硒化修饰提高大蒜多糖体外抗新城疫病毒活性的研究

Studies on selenide modification to improve the activity of garlic polysaccharides against NDV in vitro

旨在研究硒化修饰提高大蒜多糖的体外抗新城疫病毒(NDV)活性并筛选出体外抗NDV活性较好的硒化大蒜多糖。依次通过水提醇沉法、Sevage法去蛋白得到大蒜多糖(GPS),并采用硝酸-亚硒酸钠法对大蒜多糖进行硒化修饰,得到9个硒化大蒜多糖sGPS_(1)~sGPS_(9)。以鸡胚成纤维细胞(CEF)为作用对象,采用MTT法通过先加硒化大蒜多糖,或先加NDV,或两者同时加入3种方式比较9个硒化大蒜多糖抗NDV侵入、复制、直接抑制NDV的作用,同时设置细胞对照(CC)和病毒对照(VC),并计算病毒抑制率;筛选出活性较好的sGPS_(3)、sGPS_(5)、sGPS_(6)测定其对NDV感染CEF后细胞凋亡、细胞周期的影响以及其对鸡胚的存活数的影响,同时设置GPS对照、VC对照、空白对照(BC)。结果表明,GPS_(5)、sGPS_(6)组的细胞凋亡率、处于S期的细胞百分数显著低于VC组和GPS组(P<0.05);sGPS_(6)组36 h、48 h的细胞凋亡率显著小于sGPS_(3)、GPS_(5)组(P<0.05),处于S期的细胞百分数小于sGPS_(3)、sGPS_(5)组,sGPS_(3)组在36 h、sGPS_(5)组在24 h和36 h、sGPS_(6)组在3个时间点的鸡胚死亡数均显著小于GPS组(P<0.05),sGPS_(6)组在3个时间点的鸡胚死亡数均为最低。综上,硒化修饰能提高大蒜多糖抗NDV的活性,其机制可能与阻止NDV侵入、抑制NDV诱导CEF凋亡有关。其中sGPS_(6)的活性最强,可作为抗NDV的候选药。

This study was to determine selenide modification to improve the in vitro activity of garlic polysaccharides against Newcastle disease virus(NDV)and to screen selenide garlic polysaccharides with better anti-NDV activity in vitro.The GPS were obtained using the methods of water extraction and alcohol precipitation and sevage to remove proteins in turn,and they were modified using the HNO_(3)-Na2SeO_(3) method to obtain nine kinds of sGPS,i.e.sGPS_(1)-sGPS_(9).With CEF as the target,the MTT method was used to compare the effects of the nine sGPS against NDV invasion,replication,and direct inhibiting of NDV by supplementing sGPS alone,and NDV alone,and both sGPS and NDV at the same time.Meanwhile the cell control(CC)and the virus control(VC)were designed,and the virus inhibition rate was calculated.sGPS_(3),sGPS_(5),sGPS_(6) with better activity were screened to determine their effect on cell apoptosis,cell cycle and on the survival of chicken embryos after NDV being infected with CEF.The GPS control,virus control and blank control were also designed.The results showed that the cell apoptosis rate and the percentage of cell cycle in the S phase of the GPS_(5)and sGPS_(6)groups were significantly lower than those in the VC and GPS groups(P<0.05).The cell apoptosis rates of the sGPS_(6) group at 36 h and 48 h were significantly lower than those of the sGPS_(3) and GPS_(5) groups(P<0.05).The percentage of cell cycle in S phase was lower than that in the sGPS_(3) and GPS_(5) groups.The number of embryonic deaths in the sGPS_(3)group at 36 h,the sGPS_(5)group at 24 h and 36 h,and the sGPS_(6) group at the three time points were significantly lower than that of the GPS group(P<0.05),and the number of chicken embryo deaths in the sGPS_(6) group was the lowest at the three time points.In summary,selenide modification enhanced the anti-NDV activity of GPS,and its mechanism might be related to preventing NDV invasion and inhibiting NDV induced CEF apoptosis.s GPS6 showed the strongest activity and might be used as a candidate drug against NDV.