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十二烷酰肉碱和肉豆蔻酸对小鼠肺泡上皮细胞系MLE-12细胞功能的影响
马圆, 张婷, 姜志龙, 高佳萌, 钱宇豪, 陈智鸿
复旦学报(医学版) ›› 2025, Vol. 52 ›› Issue (03) : 333-342.
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十二烷酰肉碱和肉豆蔻酸对小鼠肺泡上皮细胞系MLE-12细胞功能的影响
Effect of dodecanoylcarnitine and myristoleic acid on the cellular function of mouse alveolar epithelial cell line of MLE-12
目的: 探究十二烷酰肉碱（dodecanoylcarnitine，DA）和肉豆蔻酸（myristoleic acid，MA）对小鼠肺泡上皮系MLE-12细胞功能的影响及其作用机制。方法: 通过IL-4刺激MLE-12细胞构建炎症模型，采用ELISA检测细胞上清液中DA、MA以及鞘氨醇-1-磷酸（sphingosine-1-phosphate，S1P）表达水平；用DA和MA分别干预MLE-12细胞，RT-PCR、流式细胞术检测炎症因子IL-6和肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）表达变化及细胞内活性氧（reactive oxygen species，ROS）水平，进一步通过Western blot检测p-38 MAPK蛋白激酶（p38 mitogen activated protein kinase，p-38 MAPK）和src同源结构域2的酪氨酸磷酸酶1（src homology 2 domain containing phosphatase 1，SHP-1）等关键蛋白表达情况；使用S1PR2的抑制剂JTE-013预处理MLE-12细胞后重复上述实验，系统评估S1PR2信号通路在DA/MA介导的炎症反应及氧化应激调控中的分子机制。结果: IL-4刺激显著上调MLE-12细胞中DA、MA及S1P水平（P<0.05）。DA/MA干预组炎症因子IL-6、TNF-α表达量较对照组明显升高（P<0.05），同时ROS水平较对照组也有所增加（P<0.05）。Western blot结果显示DA/MA促进SHP-1去磷酸化及p-p38 MAPK活化，而JTE-013预处理可完全逆转上述效应（P<0.05）。结论: 哮喘相关代谢物DA和MA通过激活S1PR2受体，促进SHP-1去磷酸化和p-p38 MAPK通路活化，加剧MLE-12细胞的炎症及氧化应激反应，本研究揭示了S1PR2在该通路中的核心调控作用。
Objective: To explore the effects of dodecanoylcarnitine (DA) and myristoleic acid (MA) on the function of mouse alveolar epithelial cell line MLE-12 and their underlying mechanisms. Methods: An inflammatory model was established by stimulating MLE-12 cells with IL-4. The expression levels of DA, MA, and sphingosine-1-phosphate (S1P) in the cell supernatant were detected by ELISA. MLE-12 cells were separately intervened with DA and MA. RT-PCR and flow cytometry were used to detect the expression changes of inflammatory factors IL-6 and tumor necrosis factor-α (TNF-α) and the level of intracellular reactive oxygen species (ROS). Additionally, Western blot was performed to detect the expression of key proteins such as p38 mitogen-activated protein kinase (p-38 MAPK) and src homology 2 domain-containing phosphatase 1 (SHP-1). To explore the role of S1PR2 in the effects of DA and MA, MLE-12 cells were pretreated with the S1PR2 inhibitor JTE-013, and the above experiments were repeated. Results: IL-4 stimulation significantly upregulated the levels of DA, MA, and S1P in MLE-12 cells (P<0.05). DA/MA treatment groups exhibited significantly increased expression of IL-6 and TNF-α compared with the control group (P<0.05), along with elevated ROS levels (P<0.05). Western blot analysis revealed that DA/MA promoted SHP-1 dephosphorylation and phosphorylated p38 MAPK activation in MLE-12 cells. Notably, JTE-013 pre-treatment completely reversed these effects (P<0.05). Conclusion: Asthma-related metabolites DA and MA exacerbate the inflammatory and oxidative stress responses of MLE-12 cells by activating the S1PR2 receptor, promoting the dephosphorylation of SHP-1 and the activation of the p-p38 MAPK pathway. This study reveals the core regulatory role of S1PR2 in this pathway as well.
