TGF-β1对间歇性缺氧诱导海马神经元自噬的影响

Effect of TGF-β1 on intermittent hypoxia induced autophagy in hippocampal neurons

目的探讨TGF-β1对间歇性缺氧诱导海马神经元自噬的影响。方法体内实验:将12只SD大鼠按完全随机分组方法分为对照组(N组)和间歇性缺氧组(IH组),Morris水迷宫评估大鼠认知功能;透射电镜、免疫组织化学观察神经元的自噬改变;Western blot检测LC3、P62及TGF-β1的表达。体外实验:将小鼠海马神经元细胞系HT22细胞分为对照组(N组)、间歇性缺氧组(IH组)、间歇性缺氧+TGF-β1组(IH+TGF-β1组)和间歇性缺氧+SB431542组(IH+SB431542组),Western blot检测TGF-β1、LC3和P62的表达。结果与N组相比,IH组Morris水迷宫实验平均逃逸潜伏期延长(P<0.05),目标象限停留时间延长(P<0.05);电镜下自噬小体增多,免疫组织化学LC3染色增加;体内体外实验中Western blot结果显示LC3-Ⅱ/LC3-Ⅰ、P62表达上调(P<0.05),TGF-β1表达下调(P<0.05);与IH组相比,IH+TGF-β1组TGF-β1表达上调(P<0.05),LC3-Ⅱ/LC3-Ⅰ、P62表达降低(P<0.05),IH+SB431542组LC3-Ⅱ/LC3-Ⅰ表达增加(P<0.05)。结论间歇性缺氧导致大鼠认知功能障碍,可能与神经元自噬累积有关,TGF-β1可以减轻间歇性缺氧诱导的自噬增多。

Objective To investigate the effect of TGF-β1 on autophagy in hippocampal neurons induced by intermittent hypoxia.Methods In vivo experiment:Twelve SD rats were randomly divided as control group(N group)and intermittent hypoxia group(IH group);Their cognitive function was assessed by Morris water maze;Neuronal autophagy was observed by transmission electron microscopy and immunohistochemical assay;The expression of LC3,P62 and TGF-β1 in the hippocampus was detected by Western blotting.In vitro experiment:the mouse hippocampal neuron cell line(HT22 cells)was divided as control group(N group),intermittent hypoxia group(IH group),intermittent hypoxia+TGF-β1 group(IH+TGF-β1 group)and intermittent hypoxia+SB431542 group(IH+SB431542 group),the expression of TGF-β1,LC3 and P62 was detected by Western blotting.Results Compared with the N group,the average escape latency and the residence time in the target quadrant were both prolonged in the IH group(P<0.05).Besides,the number of autophagosomes was increased under electron microscope and the stained intensity of LC3 was enhanced in immunohistochemical assay.Western blotting indicated that the expression of LC3-Ⅱ/LC3-Ⅰand P62 was up-regulated(P<0.05),while that of TGF-β1 was down-regulated(P<0.05)in the in vitro and in vivo experiments.Compared with the IH group,the protein level of TGF-β1 was up-regulated,while that of LC3-Ⅱ/LC3-Ⅰand P62 was decreased(P<0.05)in the IH+TGF-β1 group(P<0.05);The expression of LC3-Ⅱ/LC3-Ⅰwas increased in the IH+SB431542 group(P<0.05).Conclusion Intermittent hypoxia causes cognitive dysfunction in rats,which is probably related to the accumulation of neuronal autophagy.TGF-β1 attenuates the increase of neuronal autophagy induced by intermittent hypoxia.