紫花前胡苷缓解神经病理性疼痛的机制探讨

doi:10.11958/20240971

摘要/Abstract

摘要：

目的探究紫花前胡苷（Nod）在神经病理性疼痛（NP）中的作用及机制。方法筛选并分析NP数据初级躯体感觉皮层（S1）内差异性表达基因及数据集与线粒体数据之间的重叠基因，重叠基因相互作用网络并筛选核心基因。27只小鼠随机分为假手术组、模型组及给药组（9只/组）；模型组及给药组构建坐骨神经慢性压迫损伤模型，给药组腹腔注射Nod 10 mg/kg连续1周；检测小鼠痛觉行为学和运动能力的改变；HE染色、尼氏染色检测小鼠S1区脑组织神经损伤和炎症的影响；蛋白免疫印迹分析S1脑区白细胞介素（IL）-1β、即早基因（c-Fos）、泛醇-细胞色素C还原酶复合体Ⅲ亚基（Uqcrq）和泛醌氧化还原酶亚基（Ndufb5）的表达水平；分子对接探究Nod的作用靶点。将PC12细胞分为对照组、IL-1β组（1 μmol/L IL-1β处理）和IL-1β+Nod组（1 μmol/L IL-1β+1 μmol/L Nod处理），检测各组线粒体膜电位。结果 NP数据集GSE180627中S1脑区包含293个差异性表达基因，线粒体数据包含1 082个基因，重叠基因共34个，氧化磷酸化和电子传递链相关基因被富集，蛋白相互作用网络显示核心基因包括电子传递链相关蛋白Ndufb5、Uqcrq、Ndufs8、Ndufa7、Ndufa3、Cox6b1和Mrps33。与模型组相比，给药组小鼠机械缩足阈值、热缩足反射潜伏期、转棒停留时间增加，S1组织炎症浸润细胞数量和神经元中尼氏小体数量减少，神经元c-Fos和IL-1β表达水平降低，Uqcrq和Ndufb5的表达水平升高（P<0.05）。分子对接显示Nod可结合Uqcrq和Ndufb5。与IL-1β组细胞相比，IL-1β+Nod组细胞线粒体膜电位荧光信号增强（P<0.05）。结论 Nod可改善小鼠的痛觉行为，其机制涉及改善S1内线粒体损伤。

关键词: 前胡苷, 线粒体, 炎症, 神经病理性疼痛, 初级躯体感觉皮层

Abstract:

Objective To investigate the effect and mechanism of nodakenin (Nod) in neuropathic pain (NP).Methods Differential expression genes in the primary somatsensory cortex (S1) of NP data and overlapping genes between the dataset and mitochondrial data were screened and analyzed. Overlapping gene interaction networks were overlapped and core genes were screened. A total of 27 mice were randomly divided into the sham operation group, the model group and the drug administration group (9 mice/group). The chronic compression injury model of sciatic nerve was constructed in the model group and the drug administration group. Nod 10 mg/kg was intraperitoneally injected into the drug administration group for 1 week. Changes of pain behavior and motor ability in mice were detected. HE staining and Nissl staining were used to detect effects of nerve injury and inflammation on brain tissue of S1 region of mice. The expression levels of interleukin-1β, early gene (c-Fos), panthenol-cytochrome c reductase complex III subunit (Uqcrq) and ubiquinone oxidoreductase subunit (Nduf) b5 in S1 brain region were analyzed by Western blot assay. Molecular docking was used to study the target of Nod. PC12 cells were divided into the control group, the IL-1β group (1 μmol/L IL-1β treatment) and the IL-1β+Nod group (1 μmol/L IL-1β+1 μmol/L Nod treatment), and mitochondrial membrane potential was detected in each group.Results In the NP dataset GSE180627, S1 brain region contained 293 differentially expressed genes, and the mitochondrial data contained 1 082 genes. There were 34 overlapping genes, and genes related to oxidative phosphorylation and electron transport chain were enriched. The protein interaction network showed that core genes included electron transport chain related proteins Ndufb5, Uqcrq, Ndufs8, Ndufa7, Ndufa3, Cox6b1 and Mrps33. Compared with the model group, the mechanical foot shrinkage threshold, thermal foot shrinkage reflex latency and rod rotation residence time of mice were increased in the drug administration group, the number of inflammatory infiltrating cells in S1 tissue and the number of Nislet bodies in neurons, expression levels of c-Fos and IL-1β in neurons were decreased, and expression levels of Uqcrq and Ndufb5 were increased (P<0.05). Molecular docking showed that Nod could bind Uqcrq and Ndufb5. Compared with the IL-1β group, the fluorescence signal of mitochondrial membrane potential was enhanced in the IL-1β+Nod group (P<0.05).Conclusion Nodakenin can improve pain behavior in mice, and its mechanism involves ameliorating mitochondrial damage in S1.

