4种程序性细胞死亡方式在轻型颅脑损伤中的发展探究

Exploration of development of four programmed cell death modes in mild traumatic brain injury

目的研究4种程序性细胞死亡方式(凋亡、自噬、焦亡、铁死亡)在轻型颅脑损伤中随时间的发生、发展。方法选取SPF级Wistar大鼠49只,复制轻型颅脑损伤大鼠模型。7只为空白对照组,42只为模型组;模型组分别于模型复制成功后第1天、第3天、第7天、第14天、第21天和第28天取材。采用苏木精-伊红(HE)染色、尼氏染色、铀铅双染法观察大鼠海马神经元微观结构的变化;Western blotting检测各组大鼠海马B淋巴细胞瘤-2(Bcl-2)、微管相关蛋白1轻链3B(LC3B)、铁蛋白重链(FTH)、天冬氨酸-胱氨酸特异性蛋白酶家族1(Caspase-1)蛋白表达。结果光学显微镜可见,模型组有神经元皱缩等现象。电镜及Western blotting结果显示,与空白对照组比较,模型组第1天神经元细胞轻度固缩,Bcl-2蛋白相对表达量较高(P<0.05);模型组第3天、第7天的细胞膜多处破损,细胞器部分肿胀;模型组第3天的Caspase-1蛋白相对表达量达到最高峰(P<0.05);模型组第14天的神经元线粒体嵴多局部断裂,FTH蛋白相对表达量也较高(P<0.05);模型组第28天的FTH蛋白相对表达量仍在升高(P<0.05);模型组第28天胞内可见少量初级溶酶体或自噬溶酶体,但模型组第28天的LC3B蛋白相对表达量较模型组第21天降低(P<0.05)。结论轻型颅脑损伤后较多神经元细胞第一时间选择凋亡,焦亡紧随其后,铁死亡较晚发生,自噬持续性进行。

Objective To investigate the occurrence and development of four programmed cell death modes(apoptosis,autophagy,pyroptosis,and ferroptosis)in mild traumatic brain injury with time.Methods 49 SPF Wistar rats were used to replicate the rat model with mild traumatic brain injury.7 were blank group and 42 were model group.Samples were collected at day 1,day 3,day 7,day 14,day 21 and day 28 after the successful replication of the model.The microstructure of rat hippocampal neurons was observed by hematoxylin-eosin(HE),Nissl's staining,uranium lead double staining,respectively.Western blotting was used to detect the expressions of Bcl-2,LC3B,FTH and caspase1 proteins in the hippocampus of each group of rats.Results Optical microscopy showed that the neurons in the model group were crumpled.The results of electron microscopy and Western blotting showed that compared with the blank control group,the neurons in the model group were slightly contracted at day 1,and the relative expression of Bcl-2 protein was higher(P<0.05).In the model group,the cell membrane was damaged in many places and the organelles were swollen at day 3 and day 7.The expression of caspase1 protein in the model group reached the highest level at day 3(P<0.05).On the 14th day of the model group,the mitochondrial ridge of neurons was broken more locally,and the relative expression of FTH protein was also higher(P<0.05).The relative expression of FTH protein in the model group was still increased on day 28(P<0.05);There was a small amount of primary lysosome or autophagic lysosome in the cells of the model group on day 28,but the relative expression level of LC3B protein in the model group on day 28 was lower than that on day 21(P<0.05).Conclusion After mild traumatic brain injury,more neurons chose apoptosis at the first time,pyroptosis followed,ferroptosis occurred later,and autophagy continued.