Cui Changmeng, Cui Jianzhong, Jin Feng, Cui Ying, Li Ran, Jiang Xiaohua, Tian Yanxia, Wang Kaijie, Jiang Pei, Gao Junling
Department of Neurosurgery, Affiliated Hospital of Jining Medical University, Jining, China.
Institute of Clinical Pharmacy and Pharmacology, Jining First People's Hospital, Jining Medical University, Jining, China.
Cell Physiol Biochem. 2017;42(5):1888-1896. doi: 10.1159/000479571. Epub 2017 Aug 3.
BACKGROUND/AIMS: Traumatic brain injury (TBI) is a major public health problem in the world and causes high rates of mortality and disability. Recent evidence suggests that vitamin D (VD) has neuroprotective actions and can promote function recovery after TBI. In vitro and in vivo studies have demonstrated that autophagy could be enhanced following supplementation with an active metabolite of VD (calcitriol). However, it is unclear whether autophagy participates in the protective effects of calcitriol after TBI. To test this hypothesis, we examined the protective effects of calcitriol on TBI-induced neurological impairment and further investigated whether calcitriol could modulate autophagy dysfunction-mediated cell death in the cortex region of rat brain.
Eighty-five male rats (250-280 g) were randomly assigned to sham (n=15), TBI model (TBI, n=35) and calcitriol treatment (calcitriol, n=35) groups. Rats were injected intraperitoneally with calcitriol (1 µg/kg) at 30 min, 24 h and 48 h post-TBI in the calcitriol group. The lysosomal inhibitor, chloroquine (CQ), was used to evaluate autophagic flux in the TBI and calcitriol groups. Neurological functions were evaluated via the modified neurological severity score test at 1-7 days after TBI or sham operation, and the terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick-end labeling method was used to evaluate the ability of calcitriol to inhibit apoptosis. The expression of VDR, LC3 and p62 proteins was measured by western blot analysis at 1, 3 and 7 days post-injury Results: Calcitriol treatment attenuated mNSS at 2-7 days post-TBI (P < 0.05 versus TBI group). Calcitriol dramatically increased VDR protein expression compared with the untreated counterparts at 1, 3 and 7 days post-TBI (P < 0.05). The rate of apoptotic cells in calcitriol-treated rats was significantly reduced compared to that observed in the TBI group (P < 0.05). The LC3II/LC3I ratio was decreased in the cortex region at 1, 3 and 7 days post-TBI in rats treated with calcitriol (p < 0.05 versus TBI group), and the p62 expression was also attenuated (p < 0.05 versus TBI group). The LC3II/LC3I ratio in the calcitriol group was significantly increased when pretreated with CQ (P < 0.05).
Calcitriol treatment activated VDR protein expression and attenuated neurological deficits in this rat TBI model. The protective effects might be associated with the restoration of autophagy flux and the decrease in apoptosis in the cortex region of rat brain.
背景/目的：创伤性脑损伤（TBI）是全球主要的公共卫生问题，导致高死亡率和高致残率。最近的证据表明，维生素D（VD）具有神经保护作用，可促进TBI后的功能恢复。体外和体内研究表明，补充VD的活性代谢物（骨化三醇）后自噬可增强。然而，尚不清楚自噬是否参与骨化三醇对TBI的保护作用。为验证这一假设，我们研究了骨化三醇对TBI诱导的神经功能障碍的保护作用，并进一步探讨骨化三醇是否能调节自噬功能障碍介导的大鼠脑皮质区细胞死亡。
85只雄性大鼠（250 - 280 g）随机分为假手术组（n = 15）、TBI模型组（TBI，n = 35）和骨化三醇治疗组（骨化三醇，n = 35）。骨化三醇组大鼠在TBI后30分钟、24小时和48小时腹腔注射骨化三醇（1 μg/kg）。溶酶体抑制剂氯喹（CQ）用于评估TBI组和骨化三醇组的自噬通量。在TBI或假手术后1 - 7天通过改良神经功能缺损评分试验评估神经功能，采用末端脱氧核苷酸转移酶介导的FITC-dUTP缺口末端标记法评估骨化三醇抑制细胞凋亡的能力。在损伤后1、3和7天通过蛋白质免疫印迹分析检测VDR、LC3和p62蛋白的表达。结果：骨化三醇治疗可减轻TBI后2 - 7天的mNSS（与TBI组相比，P < 0.05）。与未治疗的大鼠相比，TBI后1、3和7天骨化三醇显著增加VDR蛋白表达（P < 0.05）。与TBI组相比，骨化三醇治疗的大鼠凋亡细胞率显著降低（P < 0.05）。TBI后1、3和7天，骨化三醇治疗的大鼠皮质区LC3II/LC3I比值降低（与TBI组相比，p < 0.05），p62表达也减弱（与TBI组相比，p < 0.05）。用CQ预处理后，骨化三醇组的LC3II/LC3I比值显著增加（P < 0.05）。
在该大鼠TBI模型中，骨化三醇治疗激活了VDR蛋白表达并减轻了神经功能缺损。其保护作用可能与大鼠脑皮质区自噬通量的恢复和细胞凋亡的减少有关。
