FGF21/β-klotho/FGFR1通路在2型糖尿病局灶性脑缺血模型大鼠脑损伤中的作用机制研究

Mechanism of FGF21/β-klotho/FGFR1 pathway in brain injury in type 2 diabetic model rats with focal cerebral ischemia

目的研究在2型糖尿病(T2DM)局灶性脑缺血模型大鼠脑损伤中成纤维细胞生长因子21(FGF21)/β-klotho/成纤维细胞生长因子1型受体(FGFR1)通路的作用。方法 SD大鼠采用高脂饲养结合腹腔链脲佐菌素(STZ)注射建立T2DM大鼠模型,采用线栓法建立大鼠大脑中动脉栓塞(MCAO)模型,实验分为正常对照组(Control组)、单纯脑缺血组(MCAO组)、T2DM脑缺血组(T2DM+MCAO组)、T2DM脑缺血FGFR1抑制剂PD173074干预组(PD173074组)及T2DM脑缺血FGFR1激动剂PF05231023干预组(PF05231023组),每组12只,PD173074组、PF05231023组分别于MCAO术前30 min尾静脉注射PD173074 5 mg/kg、PF05231023 5 mg/kg。MCAO 24 h后进行神经功能缺损评分,随后处死大鼠取脑组织标本。采用原位末端标记法(TUNEL)检测脑组织神经细胞凋亡,实时荧光定量PCR(RT-qPCR)检测脑组织FGF21、β-klotho、FGFR1 mRNA表达,免疫印迹(Western blot)检测脑组织FGF21、β-klotho、FGFR1蛋白及血小板内皮细胞粘附分子(CD31)、内皮素-1(ET-1)及血管内皮生长因子(VEGF)蛋白表达。结果与Control组比较,MCAO组和T2DM+MCAO组大鼠神经功能缺损评分、神经细胞凋亡率、FGF21、β-klotho、FGFR1 mRNA及蛋白水平、CD31、ET-1、VEGF蛋白水平显著增加(P<0.05);与T2DM+MCAO组比较,PD173074组大鼠神经功能缺损评分、神经细胞凋亡率、CD31、ET-1、VEGF蛋白水平显著增加,FGF21、β-klotho、FGFR1 mRNA及蛋白水平显著降低(P<0.05),PF05231023组大鼠神经功能缺损评分、神经细胞凋亡率、CD31、ET-1、VEGF蛋白水平显著降低(P<0.05),FGF21、β-klotho、FGFR1 mRNA及蛋白水平显著增加(P<0.05)。结论 FGF21/β-klotho/FGFR1通路激活可能在T2DM局灶性脑缺血模型大鼠中发挥重要保护作用。

Objective To investigate the mechanism of the fibroblast growth factor 21(FGF21)/β-klotho/fibroblast growth factor type 1 receptor(FGFR1) pathway in brain injury of type 2 diabetes mellitus(T2 DM) model rats with focal cerebral ischemia. Methods SD rats were fed a high fat diet and injected intraperitoneally with streptozotocin(STZ) to establish the T2 DM rat model. The middle cerebral artery occlusion(MCAO) model was established by the suture method. Rats were divided into normal control group(Control group), simple cerebral ischemia group(MCAO group), T2 DM cerebral ischemia group(T2 DM + MCAO group), T2 DM cerebral ischemia FGFR1 inhibitor PD173074 treatment group(PD173074 group) and T2 DM cerebral ischemia FGFR1 agonist PF05231023 treatment group(PF05231023 group) with 12 rats in each group. PD173074 and PF05231023 groups were injected with 5 mg/kg PD173074 and 5 mg/kg PF05231023 respectively at 30 minutes before MCAO. After MCAO for 24 hours, the neurological deficit score was calculated and the rats were sacrificed to obtain brain tissue samples. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect neuronal apoptosis. Real-time quantitative PCR was used to detect mRNA expression of FGF21, β-klotho and FGFR1. Protein expression of FGF21, β-klotho, FGFR1, platelet endothelial cell adhesion molecule(CD31), endothelin-1(ET-1) and vascular endothelial growth factor(VEGF) was detected by Western blot. Results Compared with the control group, the neurological deficit score, apoptosis rate of nerve cells, mRNA and protein levels of FGF21, β-klotho, FGFR1 and protein levels CD31, ET-1, VEGF were significantly increased in MCAO and T2 DM + MCAO groups(P<0.05). Compared with the T2 DM + MCAO group, the neurological deficit score, apoptosis rate of nerve cells, protein levels of CD31, ET-1 and VEGF were increased in the PD173074 group, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were decreased(P<0.05). Moreover, the neurological deficit score, apoptosis rate of nerve cells and the protein levels of CD31, ET-1 and VEGF in the PF05231023 group were decreased, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were increased(P<0.05). Conclusions Activation of the FGF21/β-klotho/FGFR1 pathway may play an important protective role in focal cerebral ischemia model rats with T2 DM.