Fig 2: Additional ATP supplementation ameliorated ATPIF1- or sevoflurane-induced inflammation and caspase-3 activation in BV2 cells. (A) Overexpression of ATPIF1 decreased the intracellular ATP concentrations in BV2 cells. (B) ATP (50 μg/ml) ameliorated the ATPIF1 overexpression induced inflammation and caspase-3 activation in BV2 microglia. (C) Statistics of the relative gray value indicating the protein expression in panel (B). (D) ATP also ameliorated sevoflurane treatment- induced inflammation and caspase-3 activation in BV2 microglia. (E) Statistics of the relative gray value indicating the protein expression in panel (D). Ctrl means control. β-Actin served as a loading control. One-way ANOVA with repeated measurement were used to determine differences in panels (C,E). The data shown are the means ± SD, n = 3, * or # means p < 0.05, ** or ## means p < 0.01. ns means no significant difference.

Fig 3: Inflammation regulates ATPIF1 expression and is attenuated by additional ATP supplementation in microglia. (A) LPS treatment upregulates ATPIF1 expression in microglia. (B) Statistics of the relative gray value indicating the ATPIF1 protein expression in panel (A). (C) Methylprednisolone sodium succinate (MPSS) attenuated the COX-2 and TNF-α upregulation and promotion of caspase-3 activation caused by sevoflurane. (D) Statistics of the relative gray value indicating the protein expression in panel (C). (E) Dexamethasone (DXM) also attenuated the COX-2 and TNF-α upregulation and promotion of caspase-3 activation caused by sevoflurane. (F) Statistics of the relative gray value indicating the protein expression in panel (E). (G) ATP (50 μg/ml) ameliorated LPS induced inflammation in mouse primary microglia. (H) Statistics of the relative gray value indicating the protein expression in panel (G). (I) ATP (50 μg/ml) ameliorated TNF-α-induced inflammation in mouse primary microglia. (J) Statistics of the relative gray value indicating the protein expression in panel (I). Ctrl means control. β-Actin served as a loading control. Student’s t-test was used to determine differences in panel (B). One-way ANOVA with repeated measurement were used to determine differences in panels (D,F,H,J). The data shown are the means ± SD, n = 3. * or # means p < 0.05, ** or ## means p < 0.01, *** or ### means p < 0.001. ns means no significant difference.

Fig 4: (See previous page.) Mitochondrial DNA release following infection and requirement for mitochondria for inflammasome activation by P. aeruginosa. (A) and (B), qPCR analysis of cytosolic mitochondrial DNA (mtDNA) relative to nuclear DNA in macrophages pretreated (A) with Mito-TEMPO (500 μM) or 3-MA (10 mM) or control or Lc3b siRNA (B) and infected with PA103ΔUΔT (MOI 25) for 4 h or uninfected (Basal) as shown. Columns show means of 3 independent determinations; error bars are SEM. (C) Mitochondrial content of J774A.1 cells exposed to ethidium bromide (EtBr) at the indicated concentration (ng/ml) measured by qPCR (normalized to untreated cells; upper panel) and immunoblot for the mitochondrial protein ATPIF1 (lower panel) at low and high exposure time; TUBB5 is shown as a loading control. (D) Mitochondrial content of control or ethidium bromide-treated J774A.1 cells (ρJ774A.1) assayed by flow cytometry of MitoTracker Green stained cells. (E) Flow cytometry of J774A.1 and ρ°J774A.1 cells left uninfected (Basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and stained with MitoSOX Red. (F) J774A.1 cells grown in the absence or presence of 500 ng/ml ethidium bromide (EtBr) were left untreated (basal) or infected with PA103ΔUΔT (MOI 25) for 4 h and analyzed as described in Figure 1A. *** indicates significant differences between the levels in the presence and absence of the EtBr (500 ng/ml), P < 0.001. All data are representative of results from 2 or 3 independent experiments.

Fig 5: The hypothesized pathway indicating sevoflurane regulates microglial inflammatory responses and caspase-3 activation. Proposed mode pathway indicates that the sevoflurane promotes ATPIF1 expression in microglia to induce inflammatory factor upregulation and capsase-3 activation and decrease ATP level. Supplementation of ATP alleviated sevoflurane induced anxiety behavior and memory damage in POD mice.