RhoA/cofilin通路激活破坏海马突触可塑性参与铝中毒致学习记忆障碍的机制研究

A study on mechanisms of activating the RhoA/cofilin pathway to disrupt hippocampal synaptic plasticity to participate in aluminum toxicity-induced learning and memory impairments

目的探讨RhoA/cofilin通路激活对海马突触可塑性的影响,及其在铝中毒致学习记忆障碍中的作用机制。方法从30只无特定病原体SD大鼠中随机选20只,予麦芽酚铝溶液腹腔注射2个月构建慢性铝中毒大鼠模型。将中毒模型大鼠分为铝中毒模型组(10只)和RhoA抑制剂组(10只),后者予Rhosin盐酸盐腹腔注射30 d。剩余的10只正常大鼠作为空白对照组。通过Morris水迷宫实验检测大鼠的学习及记忆能力,应用透射电子显微镜观察大鼠海马CA1区突触超微结构的改变,通过实时荧光定量聚合酶链式反应(RT-qPCR)和免疫组化染色检测大鼠海马组织CA1区中RhoA、cofilin、PSD-95、SYN的定位表达情况。结果Morris水迷宫实验结果显示,铝中毒模型组大鼠潜伏期较空白对照组显著延长(P<0.05),RhoA抑制剂组大鼠的潜伏期较铝中毒模型组显著缩短(P<0.05)。RT-qPCR结果显示,与空白对照组相比,铝中毒模型组海马CA1区组织RhoA mRNA表达水平升高,cofilin mRNA、PSD-95 mRNA、SYN mRNA表达水平降低,差异有统计学意义(P<0.05);与铝中毒模型组相比,RhoA抑制剂组大鼠海马CA1区组织RhoA mRNA表达水平降低,cofilin mRNA、PSD-95 mRNA、SYN mRNA表达水平升高,差异有统计学意义(P<0.05)。免疫组化染色结果显示,与空白对照组相比,铝中毒模型组海马CA1区RhoA阳性细胞率增高,cofilin、PSD-95和SYN阳性细胞率降低,差异有统计学意义(P<0.05);与铝中毒模型组相比,RhoA抑制剂组海马CA1区RhoA阳性细胞率降低,cofilin、PSD-95和SYN阳性细胞率增高,差异有统计学意义(P<0.05)。透射电子显微镜观察结果显示,与空白对照组相比,铝中毒模型组中突触数量减少,突触后致密物质厚度变薄,突触间隙宽度变窄,差异有统计学意义(P<0.05);与铝中毒模型组相比,RhoA抑制剂组突触数量增多,突触后致密物质厚度增加,突触间隙宽度增加,差异有统计学意义(P<0.05)。相对于空白对照组,铝中毒模型组线粒体形态发生显著变化,RhoA抑制剂组的线粒体轻微膨胀,膜结构保持完好,线粒体形态及突触超微结构好于铝中毒模型组。结论铝中毒可通过激活RhoA/cofilin信号通路破坏海马突触可塑性,进而影响学习记忆能力。

Objective To explore the effect of Ras homolog gene family member A(RhoA)/cofilin pathway activation on hippocampal synaptic plasticity and the mechanistic role of RhoA/cofilin pathway activation in aluminum toxicity-induced learning and memory impairments.Methods Twenty rats were randomly selected from 30 specific pathogen-free Sprague-Dawley rats and were injected intraperitoneally with maltol-aluminum solution for 2 months to establish a rat model of chronic aluminum intoxication.The aluminum-intoxicated model rats were divided into aluminum intoxication model group(10 rats)and RhoA inhibitor group(10 rats),and the latter group was injected intraperitoneally with Rhosin hydrochloride for 30 days.The remaining 10 normal rats from the 30 specific pathogen-free SD rats were used as blank control group.The learning and memory abilities of the rats were tested by Morris water maze experiment.Transmission electron microscopy was used to observe changes in the ultrastructure of synapses in the CA1 region of the hippocampus of the rats.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and immunohistochemical staining were used to detect the localized expressions of RhoA,cofilin,post-synaptic density protein-95(PSD-95)and synaptophysin(SYN)in the CA1 region of the hippocampal tissues of the rats.Results The results of Morris water maze experiment showed that the latency period of the rats in the aluminum intoxication model group was significantly longer than that in the blank control group(P<0.05),and the latency period of the rats in the RhoA inhibitor group was significantly shorter than that in the aluminum intoxication model group(P<0.05).The results of RT-qPCR showed that compared with those in the blank control group,the level of RhoA mRNA expression in CA1 region of the hippocampal tissues in the aluminum intoxication model group increased,while the levels of cofilin mRNA,PSD-95 mRNA and SYN mRNA expressions decreased,and the differences were statistically significant(P<0.05).Compared with those in the aluminum intoxication model group,the level of RhoA mRNA expression in the CA1 region of the hippocampal tissues of the rats in the RhoA inhibitor group decreased,and the levels of cofilin mRNA,PSD-95 mRNA and SYN mRNA expressions increased,and the differences were statistically significant(P<0.05).The results of immunohistochemical staining showed that compared with the blank control group,the aluminum intoxication model group had an increase in the percentage of RhoA-positive cells in the CA1 region of the hippocampus,while the aluminum intoxication model group had decreases in the percentages of cofilin-,PSD-95-,and SYN-positive cells,and the differences were statistically significant(P<0.05).Compared with the aluminum intoxication model group,the RhoA inhibitor group had a decrease in the percentage of RhoA-positive cells in the CA1 region of the hippocampus,while the RhoA inhibitor group had increases in the percentages of cofilin-,PSD-95-,and SYN-positive cells,and the differences were statistically significant(P<0.05).The results of the observations by using transmission electron microscopy revealed that compared with those in the blank control group,the number of synapses was less,and the thickness of the postsynaptic dense substance became thinner,and the width of the synaptic cleft became narrower in the aluminum intoxication model group,and the differences were statistically significant(P<0.05).The results of the observations by using transmission electron microscopy revealed that compared with those in the aluminum intoxication model group,the number of synapses was more,and the thickness of the postsynaptic dense substance became thicker,and the width of the synaptic cleft became wider in the RhoA inhibitor group,and the differences were statistically significant(P<0.05).Compared with those in the blank control group,the mitochondrial morphology in the aluminum intoxication model group changed significantly,and the mitochondria in the RhoA inhibitor group slightly dilated and the membrane structure remained intact,and the mitochondrial morphology and synaptic ultrastructure in the RhoA inhibitor group were better than those in the aluminum intoxication model group.Conclusion Aluminum intoxication can disrupt hippocampal synaptic plasticity through the activation of the RhoA/cofilin signaling pathway,subsequently affecting learning and memory abilities.