乳酸调节巨噬细胞向M2型极化的作用

Lactate Regulates Macrophage Polarization through PI3K/Akt Path- way

目的探讨乳酸对巨噬细胞极化作用的影响，以及P13K／Akt信号通路在上述过程中的作用。方法体外培养小鼠单核巨噬细胞株RAW264．7。分为阴性对照组和实验组（乳酸干预浓度分别为1、10、100μmol／L），培养24h后，RT．PCR方法检测各组细胞Argl、CD206、NOS2基因mRNA表达水平的变化，Western印迹方法检测各组细胞Argl、CD206、NOS2、P13K、Akt蛋白表达水平的变化。ELISA法检测各组细胞因子的表达。结果乳酸诱导RAW264．7细胞内M2型巨噬细胞标志分子Argl、CD206表达上调，M1型巨噬细胞标志分子NOS2表达下调，此作用且呈浓度依赖性。RT-PCR结果显示，与阴性对照组相比，100μmol／L乳酸对Argl、CD206基因mRNA上调作用最为明显，分别增加了2．37倍（t=7．901，P〈0．05）和3．21倍（t=11．225，P〈0．05）。100μmol／L乳酸对NOS2基因mRNA下调作用最为明显，其表达下降了（76．35±4．21）％（t=13．475，P〈0．05）。Western印迹结果显示，100μmol／L乳酸对Argl、CD206蛋白表达水平上调作用最为明显，分别增加了2．69倍（t=8．922，P〈0．05）和3．77倍（t=10．737，P〈0．05）。100μmol／L乳酸对NOS2蛋白表达水平下调作用最为明显，其表达下降了（80．12±5．21）％（t=9．563，P〈0．05）。ELISA结果显示，乳酸诱导培养上清液中M1型细胞因子TNF．d、IFN．1和IL．1仅表达下调，M2型细胞因子IL-10和IL4表达上调。Western印迹结果显示P13K、Akt基因蛋白表达水平下调，且呈浓度依赖性。与阴性对照组相比，100μmol／L乳酸对P13K、Akt基因蛋白表达水平下调最为明显，分别下降了（83．63±3．75）％（t=12．256，P〈0．05）和（75．17±4．32）％（t=10．261，P〈0．05）。结论乳酸能诱导巨噬细胞极化为M2型，机制可能包括P13K／Akt信号通路参与。

Objective To investigate the regulating effect of lactate on the macrophage polari-zation, and explore the role of PI3K/Akt signaling pathway in this process. Methods The mouse macrophage cell line RAW264. 7 was cuhuredin vitro, with different concentrations of lactate （1 μmol/L, 10 ixmol/L, 100 μmol/L） for 24 hours. The mRNA expression of Argl, CD206 and NOS2 was detected by RT-PCR. The protein expression of Argl, CD206, NOS2, PI3K and Akt was detected by Western blot method. The expression of cytokines was detected by ELISA. Results M2 macrophages markers such as Argl and CD206 were up-regulated after induction with lactate in RAW264.7 cells. However, M1 macrophages marker NOS2 was down-regulated. This regulating effect was dose-dependent. After treatment with lactate at 100 μmol/L, the mRNA expression of Argl and CD206 was up-regulated most obviously, with a 2. 37-fold increase (t = 7. 901, P 〈 0. 05) and a 3.21-fold increase (t = 11. 225, P 〈 0. 05) , respectively. Meanwhile, the mRNA expression of NOS2 was down-regulated most strikingly, being decreased by （76.35 ± 4.21) % （t=13.475, P 〈0.05） After treatment at 100 μmol/L, the protein expression of Argl and CD206 was increased by 2.69-fold （t--8.922, P 〈0.05） and 3.77-fold （t = 10. 737, P 〈 0. 05), respectively. The protein expression of NOS2 was decreased, being reduced by （80. 12 ± 5.21) % （t = 9. 563, P 〈 0. 05） . ELISA results showed that the Ml-type eytokines such as TNF-α, IFN-＇γand IL-1α were decreased, and the M2-type eytokines such as IL-10 and IL-4 were ele-vated. After treatment at 100μmol/L, the protein expression of PI3K and Akt was down-regulated most significantly, withadeeline of （83.63 ± 3.75）% （t=12.256, P〈0.05） and （75.17 ± 4.32） % (t = 10.261, P 〈 0. 05）, respectively. Conclusion Lactate can induce the M2 phe-notype shift in the maerophage, and the mechanism may involve the PI3K/Akt signal pathway.