铅致大鼠海马神经发生的抑制作用

Neurogenesis impairment in hippocampus of rats induced by lead

[背景]铅是一种常见的神经毒物,长期低剂量铅暴露可造成神经系统损害。已有研究表明铅暴露可致神经细胞凋亡增加,致使受损神经元细胞不能得到补偿,进而影响大脑的正常功能。成年哺乳动物脑内海马齿状回颗粒下区(SGZ)终生存在有神经干细胞,在某些生理或病理因素刺激下,可增殖、分化为神经元和神经胶质细胞,迁移到损伤的脑区替换受损细胞,从而建立新的神经回路,维护大脑的正常功能。研究发现,干扰素γ(IFN-γ)可抑制神经干细胞的增殖及分化。转化生长因子β(TGF-β)也是调控细胞发育和细胞周期的关键分子,可促进神经发生过程。[目的]初步探讨IFN-γ和TGF-β在铅致大鼠SGZ区神经发生损伤中的可能调控作用。[方法 ]雄性成年Fisher 344大鼠45只,随机分为对照组、低铅暴露组、高铅暴露组。低铅暴露组和高铅暴露组大鼠分别饮用含有300、600 mg/L的醋酸铅溶液,对照组大鼠饲以含有600 mg/L的醋酸钠溶液,暴露时间为9周。应用Morris水迷宫试验检测大鼠空间学习和记忆能力;以Ki67+-DCX+双标染色方法,应用激光扫描共聚焦显微镜观察大鼠SGZ区神经干细胞增殖及分化情况。应用real-time PCR技术检测大鼠海马齿状回(DG)区Ki67、DCX mRNA的表达水平;采用ELISA法检测大鼠海马DG区IFN-γ与TGF-β1水平。[结果 ]高铅暴露组和低铅暴露组大鼠血铅水平分别为(0.34±0.10)、(0.19±0.04)μg/L,均高于对照组[(0.09±0.03)μg/L];高铅暴露组和低铅暴露组大鼠海马DG区铅水平分别为(0.53±0.06)、(0.48±0.08)μg/g,亦均高于对照组[(0.40±0.05)μg/g]。神经行为学试验检测结果显示,高铅暴露组和低铅暴露组大鼠第3天逃避潜伏期分别为(38.37±10.37)、(31.62±9.24)s,均高于对照组[(21.86±6.45)s],且高铅暴露组大鼠逃避潜伏期高于低铅暴露组(P <0.05)。高铅暴露组大鼠的平均穿越平台次数为(2.13±0.92)次,低于对照组[(4.27±1.16)次]。铅暴露大鼠海马CA1区神经元细胞呈不同程度减少,排列不规则,可见鬼影细胞及固缩神经元细胞,高铅暴露组损伤更为明显。高铅暴露组大鼠海马DG区Ki67和DCX的mRNA表达水平分别为0.68±0.10和0.78±0.12,均低于对照组(均为1.00±0.16);高铅暴露组大鼠SGZ区(Ki67+-DCX+)/Ki67+值为对照组的56.4%(20.87%vs. 37.00%),亦低于低铅暴露组,为其61.2%(20.87%vs. 34.11%),差异有统计学意义(P <0.05)。高铅暴露组和低铅暴露组大鼠海马DG区IFN-γ水平[分别为(1.22±0.09)、(1.20±0.11)ng/mg(以蛋白计,下同)],均高于对照组[(1.08±0.08)ng/mg],差异有统计学意义(P <0.05)。高铅暴露组和低铅暴露组大鼠海马DG区TGF-β1表达水平分别为(3.15±0.24)、(3.36±0.32)ng/mg,均低于对照组[(3.65±0.37)ng/mg],差异有统计学意义(P <0.05)。[结论 ]铅暴露可能导致IFN-γ水平升高,进而抑制TGF-β1的表达水平,影响神经干细胞的分化,致使铅暴露组大鼠新生神经元细胞减少。不能代偿受损神经细胞,这可能是导致成年大鼠神经行为改变,学习记忆能力下降的原因之一。

