Structural Characterization Of Pinus Koraiensis Pine Nut Polysaccharide And Its Hepatoprotection Mechanisms Against Chemical Induced Liver Injury

With the development of industrial technology,modern science has not only greatly improved people’s living standards,but also markedly enhanced the exposure to hepatotoxicants.As a result,the incidence of liver injury has increased year by year,seriously threatening people’s health and quality of life.As the largest digestive and metabolic organ in human body,liver tissue is mainly responsible for detoxification.Thus,liver tissue is extremely vulnerable to damage by toxic substances.Various factors such as poor air quality,serious environmental pollution,excessive alcohol consumption and drug abuse can cause chemical liv er injury.Long-term or short-term severe liver injury will further cause fatty liver,hepatitis,cirrhosis and even liver cancer.Currently,the existing drugs for prevention and treatment of liver diseases generally have disadvantages such as poor efficacy and strong side effects.Therefore,one of the important measures to protect from liver injury and decrease the incidence of liver diseases is to develop drugs or functional foods with good efficacy and low side effects for hepatoprotection.In this study,we extracted polysaccharides from Pinus koraiensis pine nuts by the method of water extraction and alcohol precipitation.The extraction process of pine nut polysaccharide was optimized by response surface design.PNP40c-1 was screened as the component with the strongest hepatoprotective activity in pine nut polysaccharides by tracking their hepatoprotection in vitro,and its structural features were then characterized and analyzed.In addition,the protecti ve effects of the in vitro simulated digestion product of PNP40c-1 on carbon tetrachloride(CCl4)-,ethanol-and drug-induced liver injury were systematically studied in this research.By comparison,it was determined that the in vitro simulated digestion product of PNP40c-1 exerted the strongest hepatoprotective activity against CCl4-induced liver injury.And the detailed action mechanisms of its hepatoprotection were also revealed by combining with the animal experimental results.Box-Behnken response surface optimization was used to determine the optimal pine nut polysaccharide extraction parameters: ratio of raw materials to water 1:40(g/m L),extraction temperature 95 ℃,single extraction time 1.5 h,and extraction times 3,and the yield of polysaccharide was 9.26 ± 0.10%.DEAE-52 and Sephacryl S400 were sequentially utilized to purify the crude pine nut polysaccharide.L-02 cell liver injury model was established to screen and identify the main hepatoprotective component in pine nut polysaccharide,PNP40c-1.HPGPC,GC-MS,FT-IR,1D-NMR and 2D-NMR were applied to characterize the structure of PNP40c-1.Results showed that PNP40c-1 was a homogeneous polysaccharide with an average molecular weight of 2.06 × 105 Da,which was mainly composed of arabinose,rhamnose and glucose at a molar ratio of 2.98: 1.00: 0.52.The bone structure of PNP40c-1 consisted of →3,4)-α-L-Arap(1→,→4)-α-L-Arap3Me(1→,→3)-α-L-Rhap(1→ and →6)-β-D-Glcp(1→,and the branch structure is β-D-Glcp-(1→,connected to the C4 position of →4)-α-L-Arap3Me(1→.In the simulated digestion in vitro,the molecular weight of PNP40c-1 did not change obviously after the saliva digestion,but was significantly decreased after the simulated gastric and intestinal digestion.Meanwhile,the contents of reducing ends were also incaresed markedly.However,during the whole simulated digestion,no free monosaccharide was detected,suggesting that the decrease in the molecular weight of PNP40c-1 was mainly due to the breakage of glycosidic bonds.Based on the L-02 cell experiment,we compared and determined that the in vitro digestion product of PNP40c-1 exerted the strongest hepatoprotective effects against CCl4-induced liver injury.Thus,we further studied the in vitro protective effect of PNP40c-1 digestion product on CCl4-induced liver injury,and discovered that it could not only activate the KEAP1/NRF2/ARE signaling pathway to protect the oxidative damage in hepatocytes,promoting the expression of targeted genes HO-1,GCLC and NQO1,increasing the antioxidant enzymes activities and antioxidants contents,reducing the level of lipid peroxidation and ROS production in L-02 cells,but also reduce the expression of cytochrome P450 and inhibit the apoptotic protein Caspase-3 activation in hepatocytes,hindering cell apoptotic pathway.Additionally,the digestion product of PNP40c-1 in vitro was also proved to possess anti-inflammatory activity,significantly inhibiting the secretion of inflammatory factors in LPS-stimulated RAW264.7 macrophages,and alleviating inflammatory responses.To investigate the hepatoprotective effects of PNP40c-1 against CCl4-induced liver injury in vivo and explore the possible action mechanisms for its hepatoprotection,a mice model was established.Results showed that PNP40c-1 exhibited significantly protective effect on CCl4-induced liver injury in vivo.On one hand,PNP40c-1 regulated the intracellular redox state through the NRF2/ARE signaling pathway,promoting the expression of antioxidant genes,increasing the anti-oxidative stress capacity in hepatocytes,and protecting the oxidative damage of hepatocytes caused by CCl4.On the other hand,it also promoted MKP1 protein expression via NRF2/ARE/MKP1/JNK pathway.The increased MKP1 expression could effectively inhibit the activation of JNK pathway,inhibiting the hepatocytes apoptosis,reducing the expression of pro-apoptotic proteins Bax and cleaved-Caspase-3 in liver cells,upregulating the expression of anti-apoptotic protein Bcl2,and reducing CCl4-induced liver cell apoptosis.Furthermore,PNP40c-1 could also inhibit the phosphorylation of IKKβ and IκBα,reduce NF-κB nuclear transfer and prevent NF-κB from binding to targeted genes in nucleus,thereby reducing excessive secretion of inflammatory mediators,and alleviating liver inflammation to help liver injury induced by CCl4.In this research,the polysaccharide PNP40c-1 with hepatoprotective activity was isolated and purified from Pinus koraiensis pine nuts and its molecular structure was determined.We further proved its protective effects on CCl4-induced liver injury in vivo and in vitro,and revealed the detailed molecular mechanisms.This study is expected to provide a theoretical basis for the development and comprehensive utilization of Pinus koraiensis resources,and it is also important for the research and development of natural hepatoprotective drugs and functional foods with high efficiency and low toxic.