| Ionizing radiation could induce the body to produce excessive of ROS, whichattack the biological substances such as proteins, lipids and carbohydrates that cancause a chain reaction and damage the body homeostasis. Studies have shown thatlong-term exposure to ionizing radiation environment could induce the body toproduce a variety of diseases such as aging, inflammation, cardiovascular andcerebrovascular diseases, and even cancer. Existing chemical radioprotectors werenot suitable for long-term use due to their side effects on normal tissues and organs.Therefore, screening natural radioprotectors with efficiency and low toxicitybecome a focus. This subject sreened neutral polysaccharide of Hohenbueheliaserotina from8kinds of mushroom through multiple antioxidant model in vitro asthe object; optimized the extraction process using RSM and characterized itsstructure, systematically studied the in vivo and in vitro protective effect of NTHSPon radiation induced oxidative stress and revealed the mechanism of radiationprotection; the main findings were as follows:23kinds of polysaccharides were isolated from eight kinds of mushrooms withthree kinds of solvents. There was a small amout of protein and no polyphnol inpolysaccharides. By establishing multiple antioxidant model in vitro, we studied thescavenging activity on physiological hydroxyl radicals and non-physiological ABTSradical, as well as lipid peroxidation inhibition activities of23kinds of ediblefungus polysaccharides. The results indicated that HSP had the most significantantioxidant activities in vitro. The ferric reducing ability and superoxide radicalscavenging activity of three kinds of HSP were determined. The results showed thatNTHSP could effectively supply electron and suppress radical chain reaction whichwas the best fraction for antioxidant activities.An ultrasonic-assisted procedure for the extraction of polysaccharides from thefruiting body of Hohenbuehelia serotina was investigated using response surfacemethodology. The effect of four factors on the yield of polysaccharides were studied.The optimized conditions for maximum yield were extraction temperature of94°C,extraction time of3.0h, water to raw material ratio of110:1and ultrasonic power of480W. Under these conditions, the experimental yield of polysaccharides was17.45±0.18%, which was higher than the yield of polysaccharide extracted bytraditional method (10.21±0.36%). Then, the structural features of untreatedmaterials, hot water extraction residue and ultrasonic-assisted extraction residuewere compared by SEM. The results indicated that ultrasonic-assisted extraction technology could be an effective and advisable technique. The NTHSP was purifiedby DEAE-52and Sephadex G-100. The structure of NTHSP-A1was characterizedby HPCPC, GC-MS, FT-IR, NMR and AFM. The results suggested that NTHSP wasa glucan with high branches. The molecular weigh is8.09×103. The polysaccharidewas composed of arabinose, mannose, glucose and galactose with a ratio of3.8:15.8:28.4:10.5. The main chain of NTHSP-A1wasâ†'3,6)-α-D-Glcp-(1â†', andthe side chainsâ†'2)-α-L-Arap-(1â†', α-D-Manp-(1â†'andâ†'6)-β-D-Galp-(1â†'werelinked at3-position,3-position and6-position ofâ†'3,6)-α-D-Glcp-(1â†'respectively.Furthermore, methyl groups were respectively connected withâ†'2)-α-L-Arap-(1â†'andâ†'6)-β-D-Galp-(1â†'at2-position and6-position.By the established model of radiation inducing oxidative stress in cells, westudied the radioprotective effect of HSPs on rat peripheral blood white blood cells,rat spleen cells and human umbilical vein endothelial cells. The results indicatedthat HSPs were able to reduce the number of atypical lymphocyte, leaf granulocytecotaining vacuoles and degenerative cell, and effectively protect the peripheralblood leukocytes. Three kinds of HSP also could promote the proliferation of ratsplenocytes and endothelial cells, significantly improved the survival of thesplenocytes and endothelial cells after radiation. NTHSP possessed the mostsignificant effect; SOD and CAT activities and MDA level in spleen cells afterradiation were investigated. The results showed that NTHSP could significantlyincrease the activities of SOD and CAT, and inhibit the occurrence of lipidperoxidation. DNA ladder study further demonstrated that NTHSP could play aprotective effect on ionizing radiation induced DNA damage.The oxidative damage model of mice induced by60Co-γ radiation wasestablished to investigate the radioprotective effect of NTHSP. The results showedthat NTHSP could effectively increase the activities of SOD, CAT and GSH-Px aswell as enhance the synthesis of antioxidants (GSH, Vitamin E and ceruloplasminprotein) and reduce the level of MDA. The results showed that NTHSP can enhancethe activity of anti-oxidative system after radiation. By studying the specificimmunity and non-specific immunity, it was found that NTHSP could significantlyincrease the immune organ index, promote spleen lymphocyte proliferation andenhance monocyte phagocytosis. By studying the mouse micronucleus,chromosome aberrations and DNA content in mice bone marrow, it was obeservedthat NTHSP could protect DNA against damage induced by radiation.The protective mechanism of NTHSP on radiation induced oxidative stress wasanalyzed by the methods of flow cytometry and immunohistochemistry. The resultssuggested that NTHSP could protect splenocytes against the damage caused by radiation through inhibiting more cells arrest into G0/G1phase and restore thephysiology metabolic processes. NTHSP could also play a protective effect for theapoptosis of splenocytes induced by radiation. The related proteins of apoptosis inmitochondrial pathway were determined and the results showed that NTHSP couldsignificantly inhibit the expression of pro-apoptosis protein Bax, Cytochrome c andCaspase-3and as well as promote the expression of anti-apoptosis protein Bcl-2,and then effectively protect the splenocytes away from oxidative damage inducedby radiation.These results indicated that NTHSP could prevent the radiation induceddamage by reducing the production of ROS and regulating the redox state, and theneffectively inhibiting the mitochondrial apoptotic signaling pathway. The presentstudy will play an important theoretical and practical role in revealing protectivemechanism of radiation and exploiting the natural and efficient raidioprotectors. |
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