胰高血糖素样肽-1受体调控SNI大鼠痛觉敏化的机制研究

The role of GLP-1 receptor in the hyperalgesia of SNI rats

目的探究胰高血糖素样肽-1(GLP-1)受体对选择性坐骨神经损伤(SNI)大鼠痛觉敏化的调控作用及机制。方法将24只SD大鼠随机分为假手术组、SNI术后3 d组、SNI术后7 d组、SNI术后14 d组,每组6只;采用免疫荧光染色和Western blot实验观察各组大鼠神经损伤侧脊髓背角中GLP-1受体的表达情况。另将48只大鼠随机分为Sham+NS组、SNI+NS组、SNI+Exendin 4组、SNI+Exendin 9-39组,每组12只;分别于SNI术前1 d及术后3、7、14 d,检测各组大鼠的机械缩足反射阈值(PWT)和热缩足反射潜伏期(TWL)值;SNI术后14 d,采用免疫荧光染色和Western blot实验观察各组大鼠神经损伤侧脊髓背角中离子钙接头蛋白1(Iba-1)的表达水平,采用ELISA实验检测各组大鼠神经损伤侧脊髓背角中炎症因子γ-干扰素(IFN-γ)和白细胞介素-6(IL-6)的释放情况。结果免疫荧光染色结果表明,与假手术组大鼠比较,SNI术后7 d组及14 d组大鼠脊髓背角中GLP-1受体光密度值明显增加,差异均有统计学意义(P均<0.05);各组间GLP-1受体光密度值比较差异有统计学意义(F=60.25,P<0.05)。Western blot结果表明,与假手术组大鼠比较,SNI术后7 d组及14 d组大鼠脊髓背角GLP-1受体蛋白表达明显增加,差异均有统计学意义(P均<0.05);各组间GLP-1受体蛋白表达比较差异有统计学意义(F=90.98,P<0.05)。SNI术后14 d,免疫荧光双标发现GLP-1受体主要定位于小胶质细胞。SNI术后3、7、14 d,与Sham+NS组比较,SNI+NS组大鼠右后肢PWT和TWL明显降低,差异均有统计学意义(P均<0.05);SNI术后7、14 d,与SNI+NS组比较,SNI+Exendin 4组大鼠右后肢PWT和TWL明显增加,SNI+Exendin 9-39组大鼠右后肢PWT和TWL明显降低,差异均有统计学意义(P均<0.05);SNI术后3、7、14 d,各组间PWT和TWL比较差异均有统计学意义(术后3 d:F_(PWT)=26.73,F_(TWL)=12.37;术后7 d:F_(PWT)=49.65,F_(TWL)=32.65;术后14 d:F_(PWT)=152.82,F_(TWL)=70.33;P均<0.05)。SNI术后14 d,与Sham+NS组比较,SNI+NS组大鼠脊髓背角Iba-1、IFN-γ、IL-6表达量明显增加,差异均有统计学意义(P均<0.05);与SNI+NS组比较,SNI+Exendin 4组大鼠脊髓背角Iba-1、IFN-γ、IL-6表达明显降低,SNI+Exendin 9-39组大鼠脊髓背角Iba-1、IFN-γ、IL-6表达明显增加,差异均有统计学意义;各组间脊髓背角Iba-1、IFN-γ、IL-6表达比较差异均有统计学意义(F_(Iba-1)=60.25,F_(IFN-γ)=44.51,F_(IL-6)=62.52;P均<0.05)。结论SNI大鼠脊髓背角中GLP-1受体表达上调,而GLP-1受体通过抑制脊髓背角小胶质细胞活化及神经炎症进展发挥镇痛作用。

Objective To investigate the role of glucagon-like peptide-1(GLP-1)receptor in spared nerve injury(SNI)-induced hyperalgesia and its mechanism.Methods A total of 24 SD rats were randomly divided into Sham group,SNI-3d group,SNI-7d group,SNI-14d group,6 rats in each group.Immunofluorescence staining and Western blot essay were used to detect the expression of GLP-1 receptor in ipsilateral spinal dorsal horn of rats.A total of 48 SD rats were divided into Sham+NS group,SNI+NS group,SNI+Exendin 4 group,SNI+Exendin 9-39 group,12 rats in each group.Paw withdrawal threshold(PWT)and thermal withdrawal lantency(TWL)of the rats were detected at preoperative day 1 and postoperative day 3,7,14.At postoperative day 14,immunofluorescence staining and Western blot were used to detect the expression of ionized calcium binding adapter molecule 1(Iba-1)in ipsilateral spinal cords of rats in four groups.Meanwhile,ELISA essay was used to investigate the release of interferon-γ(IFN-γ)and interleukin-6(IL-6)in the ipsilateral spinal dorsal horn of rats in four groups.Results The results of immunofluorescence staining showed that,compared with Sham group,the optical density of GLP-1 receptor in the ipsilateral spinal dorsal horn of rats SNI-7d group and SNI-14d group was significantly upregulated,the differences were statistically significant(all P<0.05);and the optical density of GLP-1 receptorn was significant difference among the groups(F=60.25,P<0.05).The results of Western blot showed that,compared with Sham group,the protein expression of GLP-1 receptor in the ipsilateral spinal dorsal horn of rats of SNI-7d group and SNI-14d group was significantly upregulated,the differences were statistically significant(all P<0.05);and the expression of GLP-1 receptor protein was significant difference among the groups(F=90.98,P<0.05).At postoperative day 14,immunofluorescence double staining showed that GLP-1 receptor was mainly expressed in microglia.At postoperative day 3,7,14,compared with Sham+NS group,the PWT and TWL of right hind paw of rats in SNI+NS group were obviously decreased,the differences were statistically significant(all P<0.05);compared with SNI+NS group,PWT and TWL of right hind paw of rats in SNI+Exendin 4 group significantly increased,while the PWT and TWL of right hind paw of rats in SNI+Exendin 9-39 group were significantly decreased,the differences were statistically significant(all P<0.05);and PWT and TWL were significant differences among groups at 3,7 and 14 days after SNI(3 d:F_(PWT)=26.73,F_(TWL)=12.37;7 d:F_(PWT)=49.65,F_(TWL)=32.65;14 d:F_(PWT)=152.82,F_(TWL)=70.33;all P<0.05).At postoperative day 14,compared with Sham+NS group,the protein levels of Iba-1,IFN-γ,IL-6 in ipsilateral spinal dorsal horns of rats in SNI+NS group were increased,the differences were statistically significant(all P<0.05);compared with SNI+NS group,the protein levels of Iba-1,IFN-γ,IL-6 in ipsilateral spinal dorsal horns of rats in SNI+Exendin 4 group were decreased,while the protein levels of Iba-1,IFN-γ,IL-6 in ipsilateral spinal dorsal horns of rats in SNI+Exendin 9-39 group were increased,the differences were statistically significant(all P<0.05);and the expressions of Iba-1,IFN-γand IL-6 in spinal dorsal horn were significantly different among the groups(F_(Iba-1)=60.25,F_(IFN-γ)=44.51,F_(IL-6)=62.52;all P<0.05).Conclusions Nerve injury induced the upregulation of GLP-1 receptor in the ipsilateral spinal dorsal horn of SNI rats.In addition,the inhibition of microglia and neuroinflammation might contribute to the analgesic effect of GLP-1 receptor.