细胞色素P4501A1对巨噬细胞吞噬功能的调控作用及机制研究

Effect and mechanism of cytochrome P4501A1 on regulating phagocytosis of macrophage

目的探讨细胞色素P4501A1(CYP1A1)对巨噬细胞吞噬大肠杆菌(E.coli)能力的调控作用及机制。方法①体外培养小鼠单核/巨噬细胞白血病细胞株RAW264.7(RAW),用感染复数(MOI)为30的E.coli刺激细胞40 min,并设甘油对照组,观察感染时CYP1A1的表达变化。②体外培养CYP1A1过表达RAW细胞(CYP1A1/RAW)、CYP1A1敲除RAW细胞(CYP1A1 KO/RAW),用MOI为30的E.coli刺激细胞40 min,并以阴性对照RAW细胞(NC/RAW)为对照,观察细胞吞噬功能与CYP1A1表达的关系,以及CYP1A1对吞噬受体〔清道夫受体A(SR-A)〕及其信号通路〔丝裂素活化蛋白激酶(MAPK)通路〕的调控作用。③体外培养NC/RAW、CYP1A1 KO/RAW细胞,用1μmol/L细胞外信号调节激酶(ERK)抑制剂U0126预处理2 h后,再用MOI为30的E.coli刺激细胞40 min,并设磷酸盐缓冲液(PBS)对照组,观察CYP1A1是否通过调节MAPK通路调控巨噬细胞的吞噬功能。④体外培养RAW细胞,用100 nmol/L的CYP1A1羟化酶活性产物12(S)-羟基二十碳四烯酸〔12(S)-HETE〕预处理2 h后,再用MOI为30的E.coli刺激细胞40 min,并设PBS对照组,观察巨噬细胞的吞噬功能是否与CYP1A1羟化酶代谢产物有关。⑤体外培养CYP1A1过表达且羟化酶活性位点突变的RAW细胞(CYP1A1m/RAW),用MOI为30的E.coli刺激细胞40 min,并设CYP1A1/RAW对照组,观察巨噬细胞的吞噬功能是否与CYP1A1羟化酶活性有关。结果①与甘油对照组相比,E.coli刺激后RAW细胞中CYP1A1 mRNA表达显著增加(2^(-ΔΔCt):7.79±0.71比1.00±0.00,P<0.05),提示CYP1A1可能参与调控感染进程。②与NC/RAW细胞相比,E.coli刺激40 min后CYP1A1/RAW细胞吞噬E.coli菌落数明显降低(×10^(3) CFU/mL:4.67±3.06比15.67±5.03,P<0.05),而CYP1A1 KO/RAW细胞吞噬E.coli菌落数则显著增加(×10^(3) CFU/mL:46.00±5.29比15.67±5.03,P<0.05),说明CYP1A1可负性调控巨噬细胞吞噬功能。同时,与NC/RAW细胞相比,CYP1A1/RAW细胞中SR-A mRNA表达明显下调(2^(-ΔΔCt):0.31±0.03比1.00±0.00,P<0.05),且ERK的活化水平明显降低;而CYP1A1 KO/RAW细胞中SR-A mRNA表达明显上调(2^(-ΔΔCt):3.74±0.25比1.00±0.00,P<0.05),且ERK活化增强,说明CYP1A1可以负性调控吞噬受体及其信号通路。③与PBS相比,U0126预处理可显著抑制CYP1A1敲除诱导的SR-A mRNA表达上调(2^(-ΔΔCt):0.62±0.05比4.38±0.39,P<0.05),ERK活化水平也被抑制,同时可阻遏CYP1A1敲除导致的巨噬细胞吞噬能力增强〔细胞吞噬E.coli菌落数(×10^(3) CFU/mL):12.67±1.15比45.33±4.16,P<0.05〕,说明CYP1A1可通过抑制ERK活化而减弱巨噬细胞的吞噬功能。④与PBS相比,12(S)-HETE预处理后,RAW细胞的吞噬功能并无明显变化〔细胞吞噬E.coli菌落数(×10^(3) CFU/mL):17.00±1.00比16.33±2.52,P>0.05〕,说明CYP1A1对巨噬细胞吞噬功能的调控作用可能并非通过其羟化酶代谢产物12(S)-HETE实现。⑤与单纯过表达CYP1A1的RAW细胞相比,CYP1A1m/RAW细胞的吞噬功能并无明显变化〔细胞吞噬E.coli菌落数(×10^(3) CFU/mL):3.67±1.15比3.33±0.58,P>0.05〕,说明CYP1A1也并非通过其羟化酶活性来调控巨噬细胞的吞噬功能。结论CYP1A1可通过抑制ERK活化降低SR-A表达,从而负性调控巨噬细胞的吞噬功能,但此调控效应与CYP1A1羟化酶活性及其致炎产物12(S)-HETE均无关。

Objective To explore the effect and mechanism of cytochrome P4501A1(CYP1A1)on regulating phagocytosis of macrophage treated with Escherichia coli(E.coli).Methods①The 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.②The 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].③NC/RAW and CYP1A1 KO/RAW cells were cultured in vitro and pretreated with 1μmol/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.④The 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.⑤The 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.Results①Compared 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.②Compared 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.③Compared 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.④Compared 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.⑤Compared 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.