南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (12): 2561-2572.doi: 10.12122/j.issn.1673-4254.2025.12.04
夏金枝1,5(
), 陈悦3,5, 任侣3,5, 李静1,2, 宋雪2,4, 陶露2,4, 胡建国1,2(
)
收稿日期:2025-05-16
出版日期:2025-12-20
发布日期:2025-12-22
通讯作者:
胡建国
E-mail:Jinzhixia0511@163.com;jghu9200@bbmu.edu.cn
作者简介:夏金枝,在读硕士研究生,E-mail: Jinzhixia0511@163.com
基金资助:
Jinzhi XIA1,5(
), Yue CHEN3,5, Lü REN3,5, Jing LI1,2, Xue SONG2,4, Lu TAO2,4, Jianguo HU1,2(
)
Received:2025-05-16
Online:2025-12-20
Published:2025-12-22
Contact:
Jianguo HU
E-mail:Jinzhixia0511@163.com;jghu9200@bbmu.edu.cn
Supported by:摘要:
目的 探究咖啡豆醇(Kah)在脊髓损伤后通过抑制小胶质细胞活化介导的炎症反应对运动功能恢复的影响,并阐明其作用机制。 方法 随机将54只8~10周龄的C57BL/6J小鼠分为Sham组(只进行椎板剥离), 脊髓损伤(SCI)组(剥离椎板并撞击脊髓),Kah组(术后每日腹腔注射20 mg/kg的Kah),18只/组。采用BMS评分、足迹分析、游泳实验评估小鼠的运动功能。通过HE、快蓝染色及尼氏染色评估脊髓损伤区域、髓鞘完整性和神经元存活。使用脂多糖刺激BV2细胞建立体外炎症模型及BV2/HT22神经元共培养体系,利用不同浓度Kah干预,通过免疫荧光对体内外活化的小胶质细胞及神经元凋亡的数量进行评估;通过Western blotting检测凋亡相关蛋白(Bax/Bcl-2/cleaved caspase3)及IκBα/NF-κB通路蛋白。通过qRT-PCR 、ELISA检测炎症因子(TNF-α、IL-6、IL-1β)水平。使用NF-κB通路激活剂PMA于体外实验进行干预,分析Kah的作用机制。 结果 Kah治疗改善了SCI小鼠的运动功能;组织学检测显示,Kah组脊髓组织病理性损伤区域较SCI组缩小(P<0.001),同时髓鞘化面积及神经元数目增加(P<0.001);体内外实验显示,Kah通过抑制小胶质细胞的活化并减少其释放炎症因子缓解神经元凋亡;Western blotting结果显示,Kah可降低NF-κB及IκBα的磷酸化水平。体外免疫荧光结果证实,与Kah干预组相比,给予PMA后,活化的BV2细胞及凋亡的神经元增加(P<0.05)。 结论 Kah通过抑制NF-κB通路调控小胶质细胞活化来促进SCI后运动功能的恢复,为临床治疗提供了潜在新策略。
夏金枝, 陈悦, 任侣, 李静, 宋雪, 陶露, 胡建国. 咖啡豆醇通过调控IκBα/NF-κB通路抑制小胶质细胞活化改善脊髓损伤后小鼠的运动功能[J]. 南方医科大学学报, 2025, 45(12): 2561-2572.
Jinzhi XIA, Yue CHEN, Lü REN, Jing LI, Xue SONG, Lu TAO, Jianguo HU. Kahweol improves motor function of mice with spinal cord injury by inhibiting microglial activation via regulating the IκBα/NF-κB pathway[J]. Journal of Southern Medical University, 2025, 45(12): 2561-2572.
