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中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (04) : 193 -203. doi: 10.3877/cma.j.issn.2095-1221.2024.04.001

论著

姜黄素通过调节NF-κB/NLRP3通路减轻LPS诱导小胶质细胞神经炎症损伤
季加翠1, 孙春斌2, 罗恩丽3,†()   
  1. 1. 250014　济南，山东省精神卫生中心
    2. 100083，北京科技大学化学与生物工程学院
    3. 518116　深圳，广东深圳大学附属华南医院中医二科
  • 收稿日期:2024-02-07 出版日期:2024-08-01
  • 通信作者: 罗恩丽
  • 基金资助:
    国家自然科学基金(82374567); 广东省自然基金面上项目(2023A1515012779); 深圳市科技创新委员会基础研究面上项目(JCYJ20210324093213035)

Curcumin alleviates LPS-induced neuroinflammatory damage of microglia by regulating the NF-κB/NLRP3 pathway

Jiacui Ji1, Chunbin Sun2, Enli Luo3,†()   

  1. 1. Shandong Mental Health Center, Jinan 250014, China
    2. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China
    3. Second Department of Traditional Chinese Medicine, South China Hospital, Shenzhen University, Shenzhen 518116, China
  • Received:2024-02-07 Published:2024-08-01
  • Corresponding author: Enli Luo
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引用本文：

季加翠, 孙春斌, 罗恩丽. 姜黄素通过调节NF-κB/NLRP3通路减轻LPS诱导小胶质细胞神经炎症损伤[J]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 193-203.

Jiacui Ji, Chunbin Sun, Enli Luo. Curcumin alleviates LPS-induced neuroinflammatory damage of microglia by regulating the NF-κB/NLRP3 pathway[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(04): 193-203.

目的

研究姜黄素对脂多糖（LPS）诱导的神经细胞炎症的抑制作用，并探讨姜黄素抑制炎症的分子机制。

方法

以小胶质细胞（BV2）为研究对象，对照不做任何干预，模型组给予100 ng/mL LPS干预24 h构建神经炎症细胞模型，1、5、10 μmol/L姜黄素+ 100 ng/mL LPS干预24 h，应用细胞免疫荧光法检测BV2细胞内白细胞介素6 （IL-6）、IL-10、p-NF-κB、NLRP3、Bax和Bcl-2蛋白表达水平；应用Western blot检测NF-κB/NLRP3信号通路蛋白表达水平；采用ELISA法检测各组细胞上清肿瘤坏死因子α （TNF-α）、IL-1β和iNOS含量。给予NF-κB抑制剂BAY 11-7082后，检测BV2细胞内IL-6、IL-10、Bax和Bcl-2蛋白的表达水平。实验数据采用t检验、单因素方差分析和Dunnett-t检验方法分析。

