甜叶悬钩子苷对脊髓损伤小鼠运动功能障碍和神经炎症的改善作用及其机制

doi:10.13481/j.1671-587X.20240205

摘要/Abstract

摘要：

目的探讨甜叶悬钩子苷（RUB）对小鼠脊髓损伤（SCI）和神经炎症的影响，并阐明其作用机制。方法将48只雌性昆明小鼠随机分为假手术组、SCI组、SCI+低剂量RUB组和SCI+高剂量RUB组，每组12只；采用脊髓损伤行为学（BBB）评分法评估SCI小鼠后肢运动功能，脊髓组织含水量法检测SCI小鼠脊髓水肿情况，实时荧光定量PCR（RT-qPCR）法检测各组小鼠促炎细胞因子环氧化酶2（COX-2）、白细胞介素1β（IL-1β）和肿瘤坏死因子α（TNF-α）mRNA表达水平，ELISA法检测各组小鼠血清中炎症因子水平，HE染色观察各组小鼠脊髓组织病理形态学，免疫荧光法检测各组小鼠脊髓组织中小胶质细胞活化情况，Western blotting法检测SCI小鼠脊髓组织中相关蛋白表达水平。结果 BBB评分，与假手术组比较，SCI组小鼠评分低至0分；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠BBB评分逐步升高。脊髓组织含水量法检测，与假手术组比较，SCI组小鼠脊髓组织含水量明显升高（P<0.01）；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠脊髓组织含水量明显降低（P<0.01）。RT-qPCR法检测，与假手术组比较，SCI组小鼠脊髓组织中COX-2、IL-1β和TNF-α mRNA表达水平明显升高（P<0.001）；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠脊髓组织中COX-2、IL-1β和TNF-α mRNA表达水平明显降低（P<0.001）。ELISA法检测，与假手术组比较，SCI组小鼠血清中IL-1β（P<0.01）和TNF-α（P<0.001）水平升高；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠血清中IL-1β和TNF-α水平降低（P<0.001）；Western blotting法检测，与假手术组比较，SCI组小鼠脊髓组织中核因子κB（NF-κB）抑制因子α（IκB-α）、磷酸化IκB-α（p-IκB-α）、磷酸化p65（p-p65）、p-65、磷酸化p38（p-p38）、磷酸化细胞外调节蛋白激酶（p-ERK）和磷酸化c-Jun氨基末端激酶（p-JNK）蛋白表达水平明显升高（P<0.05或P<0.001）；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠脊髓组织中IκB-α、p-IκB-α、p-p65、p-65、p-p38、p-ERK和p-JNK蛋白表达水平明显降低（P<0.001）。HE染色观察，与假手术组比较，SCI组小鼠脊髓组织中可见组织疏松，有空泡形成，脊髓中间有一较大的坏死空洞区域；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠脊髓中央坏死空洞区域明显减小（P<0.05）。免疫荧光法检测，与假手术组比较，SCI组小鼠脊髓组织中小胶质细胞阳性细胞数明显增加（P<0.001）；与SCI组比较，SCI+低剂量RUB组和SCI+高剂量RUB组小鼠脊髓组织中小胶质细胞阳性细胞数明显减少（P<0.001）。结论 RUB可改善SCI小鼠运动功能障碍，减轻脊髓组织神经炎症，抑制小胶质细胞活化，其机制可能与下调脊髓组织中NF-κB和丝裂原活化蛋白激酶（MAPK）信号通路相关蛋白表达有关。

关键词: 甜叶悬钩子苷, 脊髓损伤, 神经炎症, 核因子κB, 丝裂原活化蛋白激酶

Abstract:

