鞘内注射瞬时受体电位通道A1 shRNA对部分坐骨神经结扎小鼠神经病理性疼痛的作用及其机制

doi:10.13481/j.1671-587X.20210619

摘要/Abstract

摘要： 目的

探讨瞬时受体电位通道 A1（TRPA1）在部分坐骨神经结扎（pSNL）小鼠神经病理性疼痛模型中的作用，阐明其作用机制。

方法

30只SPF级雄性C57BL/6小鼠随机分为对照组（n=6）、假手术组（n=6）和pSNL组（n=18）。pSNL组小鼠鞘内置管成功后随机分为pSNL组、pSNL+NC shRNA组（于术后第 7 天鞘内注射NC shRNA）和pSNL+TRPA1 shRNA组（于术后第7天鞘内注射TRPA1 shRNA）。检测注射前和注射后l、7、12和24 h各组小鼠后肢机械缩足反射阈值（MWT）和热阈值（TWL）。最后一次检测后2 h处死小鼠，采用Western blotting法检测各组小鼠术侧背根神经节（DRG）中TRPA1蛋白激酶Cε型（Prkce）和星形胶质细胞激活标记物胶质纤维酸性蛋白（GFAP）的表达， ELISA法检测各组小鼠细胞上清和血清中肿瘤坏死因子α（TNF-α）和单核细胞趋化蛋白1（MCP-1）水平。分离培养pSNL模型小鼠的原代星形胶质细胞，过表达或敲低TRPA1，检测星形胶质细胞中TRPA1、Prkce和GFAP蛋白表达水平以及细胞上清中TNF-α和 MCP-1水平。采用STRING数据库和免疫共沉淀（Co-IP）法预测并验证TRPA1和Prkce相互作用。检测过表达或敲低TRPA1后空质粒组、Ad-TRPA1、sh-NC组和sh-TRPA1组星形胶质细胞中Prkce和GFAP蛋白表达水平以及细胞上清中TNF-α和 MCP-1水平。将TRPA1 shRNA单独转染或与Ad-Prkce共转染星形胶质细胞，分为sh-TRPA1+empty vector组（转染空载体）、sh-TRPA1+Ad-Prkce组（转染TRPA1过表达载体）和sh-TRPA1+Ad-Prkce组（转染Prkce过表达载体）。采用Western blotting法检测各组细胞中TRPA1、Prkce和GFAP蛋白表达水平， ELISA法检测细胞上清中TNF-α和MCP-1水平。

结果

与对照组比较，pSNL组小鼠MWT和TWL降低（P<0.01）；与pSNL+NC中shRNA组比较，pSNL+TRPA1 shRNA组小鼠MWT和TWL升高（P<0.01）。与假手术组比较，术后第7天pSNL组小鼠DRG中TRPA1和GFAP蛋白表达水平升高（P<0.05），血清中TNF-α和MCP-1水平升高（P<0.05）。与pSNL+NC shRNA组比较， pSNL+TRPA1 shRNA组小鼠DRG中TRPA1和GFAP蛋白表达水平及血清中TNF-α和MCP-1水平降低（P<0.05）。与空质粒组比较，Ad-TRPA1组细胞中GFAP蛋白表达水平明显升高（P<0.01），细胞上清中TNF-α和MCP-1水平明显升高（P<0.05）；与sh-NC组比较，sh-TRPA1组细胞中GFAP蛋白表达水平明显降低（P<0.05），细胞上清中TNF-α和MCP-1水平明显降低（P < 0.05）。与sh-TRPA1+empty vector组比较，sh-TRPA1+Ad-Prkce组细胞中TRPA1、Prkce和GFAP蛋白表达水平升高（P<0.01），细胞上清中TNF-α和MCP-1水平明显升高（P<0.05）。

结论

TRPA1通过与Prkce相互作用参与pSNL模型小鼠星形胶质细胞的激活以及神经病理性疼痛发生发展过程。

关键词: 瞬时受体电位通道 A1, 蛋白激酶Cε型, 部分坐骨神经结扎, 神经性疼痛, 星形胶质细胞

Abstract:

Objective： To investigate the role of transient receptor potential cation channel subfamily A member 1 （TRPA1） in the neuropathic pain models induced by partial sciatic nerve ligation （pSNL） in the mice， and to charify its mechanism.

Methods

Thirty SPF male C57BL/6 mice were randonly divided into control group （n=6）， sham operation group （n=6）， and pSNL group （n=18）. The mice in pSNL group were randomly divided into pSNL group （n=6）， pSNL+NC shRNA group （the NC shRNA was injected intrathecally on the 7th day， n=6）， and pSNL+ TRPA1 shRNA group （the TRPA1 shRNA was injected intrathecally on the 7th day， n=6） after successful intrathecal cathetering. The mechanical withdrawal threshold （MWT） and the thermal withdrawal latency （TWL） of hind limbs of the mice were measured before and 1， 7， 12， and 24 h after injection. The mice were sacrificed 2 h after the last test. The expression levels of TRPA1 protein kinase C epsilon type（Prkce），and the astrocyte activation marker glial fibrillary acidic protein （GFAP） in dorsal root ganglion （DRG） of the mice in various groups were detected by Western blotting method， and the levels of tumor necrosis factor-α（TNF-α） and monocyte chemoattractant protein-1 （MCP-1） in serum and cell superatant were detected by ELISA method. The primary astrocytes of pSNL model mice were isolated and cultured. TRPA1 was over-expressed or knocked down. The expression levels of TRPA1， Prkce， and GFAP proteins in the astrocytes and the levels of TNF-α and MCP-1 in cell supernatant were detected.The interaction between TRPA1 and Prkce was predicted and verified by STRING database and immunoprecipitation （Co-IP） method.After over-expression or knockdown of TRPA1， the expression levels of Prkce and GFAP proteins in the astrocytes in empty plasmid group， Ad-TRPA1 group， sh-NC group and sh-TRPA1 group and the levels of TNF-α and MCP-1 in the cell supernatant were detected. The astrocytes were transfected with TRPA1 shRNA alone or co-transfected with Ad-Prkce and divided into sh-TRPA1+empty vector group （transfected with empty vector）， sh-TRPA1+Ad-Prkce group （transfected with TRPA1 over-expression vector） and sh-TRPA1+Ad-Prkce group （transfected with Prkce over-expression vector）. The expression levels of TRPA1， Prkce，and GFAP proteins in the cells in various groups were detected by Western blotting method， and the levels of TNF- α and MCP-1 in the cell supernatant in various groups were detected by ELISA method.