结果

与对照比较，模型组炎症因子TNF-α（398.80 ± 11.39比113.00 ± 5.93）、IL-1β （592.40 ± 11.32比206.70 ± 6.81）、IL-6 （1.73 ± 0.09比0.99 ± 0.03）、iNOS（88.39 ± 2.14比32.48 ± 1.71）、p-NF-κB （1.92 ± 0.02比1.02 ± 0.05）、NLRP3蛋白表达水平（10.33 ± 0.61比1.02 ± 0.05）和凋亡相关蛋白Bax表达水平（2.68 ± 0.10比1.05 ± 0.08）升高，IL-10蛋白表达水平（0.82 ± 0.03比1.01 ± 0.06）和Bcl-2蛋白表达水平（0.70 ± 0.02比1.11 ± 0.06）降低（P均< 0.05）。Western blot结果表明，与对照比较，模型组p-NF-κB （2.65 ± 0.06比1.04 ± 0.05）和NLRP3蛋白表达水平（4.380 ± 0.08比1.07 ± 0.04）升高（P < 0.001）。与模型组相比，高浓度姜黄素处理后TNF-α （207.90 ± 7.37比398.80 ± 11.39）、IL-1β （405.40 ± 6.56比592.40 ± 11.32）、IL-6 （1.09 ± 0.05比1.73 ± 0.09）、iNOS （39.52 ± 1.32比88.39 ± 2.14）、p-NF-κB （01.21 ± 0.05比1.92 ± 0.02）和NLRP3蛋白表达水平（1.51 ± 0.11比10.33 ± 0.61）降低，IL-10蛋白表达水平（2.61 ± 0.03比0.82 ± 0.03）升高；与模型组相比，高浓度姜黄素处理后Bax蛋白表达水平（0.80 ± 0.04比2.68 ± 0.10）降低，Bcl-2蛋白表达水平（1.93 ± 0.02比0.70 ± 0.02）升高（P < 0.001）。与模型组相比，NF-κB抑制剂作用后，IL-6蛋白表达水平（0.88 ± 0.06比2.70 ± 0.12）和Bax蛋白表达水平（1.14 ± 0.04比3.63 ± 0.16）降低，IL-10蛋白表达水平（1.58 ± 0.03比0.52 ± 0.04）和Bcl-2蛋白表达水平（1.48 ± 0.11比0.53 ± 0.05）升高（P均< 0.001）。Western blot结果表明，与模型组相比，1、5、10 μmol/L姜黄素处理后p-NF-κB和NLRP3蛋白表达水平降低，且呈浓度依赖性（0.40 ± 0.02比2.65 ± 0.06、0.21 ± 0.02比4.38 ± 0.08，P < 0.001）。

结论

姜黄素可降低LPS诱导的小胶质细胞神经炎症损伤，其机制可能与下调NF-κB/NLRP3信号通路，抑制炎症和细胞凋亡有关。

关键词: 姜黄素, NF-κB/ NLRP3信号通路, 炎症损伤, 细胞凋亡
Objective

To investigatethe inhibitory effect and mechanisms of curcumin on lipopolysaccharide (LPS) -induced inflammation of nerve cells.

Methods

Microglia (BV2) was used in current study and named control group (without any treatment), model group (treated with LPS 100 ng/mL for 24 h) and treatment group (curcumin at 1, 5, 10 μmol/L concentration plus 100 ng/ml LPS for 24 h). The expression levels of interleukin-6 (IL-6), IL-10, p-NF-κB, NLRP3, Bax and Bcl-2 in BV2 cells were detected by immunofluorescence assay. Western blot was used to detect the protein expression of NF-κB/NLRP3 signaling pathway. The contents of tumor necrosis factor α (TNF-α), IL-1β and iNOS were detected by ELISA. In addition, the expression levels of IL-6, IL-10, Bax and Bcl-2 proteins in BV2 cells were detected after administration of NF-κB inhibitor BAY 11-7082. The experimental data were analyzed by t test, one-way ANOVA and Dunnett-t test.

