The Structural Analysis Of Polysaccharide HDPS-2Ⅱ From Holotrichia Diomphalia Bates And Its Protective Effect On DNA Damage

DNA is a significant genetic material in organisms and plays an important role in the process of growth and development,but DNA is very susceptible to damage.Both endogenous and exogenous factors can cause DNA damage easily,especially when the chemotherapy drugs used in cancer exert their own powerful anti-tumor effects,so they often have serious side effects.Most chemotherapy drugs can induce DNA damage by enhancing intracellular oxidative stress,resulting dramatic injure to normal tissues.It has been found that many polysaccharides from traditional Chinese medicine have antioxidant effects,and can protect DNA by inhibiting exogenous oxidative stress.Our previous studies have found that the polysaccharide named HDPS-2Ⅱ,isolated from Holotrichia diomphalia Bates,had a protective effect on acute liver injury caused by the chemical inducer CCl₄ in mice.However,its structure and DNA protective activity has not been studied in depth.Therefore,this study aims to analyze the structure of HDPS-2Ⅱ and to clarify whether HDPS-2Ⅱ has a protective effect on cellular DNA,and further explore the potential of HDPS-2Ⅱ as a DNA protector.Methods:1.The total polysaccharides,named HDPS,from Holotrichia diomphalia grown in Shaanxi and Anhui provinces were separated and purified using the Sephadex A-25and Sephadex G-100 columns.Two kinds of homogeneous polysaccharide HDPS-2Ⅱ were obtained.2.The monosaccharide composition of HDPS-2Ⅱ from two localities of growth were determined by high performance liquid chromatography（HPLC）;acid hydrolysates of HDPS-2Ⅱ were prepared by trifluoroacetic acid and the glycosidic bond types of HDPS-2Ⅱ were analyzed by methylation and GC-MS.The glycosidic bond connection mode was determined by 1D and 2D NMR.The fatty acids in HDPS-2Ⅱ were extracted with ether and derivatized with sulfuric acid-methanol and then analyzed by GC-MS.3.The effects of HDPS-2Ⅱ on cell growth inhibition and chromosome aberration in Chinese hamster lung cells were investigated by MTT assay and the conventional Giemsa staining method,respectively.Using 3.5μM doxorubicin（ADM）and 50μM hydrogen peroxide（H₂O₂）as DNA damage inducers,and explore the protective effects of HDPS-2Ⅱ（2.5 mg/L,5 mg/L,10 mg/L）on chromosomal aberrations induced by ADM and H₂O₂,respectively.4.The CHL cell injury model induced by ADM and H₂O₂ were treated with HDPS-2Ⅱ（2.5 mg/L,5 mg/L,10 mg/L）The comet assay was used to analyze the comet tail length of the cells,and the immunofluorescence staining assay was used to detect the expression ofγ-H2AX.Reactive oxygen species（ROS）,malondialdehyde（MDA）,and superoxide dismutase（SOD）kits were used to detected the levels of ROS,MDA,and SOD in CHL cells.Results:1.Two kind of homogeneous HDPS-2Ⅱ from Shaanxi and Anhui were obtained;their molecular weights were 8.7 k Da and 5.9 k Da,respectively;their total sugar contents were 88.0%and 89.1%,the uronic acid contents were 1.6%and 3.1%,and the protein contents were 2.0%and 2.2%,respectively.The FT-IR spectra of HDPS-2Ⅱ showed the polysaccharides characteristic absorption peaks of O-H and C-H.2.The monosaccharide composition and molar ratio of HDPS-2Ⅱ from Shaanxi were Man∶Glc∶Gal∶Ara（2.93∶22.25∶1.49∶1.00）,and its main sugar residue types and molar ratio were T-Araf∶1,2-Manp∶1,3-Araf∶1,6-Manp∶T-Glcp∶1,4-Galp∶1,6-Glcp（1.11∶1.00∶1.01∶2.18∶8.44∶1.74∶17.92）.The monosaccharide composition