Study On Extraction Of Polygonatum Sibiricum Polysaccharides And Its Protective Effect On CCl₄-Induced Acute Liver Injury In Rats

Liver injury is one of the clinical diseases threatening human and animal health.In recent years,due to drug poisoning,feed moldy,pathogenic microorganism infection and so on,animal liver damage has become more and more common.Drug therapy is unable to cure liver diseases due to the toxic effects of some components to the liver or the destruction of liver cells by its derivatives.Traditional Chinese medicine has a good effect on protecting liver.The efficacy of traditional Chinese medicine has the advantages of small side effect,which has obvious effect on improving the liver antioxidant level and repairing liver injury.At present,many kinds of Chinese medicine ingredients have been used in the treatment of liver diseases,and achieved good results.Polygonatum sibiricum polysaccharides is the main functional ingredient of the Polygonatum sibiricum.It has been reported that it has a therapeutic effect on heart,lung and kidney injury,and has the function of antioxidation and immunity,but it has not been reported in the treatment of liver injury.In this experiment,the response surface method was used to optimize the extraction process of Polygonatum sibiricum Polysaccharides,and the protective effect of PSP on CCl₄ liver injury was explored from the biochemical indexes of liver function,antioxidation pathway,inflammatory gene and anti apoptotic gene,to provide experimental basis for the clinical application of Polygonatum sibiricum polysaccharides protecting liver.The extraction process and extraction rate of Polygonatum sibiricum polysaccharides are clarified with the extraction process optimized and the influence of different extraction conditions on the extraction rate analyzed in this experiment.A CCl₄-induced acute liver injury model was established with using the prevention of by Polygonatum sibiricum polysaccharides subsequently.The effects of Polygonatum sibiricum polysaccharides on the biochemical indexes of liver function in rats and the regulation of gene expression of inflammatory factors were explored.（1）Optimization of Polygonatum sibiricum polysaccharides extraction process by response surface analysisFirstly,three main factors affecting the extraction rate of Polygonatum sibiricum polysaccharides（liquid and material ratio 10:1,15:1,20:1,25:1,30:1,time 1,2,3,h,etc.）were tested by single factor test.Then,the extraction process was optimized by response surface analysis with the extraction rate used as the response value.The results showed that（1）the optimum extraction conditions of Polygonatum sibiricum polysaccharides were as follows:liquid material ratio was 19.13:1,temperature was 73.33℃and extraction time was 3.49 h.The extraction rate of PSP is 10.107%.（2）The protective effect of Polygonatum sibiricum polysaccharides on the CCl₄-induced acute liver injuryBiochemical results showed that compared with the control group,the activities of ALT,AST and ALP in the serum of model group were significantly improved（P<0.05）,and the contents of DBIL and TBIL increased significantly（P<0.05）.Compared with the model group,the activity of ALT,AST and ALP in the serum decreased significantly（P<0.05）,and the content of DBIL and TBIL decreased significantly（P<0.05）.PSP could improve the disorder of biochemical indexes of liver injury caused by CCl₄.Quantitative PCR results showed that PSP significantly lowered the Keap1 gene and significantly up-regulated the Nrf2 and NQO1 genes（P<0.05）,suggesting that PSP activates the downstream antioxidant gene NQO1 by activating the Nrf2/ARE signaling pathway to play an antioxidant role.PSP significantly inhibited the down regulation of TNF-αand IL-6genes（P<0.05）,and significantly up-regulated the Bcl-2 and Bcl-xl genes（P<0.05）,indicating that PSP prevents inflammation of the liver by inhibiting the expression of inflammatory factors and improves the antioxidant level of liver cells by promoting the expression of anti apoptotic genes.The results of immunoblotting showed that PSP decreased the expression of Keap1 protein and increased the expression of Nrf2 and NQO1protein.PSP could increase the synthesis of antioxidant protein NQO1 by activating the Keap1-Nrf2-ARE pathway to resist the oxidation of CCl₄ to the liver.Conclusion:（1）The response surface method can optimize the extraction process of Polygonatum sibiricum polysaccharides;（2）By activating antioxidant pathway Keap1-Nrf2-ARE,PSP up-regulated the expression of antioxidant gene NQO1,anti apoptotic gene Bcl-2 and Bcl-xl,down regulated the expression of IL-6 and TNF-α,enhanced the level of liver antioxidant and anti apoptosis,and inhibited the occurrence of inflammatory reaction.