Results

Compared with the control group, the inflammatory cytokines TNF-α (398.80 ± 11.39 vs 113.00 ± 5.93), IL-1β (592.40 ± 11.32 vs 206.70 ± 6.81), IL-6 (1.73 ± 0.09 vs 0.99 ± 0.03), iNOS (88.39 ± 2.14 vs 32.48 ± 1.71), p-NF-κB (1.92 ± 0.02 vs 1.02 ± 0.05), INOS (88.39 ± 2.14 vs 32.48 ± 1.71), NLRP3 (10.33 ± 0.61 vs 1.02 ± 0.05) and apoptosis-related protein Bax (2.68 ± 0.10 vs 1.05 ± 0.08) in the model group were increased. IL-10 (0.82 ± 0.03 vs 1.01 ± 0.06) and Bcl-2 protein expression levels (0.70 ± 0.02 vs 1.11 ± 0.06) were decreased (P < 0.05). Western blot results showed that compared with the control group, the protein expression levels of p-NF-κB (2.65 ± 0.06 vs 1.04 ± 0.05) and NLRP3 (4.38 ± 0.08 vs 1.07 ± 0.04) in the model group were increased (P < 0.001). Compared to the model group, the expression levels of TNF-α (207.90 ± 7.37 vs 398.80 ± 11.39), IL-1β (405.40 ± 6.56 vs 592.40 ± 11.32), IL-6 (1.09 ± 0.05 vs 1.73 ± 0.09), iNOS (39.52 ± 1.32 vs 88.39 ± 2.14), p-NF-κB (01.21 ± 0.05 vs 1.92 ± 0.02) and NLRP3 (1.51 ± 0.11 vs 10.33 ± 0.61) were decreased, and the expression levels of IL-10 (2.61 ± 0.03 vs 0.82 ± 0.03) were increased after treatment with high concentration of curcumin. Compared with the model group, the expression level of Bax protein was decreased (0.80 ± 0.04 vs 2.68 ± 0.10), while the expression level of Bcl-2 protein was increased after high concentration curcumin treatment (1.93 ± 0.02 vs 0.70 ± 0.02) (P < 0.001). Compared with the model group, the expression levels of IL-6 (0.88 ± 0.06 vs 2.70 ± 0.12) and Bax protein (1.14 ± 0.04 vs 3.63 ± 0.16) were decreased, while the expression levels of IL-10 (1.58 ± 0.03 vs 0.52 ± 0.04) and Bcl-2 protein (1.48 ± 0.11 vs 0.53 ± 0.05) were increased after NF-κB inhibitor treatment (P < 0.001). Western blot results showed that compared with the model group, the expression levels of p-NF-κB and NLRP3 protein were decreased after curcumin treatment with 1, 5 and 10 μmol/L concentration in dose-dependent manner (0.40 ± 0.02 vs 2.65 ± 0.06, 0.21 ± 0.02 vs 4.38 ± 0.08, P < 0.001) .

Conclusion

Curcumin could reduce LPS-induced neuroinflammatory damage in microglia, and its mechanism may be related to down-regulating NF-κB/NLRP3 signaling pathway, inhibiting inflammation and apoptosis.

Key words: Curcumin, NF-κB/NLRP3 signaling pathway, Inflammatory damage, Apoptosis
图1 不同浓度的姜黄素对BV2小胶质细胞增殖的影响注：与0 μmol/L比较，aP < 0.01，bP < 0.001；ns表示差异无统计学意义
图2 IL-6和IL-10荧光定量分析与模型比较，aP < 0.001，bP < 0.05
图3 荧光显微镜观察BV2细胞IL-6和IL-10蛋白免疫荧光结果（×20）
图4 ELISA检测BV2小胶质细胞上清中TNF-α、IL-1β和iNOS的含量注：a ~ c分别TNF-α、IL-1β和iNOS的含量；与模型比较，aP < 0.001
图5 p-NF-κB和NLRP3蛋白荧光定量分析注：与模型比较，aP < 0.001
图6 荧光显微镜观察BV2细胞p-NF-κB和NLRP3蛋白免疫荧光结果（×20）
图7 BV2细胞p-NF-κB和NLRP3蛋白表达注：a图为BV2细胞p-NF-κB和NLP3蛋白表达水平；b、c图分别为p-NF-κB和NLRP3蛋白相对表达水平；与模型比较，aP < 0.001
图8 荧光显微镜观察BV2细胞Bax和Bcl-2蛋白免疫荧光结果（×20）
图9 Bax和Bcl-2蛋白荧光定量分析注：与模型比较，aP < 0.001，bP < 0.01
图10 IL-10和IL-6蛋白荧光定量分析注：与模型比较，aP < 0.001，bP < 0.01
图11 荧光显微镜观察BV2细胞IL-10和IL-6蛋白免疫荧光结果（×20）
图12 荧光显微镜观察BV2细胞Bax和Bcl-2蛋白免疫荧光结果（×20）
图13 Bax和Bcl-2蛋白荧光定量分析注：与模型比较，aP < 0.001，bP < 0.01，c P < 0.05
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