Effect and mechanism of cytochrome P450 1A1 on regulating phagocytosis of macrophage
10.3760/cma.j.cn121430-20220825-00785
- VernacularTitle:细胞色素P450 1A1对巨噬细胞吞噬功能的调控作用及机制研究
- Author:
Lixing TIAN
1
;
Linghui WAN
;
Junyu ZHU
;
Huaping LIANG
Author Information
1. 陆军军医大学边防卫勤训练大队军事预防医学教研室，新疆维吾尔自治区呼图壁 831200
- Keywords:
Cytochrome P450 1A1;
Escherichia coli;
Macrophage;
Phagocytosis
- From:
Chinese Critical Care Medicine
2023;35(2):158-163
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect and mechanism of cytochrome P450 1A1 (CYP1A1) on regulating phagocytosis of macrophage treated with Escherichia coli ( E.coli). Methods:① The mouse leukemia cells lines of monocyte macrophage RAW264.7 (RAW) were cultured in vitro and treated with 30 multiplicity of infection (MOI) dosages of E.coli for 40 minutes, glycerin control group was set up to observe the change of CYP1A1 during infection. ② The RAW cells with CYP1A1 overexpression (CYP1A1/RAW) and knock out (CYP1A1 KO/RAW) were cultured in vitro and treated with 30 MOI E. coli for 40 minutes, while the negative controlled RAW cells (NC/RAW) were established as control to observe the relationship between cell phagocytosis and CYP1A1 expression, and the effect of CYP1A1 on phagocytic receptor [scavenger receptor-A (SR-A)] and its signal pathway [mitogen-activated protein kinase (MAPK) pathway]. ③ NC/RAW and CYP1A1 KO/RAW cells were cultured in vitro and pretreated with 1 μmol/L extracellular signal-regulated kinase (ERK) inhibitor (U0126) for 2 hours, and then treated with 30 MOI E.coli for 40 minutes, phosphate buffered solution (PBS) control group was set up to observe whether the effect of CYP1A1 on phagocytosis through controlled the MAPK pathway. ④ The RAW cells were cultured in vitro and pretreated with 100 nmol/L CYP1A1 hydroxylase active product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] for 2 hours, and then treated with 30 MOI E.coli for 40 minutes, and PBS control group was set up to observe whether the effect of CYP1A1 on phagocytosis was related to CYP1A1 hydroxylating metabolite. ⑤ The RAW cells with overexpression CYP1A1 hydroxylase-activity mutation (CYP1A1m/RAW) were cultured in vitro and treated with 30 MOI E.coli for 40 minutes, the CYP1A1/RAW cells were set up as control group to observe whether the effect of CYP1A1 on phagocytosis was related to CYP1A1 hydroxylase-activity. Results:① Compared with glycerin control group, CYP1A1 mRNA expression was significantly increased by E.coli stimulation (2 -ΔΔCt: 7.79±0.71 vs. 1.00±0.00, P < 0.05), indicating that CYP1A1 might participate in regulating infection progress. ② Compared with NC/RAW cells, the number of E.coli colonies phagocytized by CYP1A1/RAW cells was significantly decreased after 40 minutes of E.coli stimulation (×10 3 CFU/mL: 4.67±3.06 vs. 15.67±5.03, P < 0.05), while CYP1A1 KO/RAW cells had a significant increase in the number of E.coli colonies phagocytized (×10 3 CFU/mL: 46.00±5.29 vs. 15.67±5.03, P < 0.05), suggesting that CYP1A1 might negatively control macrophage phagocytosis function. Meanwhile, compared with NC/RAW cells, the expression of SR-A mRNA in CYP1A1/RAW cells was significantly down-regulated (2 -ΔΔCt: 0.31±0.03 vs. 1.00±0.00, P < 0.05), and the activation level of ERK was significantly reduced. However, the expression of SR-A mRNA in CYP1A1 KO/RAW cells was significantly up-regulated (2 -ΔΔCt: 3.74±0.25 vs. 1.00±0.00, P < 0.05), and the activation of ERK was enhanced, indicating that CYP1A1 could negatively regulate phagocytic receptors and their signaling pathways.③ Compared with PBS, U0126 pretreatment significantly inhibited the CYP1A1 knockout induced upregulation of SR-A mRNA expression (2 -ΔΔCt: 0.62±0.05 vs. 4.38±0.39, P < 0.05) and ERK activation, and inhibited the enhancement of phagocytosis in macrophages induced by CYP1A1 knock out [ E.coli colonies phagocytized by cells (×10 3 CFU/mL): 12.67±1.15 vs. 45.33±4.16, P < 0.05], suggesting that CYP1A1 inhibited macrophage phagocytosis function by regulating ERK activation. ④ Compared with PBS, the phagocytosis of RAW cells pretreated with 12(S)-HETE did not change significantly [ E.coli colonies phagocytized by cells (×10 3 CFU/mL): 17.00±1.00 vs. 16.33±2.52, P > 0.05], suggesting that CYP1A1 might not control phagocytosis function by its hydroxylase-activity metabolism 12(S)-HETE. ⑤ Compared with CYP1A1/RAW cells, there was no significant change in the phagocytic function of CYP1A1m/RAW cells [ E.coli colonies phagocytized by cells (×10 3 CFU/mL): 3.67±1.15 vs. 3.33±0.58, P > 0.05], suggesting that CYP1A1 might not control phagocytosis function by its hydroxylase-activity. Conclusion:CYP1A1 can negatively regulate the phagocytosis of macrophages by inhibiting the activation of ERK and reducing the expression of SR-A, but this regulatory effect is not related to the activity of CYP1A1 hydroxylase and its pro-inflammatory metabolism 12(S)-HETE.
