| Ionizing radiation is a natural resource that penetrates all aspects of our life,and it is also an important therapeutic and diagnostic tool in the field of medicine.High dosage or prolonged exposure to ionizing radiation can cause harm to the body,which would induce three types of radiation injury of disease involving gastric bowel syndrome,blood-spinal cord syndrome and central nerve syndrome.Although some drugs usually adopted in radiation therapy such as sulfur-containing compounds,biogenic amine compounds and many other drugs have been proved to be beneficial to radioresistance,the toxicity and the gradually appeared tolerance of the drugs have limited their use.So the hotspot of scientific research has gradually focused on the development active ingredients with the function of radiation protection agent or adjuvant therapy from natural plants for their efficient and non-toxic nature.Phellodendron amurense Rupr pollen polysaccharide was screened as the research object from different raw pollens by two approaches involving the vitro antioxidant activity and correlation analysis.The Box-Behnken response surface design and some analysis technology including FT-IR,HPGPC,GC-MS and NMR were used together to optimize the extraction process and characterize the main component structure.The effect and mechanism of AC-PARPP protection from oxidative stress damage induced by 60Co-γ radiation was researched with the vivo model by using HE staining,determination of enzyme activity,ELISA,Western blot and IHC protein quantitative analysis method.Phellodendron amurense Rupr,Sorbaria sorbifolia and Brassica campestris pollen showed strong scavenging free radicals ability of DPPH,O2·-and OH·as well as FRAP reduction ability among 11 kinds of pollens.Composition analysis showed that in all of pollens,the lowest content of polysaccharide was 50mg/g CE which was significantly higher than that of the other components.Especially in Phellodendron amurense Rupr pollen the polysaccharide content can be up to 144.05±1.85 mg/g.The highest correlation coefficient R2 value(0.8680)showed that the polysaccharide content and antioxidant activity were positively correlated.So the Phellodendron amurense Rupr pollen polysaccharide was choosed as the research object according to the above results.Three kinds of polysaccharide involving acid,alkaline and neutral polysaccharide were extracted from Phellodendron amurense Rupr pollen material,respectively.The polysaccharide content can be up to 518.27±0.91 mg/g,and the scavenging ability of OH·as well as FRAP reduction ability was the strongest among three polysaccharides.The subsequent matter of cell proliferation of spleen cells and ECV304 treated with AC-PARPP in the vitro cell oxidative damage model induced by the radiation further verified our right choice of extracting acid polysaccharide from Phellodendron amurense Rupr pollen.The extraction yield of 4.60±0.03% can be received from ultrasonic-assisted extraction of acid polysaccharides after optimization with the response surface method under the following optimal conditions: ultrasonic temperature was 46 ℃,ultrasonic time was 31 min,Na OH concentration was 0.41% and the ratio of liquid to raw materal was 12:1.Primary removing impurities and secondary column chromatography were used respectively to purify AC-PARPP,and the optimal conditions of purification process was as follows: papain hydrolysis temperature was 61 ℃,p H was 7.10,[E]/[S]% was 1.20%,under above conditions the polysaccharide content of AC-PARPP was 82.74±0.32%.The main component obtained from AC-PARPP after successively purification by DEAE-52 and Sephadex G-200 column chromatography was identifed as a kind of RG-I pectin polysaccharide with high branch structure named as AC-PARPP-3,and the content among all components was 72.23%.In this process FT-IR,HPGPC,GC-MS,periodate oxidation and Smith degradation,NMR were used to characterize the AC-PARPP-3 structure.The result suggested that the molecular weight was 6.52×104,and the monosaccharide composition was: rhamnose: arabinose: galacturonic acid: galactose was 8.94: 16.51: 9.03: 5.27,respectively.The main chain of AC-PARPP-3 was → 4,1)-β-Galp A-(4 → and →1)-α-1-Rhap-(2 → arranged alternately emerging.It belongs to evident characteristics of RG-I pectic polysaccharide.The branch point on the α-1,2,4-Rhap was at C-4 position,which connected with the structure of →4,1)-α-Araf-(5 →,→ 5,1)-α-Galp-(4 → → 1)-α-Araf,ending at α-Araf nonreductive terminus.AC-PARPP had the protective effect on the immune organs by increasing organs index and phagocytic index of mice suffered with 60Co-γ-induced oxidative stress demage.The decreasing of blood cells in vivo and hemoglobin concentration were improved,AC-PARPP also protect splenocytes and small intestine against the damage caused by radiation.The activity of antioxidant enzymes SOD,CAT and GSH-Px was increased and the lipid peroxidation products MDA content was decreased,which indicated that AC-PARPP enhanced the body’s antioxidant system.Promotion the secretion of TNF-α、INF-γ、Ig M and secretoinhibitory of TGF-β was another way of AC-PARPP in improving the immunity to resist radiation damage in vivo.On the molecular expression level,it was found that p-Akt expression level and the Bcl-2/Bax ratio was increased,which indicated the opening of Akt/Bcl-2 signal pathways by the regulation of AC-PARPP.The Cyt C and Caspase-9 expression in the mitochondrial-dependent apoptosis pathway as well as Caspase-8 expression in the death receptor pathway were all decreased,the resulting in the decrease of the Caspase-3 expression.It was important to confirm that two different pathway of the mitochondria-dependant apoptosis and death receptor was AC-PARPP used to protect cell against apoptosis induced by radiation.This research found that AC-PARPP as a good antioxidant can enhance immune capacity and protect against 60Co-γ-induced oxidative injury by two different mechanisms involving the mitochondrial apoptosis pathway and death receptor pathway in vivo.The radioprotection function of pollen polysaccharide extracted from Phellodendron amurense Rupr was further confirmed,which has the theoretical and practical significance for development and utilization of PARPP to decrease the side effects of radiation injury. |
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