| Gene (mice) | Forward primer | Reverse primer |
|---|---|---|
| GAPDH | TGGCCTTCCGTGTTCCTAC | GAGTTGCTGTTGAAGTCGCA |
| TNF-α | CAGGCGGTGCCTATGTCTC | CGATCACCCCGAAGTTCAGTAG |
| IL-6 | TCTATACCACTTCACAAGTCGGA | GAATTGCCATTGCACAACTCTTT |
| IL-1β | GAAATGCCACCTTTTGACAGTG | TGGATGCTCTCATCAGGACAG- |
表1 引物序列
Tab.1 Primer sequences (5' to 3') for RT-qPCR
| Gene (mice) | Forward primer | Reverse primer |
|---|---|---|
| GAPDH | TGGCCTTCCGTGTTCCTAC | GAGTTGCTGTTGAAGTCGCA |
| TNF-α | CAGGCGGTGCCTATGTCTC | CGATCACCCCGAAGTTCAGTAG |
| IL-6 | TCTATACCACTTCACAAGTCGGA | GAATTGCCATTGCACAACTCTTT |
| IL-1β | GAAATGCCACCTTTTGACAGTG | TGGATGCTCTCATCAGGACAG- |
图1 Kah可改善SCI小鼠的运动功能及SCI后脊髓组织的病理损伤
Fig.1 Kahweol (Kah) improves motor function and alleviates spinal cord tissue pathologies of SCI mice. A: BMS Score. B, C: Footprint score. D, E: Swimming experiments. F: Dorsal view of fresh spinal cords on days 7. G: HE staining. H: Quantitative analysis of the lesion areas in the 3 groups. I: LFB staining. J: Quantitative analysis of residual myelination in the 3 groups. K: Nissl staining. L: Quantitative analysis of the number of motor neurons in the 3 groups. n=5 in each group. *P<0.05, **P<0.01.
图2 Kah可减轻脊髓损伤后神经元的凋亡
Fig.2 Kah attenuates neuronal apoptosis in the spinal cord of SCI mice. A: Immunofluorescent staining for NeuN and cleaved caspase3. B: Quantification analysis of the number of NeuN+ cleaved caspase3+ cells. C-F: Western blotting and quantitative analysis of protein expression levels. n=3 in each group. *P<0.05, **P<0.01.
图3 Kah减轻了脊髓损伤后炎症相关因子的表达并抑制小胶质细胞的活化
Fig. 3 Kah reduces the expressions of inflammation-related factors and inhibits microglial activation in the spinal cord of SCI mice. A: Immunofluorescent staining for CD11b and CD68. B: Quantification analysis of the number of the CD11b+ CD68+ cells. C-E: Effect of Kah on mRNA levels of TNF‑α, IL-6 and IL-1β. F-H: Quantification of protein levels of TNF-α, IL-6 and IL-1β. n=3 in each group. **P<0.01.
图4 Kah对SCI的保护性作用可能与NF-κB信号通路有关
Fig. 4 The protective effect of Kah in SCI mice may be related to the NF-κB signaling pathway. A-C: Proteins expressions of NF-κB, p-NF-κB, IκBα and p-IκBα in the spinal cord of SCI mice detected by Western blotting and quantitative analysis of protein levels. n=3 in each group. *P<0.05.
图5 Kah抑制LPS诱导的BV2细胞活化和炎症因子的表达
Fig.5 Kah inhibits LPS-induced microglial activation and expressions of inflammatory cytokines. A: Immunofluorescent staining for CD11b and CD68. B: Quantification analysis of the number of the CD11b+ CD68+ cells. C-E: Effect of Kah on mRNA levels of TNF‑α, IL-6 and IL-1β. F-H: Quantification of protein levels of TNF-α, IL-6 and IL-1β. n=3 in each group. *P<0.05, **P<0.001.
图6 Kah减轻BV2细胞活化引起的神经元凋亡
Fig.6 Kah attenuates neuronal apoptosis caused by microglial activation. A: Immunofluorescent staining for NeuN and cleaved caspase-3. B: Quantification analysis of the number of the NeuN+/cleaved caspase-3+ cells. C-F: Western blotting analysis and quantitative analysis of protein expression levels. n=3 in each group. *P<0.05, **P<0.001.
图7 Kah抑制NF-κB信号通路
Fig.7 Kah inhibits the NF-kB signaling pathway. A-C: Proteins expressions of NF-κB, p-NF-κB, IκBα and p-IκBα in BV2 cells detected by Western blotting and quantitative analysis of protein levels. n=3 in each group. *P<0.05, **P<0.001.
图8 Kah通过抑制NF-κB通路减轻BV2细胞活化及神经元凋亡
Fig.8 Kah attenuates microglial activation and neuronal apoptosis by inhibiting the NF-κB pathway. A: Immunofluorescent staining for CD11b and CD68. B: Quantification analysis of the number of CD11b+CD68+ cells. C: Immunofluorescent staining for NeuN and cleaved caspase3. D: Quantification analysis of the number of the NeuN+ cleaved caspase3+ cells. E, F: Western blotting and quantitative analysis of protein expression levels. n=3 in each group. *P<0.05, **P<0.001.
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