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.

Objective:To explore the influences of neutrophilic granule protein (NGP) on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages and the regulatory mechanism.Methods:NGP highexpression RAW264.7 cells (NGP/RAW) and negative control empty vector cells (NC/RAW), NGP knockout RAW264.7 cells (NGP KO/RAW) and wild-type cells (WT/RAW) were cultured in vitro. Cells in logarithmic phase were stimulated with 10 mg/L LPS (LPS group) or phosphate buffered saline (PBS group) respectively. The content of NO in the supernatant was detected by Griess method. The mRNA expression of inducible nitric oxide synthase (iNOS) was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expressions of iNOS and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) were detected by Western blotting.Results:Compared with PBS group, iNOS mRNA and NO expression were significantly increased at different time after LPS stimulation, the mRNA expression of iNOS peaked at 12 hours after LPS stimulation (2 -ΔΔCt: 38.45±1.34 vs. 1.00±0.00 in NC/RAW cells, 56.24±2.41 vs. 1.45±0.30 in NGP/RAW cells, 37.84±1.52 vs. 1.00±0.00 in WT/RAW cells, 5.47±0.62 vs. 0.98±0.40 in NGP KO/RAW cells, all P < 0.05), and the production of NO peaked at 24 hours after LPS stimulation (μmol/L: 24.15±1.26 vs. 0.15±0.04 in NC/RAW cells, 58.80±2.11 vs. 0.18±0.02 in NGP/RAW cells, 25.04±1.80 vs. 0.16±0.02 in WT/RAW cells, 2.42±0.38 vs. 0.12±0.03 in NGP KO/RAW cells, all P < 0.05). After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP/RAW cells were increased significantly compared with NC/RAW cells [iNOS mRNA (2 -ΔΔCt): 8.42±0.59 vs. 4.63±0.37 at 2 hours, 27.16±1.60 vs. 14.25±1.02 at 6 hours, 56.24±2.41 vs. 38.45±1.34 at 12 hours; NO (μmol/L): 4.12±0.25 vs. 2.23±0.17 at 6 hours, 16.50±1.52 vs. 6.35±0.39 at 12 hours, 58.80±2.11 vs. 24.15±1.26 at 24 hours, all P < 0.05]. At the same time, the protein expressions of p-STAT1 and iNOS were also significantly enhanced (p-STAT1/GAPDH: 4.26±1.84 vs. 1.00±0.32 at 0 hours, 20.59±4.97 vs. 0.93±0.21 at 2 hours, 141.99±10.99 vs. 11.17±2.11 at 6 hours; iNOS/GAPDH: 1.27±0.86 vs. 1.00±0.22 at 0 hours, 7.94±1.94 vs. 2.01±0.92 at 2 hours, 24.24±4.88 vs. 3.72±1.11 at 6 hours, all P < 0.05), indicating that NGP might increase the expression of iNOS by promoting the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) pathway, thereby increasing the production of NO. After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP KO/RAW cells were significantly lower than that of WT/RAW cells [iNOS mRNA (2 -ΔΔCt): 2.46±0.31 vs. 4.22±0.18 at 2 hours, 3.61±0.44 vs. 13.02±1.34 at 6 hours, 5.47±0.62 vs. 37.84±1.52 at 12 hours; NO (μmol/L): 1.22±0.19 vs. 2.01±0.12 at 6 hours, 1.60±0.44 vs. 5.15±0.62 at 12 hours, 2.42±0.38 vs. 25.04±1.80 at 24 hours, all P < 0.05]. It showed that iNOS activation was reduced after NGP knockout, which in turn reduced NO production. Conclusion:NGP can positively regulate NO production in activated macrophages by activating the STAT1/iNOS pathway.