Results

Compared with control group， the MWT and TWL of the mice in pSNL group were decreased （P<0.01）；compared with pSNL+NC shRNA group， the MWT and TWL of the mice in pSNL+TRPA1 shRNA group were increased （P<0.01）. Compared with sham operation group， the expression levels of TRPA1 and GFAP proteins in DRG and the levels of TNF-α and MCP-1 in serum of the mice in pSNL group were increased 7 d after operation （P<0.05）. Compared with pSNL+NC shRNA group， the expression levels of TRPA1 and GFAP proteins in DRG and the levels TNF- α and MCP-1 in serum of the mice in pSNL+TRPA1 shRNA group were decreased （P<0.05）.Compared with empty plasmid group， the expression level of GFAP protein in the cells in Ad-TRPA1 group was significantly increased （P<0.01）， and the levels of TNF-α and MCP-1 in the cell supernatant were increased significantly （P<0.05）； compared with sh-NC group， the expression level of GFAP protein in the cells in sh-TRPA1 group was decreased significantly （P<0.05），and the levels of TNF-α and MCP-1 in the cell supernatant were decreased significantly （P<0.05）. Compared with sh-TRPA1+empty vector group， the expression levels of TRPA1， Prkce，and GFAP proteins in the cells in sh-TRPA1+Ad-Prkce group were increased （P<0.01），and the levels of TNF-α and MCP-1 in the cell supernatant were increased significantly （P<0.05）.

Conclusion

TRPA1 participates in the activation of astrocytes and the production and maintenance of neuropathic pain in the pSNL model mice through the interaction with Prkce.

Key words: transient receptor potential cation channel subfamily A member 1, protein kinase C epsilon type, partial sciatic nerve ligation, neuropathic pain, astrocytes

中图分类号:

R741

赵峰,樊少卿,程晓燕,李小娜,李长生,马浩杰. 鞘内注射瞬时受体电位通道A1 shRNA对部分坐骨神经结扎小鼠神经病理性疼痛的作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(6): 1485-1494.

Feng ZHAO,Shaoqing FAN,Xiaoyan CHENG,Xiaona LI,Changsheng LI,Haojie MA. Effect of intrathecal injection of transient receptor potential cation channel subfamily A member 1 shRNA on neuropathic pain in mice with partial sciatic nerve ligation and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(6): 1485-1494.

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Group	MWT
Group	Before injection	1 h after injection	7 h after injection	12 h after injection	24 h after injection
Control	11.51±0.80	11.70±0.21	11.06±0.45	10.81±0.35	11.62±0.34
Sham operation	11.20±0.68	11.82±0.25	10.80±0.31	11.22±0.43	11.31±0.14
pSNL	5.62±0.33^*	5.53±0.25^*	5.72±0.13^*	5.51±0.32^**	4.84±0.69^*
pSNL+NC shRNA	5.82±0.42^*	5.73±0.33^*	5.52±0.43^*	5.81±0.63^**	4.55±0.38^*
pSNL+TRPA1 shRNA	5.53±0.28^*	9.31±0.13^*△#	10.22±0.42^△#	10.73±0.48^△#	10.90±0.13^△#

Group	TWL
Group	Before injection	1 h after injection	7 h after injection	12 h after injection	24 h after injection
Control	18.52±0.90	17.71±0.19	17.20±0.25	16.83±0.51	17.64±0.43
Sham operation	18.20±0.76	17.81±0.65	17.80±0.11	17.22±0.46	18.03±0.12
pSNL	11.04±0.22^*	10.91±0.12^*	10.33±0.35^*	10.52±0.65^*	8.73±0.52^*
pSNL+NC shRNA	10.81±0.32^*	10.74±0.31^*	10.40±0.53^*	10.31±0.53^*	8.53±0.34^*
pSNL+TRPA1 shRNA	10.50±0.18^*	16.33±0.23^*	16.52±0.51^*△#	16.81±0.55^△#	17.63±0.63^△#

Group	TNF-α	MCP-1
Sham operation	65.50±4.63	49.35±2.68
pSNL	239.54±8.35^*	156.18±4.38^*
pSNL+NC shRNA	248.49±3.28	152.47±7.33
pSNL+TRPA1 shRNA	85.56±4.38^△	43.64±6.23^△

Group	TNF-α	MCP-1
Empty vector	77.34±4.38	43.59±4.82
Ad-TRPA1	210.42±5.36^*	148.26±7.85^*
sh-NC	75.23±8.37	53.65±2.34
sh-TRPA1	28.53±4.39^△	13.48±2.33^△

Group	TNF-α	MCP-1
Empty vector	80.21±3.46	45.91±6.29
Ad-Prkce	240.25±5.62^*	179.65±8.53^*
sh-NC	75.23±8.39	42.78±6.47
sh-Prkce	25.38±4.92^△	12.81±5.38^△