Objective To discuss the effect of rubusoside （RUB） on the spinal cord injury （SCI） and neuroinflammation in the mice， and to clarify its mechanism. Methods A total of 48 female Kunming mice were randomly divided into sham operation group， SCI group， SCI+low dose of RUB group， and SCI+high dose of RUB group， and there were 12 mice in each group. Spinal cord injury behavior （BBB） scoring method was used to assess the motor function of the hind limbs of the SCI mice； water content method was used to detect the spinal cord edema of the SCI mice； real-time fluorscence quantitative PCR （RT-qPCR） method was used to detect the expression levels of pro-inflammatory cytokines cyclooxygenase-2 （COX-2）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） mRNA of the mice in various groups； ELISA method was used to detect the levels of inflammatory factors in serum of the mice in various groups； HE staining was used to observe the pathomorphology of spinal cord tissue of the mice in various groups；immunofluorescence was used to detect the activation of microglia in spinal cord tissue of the mice in various groups； Western blotting method was used to detect the expression levels of related proteins in spinal cord tissue of the mice in various groups. Results The BBB score analysis results showed that compared with sham operation group， the score of the mice in SCI group was decreased to 0； compared with SCI group， the BBB scores of the mice in SCI+low dose of RUB group and SCI+high dose of RUB group were gradually increased. The spinal cord tissue water content detection results showed that compared with sham operation group， the water content in spinal cord tissue of the mice in SCI group was significantly increased （P<0.01）； compared with SCI group， the water contents in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.01）. The RT-qPCR results showed that compared with sham operation group， the expression levels of COX-2， IL-1β， and TNF-α mRNA in spinal cord tissue of the mice in SCI group were significantly increased （P<0.001）. Compared with SCI group， the expression levels of COX-2，IL-1，and TNF-α mRNA in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. The ELISA results showed that compared with sham operation group，the levels of IL-1β （P<0.01） and TNF-α （P<0.001） in serum of the mice in SCI group were increased； compared with SCI group， the levels of IL-1β and TNF-α in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were decreased （P<0.001）.The Western blotting results showed that compared with sham operation group， the expression levels of nuclear factor kappa B （NF-κB） inhibitor protein-α （IκB-α）， phosphorylated IκB-α （p-IκB-α）， phosphorylated p65 （p-p65）， p65， phosphorylated p38 （p-p38）， phosphorylated extracellular regulated protein kinase （p-ERK）， and phosphorylated c-Jun N-terminal kinase （p-JNK） proteins in spinal cord tissue of the mice in SCI group were significantly increased （P<0.05 or P<0.001）； compared with SCI group， the expression levels of IκB-α， p-IκB-α， p-p65， p65， p-p38， p-ERK， and p-JNK proteins in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. The HE staining results showed that compared with sham operation group， the spinal cord tissue of the mice in SCI group had loose organization with the formation of vacuoles， and a large necrotic cavity was present in the center of the spinal cord； compared with SCI group， the central necrotic cavity area of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group was significantly decreased （P<0.05）.The immunofluorescence results showed that compared with sham operation group， the number of positive microglia cells in spinal cord tissue of the mice in SCI group was significantly increased （P<0.001）； compared with SCI group， the numbers of positive microglia cells in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased （P<0.001）. Conclusion Rubusoside can improve the motor function impairment in the SCI mice，mitigate the neuroinflammation in spinal cord tissue，and inhibit the activation of microglia. The mechanism may be related to the downregulation of expression of proteins associated with the NF-κB and mitogen-activated protein kinase （MAPK） signaling pathways in spinal cord tissue.

Key words: Rubusoside, Spinal cord injury, Neuroinflammation, Nuclear factor kappa-B, Mitogen-activated protein kinase

中图分类号:

R363.1

杨爽,许娜,张剑旭,孙成彪,王燕,董明鑫,刘文森. 甜叶悬钩子苷对脊髓损伤小鼠运动功能障碍和神经炎症的改善作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(2): 326-335.

Shuang YANG,Na XU,Jianxu ZHANG,Chengbiao SUN,Yan WANG,Mingxin DONG,Wensen LIU. Improvement effect of rubusoside on motor dysfunction and neuroinflammation in mice with spinal cord injury and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 326-335.

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Name	Primer sequence
IL-1β	5'-CTTCAGGCAGGCAGTATC-3'
IL-1β	5'-CAGCAGGTTATCATCATCATC-3'
TNF-α	5'-GTGGAACTGGCAGAAGAG-3'
TNF-α	5'-TAGACAGAAGAGCGTGGT-3'
COX-2	5'-GAACTGTAGCACAGCACAGGAAAT-3'
COX-2	5'-CGTACCGGATGAGCTGTGAAT-3'
β-actin	5'-AACAGTCCGCCTAGAAGCAC-3'
	5'-CGTTGACATTAAAGACC-3'

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