Objective:To determine the effects of cytochrome P450 1A1 (CYP1A1) on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages and the underlying mechanism.Methods:The peritoneal macrophages (PMs) were isolated from healthy C57BL/6 mice and stimulated with 10 mg/L LPS to establish inflammatory response model. The CYP1A1 mRNA and protein expressions in the cells were determined. The mouse macrophages RAW264.7 cell line with CYP1A1 overexpression (CYP1A1/RAW) were cultured in vitro, and they were stimulated by 10 mg/L LPS at logarithmic phase. The negative control-expressed RAW264.7 cells (NC/RAW) were established. The protein and mRNA expressions of activator protein-1 (AP-1) and inducible nitric oxide synthase (iNOS) in the cells as well as the content of NO in the cell supernatant were determined. The RAW264.7 cells were cultured in vitro, and they were stimulated by 10 mg/L LPS and 100 nmol/L 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] only or in combination at logarithmic phase. The blank control group was set up. The expression of iNOS mRNA in the cells and NO content in the cell supernatant were determined to observe whether the effect of CYP1A1 on LPS induced NO production in macrophages was related to 12(S)-HETE produced by metabolism. The RAW264.7 cells with CYP1A1 overexpression and hydroxylase activity mutation (CYP1A1m/RAW) were cultured in vitro, and they were stimulated by 10 mg/L LPS at logarithmic phase. The CYP1A1/RAW cell control group was set up. The iNOS mRNA expression in the cells and NO content in the cell supernatant were determined to observe the effect of hydroxylase activity of CYP1A1 in regulating NO production in macrophages. Results:Compared with the phosphate buffered saline (PBS) control group, the CYP1A1 mRNA expressions were elevated significantly from 2 hours after LPS stimulation and reached a peak at 12 hours [CYP1A1 mRNA (2 -ΔΔCt): 6.41±0.98 vs. 1.00±0.00, P < 0.05], while CYP1A1 protein expressions were increased from 6 hours after LPS stimulation and reached a peak at 24 hours, suggesting that CYP1A1 expression might be involved in LPS-induced macrophage over-activation. Compared with NC/RAW+LPS group, the iNOS mRNA expressions and NO contents both increased in CYP1A1/RAW+LPS group and reached a peak after 12 hours and 24 hours, respectively [12-hour iNOS mRNA (2 -ΔΔCt): 54.42±8.21 vs. 24.22±3.89, 24-hour NO (μmol/L): 66.52±4.09 vs. 41.42±2.09, both P < 0.05], while the iNOS protein expression and AP-1 phosphorylation also enhanced, suggesting that CYP1A1 might increase NO production by promoting AP-1 activation and iNOS expression. LPS and 12(S)-HETE stimulation only or in combination had no effect on iNOS mRNA expression and NO production, and no significant difference was found between the 12 (S)-HETE+LPS group and LPS group [12-hour iNOS mRNA (2 -ΔΔCt): 34.24±4.07 vs. 34.35±4.01, 24-hour NO (μmol/L): 44.02±3.14 vs. 44.56±3.21, both P > 0.05], suggesting that the regulation of CYP1A1 on NO production might not be induced by 12 (S)-HETE. There was no significant difference in the iNOS mRNA expressions or NO content between the CYP1A1m/RAW+LPS group and CYP1A1/RAW+LPS group [12-hour iNOS mRNA (2 -ΔΔCt): 52.11±6.84 vs. 50.21±5.19, 24-hour NO (μmol/L): 60.42±4.14 vs. 52.01±5.12, both P > 0.05], suggesting that CYP1A1 hydroxylase activity deficiency showed no effect on NO production. Conclusions:LPS stimulation significantly increases CYP1A1 expression in macrophages. CYP1A1 overexpression promotes NO production by activated macrophages through AP-1/iNOS pathway, while hydroxylase-deficiency or 12(S)-HETE has no effect on this regulation.

