Polygonatum Polysaccharide Alleviate CCl₄-induced Acute Liver Injury In Rats Via PI3K/AKT/mTOR Pathway

The liver is the main organ involved in detoxification and metabolism of endogenous and exogenous substances.Various chemical substances,drug abuse,and virus infections can cause liver damage,and the development of liver-protective drugs is of great importance.A variety of chemical components in Chinese medicine such as polysaccharides,flavonoids and terpenoids have good hepatoprotective activity and are a natural treasure trove for screening hepatoprotective drugs.Polygonatum polysaccharide is the main active constituent of polygonatum,which has pharmacological effects such as antioxidant,immunomodulatory,anti-inflammatory,and blood sugar regulation.This study induced acute liver injury in rats with a single intraperitoneal injection of carbon tetrachloride（CCl₄）and treated with polygonatum polysaccharides by gavage.The anti liver injury effect and mechanism of polygonatum polysaccharides were explored from the PI3K/AKT/m TOR pathway,providing a theoretical basis for the clinical prevention and treatment of liver injury with polygonatum polysaccharides.Methods:Thirty-two male SD rats were randomly divided into 4 groups（n=8）,namely the control group,model group,polygonatum polysaccharide treatment group(400mg·kg^-1·d^-1),and positive control group(150 mg·kg^-1·d^-1,silymarin).The control group and model group were given ultrapure water by gavage,while the silymarin group was given a solution of silymarin by gavage,while the polygonatum polysaccharide group was given a solution of polygonatum polysaccharide by gavage for 7 consecutive days.After the last gavage for 1 hour,all groups of rats,except for the control group,were intraperitoneally injected with 50%CCl₄soybean oil solution(2.5 m L·kg^-1).The control group was intraperitoneally injected with an equal dose of soybean oil solution.After 24 h,the rats were weighed and anesthetized,and blood and liver samples were collected.Observe the pathological changes of the liver with the naked eye and weigh it;Detection of liver function indicators and oxidative stress indicators in rats using a reagent kit;H&E staining was used to observe the pathological changes of liver tissue;TUNEL staining was used to detect the level of liver cell apoptosis;Transmission electron microscopy was used to observe the ultrastructure of liver cells;Western blot was used to detect the expression and phosphorylation levels of autophagy related proteins and phosphatidylinositol 3-kinase（PI3K）/protein kinase B（AKT）/mammalian rapamycin target protein（m TOR）pathway proteins;Immunofluorescence staining was used to detect the expression of LC3microtubule associated protein 1A/1B light chain 3（LC3 II）.Results:（1）Anatomical changes of the liverThe liver of rats in the control group was dark red with smooth surface,sharp edges and no swelling.The liver of rats in the model group was dull and yellowish with rough surface,adhering to gastrointestinal tissues and swollen.Compared with the model group,the liver lesions of the rats in the polygonatum polysaccharide and silymarin groups were significantly improved.（2）Changes in liver indexCompared with the control group,the liver index was significantly increased（P<0.01）.Compared with the model group,the liver index of the polygonatum polysaccharide group and the silymarin group decreased significantly（P<0.01）.（3）Changes in liver function indicatorsCompared with the control group,the levels of aspartate transaminase（AST）,alanine transaminase（ALT）and alkaline phosphatase（AKP）in the model group were significantly higher（P<0.01）,and the levels of albumin（ALB）were significantly lower（P<0.01）.Compared with the model group,the AST,ALT,and AKP levels in the polygonatum polysaccharide group and silymarin group were significantly reduced（P<0.01）,while the ALB level was significantly increased（P<0.01）.（4）Changes in liver oxidative stress indicatorsCompared with the control group,the levels of malondialdehyde（MDA）and reactive oxygen species（ROS）in the model group were significantly higher（P<0.01）,and the levels of superoxide dismutase（SOD）and glutathione（GSH）were significantly lower（P<0.01）.Compared with the model group,the levels of MDA and ROS in the polygonatum polysaccharide group and silymarin group were significantly reduced（P<0.01）,while the levels of GSH and SOD（P<0.05）were significantly increased.（5）Histopathology changes of liverCompared with the control group,the hepatocytes in the model group had vacuolar degeneration,necrosis and disorganized arrangement.Compared with the model group,liver cells in the polygonatum polysaccharide group were more neatly arranged,and necrotic and inflammatory cells were significantly reduced;liver cells in the silymarin group were neatly arranged,and necrotic and inflammatory cells were significantly reduced.（6）Changes in liver cell apoptosis levelsTUNEL positive cells in the model group were extremely significantly higher than those in the control group（P<0.01）.Compared with the model group,the TUNEL positive cell poles were significantly lower in thepolygonatum polysaccharide and silymarin groups（P<0.01）.（7）Changes in autophagy levels of liver cellsThe number of autophagosomes in the model group was less than that in the control group,and the fluorescence intensity of LC3Ⅱpositive cells was highly significant lower（P<0.01）,LC3II/LC3Ⅰprotein expression was highly significant lower（P<0.01）,and p62expression was highly significant higher（P<0.01）.Compared with the model group,the number of autophagosomes was significantly increased,the fluorescence intensity of LC3II positive cells was highly significant（P<0.01）,LC3II/LC3Ⅰprotein expression was highly significant（P<0.01）and p62 expression was highly significant（P<0.01）in the polygonatum polysaccharide group and silymarin group.（8）Protein expression associated with the PI3K/AKT/m TOR pathwayThe expression levels of p-PI3K/PI3K,p-AKT/AKT and p-m TOR/m TOR proteins were extremely significantly higher in the model group than in the control group（P<0.01）.Compared with the model group,the p-PI3K/PI3K,p-AKT/AKT and p-m TOR/m TOR protein expression levels were highly significant lower in the xanthin polygonatum polysaccharide and silymarin groups（P<0.01）.Conclusion:Polygonatum polysaccharide exerts a protective effect on rat liver by regulating the PI3K/AKT/m TOR pathway to activate autophagy.