The Structural Characteristics And Anti-Hepatoma Mechanism Of Polysaccharides From Penthorum Chinense Pursh

Penthorum chinense Pursh is a perennial herbaceous plant in the Saxifragaceae family,which is widely distributed in East Asia and has excellent medicinal value.For thousands of years,P.chinense has been regarded as"immortal herb"by the Hmong people because of its excellent hepatoprotective effect.At present,various bioactive compounds have been isolated from P.chinense,such as flavonoids,polyphenols,organic acids,sterols,and polysaccharides from P.chinense,which exhibit anti-hepatoma,anti-inflammatory,hypoglycemic,liver protection,antioxidant,and antiviral activities.Polysaccharides,as one of the functional substances of P.chinense,have attracted much more attention in recent years.It has been reported that variations in extraction temperature can result in changes of the chemical structure of polysaccharides,thereby affecting their biological activities.At present,the extraction of P.chinense polysaccharides is mainly carried out by water diffusion or enzyme-assisted extraction at a single temperature.In addition,there is little data on the mechanism of action of P.chinense polysaccharides on liver cancer.Therefore,the polysaccharides were prepared at different temperatures,and the effects of temperature on the structural properties and anti-hepatoma activity of polysaccharides were systematically investigated,as well as the mechanism which the polysaccharide against different species of hepatoma cells（murine and human）in vitro and in vivo were thoroughly discussed in this study.This study firstly analyzed the effect of temperature on the structural properties and anti-hepatoma activity of the P.chinense polysaccharides.Two purified polysaccharides,named PCP-4 and PCP-100,were successfully obtained from P.chinense at 4°C and 100°C via a series of separation and purification steps,respectively.High-performance gel permeation chromatography showed that PCP-4 and PCP-100 were homogeneous polysaccharides,and the molecular weight decreased with increasing temperature,which was 1.83×10⁶ Da and1.46×10⁶Da,respectively.Combined with Fourier transform infrared spectroscopy,ion chromatography,methylation,and nuclear magnetic resonance analysis,PCP-4 was found to have rhamnogalacturonan-I which is composed of→2,4)-α-L-Rhap-（1→and→4）-α-Galp A-（1→.And the neutral sugar chains of PCP-4 composed of→3,5）-α-L-Araf-(1→,α-L-Araf-（1→,→3）-β-D-Galp-（1→,→5）-α-L-Araf-（1→,→6）-β-D-Galp-（1→,→3,6）-β-D-Galp-（1→,→3）-α-L-Araf-（1→,→4）-β-D-Galp-(1→.In contrast,PCP-100,which was extracted at high temperature,showed significantly higher galacturonic acid content and homogalacturonan compared with PCP-4,while the proportion ofβ-D-Galp andα-L-Araf in the neutral sugar chain region decreased.The results of scanning electron microscopy,atomic force microscopy and X-ray diffraction analysis also further confirmed that temperature resulted in complex changes to the structural properties of polysaccharides.Additionally,MTT results showed that PCP-4 could significantly inhibit the proliferation of mouse H22 ascites tumor cells and human HepG2 liver cancer cells,and its tumor growth inhibition was far greater than those of PCP-100.In conclusion,high-temperature treatments destroyed the unstable neutral sugar chain,leading to changes in the structural properties of polysaccharides,which in turn led to a significant weakening of their antitumor activity.The mouse H22 ascites tumor cells were used to investigate the pro-apoptotic effect and related mechanism of PCP-4.The proliferation status of H22 cells,as suspension-type hepatocellular carcinoma cells divide and proliferate in the peritoneal cavity of mice,can assess the growth status and adaptive capacity of tumor cells under internal environmental conditions.MTT assay showed that PCP-4 significantly inhibited the growth and proliferation of H22 cells in a time-and dose-dependent manner.Further,it was found by PI staining that PCP-4 could interfere with S phase transition and prevent mitosis,resulting in effective inhibition of H22 cell proliferation.Morphological observations showed that PCP-4 induced H22 cells to show typical morphological features of apoptosis,such as cell shrinkage,membrane blebbing,nuclear chromatin sequestration and cellular debris increased.Annexin V-FITC/PI double staining further confirmed that PCP-4 induced apoptosis of H22cells in a dose-dependent manner.Combined the results from Rhodamine 123 staining,DCFH-DA staining,and western blot analysis,PCP-4 was found to significantly increase the expression of Bax,inhibit the expression of Bcl-2,decrease the mitochondrial membrane potential,and promote the accumulation of intracellular reactive oxygen species,resulting in mitochondrial dysfunction and increased membrane permeability.Subsequently,the cytochrome c is released into the cytoplasm and binds to Apaf-1,which activates caspase-9and caspase-3 to induce apoptosis in H22 cells via the mitochondrial pathway.This study explored the changes and differential gene expressions in human HepG2hepatocellular carcinoma cells,so as to comprehensively assess the anti-hepatoma effects and related targets of PCP-4.Combining MTT analysis,inverted microscope observation,Hoechst 33258 staining,Annexin V-FITC/PI staining and PI single staining results,it can be seen that the proliferation of HepG2 cells were significantly inhibited after PCP-4 treatment,and the cell cycle was blocked in the G₂/M phase,while the cells showed a change from early to late apoptosis and finally to death changes.The results of PCR array showed that the expressions of PYCAD,TNFRSF8,BNIP2,BCL2L10,GADD45A,HRK and BNIP3L were upregulated after PCP-4 treatment,while the expressions of CIDEB,AKT1,BCL2L1 and IL10 were downregulated.These differential genes can be classified as pro-apoptotic or anti-apoptotic genes,mainly involving Bcl-2,Caspase,Card,IAP,TNF ligand/TNF receptor,death domain/death effector domain,DNA damage/p53 and NF-κB family.Although PCP-4treatment may trigger multiple targets and cause multiple apoptotic pathways,the death receptor-mediated signaling pathway and the mitochondrial pathway are the main pathways through which PCP-4 induces apoptosis in HepG2 cells.Finally,this study assessed the in vivo anti-hepatoma effect and mechanism of PCP-4using H22 tumor-bearing mice as an animal model.After the gavage of PCP-4,the tumor volume and weight of mice were significantly decreased,and the tumor growth inhibition rate was up to 41.38%.Compared with 5-Fu,PCP-4 has not only played an anti-tumor effect,but also has no toxic or side effects on the body.Meanwhile,combined with the results of organ index and lymphocyte distribution,PCP-4 was found to protect the immune organs of tumor-bearing mice,alleviate spleen enlargement and thymus atrophy,enhance the proliferation of lymphocytes,and increase the proportion of CD19⁺,CD3⁺,CD4⁺,CD8⁺lymphocytes in the spleen and peripheral blood.In addition,PCP-4 can significantly enhance the phagocytic ability of macrophages and the killing ability of NK cells.These data indicated that PCP-4 inhibits tumor growth in vivo by regulating the body’s immune capacity.H&E staining of liver tissues and detection of oxidative stress showed that PCP-4 could protect the liver tissue,up-regulate peroxidase activity,enhance the body’s antioxidant defense capacity,and restore internal environment stability,thus more effectively suppressing the growth of the tumor.The observation of solid tumor tissues and cell suspensions of mice by H&E staining indicated that the tumor cells in the PCP-4 group showed the typical morphological features of apoptotic cells such as lighter cytoplasmic staining,nuclei lysis and increased cell fragmentation.Annexin V-FITC/PI double staining further confirmed that PCP-4 dose-dependently induced cell apoptosis in H22 tumor-bearing mice,thereby inhibiting tumor growth and achieving an anti-hepatoma effect in vivo.