BACKGROUND: Hemorrhoids are one of the most common conditions that lead to surgery, and until now surgical hemorrhoidectomy has been the major effective treatment. Post-operative pain from hemorrhoidectomy has been experienced by thousands of patients and remains a major inconvenience of the operation. OBJECTIVE: This study evaluates the clinical efficacy of the pestle needle therapy, an acupoint stimulation method, for relief of post-hemorrhoidectomy pain. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: This was a single-center, patient-assessor-blinded and randomized controlled trial with 154 patients receiving Milligan hemorrhoidectomy surgery. Eligible patients were randomly assigned to either a treatment group or a control group at a ratio of 1:1. The treatment group received the pestle needle therapy, with manual stimulation at Yaoshu (DU2), Mingmen (DU4), Changqiang (DU1), Chengshan (BL57), Erbai (EX-UE2) and the perianal points (1, 3, 5, 7, 9, and 11o'clock around the lesion); while the control group received a sham treatment with very light pressure. Three sessions of treatment were performed at 30 min, 4 h and 12 h after the surgery, and each lasted for 15 min. MAIN OUTCOME MEASURES: The primary outcome was post-operative pain measured with the visual analogue scale (VAS) at 12 h after surgery. The secondary outcomes included the VAS scores measured at 0.5, 2, 4, 6, 8, 24 and 48 h after surgery, the analgesic dose, the time and the VAS score of the patients' first defecation after surgery, as well as the Hamilton Rating Scale for Anxiety (HAMA) evaluated before discharge. RESULTS: The mean pain score of the treatment group was significantly lower than that of the control group (3.10 ± 1.27 vs 4.82 ± 1.29; P < 0.001) at 12 h after surgery. Compared with the control group, patients in the treatment group needed a smaller dose of analgesic within the first 24 hours after surgery (P = 0.002); and their HAMA scores before discharge were lower (4.07 ± 2.40 vs 5.10 ± 2.45, P = 0.009). Compared to the treatment group, patients in the control group had a greater time to the first defecation after surgery ([52.34 ± 15.72] h vs [27.08 ± 13.68] h; P < 0.001), but there was no difference in their VAS scores at the first defecation (P = 0.092). CONCLUSION The pestle needle therapy was effective for relieving pain, reducing anxiety and improving bowel function after hemorrhoidectomy, and it is worthy of clinical application.

12-HETE is a metabolite of arachidonic acid (AA). AA is normally present in membrane phospholipids. The exposure to different stimuli can trigger the release of AA through the activity of phospholipase A₂ (PLA₂) by cells. An important metabolic pathway which utilizes AA as its substrate is 12-Lipoxygenase (12-LOX), resulting in the formation of 12-HETE. 12-HETE plays an important role in many diseases such as cancer, diabetes, hypertension, and participates in the pathogenesis of inflammation and oxidative stress and other pathological processes.Current research shows that it participates in metamorphism and exudation in the process of inflammation. This review is aimed at summarizing its role in inflammation and oxidative stress, with improved understanding of 12-HETE.

Objective: To investigate the potential effects of cytochrome P450 1A1 (CYP1A1) in regulating macrophages polarize to M2 type and explore the molecular mechanism. Methods: All trials were completely randomized. ① Experiment 1: 6-8 weeks old healthy male C57BL/6J mice were collected, and primary peritoneal cells were extracted, then the cells were divided into phosphate buffered saline (PBS) group and interleukin-4 (IL-4) group. The cells in the IL-4 group were stimulated with 10 mg/L IL-4 (M2 macrophage inducer); and those in the PBS group were given with an equal amount of PBS. The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1 (Arg-1) and chitinase 3 like protein 1 (YM1) at 2, 4, 6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6 (JAK1/STAT6) signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6, 12, 24 hours after IL-4 challenge were determined by Western Blot. ② Experiment 2: RAW264.7 cells with high expression CYP1A1 (CYP1A1/RAW) and their negative control cells (NC/RAW) were cultured in vitro. The cells in logarithmic growth phase were collected, and then they were divided into PBS control group and IL-4 group. The treatment method was the same as experiment 1. The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot. The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot, respectively. Results: ① Experiment 1: after IL-4 challenge for 2 hours, the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group [Arg-1 mRNA (2^-ΔΔCt): 1.75±0.82 vs. 1.00±0.21; YM1 mRNA (2^-ΔΔCt): 2.58±0.53 vs. 1.00±0.20, both P < 0.05] which indicated that IL-4 induced macrophage to M2 type polarization successfully. Meanwhile, the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group, and peaked at 4 hours [CYP1A1 mRNA (2^-ΔΔCt): 2.25±0.69 vs. 1.00±0.17, P < 0.01]. The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type. Compared with the PBS control group, the bands of phosphorylated JAK1 (p-JAK1), phosphorylated STAT6 (p-STAT6), Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group, and reached the peak value at 12 hours then gradually decreased. This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1, and the pathway was activated. ② Experiment 2: after IL-4 challenge for 2 hours, the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells (2^-ΔΔCt: 3.02±0.60 vs. 1.47±0.43, P < 0.05), and the protein band signal was also stronger, and both peaked at 4 hours. This indicated that CYP1A1 could promote the polarization of macrophages to M2. After IL-4 challenge for 2 hours, the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group, but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells, indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway. In the meantime, the protein band of Akt, a downstream protein of the PI3K/Akt pathway, in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells, indicating that CYP1A1 did not promote macrophage polarization through this pathway. Conclusions CYP1A1 promotes the polarization of macrophage to M2 type. The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.

