Borrelidin对小鼠脾窦内皮细胞Bid/Bcl-xl和Caspase-8的作用实验研究

Experimental Study on Borrelidin's Function to the Bid/Bcl-xl and Caspase-8 in Endothelial Cells of Mice Spleen Sinus

目的:评价小鼠脾窦内皮细胞在Borrelidin作用前后Bid/Bcl-xl比值和Caspase-8的变化。方法:60只9周龄SPF雄鼠随机分为4组。NC组小鼠正常饲养不做处理,NS组脾脏直接注射生理盐水0.5mL,BO3d组和BO7d组脾脏注射Borrelidin注射液0.5mL,第3天处死NC组、NS组、BO3d组小鼠,第7天处死BO7d组小鼠,TUNEL法标记凋亡细胞计算凋亡指数(AI);SABC法检测Caspase-8、Bid和Bcl-xl的表达,比较积分光密度,计算Bid/Bcl-xl的比值,分析三者间关系。结果:NC组与NS组仅有微量凋亡细胞表达,BO3d、BO7d组凋亡广泛表达于脾窦内皮细胞,与NC组差异显著,BO7d组凋亡阳性率最高。NC组与NS组Bcl-xl有一定量表达,无统计学差异,BO7d组与NC组有显著差异;Caspase-8、Bid的表达及Bid/Bcl-xl与凋亡的阳性表达趋势一致。Bcl-xl的表达趋势与AI呈反相关,Caspase-8、Bid表达趋势及Bid/Bcl-xl比值与AI呈正相关。结论:Borrelidin可通过Caspase-8的启动激活Bid蛋白,在较大程度上拮抗Bcl-xl的抑凋亡作用,提升Bid/Bcl-xl比值,诱导小鼠脾窦内皮细胞的凋亡。

Objective： To evaluate the ratio of Bid/Bcl-xl and change of Caspase-8 before and after Borrelidin＇s effect on the spleen sinus endothelial cells of mice.Methods： 60 SPF male mice aging nine weeks are divided into four groups： NC group,NS group,BO3d group and BO7d group.Mice in NC group are normally raised without any treatment.Mice in NS group are injected saline.Mice in BO3d and BO7d group are injected 0.5ml Borrelidin.After 3 days mice in NC,NS and BO3d groups are executed and mice in BO7d group are executed after 7 days.The apoptotic cells are marked by TUNEL to get AI.Immunohistochemical method is used to detect the expression of apoptosis related proteins Bid,Bcl-xl and Caspase-8.Image analysis technique is used to compare integral light density,calculate the ratio of Bid/Bcl-xl and analyze the relationship between them.Results： The apoptosis in BO3d and BO7d group are widely present in the spleen sinus endothelial cells.The expression of Bcl-xl is present in NC and NS groups with certain amount but no significant difference.Expression of Bcl-xl is slightly declined as drug functions continue.There is significant difference between BO7d and NC groups.The expression of Caspase-8 and Bid is consistent with the positive expression of Bid/Bcl-xl and apoptosis.The expression of Bcl-xl is negatively correlated with AI.The expression of Caspase-8 and Bid and the ratio of Bid/Bcl-xl is positively correlated with AI.Conclusion： Borrelidin is capable of activating the Bid protein through starting Caspase-8,which in a certain extent antagonizes inhibiting apoptosis roles of Bcl-xl,promotes the Bid/Bcl-xl ratio,and induces overall apoptosis trends of spleen sinus endothelial cell of mice.