铁调素在创伤性脑损伤大鼠皮质中的表达特性

Expression characteristics of hepcidin in rat cortex after traumatic brain injury

目的研究铁调素在创伤性脑损伤大鼠皮质中的表达特性。方法建立大鼠左侧控制性皮质撞击(controlled corticalimpact,CCI)模型,采用去铁胺干预,研究创伤性脑损伤早期(72 h内)大鼠皮质内铁调素的变化情况、铁浓度与铁调素表达的关系及铁调素与神经元的共定位情况。通过实时定量逆转录聚合酶链反应评估铁调素mRNA转录情况,通过酶联免疫吸附测定评估铁调素浓度变化情况,通过铁浓度检测试剂盒评估非血红素总铁含量,通过蛋白质印迹法评估BMPs/Smad1/5/8信号通路激活情况。通过免疫荧光评估铁调素在脑组织细胞中的信号强度,并进行铁调素与神经元和星形胶质细胞的共定位分析。结果与Sham组相比,CCI 6 h组大鼠脑皮质中的铁含量、铁调素mRNA相对表达量、铁调素浓度及pSmad1/5/8/Smad1/5/8比值均无明显变化(均P>0.05)。CCI 24 h组和CCI 72 h组大鼠脑皮质中的铁含量、铁调素mRNA相对表达量、铁调素浓度及pSmad1/5/8/Smad1/5/8比值均显著高于Sham组(均P<0.01);CCI 72 h+去铁胺组大鼠脑皮质中的铁含量、铁调素mRNA相对表达量、铁调素浓度及pSmad1/5/8/Smad1/5/8比值均显著低于CCI 72 h组(均P<0.05)。相较于Sham组,CCI 72 h组大鼠脑皮质DAPI阳性细胞核减少,脑细胞内的荧光强度显著增强(P<0.0001)。共定位分析显示神经元与铁调素信号共标现象增多,而未见明显的星形胶质细胞与铁调素共标现象。结论创伤性脑损伤后脑组织非血红素总铁水平显著升高,并可能通过BMPs/Smad1/5/8信号通路引起脑组织铁调素表达上调。神经元中铁调素水平升高而星形胶质细胞内未见明显铁调素水平变化,说明创伤性脑损伤后铁调素对所作用的脑细胞具有选择性。

Objective To investigate the expression characteristics of hepcidin in rat cortex after traumatic brain injury. Method The rat model of controlled cortical impact(CCI) was established to study the changes of hepcidin in the cortex of rats in the early stage of traumatic brain injury(within 72 h), the relationship between iron concentration and hepcidin level, and the expression of hepcidin in neuron in rats’ brain within 72 h after traumatic brain injury were analyzed. Hepcidin m RNA was evaluated by quantitative reverse transcriptase-mediated PCR(q RT-PCR);the concentration of hepcidin was evaluated by enzyme-linked immunosorbent assay(ELISA);the concentration of the total non-heme iron was evaluated by Iron Assay Kit;the activation of the BMPs/Smad1/5/8 signal pathway was evaluated by Western blotting;the signal strength of hepcidin in the brain tissue cells was evaluated by immunofluorescence, and the colocalization relationship of hepcidin with neurons(Neu N) or astrocytes(GFAP) was analyzed. Result Compared with Sham group, the concentration of iron, the relative expression of hepcidin m RNA, the concentration of hepcidin and the ratio of p Smad1/5/8/Smad1/5/8 in CCI 6 h group had no significant changes(all P >0.05). The concentration of iron, the relative expression of m RNA, the concentration of hepcidin and the ratio of p Smad1/5/8/Smad1/5/8 in CCI 24 h group and CCI 72 h group were significantly higher than those in Sham group(all P<0.01). The concentration of iron, the relative expression of mRNA, the concentration of hepcidin and the ratio of p Smad1/5/8/Smad1/5/8 in CCI 72 h+deferoxamine group were significantly lower than those in CCI 72 h group(all P<0.05). Compared with Sham group, the number of DAPI positive cells in CCI 72 h group decreased and the intracellular fluorescence intensity of hepcidin in CCI 72 h group was significantly higher than that in Sham group(P<0.0001).Co-localization analysis showed that the co-labeling of neurons and hepcidin signals increased, but there was no obvious co-labeling of astrocytes and hepcidin. Conclusion After traumatic brain injury, iron level in brain tissue is significantly increased, and it may induce the expression of hepcidin by activating BMPs/Smad1/5/8 signaling pathway. The concentration of hepcidin in neurons is upregulated, while the concentration of hepcidin in astrocytes is not changed, indicating that the location of hepcidin in brain tissue cells after traumatic brain injury has selectivity.