Key words: Nodakenin, mitochondria, inflammation, neuropathic pain, primary somatosensory cortex

中图分类号:

R965

图/表 10

图1 NP及线粒体差异表达基因分析 A：S1在NP中差异表达基因的火山图谱；B：NP与线粒体基因的韦恩图；C、D：重叠基因GO和KEGG富集分析；E：重叠基因的PPI作用网络。

Fig.1 Analysis of NP and mitochondrial differential expression genes

表1 各组小鼠行为学测试比较

Tab.1 Comparison of behavioral tests between three groups of mice （n=9，$\bar{x}$±s）

组别	PWT/g	TWL/s	转棒停留时间/s
假手术组	1.00±0.32	15.40±3.01	526.67±62.11
模型组	0.33±1.77^a	5.89±1.95^a	129.14±48.84^a
给药组	0.57±0.17^ab	10.89±1.69^ab	277.73±81.23^ab
F	19.550^＊＊	38.790^＊＊	84.826^＊＊

图2 各组小鼠S1皮层的HE染色

Fig.2 HE staining of S1 cortex of mice in each group

图3 各组小鼠S1皮层的尼氏染色

Fig.3 Nissl staining of S1 cortex of mice in each group

表2 Comparison of the number of inflammatory cells and Nissellite bodies between the three groups （n=3，个/104 μm2，$\bar{x}$±s）

Tab.2

组别	炎症细胞	尼氏小体
假手术组	6.66±0.81	8.83±1.83
模型组	14.16±1.94^a	16.00±1.78^a
给药组	9.00±0.89^ab	10.50±1.37^b
F	50.670^＊＊	29.902^＊＊

图4 各组IL-1β和c-Fos的蛋白表达印迹图

Fig.4 Protein expression of proinflammation cytokines IL-1β and c-Fos in each group

表3 各组小鼠炎性因子IL-1β和c-Fos蛋白表达水平比较

Tab.3 Comparison of proinflammation cytokines IL-1β and c-Fos protein expression between the three groups （n=3，$\bar{x}$±s）

组别	c-Fos	IL-1β
假手术组	1.00±0.08	1.00±0.06
模型组	1.32±0.07^a	2.07±0.06^a
给药组	1.09±0.07^ab	1.81±0.05^ab
F	13.113^＊＊	195.269^＊＊

图5 分子对接结果示意图和各组Uqcrq和Ndufb5的蛋白表达免疫印迹图 A：Nod与Ndufb5分子对接示意图；B：Nod与Uqcrq分子对接示意图；C：线粒体相关蛋白的免疫印迹图。

Fig.5 Schematic diagram of molecular docking results and Western blot assay of Uqcrq and Ndufb5 protein in each group

表4 各组线粒体相关蛋白Ndufb5和Uqcrq表达水平比较

Tab.4 Comparison of proinflammation cytokines Ndufb5 and Uqcrq protein expression between the three groups （n=3，$\bar{x}±s$）

组别	Ndufb5	Uqcrq
假手术组	1.00±0.05	1.00±0.04
模型组	0.68±0.05^a	0.89±0.05^a
给药组	1.14±0.05^ab	1.08±0.04^b
F	73.705^＊＊	12.909^＊＊

图6 各组细胞线粒体膜电位荧光信号强度（×400）

Fig.6 Fluorescence intensity of each group of cells (×400)

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