and inflammatory factor) involved in immune response and the development of liver fibrosis. TGF – β 1 can lead to the activation, proliferation, and fibrosis of hepatic stellate cells (HSCs). Alpha SMA can reflect the activation status of HSC and serve as a marker protein for HSC activation. It can also be used to evaluate the degree of liver fibrosis. Matrix metalloproteinases (MMPs) are key factors in the degradation and remodeling of extracellular matrix. MMP9 is one of the most relevant MMPs for degrading normal liver matrix, which can promote the development of liver fibrosis. Western blot analysis showed that the protein expression levels of TGF – β 1, α – SMA, and MMP9 in the liver tissue of the model group rats were significantly increased; Compared with the model group, the protein expression levels of TGF-21, a-SMA, and MMP9 in the liver tissues of rats in the low, medium, and high dose groups of DCP were significantly reduced. This result indicates that DCP can alleviate HF by reducing the expression of TGF – β 1 and MMP9 proteins in liver tissue induced by CCl4, slowing down the rate of collagen deposition in the liver.
The LIN28A gene can be recognized through its unique cold shock domain (CSD) and cysteine cysteine histidine cysteine zinc finger domain pairing, playing an important role in sequence specific mRNA binding, miNNA binding, miNNA pre-processing, and miNNA regulated gene silencing. Previous studies have shown that LIN28A participates in inflammatory responses through the NF-kBp65 signaling pathway, and reducing the expression of NF-kBp65 helps alleviate HF. In the experiment, compared with the normal group, the protein expression levels and mRNA relative contents of LIN28A and NF-kBp65 in the liver of the model group rats were significantly increased, and the protein expression levels and mRNA relative contents of LIN28A and NF-kBp65 in the liver tissues of rats in the low, medium, and high doses of DCP groups were significantly reduced and showed concentration dependence. The above results indicate that the process of HF inflammation may be related to the activation of LIN28A. Combining with DCP can inhibit the expression of LIN28A and NF-kBp65, as well as the expression of downstream inflammatory factors such as COX-2, iNOS, TNF – α, IL-6, and IL-1 β in NF-kBp65. It is speculated that the anti-inflammatory and therapeutic mechanisms of DCP for HF may be related to the inhibition of the LIN28A/NF kB signaling pathway.
In summary, DCP has a significant improvement effect on rat HF, and its pharmacological mechanism may be achieved by regulating the LIN28A/NF kB signaling pathway to prevent inflammatory response.