哮喘 / 代谢 / 十二烷酰肉碱(DA) / 肉豆蔻酸(MA) / JTE-013 / 小鼠 {{custom_keyword}} /
asthma / metabolism / dodecanoylcarnitine (DA) / myristoleic acid (MA) / JTE-013 / mouse {{custom_keyword}} /
表 1 qRT-PCR引物序列Tab 1 qRT-PCR primers for analysis |
| Gene | Primers（5’-3’） |
| IL-6 | Forward：CACAAGTCCGGGAGGAGAC |
| Reverse：TCCACGATTTCCCAGAGAAC | |
| TNF-α | Forward：CCAAATGGCCTCCCTCTCAT |
| Reverse：GGTGGTTTGCTACGACGTGG | |
| S1PR2 | Forward：CATCTTACTGGCTATCGTGGCTCTG |
| Reverse：GCGTCTGAGGACCAGCAACATC | |
| GAPDH | Forward：GGCAAATTCCATGGCACCGT |
| Reverse：GCATCGCCCCACTTGATTTT |
图 1 构建IL-4诱导的MLE-12细胞炎症模型Fig 1 Generation of of IL-4-induced MLE-12 cell inflammation model A and B: The expression of IL-6 in MLE-12 cells between IL-4 and control group was measured by flow cytometry and quantified; C and D: The expression of TNF-α in MLE-12 cells between IL-4 and control group was measured by flow cytometry and quantified; E and F: IL-6 and TNF-α mRNA expression was detected by qRT-PCR. n=3 per group, (1)P<0.05, (2)P<0.01 by two-tailed Student t-test, |
图 2 IL-4诱导MLE-12分泌DA、MA和S1PFig 2 The secretion of DA, MA and S1P of MILE-12 cells induced by IL-4 A: The value of MA in the supernatant of MLE-12 cells was detected by ELISA; B: The value of DA in the supernatant of MLE-12 cells was detected by ELISA; C: The value of S1P in the supernatant of MLE-12 cells was detected by ELISA. MA: Myristoleic acid; DA: Dodecanoylcarnitine; S1P: Sphingosine-1-phosphate. n=3 per group, (1)P<0.05 by two-tailed Student t-test, |
图 3 JTE-013有效抑制DA和MA诱导的MLE-12细胞S1PR2表达Fig 3 JTE-013 effectively inhibited the expression of S1PR2 in MLE-12 cells induced by DA and MA A and B: The expression of S1PR2 in MLE-12 cells among control, DA and DA+JTE-013 group was measured by flow cytometry and quantified; C and D: The expression of S1PR2 in MLE-12 cells among control, MA and MA+JTE-013 group was measured by flow cytometry and quantified; E and F: S1PR2 mRNA expression was detected by qRT-PCR. n=3 per group, (1)P<0.05, (2)P<0.01, (3)P<0.001, (4)P<0.000 1 by two-tailed Student t-test, |
图 4 JTE-013有效抑制DA和MA促进的MLE-12细胞的炎症分泌Fig 4 JTE-013 inhibited DA-induced and MA-induced allergic inflammatory responses in MLE-12 A-D: IL-6 and TNF-α mRNA expression was detected by qRT-PCR; E-H: The expression of IL-6 and TNF-α in MLE-12 cells among control, DA and DA+JTE-013 group was measured by flow cytometry and quantified; I-L: The expression of IL-6 and TNF-α in MLE-12 cells among control, MA and MA+JTE-013 group was measured by flow cytometry and quantified. n=3 per group, (1)P<0.05, (2)P<0.01, (3)P<0.001, (4)P<0.000 1 by two-tailed Student t-test, |
图 6 DA通过S1PR2/SHP-1/p38 MAPK信号通路介导炎症反应激活Fig 6 DA-mediated inflammatory activation was dependent on the S1PR2/SHP-1/p38 MAPK signaling pathway A: Protein expression level among different groups in MLE-12 was detected by Western blot; B and C: Quantitative analysis of p-SHP-1 and p-p38 protein expression in MLE-12. n=3 per group, (1) P<0.01, (2)P<0.001 by two-tailed Student t-test, |
图 7 MA通过S1PR2/SHP-1/p38 MAPK信号通路介导炎症反应激活Fig 7 MA-mediated inflammatory activation was dependent on the S1PR2/SHP-1/p38 MAPK signaling pathway A: Protein expression level among different groups in MLE-12 was detected by Western blot. B and C: Quantitative analysis of p-SHP-1 and p-p38 protein expression in MLE-12. n=3 per group, (1)P<0.05, (2) P<0.001 by two-tailed Student t-test, |
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作者贡献声明 马圆 实验实施，论文撰写和修订。张婷 实验设计，数据分析，论文撰写。姜志龙 实验方法设计。高佳萌，钱宇豪 实验数据分析。陈智鸿 实验方法设计，论文指导和修订。
利益冲突声明 所有作者均声明不存在利益冲突。
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表 1 qRT-PCR引物序列
图 1 构建IL-4诱导的MLE-12细胞炎症模型图 2 IL-4诱导MLE-12分泌DA、MA和S1P图 3 JTE-013有效抑制DA和MA诱导的MLE-12细胞S1PR2表达图 4 JTE-013有效抑制DA和MA促进的MLE-12细胞的炎症分泌图 5 JTE-013抑制DA和MA促进的MLE-12组胞氧化应激图 6 DA通过S1PR2/SHP-1/p38 MAPK信号通路介导炎症反应激活图 7 MA通过S1PR2/SHP-1/p38 MAPK信号通路介导炎症反应激活/
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