[Background]Lead is a common neurotoxic substance,and long-term exposure to low-dose lead can cause damage to the nervous system.Previous studies have shown that lead exposure can increase the apoptosis of nerve cells,and damaged nerve cells can not be compensated,which in turn affects the normal function of the brain.In adult mammals,sub-granular zone(SGZ)eventually harbours neural stem cells.Under the stimulation of certain physiological or pathological factors,neural stem cells can proliferate and differentiate into neurons and glial cells,and migrate to damaged brain regions to replace damaged cells.Thus new neural circuits are established to maintain the normal functions of the brain.Studies have found that interferon-γ(IFN-γ)can inhibit the proliferation and differentiation of neural stem cells.Transforming growth factor-β(TGF-β)is also a key molecule regulating cell development and cell cycle,and promotes the process of neurogenesis.[Objective]This experiment aims to explore the possible regulatory roles of IFN-γand TGF-β1 in lead-induced neurogenesis impairment in hippocampal SGZ of rats.[Methods]Forty-five adult male Fisher 344 rats were randomly divided into control group,low-dose lead exposure group,and high-dose lead exposure group.Rats in the low-dose and high-dose lead exposure groups were given 300 mg/L and 600 mg/L lead acetate solution,respectively.Rats in the control group were treated with 600 mg/L sodium acetate solution.The exposure lasted for 9 weeks.Morris water maze test was applied to test the spatial learning and memory ability of the rats.The proliferation and differentiation of neural stem cells in SGZ were observed by laser scanning confocal microscopy with Ki67+-DCX+staining.Real-time PCR was used to detect the expressions of Ki67 and DCX mRNA in hippocampal dentate gyrus(DG)region of rats.ELISA assay was used to detect the levels of IFN-γand TGF-β1 in the hippocampal DG of rats.[Results]The blood lead levels in the high-dose and low-dose lead exposure groups were(0.34±0.10)μg/L and(0.19±0.04)μg/L,respectively,which were higher than that in the control group((0.09±0.03)μg/L)The lead levels in the hippocampal DG area in the highdose and low-dose lead exposure groups were(0.53±0.06)μg/g and(0.48±0.08)μg/g,respectively,which were also higher than that in the control group((0.40±0.05)μg/g)The neurobehavioral test showed that the escape latencies in the high-dose and low-dose lead exposure groups were(38.37±10.37)s and(31.62±9.24)s,respectively,which were higher than that in the control group[(21.86±6.45)s],and the escape latency in the high-dose lead group was higher than that in the low-dose group(P<0.05).The average number of crossing platforms in the high-dose lead exposure group was(2.13±0.92)times,which was lower than that in the control group[(4.27±1.16)times].The neurons in the hippocampal CA1 area of lead-exposed rats decreased in varying degrees,the arrangement was irregular,and ghost cells and stenotic neurons were observed,especially in the high-dose lead exposure group.The mRNA expression levels of Ki67 and DCX in hippocampal DG region in high-dose lead exposure group were 0.68±0.10 and 0.78±0.12,respectively,which were lower than the levels in the control group(both were 1.00±0.16).The ratio of SGZ(Ki67+-DCX+)/Ki67+in the high-dose lead exposure group was 56.4%(20.87%vs.37.00%)in the control group,and it was also lower than the low-dose lead exposure group,which was 61.2%(20.87%vs.34.11%),showing significant difference(P<0.05).The expression level of IFN-γin hippocampal DG area of rats with high-dose lead exposure and low-dose lead exposure were[(1.22±0.09)ng/mg(in terms of protein,thereafter)and(1.20±0.11)ng/mg],which were higher than the level of the control group[(1.08±0.08)ng/mg],showing significant difference(P<0.05).The expression levels of TGF-β1 in hippocampal DG of the high-dose and lowdose lead exposure groups were(3.15±0.24)ng/mg and(3.36±0.32)ng/mg,respectively,which were lower than that of the control group[(3.65±0.37)ng/mg],showing significant difference(P<0.05).[Conclusion]Lead exposure may increase the level of IFN-γ,inhibit the expression level of TGF-β1,affect the differentiation of neural stem cells,and result in the decrease of newborn neurons in lead exposure rats.The damaged neurons can not be compensated,which may be the reason for the decline of learning and memory ability in adult rats with neurobehavioral changes.