1.Effects of heme oxygenase-1/carbon monoxide pathway on the mitochondrial fusion in rat alveolar epithelial type Ⅱ cells stimulated by lipopolysaccharide
Haojuan JIA ; Jia SHI ; Shu'an DONG ; Yuan ZHANG ; Jianbo YU
Chinese Critical Care Medicine 2018;30(3):209-213
Objective To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type Ⅱ cells (AECⅡ) stimulated by lipopolysaccharide (LPS). Methods Once the cultured in vitro rat AECⅡcells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECⅡ cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-Ⅸ (ZnPP-Ⅸ, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-Ⅸ for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-Ⅸ + LPS group (CZL group) and Hemin + ZnPP-Ⅸ + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6)and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. Results Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L):40.7±5.3, 39.4±4.3 vs. 51.8±5.1], the protein expressions of HO-1, Mfn1, Mfn2 and OPA1 were significantly up-regulated (HO-1 protein: 0.873±0.051, 0.839±0.061 vs. 0.671±0.044; Mfn1 protein: 0.673±0.037, 0.654±0.025 vs. 0.568±0.021; Mfn2 protein: 0.676±0.044, 0.683±0.035 vs. 0.571±0.043; OPA1 protein: 0.648±0.031, 0.632±0.031 vs. 0.554±0.032; all P < 0.05); while opposite effects were found after ZnPP-Ⅸ preincubation, and there were significant differences in IL-6 and TNF-α contents and protein expressions of HO-1, Mfn1, Mfn2 and OPA1 as compared with those of LPS group [IL-6 (ng/L): 69.8±5.1 vs. 58.7±2.5, TNF-α (ng/L): 61.9±3.3 vs. 51.8±5.1, HO-1 protein: 0.545±0.023 vs. 0.671±0.044, Mfn1 protein: 0.406±0.051 vs. 0.568±0.021, Mfn2 protein:0.393±0.051 vs. 0.571±0.043, OPA1 protein: 0.372±0.050 vs. 0.554±0.032; all P < 0.05]. There were no significant differences in the parameters mentioned above between HL group and CL group, as well as among LPS, CZL and HZL groups. Conclusion HO-1/CO pathway promotes mitochondrial fusion and alleviates inflammatory response in LPS-induced rat AECⅡ cells.
2.Effect of aspirin on microglia activation induced by Poly-IC and its regulatory mechanism
Haojuan WU ; Juan CHENG ; Jiang XIE ; Jiamin LI ; Hua LI ; Hua ZHU ; Hongxia LI ; Yongmei ZHOU ; Wenming XU ; Xufeng JIA
Chinese Journal of Behavioral Medicine and Brain Science 2020;29(2):114-119
Objective:To study whether aspirin has inhibitory effect on microglia activation induced by Poly-IC and its mechanism.Methods:Microglia cell line BV2 were cultured in vitro to establish a Poly-IC stimulation-induced microglia cell immune activation model. The experiment groups were divided into control group (no treatment), model group (Poly-IC 10 μg/ml), high dose aspirin group (1 mmol/L aspirin), low dose aspirin group (0.1 mmol/L aspirin), high dose aspirin pretreatment group (Poly-IC 10 μg/ml + 1 mmol/L aspirin) and low dose aspirin pretreatment group (Poly-IC 10 μg/ml + 0.1 mmol/L aspirin). The phagocytosis ability of microglia cells, reactive oxygen species (ROS) and Iba1 protein expression were detected by using immunofluorescence method. The expression of the inflammatory cytokines Il-1β, Il-6, Il-10, TNF-α and cox-2 mRNA in microglia cells were detected by real-time quantitative PCR (RT-qPCR).Results:Compared with the control group, the morphology of microglia cells in model group changed significantly, and the phagocytosis ability and production of reactive oxygen species (ROS) increased. At the meantime, the expression of Iba1 protein was strongly decreased. In the model group, The mRNA expressions of IL-1β(20.55±1.92), IL-6 (63.98±7.83), TNF-α (16.84±3.19), COX-2 (6.78±0.42) were higher than IL-1β(1.01±0.14), IL-6 (0.95±0.17), TNF-α (1.22±0.38), COX-2 (0.87±0.11) in the control group. (Il-1β ( t=26.14), Il-6 ( t=10.22), TNF-α ( t=17.06) and COX-2 ( t=37.07), all P<0.01). In the aspirin pretreatment group, the phagocytic ability of microglia cells was inhibited compared with the model group, and the production of reactive oxygen species (ROS) reduced. The expression of Iba1 protein was also partly recovered. Meanwhile, the effect of the high aspirin dose pretreatment group on pro-inflammatory factors IL-1β(9.95±0.52), IL-6 (39.64±6.89), TNF-α(1.57±0.42), COX-2 (2.47±0.14)were lower than those in the model group significantly.(IL-1β: t=14.18, IL-6: t=3.69, TNF-α: t=16.68, COX-2: t=27.03, all P<0.01). Conclusion:Aspirin has an inhibitory effect on microglial activation induced by Poly-IC, which may be related with inhibiting the expression of inflammatory factors.