Endoglin重组蛋白对SNI大鼠的镇痛作用及机制研究

Effects and mechanisms of recombinant Endoglin on SNI rats

目的观察鞘内注射内皮糖蛋白(Endoglin)重组蛋白对选择性坐骨神经损伤(SNI)大鼠疼痛、脊髓背角小胶质细胞活化和促炎因子表达的影响。方法将66只成年雄性SD大鼠随机分为三组:假手术组(Sham组)、SNI模型组(SNI组)和干预组(ENG组),每组22只,并预先进行鞘内置管。随后,SNI组大鼠构建SNI模型,并间断鞘内给予生理盐水20μL;ENG组在SNI模型的基础上给予20μL Endoglin重组蛋白(2μg/mL);Sham组则仅做SNI的伤口对照,不损伤神经,并且给予等量生理盐水。于SNI术前1 d,术后3、7、14 d采用Von Frey纤维丝测定机械缩足阈值(PWT);免疫荧光染色观察术后14 d时脊髓背角小胶质细胞的激活;同时,Western blot检测脊髓转化生长因子-β1(TGF-β1)、Smad2、p-Smad2的蛋白表达;ELISA检测脊髓肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)的释放水平。结果在术后3~14 d时,SNI组和ENG组的PWT值均明显低于Sham组,但相比于SNI组,ENG组的PWT明显增加,差异均有统计学意义(P均<0.05)。与Sham组比较,术后14 d时SNI组及ENG组脊髓背角中小胶质细胞的活化程度以及炎症因子TNF-α、IL-1β及IL-6的释放水平明显增加,而SNI组TGF-β1、p-Smad2的蛋白表达却明显降低,差异均有统计学意义(P均<0.05)。与SNI组比较,ENG组小胶质细胞的活化程度以及炎症因子TNF-α、IL-1β及IL-6的释放水平明显降低,TGF-β1、p-Smad2的蛋白表达明显增加,差异均有统计学意义(P均<0.05)。结论鞘内注射Endoglin重组蛋白可缓解SNI大鼠的痛觉过敏现象,同时抑制其脊髓背角小胶质细胞活化及炎症因子释放,并能激活脊髓中的TGF-β1/Smad2信号。

Objective To observe the effects of intrathecal injection of recombinant endoglin protein on pain,activation of microglia and expression of pro-inflammatory factors in spinal dorsal horn of spared nerve injury(SNI)rats.Methods Sixty-six adult male SD rats were randomly divided into three groups:sham group,SNI group and ENG group,22 in each group.All rats were pretreated with intrathecal catheter.SNI model of SNI group was constructed.Meanwhile,rats in SNI group were intermittently intrathecal injected with 20μL of saline.ENG group was treated with endoglin recombinant protein(2μg/mL)in 20μL on the basis of SNI model.In sham group,only SNI wound control was performed,without nerve injury,and normal saline 20μL was injected.The paw withdrawal threshold(PWT)was measured by von Frey filaments on preoperative day 1,3,7 and 14.Immunofluorescence staining was used to observe the activation of microglia in spinal dorsal horn at 14 d.Meanwhile,the protein expression of TGF-β1,Smad2 and p-Smad2 in spinal cord were detected by Western blot.The levels of TNF-α,IL-1βand IL-6 in spinal cord were detected by ELISA.Results At 3-14 d,the PWT value of SNI group and ENG group was significantly decreased compared with Sham group(P<0.05),but the PWT value of ENG group was significantly increased compared with SNI group(P<0.05).In addition,the activation of microglia in spinal dorsal horn and the release level of inflammatory factors TNF-α,IL-1βand IL-6 were significantly increased in SNI group and ENG group compared with sham group at 14 d,while the protein expression of TGF-β1 and p-Smad2 of SNI group were significantly decreased compared with sham group(P<0.05).Compared with SNI group,the activation degree of microglia and the release levels of inflammatory factors TNF-α,IL-1βand IL-6 were significantly decreased in ENG group(P<0.05),while the protein expressions of TGF-β1 and p-Smad2 were significantly increased(P<0.05).Conclusion Intrathecal injection of recombinant endoglin protein could relieve the hyperalgesia of SNI rats,inhibit its spinal microglial activation and inflammatory factor release,and activate TGF-β1/Smad2 signal.