Protective Effects And Mechanisims Of Sipunculus Nudus Polysaccharide On HUVEC And HIEC Injuried By Ionizing Radiation

Nuclear technology is widely used in industry,scientific research,medical treatment and so on.While promoting social economic development,nuclear technology has also caused harm to human health.At present,most radioprotectants have some toxic and side effects,so human beings have to find safe and effective drugs.Our previous studies on the radioprotected effects of Sipunculus nudus polysaccharide?SNP?showed that SNP had protective effects on hematopoietic and immune system injury induced by ionizing radiation in mice.On this basis,in vitro study on cells was performed to investigate the protective mechanism of SNP.The contents and results of research are as follows:1.Extraction and preparation of SNP:The dried sipunculus nudus was purchased from Beihai of Guangxi,cut and mashed,then hydrolyzed with 10%NaOH and deproteinized with trichloroacetic acid to get crude polysaccharides.Using 0.1 mg/mL glucose as standard solution,the absorbance value was determined at 490 nm wavelength of ultraviolet spectrophotometer,and the standard curve was established.The absorbance value of 1 mg/mL crude polysaccharide of sipunculus nudus polysaccharides was determined at the same wavelength.The content of polysaccharides in SNP was calculated to be 60%.2.Establishment of model of radiation injury in HUVEC and HIEC cells:Normal human umbilical vein endothelial cells?HUVEC?and human intestinal epithelial cells?HIEC?were selected as models of ¹³⁷Cs?-ray radiation damage.HUVEC cells were irradiated with 0-10 Gy,and HIEC cells were irradiated with 0-20Gy.CCK-8 assay was used to detect the cell survival rate after irradiation.After 2 Gy,4 Gy,6 Gy,8 Gy and 10 Gy irradiation,the survival rates of HUVEC cells decreased significantly,87.94%,84.69%,70.25%,65.74%and 54.78%respectively.The survival rates of HIEC irradiated with 2 Gy,4 Gy,6 Gy,8 Gy,10 Gy,15 Gy and 20Gy were 80.39%,76.23%,70.55%,70.82%,66.49%,54.66%and 53.12%respectively.The difference between the irradiated group and the normal control group was statistically significant?p<0.05,p<0.01?.The cell survival rates were negatively correlated with the irradiation dose.According to the results and related literature,8 Gy and 10 Gy were selected as the model doses of radiation damage in HUVEC and HIEC cells.3.Study on the radioprotective effects of SNP on HUVEC and HIEC cells:?1?Compared with DMSO-positive control group,SNP had no toxic effect on cells in the concentration range?The final concentration were 0.5,1,5,10,50,100,200,500?g/mL in HUVEC cells,and the final concentration were 1,10,50,100,200,500,1,000 ug/mL in HIEC cells?.?2?HUVEC and HIEC cells were irradiated with 8 Gy and 10 Gy ¹3737 Cs?-rays respectively.The effects of different concentrations of SNP on the proliferation of irradiated cells were studied.The results showed that SNP pretreatment at a certain concentration could improve the inhibitory effect of irradiation on cell proliferation.?3?Giemsa staining was used to observe the cell cloning rate.The results showed that SNP pretreatment could effectively improve the cell cloning ability after irradiation compared with the irradiation model group.?4?The result of Hoechst 33342/PI double staining and microscopic observation of apoptotic cells showed that apoptotic cells in model group of HUVEC and HIEC cell were more than those in normal control group,and apoptotic cells in SNP pretreatment group were less than those in irradiated model group.Annexin V/PI staining and flow cytometry were used to quantitatively detect the apoptotic rate of HUVEC and HIEC cells.It was found that the apoptotic rate of HUVEC irradiated with 8 Gy increased significantly?from 1.02%to 15.6%?,while the apoptotic rate of SNP pretreated decreased significantly to 6.80%?P<0.01?,and the apoptotic rate of HIEC irradiated with 10 Gy increased significantly?from 2.12%to 14.37%?while the apoptotic rate of irradiated HIEC pretreated with SNP decreased significantly to 7.18%?P<0.01?;?5?DCFH-DA probe combined with flow cytometry was used to detect ROS production in HUVEC and HIEC cells.The results showed that the ROS mean value of HUVEC cells increased from 32.63?10⁴ to 47.10?10⁴ after irradiation?p<0.01?.After SNP pretreatment,it decreased to 41.77?10⁴?p<0.05?.Afer irradiation,ROS mean value of HIEC cells increased from 4.76×10⁴ to 16.69×10⁴?p<0.01?,and it decreased to 9.81×10⁴ in SNP pretreatment group?p<0.01?;?6?The activity of antioxidant enzymes?SOD?CAT?GST?GSH-Px?,the content of lipid peroxides?MDA?and 8-hydroxydeoxyguanosine?8-OHdG?,which is a product of DNA oxidative damage,were detected in HUVEC and HIEC cell groups by kits.The results showed that the activities of SOD,CAT,GST and GSH-Px in the irradiated model group decreased comparing with the normal control group,while the contents of MDA and 8-OHdG increased.The activities of SOD,CAT,GST and GSH-Px increased and the contents of MDA and 8-OHdG decreased after SNP pretreatment.4.Study on the radiaoprotective mechanism of SNP in HUVEC and HIEC.Flow cytometry was used to detect?-H2AX,a molecular marker of DNA double-strand breaks,in HUVEC and HIEC cells.It was found that?-H2AX increased at 2 h,4 h,8h after irradiation,while?-H2AX in the SNP pretreated group was lower than that in irradiation model group?p<0.05,P<0.01?.The expressions of hOGG1 and RAD51genes were detected by RT-PCR.The results showed that the expressions of hOGG1and RAD51 were down-regulated in the irradiated model group,while the expressions of hOGG1 and RAD51 were up-regulated by SNP pretreatment.In conclusion,SNP pretreatment can promote the proliferation and cloning of irradiated cells,inhibit cell apoptosis,protect the activity of antioxidant enzymes,reduce MDA and 8-OHdG content,and DNA double strand breakage,and up-regulate the expression of hOGG1 and RAD51.It suggest that SNP has a good anti-radiation effects and the possible mechanism of SNP is to protect cells from radiation by scavenging free radicals,alleviating DNA oxidative damage,promoting DNA repair and reducing cell apoptosis.