Preliminary Investigation On Protective Effect And Mechanism Of Cyclocarya Paliurus Polysaccharide In Mice With Acute Liver Injury

As the largest metabolic organ in mammals,the liver plays an important role in maintaining homeostasis in the body.Alcohol,drugs,heavy metals and environmental toxins can cause liver damage,disrupting body functions and leading to serious public health consequences.Current drugs for liver treatment have many side effects and limited efficacy.Therefore,the search for a natural product to prevent chemical-induced liver stress damage is imminent.Cyclocarya paliurus（C.paliurus）is distributed in several provinces south of the Yangtze River in China,and is classified as a Grade II protected species of the dicotyledonous plant family Huperziaceae.C.paliurus polysaccharide（CP）is a proteoglycan consist of arabinose,rhamnose and galactose.Previous studies have shown that CP has various biological activities,such as anti-diabetic,anti-obesity,antioxidant,anti-inflammatory,hepatoprotective and immunomodulatory effects.Carbon tetrachloride（CCl₄）is a typical environmental toxicant that is often used to establish animal models of acute or chronic liver diseases.The main causes of liver injury are the induction of oxidative stress and inflammatory response in the liver.In this paper,we firstly used in vitro cellular assay to investigate the antioxidant stress damage used by CP on cells in vitro,and then used carbon tetrachloride-induced acute liver injury mouse model to investigate its protective effect and mechanism on acute liver injury,and explored the effect of CP on intestinal flora and its metabolites in mice based on 16S r RNA high-throughput sequencing technology.The main findings of this paper are as follows.1.100μg/m L,200μg/m L,400μg/m L concentrations of CP were used to induce NCTC-1469 cells,followed by oxidative damage treatment with H₂O₂,and the results showed that the cell viability of the CP group was significantly higher than that of the model group,and the GSH-Px enzyme activity was increased in the cells treated with different concentrations of CP while the MDA content decreased.These results indicated that the early treatment of mouse liver cells with CP could improve the resistance to oxidative stress damage,which was reflected in the increase of antioxidant enzyme activity and the decrease of the production of lipid peroxidation.2.Male Kunming mice were continuous gavaged of CP at 50 mg/kg b.w（CPL）,100 mg/kg b.w（CPM）and 200 mg/kg b.w（CPH）,then the model（MC）,CP and positive（PC）groups were treated with CCl₄ starting one hour after the last gavage.The liver function index（ALT and AST levels in serum）and liver pathology（H&E staining）showed that the CP-treated mice showed significant restoration of the degree of liver injury and liver function compared to the mice in the model group.In addition,CP significantly restored the extent of carbon tetrachloride-induced liver oxidative and inflammatory damage in mice,mainly by increasing the activity of antioxidant enzymes（SOD,GSH-Px）,decreasing the MDA content and decreasing the expression of inflammatory factors（NO,TNF-α,IL-1β,COX-2 and i NOS）in mouse liver.These results suggest that CP can exert hepatoprotective activity by reducing the extent of oxidative and inflammatory damage in the liver of mice caused by CCl₄.3.Western Blot assay was used to investigate the mechanism of inflammation regulation in mouse liver,and the results showed that the expression of TLR4 and the phosphorylation of three sub-kinases（JNK,ERK,p38）in MAPK pathway were significantly increased in the liver of mice treated with CCl₄.In contrast,TLR4 was inhibited in the liver of the three groups of mice treated with CP,and p-JNK/JNK,p-ERK/ERK,and p-p38/p38 were all decreased,and showed a negative correlation with CP concentration.These results indicated that CP could effectively alleviate the CCl₄-induced upregulation of TLR4/MAPK signaling pathway in mouse liver,then alleviating the inflammatory injury caused by CCl₄,and TLR4/MAPK signaling pathway was a key regulatory pathway in CP treatment of liver injury.4.16S r RNA high-throughput sequencing technology and gas chromatography analysis were used to investigate the gut microbiota and short-chain fatty acids in the feces of mice in the normal group,model group and high-dose CP group.The results showed that the contents of the four major short-chain fatty acids（acetic acid,propionic acid,butyric acid and valeric acid）in the colon contents of the model group showed significant differences from those of the normal group,while the contents of the short-chain fatty acids in the CPH group increased to a certain extent compared with those of the model group.By analyzing theαandβdiversity of the gut microbiota,the results showed that the gut microbiota communities in the normal,model and CPH groups showed some differences,but the communities in the CPH group were close to the normal group and showed significant modulation of some specific intestinal bacterial genera.These results suggest that gut microbiota and its metabolites play a key role in CP treatment of liver injury.