| Polysaccharides have received much attention as a new source of bioactive substances,among which plant-derived polysaccharides have a wide range of pharmacological activities,including immunomodulation,antitumour,hypoglycaemia,hypolipidaemia,antioxidant,anti-ageing and hepatoprotection.The roots of Hemerocallis citrina Baroni,a traditional Chinese medicinal herb with food functionality,contains multiple effective components such as flavonoids and polysaccharides.Studies both domestically and abroad have demonstrated the pharmacological activity of the roots of Hemerocallis citrina Baroni extracts.This study aims to purify an anti-tumor active polysaccharide from the roots of Hemerocallis citrina Baroni andelucidate its molecular mechanism of inhibiting the proliferation of liver cancer cells.The main findings are as follows:Part Ⅰ: Isolation,purification,and antioxidant activity study of polysaccharides from the roots of Hemerocallis citrina Baroni.The process involved de-fatting,filtering,drying,hot water extraction,alcohol precipitation,protein removal,DEAE-52,and Sephadex G-100 column chromatography to yield three purified polysaccharides,named Hc BRPS1,Hc BRPS2,and Hc BRPS3.Ultraviolet spectrum analysis revealed high purity of Hc BRPS1,Hc BRPS2,and Hc BRPS3.Monosaccharide composition analysis showed that Hc BRPS2 was composed of Ara,Gal,and Glc,with a molar ratio of 0.088∶0.293∶0.619,while Hc BRPS3 was composed of Rha,Ara,Gal,Glc,Xyl,and Gal A,with a molar ratio of 0.063∶0.219∶0.495∶0.061∶0.032∶0.130.Antioxidant assays demonstrated that the polysaccharides possessed the ability to scavenge ·OH,·O2,and ·DPPH,and removal effect Hc BRPS3>Hc BRPS2>Hc BRPS1.Part Ⅱ: Purified polysaccharide Hc BRPS3 from the roots of Hemerocallis citrina Baroni induced apoptosis in liver cancer cells via the mitochondrial dependentpathway.Liver cancer cells were treated with different concentrations(0,25,50,100,175,and 250μg/m L)of purified polysaccharides for 48 hours,and the MTT assay was used to evaluate the inhibitory effect of the three types of polysaccharides on liver cancer cell proliferation.The results showed that Hc BRPS2 and Hc BRPS3 both had inhibitory effects on liver cancer cell proliferation,and the inhibitory effect of Hc BRPS3 was stronger than that of Hc BRPS2.Hc BRPS3 was able to inhibit the number of liver cancer cell clones formed.Flow cytometry showed that Hc BRPS3 increased the rate of cell apoptosis in a dose-dependent manner.Western blot analysis indicated that Hc BRPS3 significantly increased the levels of Cytochrome c,Bax,Activated-caspase-9,and-3 proteins,while significantly down-regulating the level of Bcl-2 protein,and decreasing the Bcl-2/Bax protein ratio.These results suggested that Hc BRPS3 induces apoptosis in liver cancer cells via the mitochondrial dependentpathway.Part Ⅲ: The purified polysaccharide Hc BRPS3 induced apoptosis in liver cancer cells through the PKM2/STAT3 signaling pathway.Treatment with Hc BRPS3 did not significantly alter the total expression level of STAT3 protein,but did lower the phosphorylation level of STAT3 protein,as well as the nuclear level of p-STAT3.In addition,Hc BRPS3 treatment inhibited PKM2 protein expression and nuclear translocation,which inhibited PKM2-mediated phosphorylation of STAT3.Hepatocellular carcinoma cell lines with overexpression of PKM2 were obtained through cell transfection and treated with Hc BRPS3.Results showed that PKM2 overexpression reversed the Hc BRPS3-induced down-regulation of STAT3 phosphorylation levels.Flow cytometry indicated that PKM2 overexpression could reverse the Hc BRPS3-induced increase in apoptosis rate and changes in expression levels of apoptosis-related proteins.These results suggest that purified polysaccharides from the roots of Hemerocallis citrina Baroni can induce cell apoptosis,and the mechanism is related to the down-regulation of PKM2 expression and inhibition of downstream STAT3 phosphorylation. |
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