[Effects of histone methyltransferase inhibitor on the polarization of peritoneal macrophages in septic mice].

Objective: To investigate the effect of histone methyltransferase (EZH2) inhibitor on the polarization of peritoneal macrophages in septic mice.

Methods: Thirty-six healthy male C57BL/6J mice were divided into three groups by random number table method (n = 12): sham operated group (Sham group), sepsis model group (CLP group) and EZH2 inhibitor treatment group (CLP+3-DZNeP group). Sepsis animal model was established by cecum ligation and puncture (CLP); Sham group was challenged only by cecum traction without ligation. 3-Deazaneplanocin A (3-DZNeP) 1 mg/kg was intraperitoneal injected 24 hours before and 1 hour after CLP in CLP+3-DZNeP group. Eight mice in each group were sacrificed at 24 hours after surgery. The levels of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in peritoneal lavatory fluid were detected by high throughput liquid protein chip. The expression levels of inducible nitrogenase (iNOS) and macrophage mannose receptor (CD206) were analyzed by flow cytometry. Mouse peritoneal macrophages were isolated and purified by adherent method, the protein expressions of EZH2, peroxisome proliferator-activated receptorγ (PPARγ) were detected by Western Blot. The remaining 4 mice were sacrificed at 48 hours after surgery, the histopathological changes of lung and kidney tissue were evaluated by hematoxylin-eosin (HE) staining.

Results: Compared with Sham group, the infiltration of inflammatory cells in lung and kidney of the CLP group, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significant increased [IL-6 (ng/L): 7 794.75±405.56 vs. 78.63±74.09, TNF-α (ng/L): 147.25±25.19 vs. 18.20±5.03, both P < 0.01], the percentage of M1 type macrophages was significantly increased [iNOS F4/80: (13.18±8.80)% vs. (1.57±0.77)%, P < 0.05], and the protein expression of EZH2 was significantly increased (EZH2/GAPDH: 0.84±0.11 vs. 0.11±0.03, P < 0.01), while the protein expression of PPARγ was significantly decreased (PPARγ/GAPDH: 0.09±0.01 vs. 0.27±0.09, P < 0.01). Compared with CLP group, the histopathological changes of lung and kidney in CLP+3-DZNeP group were significantly alleviated, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significantly decreased [IL-6 (ng/L): 4 207.10±876.60 vs. 7 794.75±405.56, TNF-α (ng/L): 63.00±25.37 vs. 147.25±25.19, both P < 0.01 ], the percentage of M1 type macrophages was significantly decreased [iNOS F4/80: (3.64±0.89)% vs. (13.18±8.80)%, P < 0.05], while the percentage of M2 type macrophages was significantly increased [CD206 F4/80: (17.68±5.63)% vs. (7.60±3.17)%, P < 0.01], the protein expression of EZH2 was significantly decreased (EZH2/GAPDH: 0.53±0.09 vs. 0.84±0.11, P < 0.05), and the protein expression of PPARγ was significantly increased (PPARγ/GAPDH: 0.39±0.14 vs. 0.09±0.01, P < 0.05).

Conclusions: Sepsis induces high expression of EZH2 in peritoneal macrophages, and may induce polarization of M1 type macrophages by inhibiting the expression of PPARγ protein. EZH2 inhibitor 3-DZNeP can lessen the inflammatory cytokines release by inhibiting the M1 type macrophages polarization.