and molar ratio of HDPS-2Ⅱ from Anhui were Man∶Rha∶Glc∶Gal∶Ara（2.77∶1.00∶3.98∶2.67∶5.33）,and the main sugar residue types and molar ratio were T-Araf∶1,6-Manp∶T-Glcp∶T-Rhap∶1,4-Rhap∶1,2-Manp∶1,4-Galp∶1,6-Glcp（10.14∶4.97∶8.20∶1.39∶1.00∶3.14∶6.97∶9.49）.In addition,the composition and molar ratio of monosaccharide residues in Anhui HDPS-2Ⅱ after acid hydrolysis of 0.5 M and 1.0 M TFA were Man∶Rha∶Glc∶Gal（5.20∶1.41∶10.90∶1.00）and Man∶Rha∶Glc∶Gal（10.62∶2.52∶26.59∶1.00）,respectively.3.The fatty acids and aromatic ring groups in HDPS-2Ⅱ from Shaanxi were 5-hydroxy-2-hexanone,2-hexanol-5-methoxyacetate,3-methylamino-1,2-propanediol,5-Methyl-3-（methylamino）-2-hexanone,3-tert-butylphenol,valeric acid and hexadecanoic acid;the fatty acids and aromatic ring groups in HDPS-2Ⅱ from Anhui were 5-hydroxy-2-hexanone,2-hexanol-5-methoxyacetate,3-methylamino-1,2-propanediol,5-Methyl-3-（methylamino）-2-hexanone,3-tert-butylphenol,valeric acid,hexadecanoic acid and octadecanoic acid.4.In the concentration range of 1～300 mg/L,the inhibition rate of HDPS-2Ⅱ on CHL cell proliferation is less than 40%,and it has no chromosomal aberration effect on CHL cells.But the same concentration of HDPS-2Ⅱ produced in Anhui has less inhibitory effect on CHL cell proliferation than HDPS-2Ⅱ produced in Shaanxi.After adding HDPS-2Ⅱ（2.5 mg/L,5 mg/L,10 mg/L）,ADM-induced cell chromosomal aberration rates decreased from 52%to 30%,26%,16%,and IC₅₀ value increased by2.5%,17.2%,174.6%;and H₂O₂-induced cell chromosomal aberration rates decreased from 42%to 32%,26%,22%,and IC₅₀ value increased by 9.7%,48.3%,81.8%.indicating that HDPS-2Ⅱ significantly reduced ADM/H₂O₂-induced CHL cytotoxicity.5.The comet assays showed that the comet tail lengths of ADM-treated and H₂O₂-treated CHL cells were significantly reduced bythe treatment of HDPS-2Ⅱ（2.5 mg/L,5 mg/L,10 mg/L）.The results of immunofluorescence staining showed that theγ-H2AX fluorescence in ADM/H₂O₂-treated cells were significantly inhibited by the treatment of HDPS-2Ⅱ（2.5 mg/L,5 mg/L,10 mg/L）.Furthermore,ADM-and H₂O₂-induced ROS and MDA increases in CHL cells were markedly inhibited by HDPS-2Ⅱ,whereas the downregulations of the SOD induced by ADM and H₂O₂ were significantly increased by HDPS-2Ⅱ.These data indicated that HDPS-2Ⅱ could markedly decrease the accumulated oxidative stress products in the cells.Conclusion:1.In this research,two novel homogenous grub polysaccharides HDPS-2Ⅱ from Shaanxi and Anhui provinces were separated and obtained.The main structure of HDPS-2Ⅱ is→6)-ɑ-Manp-（1→6）-ɑ-Glcp-（1→6）-ɑ-Glcp-(1→,F1/F3 are the fatty acids and benzene ring substituents attached to the sugar chain of HDPS-2Ⅱ from Shaanxi,F1/F2/F3 are the fatty acids and benzene ring substituents attached to the sugar chain of HDPS-2Ⅱ from Anhui.F1,F2 and F3 are the fatty acid and benzene ring substituent groups connected to the glycosidic bond,respectively.F1 is 5-（（1-hydroxy-3-palmitamidopropan-2-yl）oxy）-5-oxopentan-2-yl methyl-leucinate;F2 is 1-hydroxy-3-stearamidopropan-2-yl 4-hydroxypentanoate;F3 is 5-（（5-（（2-（（5-（3-（tert-butyl）phenoxy）hexan-2-yl）oxy）-3-hydroxypropyl）amino）-3-methyl-5-oxopentan-2-yl）oxy）hexan-2-yl pentanoate.2.HDPS-2Ⅱ did not cause chromosomal aberrations in normal CHL cells,and had concentration-dependently protective effects on ADM and H₂O₂ induced DNA damage in CHL cells.HDPS-2Ⅱ could inhibit the cytotoxicity and DNA damage induced by ADM and H₂O₂ by hampering the production of ROS and MDA and increasing the activity of SOD.