L-乳酸对创伤性颅脑损伤后缺氧诱导因子-1α和突触可塑性相关蛋白的影响

Effects of L-lactate on HIF-1α and synaptic plasticity related proteins expression for traumatic brain injury

摘要:

目的 探讨L-乳酸对创伤性颅脑损伤后缺氧诱导因子-1α（HIF-1α）和突触可塑性相关蛋白——突触后密度蛋白95（PSD-95）和生长相关蛋白43（GAP-43）表达的影响。方法 健康成年雄性SD大鼠27只，采用随机数字表法将其分为假手术组（Sham组）、创伤性颅脑损伤组（颅脑损伤组）和创伤性颅脑损伤+L-乳酸组（颅脑损伤+Lac组），每组9只。神经细胞株PC12细胞系分为对照组、细胞模型组（MSC组）和细胞模型+L-乳酸组（MSC+Lac组）。采用改良的Fenney’s自由落体打击模型复制中度创伤性脑损伤模型和机械划痕细胞模型。于颅脑损伤后7 d时检测改良神经功能评分（mNSS），采用Western blotting法检测大鼠损伤侧皮层、海马和PC12细胞HIF-1α、PSD-95和GAP-43蛋白的相对表达量，qRT-PCR检测大鼠损伤侧皮层HIF-1α mRNA相对表达量，免疫荧光染色法检测大鼠损伤侧皮质和海马PSD-95的表达。结果 3组大鼠mNSS评分比较，差异有统计学意义（P <0.05）；与Sham组比较，颅脑损伤组mNSS评分升高（P <0.01），与颅脑损伤组比较，颅脑损伤+Lac组mNSS评分降低（P <0.01）。与Sham组比较，颅脑损伤组的HIF-1α蛋白、mRNA相对表达量和PSD-95、GAP-43蛋白相对表达量降低（P <0.05）；与颅脑损伤组比较，颅脑损伤+Lac组HIF-1α蛋白、mRNA相对表达量和PSD-95、GAP-43蛋白相对表达量增加（P <0.05）。与Sham组比较，颅脑损伤组大鼠损伤侧皮层和海马PSD-95蛋白免疫荧光强度降低（P <0.05），与颅脑损伤组比较，颅脑损伤+Lac组大鼠损伤侧皮层和海马PSD-95蛋白免疫荧光强度增强（P <0.05）。与对照组比较，MSC组细胞的HIF-1α、PSD-95、GAP-43蛋白相对表达量降低（P <0.05）；与MSC组比较，MSC+Lac组细胞的HIF-1α、PSD-95、GAP-43蛋白相对表达量增加（P <0.05）。结论 L-乳酸可以减轻颅脑损伤大鼠的脑损伤程度，其机制可能与上调HIF-1α表达进而促进PSD-95和GAP-43表达有关。

Abstract:

Objective To investigate the effects of L-lactate on HIF-1α and synaptic plasticity related proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43), in TBI.Methods Twenty seven healthy adult male Sprague-Dawley rats were randomly divided into three groups, including sham operation group (Sham group), traumatic brain injury (TBI group), and traumatic brain injury + L-lactate group (TBI + Lac group), 9 cases in each group. The cell experiment was divided into control group (Control group), mechanical scratch cell model group (MSC group), and L-lactate treated mechanical scratch cell group (MSC + Lac group). Feeney’s free falling method was used to establish moderate traumatic brain injury model, and mechanical scratch model were performed to mimic traumatic cell injury in vitro. The modified neurological score (mNSS) was performed at 7 days after TBI. HIF-1α, PSD-95, and GAP-43 expressions were detected by western blotting in the ipsilateral cortex, hippocampus, and PC12 cells. The HIF-1α mRNA expression of the ipsilateral cortex was measured by RT-PCR. The fluorescence intensity of PSD-95 protein was detected by immunofluorescence staining in the ipsilateral cortex and hippocampal CA1 region.Results Compared with Sham group, the mNSS scores were increased in TBI group. Meanwhile, the expression of HIF-1α, PSD-95, and GAP-43 were also decreased in TBI group. Moreover, the fluorescence intensity of PSD-95 immunoreactivity in the ipsilateral cortex and hippocampal CA1 region was decreased in the TBI group. However, the behaviors of TBI rats were improved, the expression of HIF-1α and synaptic plasticity related proteins were increased, and the fluorescence intensity of PSD-95 immunoreactivity was also increased in the TBI + Lac group. The expression of HIF-1, PSD-95, and GAP-43 protein were decreased significantly in MSC group compared with that of control group, while they were increased in MSC + Lac group.Conclusion The mechanism of L-lactate alleviating traumatic brain injury may be related to up-regulated HIF-1 α expression and promoting the expression of PSD-95 and GAP-43.