塞来昔布对脑出血模型大鼠TLR4/NF-κB信号通路及GLT-1表达的影响

Effects of Celecoxib on TLR4/NF-κB Signaling Pathway and GLT-1 in Rats with Intracerebral Hemorrhage

目的观察塞来昔布对脑出血大鼠Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路及谷氨酸转运体1(GLT-1)表达的影响。方法将90只雄性SD大鼠分为假手术组、模型组、抑制剂组和低剂量组、中剂量组、高剂量组,其中低剂量组、中剂量组、高剂量组塞来昔布剂量分别为50 mg/(kg·d)、100 mg/(kg·d)、200 mg/(kg·d)。沿大鼠右侧尾状核置入套管,通过套管向尾状核内注射生理盐水和股动脉血建立假手术和脑出血造模。造模后假手术组和模型组每日注射生理盐水2 mL,抑制剂组和各剂量组分别注射相同剂量TLR4抑制(TAK-242)和不同剂量的塞来昔布进行干预。干预后对大鼠进行神经功能评分,免疫组织化学检测脑组织GLT-1积分光密度,实时荧光定量聚合酶链式反应(RT-PCR)和免疫印迹法(Western Blotting)法检测脑组织TLR4、NF-κB、GLT-1蛋白和mRNA表达。结果与假手术组比较,模型组神经功能评分、GLT-1积分光密度及TLR4、NF-κB、GLT-1蛋白和mRNA表达量均显著升高(P<0.05)。与模型组比较,抑制剂组和低剂量组、中剂量组、高剂量组神经功能评分、GLT-1积分光密度及TLR4、NF-κB、GLT-1蛋白和mRNA表达量均显著降低(P<0.05)。与抑制剂组比较,低剂量组、中剂量组、高剂量组TLR4蛋白和mRNA表达,低剂量组、中剂量组GLT-1积分光密度、GLT-1 mRNA、NF-κB蛋白升高,低剂量组NF-κB mRNA、GLT-1蛋白升高(P<0.05);中剂量组、高剂量组神经功能评分、高剂量组积分光密度降低(P<0.05)。与低剂量组比较,中剂量组、高剂量组神经功能评分降低、TLR4和GLT-1 mRNA、NF-κB蛋白和mRNA表达量降低,高剂量组GLT-1积分光密度、TLR4和GLT-1蛋白表达量均降低(P<0.05)。与中剂量组比较,高剂量组GLT-1积分光密度、TLR4和NF-κB蛋白和mRNA表达量、GLT-1 mRNA均降低(P<0.05)。结论塞来昔布可下调脑出血大鼠TLR4/NF-κB通路进而发挥抗炎作用保护脑组织,同时控制GLT-1过表达可减轻谷氨酸神经毒性。

Objective To observe the effects of celecoxib on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway and glutamate transporter 1(GLT-1)in rats with intracerebral hemorrhage.Methods Ninety male SD rats were divided into sham group,model group,inhibitor group and low dose group,medium dose group,and high dose group[celecoxib doses were 50 mg/(kg·d),100 mg/(kg·d)and 200 mg/(kg·d)respectively].Cannula was inserted into the right caudate nucleus of the rat,through which saline and femoral artery blood were injected into the caudate nucleus for sham operation and intracerebral hemorrhage modeling.After modeling,the sham group and the model group were injected with 2 mL of normal saline everyday,while the inhibitor group and each dose group were injected with corresponding doses of TAK-242 and different doses of celecoxib respectively for intervention.After the intervention,neurological function of rats was evaluated,the integral optical density of GLT-1 in brain tissues were detected by immunohistochemistry,the protein of TLR4,NF-κB and GLT-1 in brain tissues were detected by Reverse Transcription-Polymerase Chain Reaction,the mRNA of TLR4,NF-κB and GLT-1 in brain tissues were detected by Western Blotting.Results Compared with the sham group,the neurological function score,integral optical density of GLT-1,the mRNA,and protein expressions of TLR4,NF-κB,GLT-1 in the model group significantly increased(P<0.05).Compared with the model group,the neurological function score,integral optical density of GLT-1,the mRNA and protein expressions of TLR4,NF-κB,and GLT-1 in the inhibitor group and low dose group,medium dose group and high dose group significantly decreased(P<0.05).Compared with the inhibitor group,mRNA and protein of TLR4 in the low dose group,medium dose group and high dose group,integral optical density of GLT-1,GLT-1 mRNA,and NF-κB protein in the low dose group,medium dose group significantly increased(P<0.05).Neurological function score in the medium dose group and high dose group and integral optical density of GLT-1 in the high dose group significantly decreased(P<0.05).Compared with the low dose group,the neurological function score,and mRNA of TLR4 and GLT-1,mRNA and protein of NF-κB in the medium dose group and high dose group decreased significantly(P<0.05).Compared with the medium dose group,integral optical density of GLT-1,the mRNA and protein expressions of TLR4,NF-κB,mRNA of GLT-1 in the high dose group significantly decreased(P<0.05).Conclusion Celecoxib could down-regulate the TLR4/NF-κB pathway in rats with intracerebral hemorrhage to exert anti-inflammatory effects and protect brain tissue,and control the overexpression of GLT-1 to reduce the neurotoxicity of glutamate.