Infection is one of the main causes of death in clinical patients, and multi-drug resistance leads to ineffective treatment with conventional antibiotics. Therefore, it is imperative to develop new anti-infective drugs. Antimicrobial peptides cathelicidins are cationic host defense peptides found in many organisms. It has been demonstrated by in vivo and in vitro studies that antimicrobial peptides cathelicidins not only show broad-spectrum antibacterial activity and high sensitivity to drug-resistant bacteria, but also have a good guiding effect on the immune response. This paper summarizes the reports of antimicrobial peptides cathelicidins in recent years, highlighting their research achievements in antibiosis, anti-inflammatory, chemotaxis regulation and phagocytosis, providing new ideas for the treatment of infection-related diseases.

Infectious and inflammatory diseases are important diseases threatening human health. Without timely control, a series of complications will occur in patients, such as sepsis, inflammatory factor storm, and even lead to death. It has been found that cytochrome P4501A1 (CYP1A1) plays a key role in the development of infectious and inflammatory diseases through aromatic hydrocarbon receptor (AhR) dependent and non-dependent pathways in different cells and organs induced by different substances. The non AhR dependent regulatory mechanism of CYP1A1 and the different roles of CYP1A1 in infection and inflammation is reviewed in order to provide reference for further research on the relationship between CYP1A1 and infection and inflammation.

Infection is one of the main causes of death in clinical patients, and multi-drug resistance leads to ineffective treatment with conventional antibiotics. Therefore, it is imperative to develop new anti-infective drugs. Antimicrobial peptides cathelicidins are cationic host defense peptides found in many organisms. It has been demonstrated by in vivo and in vitro studies that antimicrobial peptides cathelicidins not only show broad-spectrum antibacterial activity and high sensitivity to drug-resistant bacteria, but also have a good guiding effect on the immune response. This paper summarizes the reports of antimicrobial peptides cathelicidins in recent years, highlighting their research achievements in antibiosis, anti-inflammatory, chemotaxis regulation and phagocytosis, providing new ideas for the treatment of infection-related diseases.
Anti-Bacterial Agents ; Anti-Infective Agents ; Antimicrobial Cationic Peptides ; Bacteria ; Cathelicidins ; Humans

12-HETE is a metabolite of arachidonic acid (AA). AA is normally present in membrane phospholipids. The exposure to different stimuli can trigger the release of AA through the activity of phospholipase A₂ (PLA₂) by cells. An important metabolic pathway which utilizes AA as its substrate is 12-Lipoxygenase (12-LOX), resulting in the formation of 12-HETE. 12-HETE plays an important role in many diseases such as cancer, diabetes, hypertension, and participates in the pathogenesis of inflammation and oxidative stress and other pathological processes.Current research shows that it participates in metamorphism and exudation in the process of inflammation. This review is aimed at summarizing its role in inflammation and oxidative stress, with improved understanding of 12-HETE.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Arachidonic Acid ; Humans ; Inflammation ; Oxidative Stress