Anti-Inflammatory and Neuroprotective Effects of DIPOPA (N,N-Diisopropyl-2-Oxopropanamide), an Ethyl Pyruvate Bioisoster, in the Postischemic Brain

Abstract

Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have protective properties, which have been attributed to its anti-inflammatory, anti-oxidative, and anti-apoptotic functions. In an effort to develop better derivatives of EP, we previously synthesized DEOPA (N,N-diethyl-2-oxopropanamide, a novel isoster of EP) which has greater neuroprotective effects than EP, probably due to its anti-inflammatory and anti-excitotoxic effects. In the present study, we synthesized 3 DEOPA derivatives, in which its diethylamino group was substituted with diisopropylamino, dipropylamino, or diisobutylamino groups. Among them, DIPOPA (N,N-diisopropyl-2-oxopropanamide) containing diisopropylamino group had a greater neuroprotective effect than DEOPA or EP when administered intravenously to a rat middle cerebral artery occlusion (MCAO) model at 9 h after MCAO. Furthermore, DIPOPA had a wider therapeutic window than DEOPA and a marked reduction of infarct volume was accompanied by greater neurological and behavioral improvements. In particular, DIPOPA exerted robust anti-inflammatory effects, as evidenced by marked suppressions of microglia activation and neutrophil infiltration in the MCAO model, in microglial cells, and in neutrophil-endothelial cocultures at lower concentration, and did so more effectively than DEOPA. In particular, DIPOPA remarkably suppressed neutrophil infiltration into brain parenchyma, and this effect was attributed to the expressional inhibitions of cell adhesion molecules in neutrophils of brain parenchyma and in circulating neutrophils via NF-κB inhibition. Together, these results indicate the robust neuroprotective effects of DIPOPA are attributable to its anti-inflammatory effects and suggest that DIPOPA offers a potential therapeutic means of ameliorating cerebral ischemic injury and other inflammation-related pathologies.

Keywords: Anti-inflammation; DIPOPA; Ethyl pyruvate; NF-κB; Stroke.

Fig. 1

Infarct suppression by the 3 DEOPA derivatives, DEOPA, and EP. (a) Structures of EP, DEOPA (N,N-diethyl-2-oxopropanamide), and the 3 DEOPA derivatives, namely, N,N-diisopropyl-2-oxopropanamide (DIPOPA), N,N-dipropyl-2-oxopropanamide (DPOPA), and N,N-diisobutyl-2-oxopropanamide (DIBOPA). (b, c) DIPOPA, DPOPA, DBIOPA, DEOPA, or EP was administered intravenously (5 mg/kg) at 6 h post-MCAO, and mean infarct volumes were measured at 2 days post-MCAO by TTC staining. Representative images of infarctions in coronal brain sections (b) and quantitative results (means ± SEMs) (c). MCAO, PBS-treated MCAO control animals (n = 4); MCAO + DIPOPA, DIPOPA-administered MCAO animals (n = 4); MCAO + DPOPA, DPOPA-administered MCAO animals (n = 5); MCAO + DBIOPA, DBIOPA-administered MCAO animals (n = 4); MCAO + DEOPA, DEOPA-administered MCAO animals (n = 5); MCAO + EP, EP-administered MCAO animals (n = 4). *p < 0.05, **p < 0.01 versus the MCAO group; ##p < 0.01 versus the MCAO + EP group

Fig. 2

Neuroprotective potency of DIPOPA. (a, b) DIPOPA (1, 2, or 5 mg/kg) or DEOPA (5 mg/kg) were administered intravenously at 6 h post-MCAO, and mean infarct volumes were measured at 2 days post-MCAO by TTC staining. (c, d) DIPOPA or DEOPA (all at 5 mg/kg) were treated intravenously at 6, 9, or 12 h post-MCAO, and mean infarct volumes were measured at 2 days post-MCAO by TTC staining. Representative images of infarctions in coronal brain sections (a, c) and quantitative results (means ± SEMs) (b, d). MCAO, PBS-treated MCAO control animals (n = 4); MCAO + DIPOPA, DIPOPA (1, 2, or 5 mg/kg)-administered MCAO animals (n = 12, n = 4 per group); MCAO + DEOPA, DEOPA-administered MCAO animals (n = 5). *p < 0.05, **p < 0.01 versus the MCAO group; ##p < 0.01 versus the MCAO + DIPOPA group

Fig. 3

Prevention of neurological and motor deficits by DIPOPA. (a) DIPOPA, DEOPA, or EP (5 mg/kg) was administered 6 or 9 h post-MCAO, and neurological deficits were evaluated using modified neurological severity scores at 2 days post-MCAO. (b, c) The rota-rod test was performed at 10 or 15 rpm at 2 days post-MCAO on animals (with a 1-h rest period between tests) administered DIPOPA, DEOPA, or EP (5 mg/kg) at 6 or 9 h post-MCAO. (d-f) DIPOPA, DEOPA, or EP (5 mg/kg) was administered 6 h post-MCAO, and neurological deficits were evaluated (d) and the rota-rod test was performed at 10 or 15 rpm (e, f) at 2, 4, 6, 8, 10, 12, or 14 days post-MCAO. Sham, sham-operated animals (n = 8); MCAO + PBS, PBS-treated MCAO control animals (n = 9); MCAO + DIPOPA, the DIPOPA-administered MCAO animals (n = 13); MCAO + DEOPA, the DEOPA-administered MCAO animals (n = 9); MCAO + EP, the EP-administered MCAO animals (n = 9). *p < 0.05, **p < 0.01 versus the MCAO group; #p < 0.05, ##p < 0.01 versus the MCAO + DIPOPA group

Fig. 4

Suppression of inflammatory processes by DEOPA in the postischemic brain. DIPOPA, DEOPA, or EP (5 mg/kg) was administered 6 h post-MCAO, and immunohistochemistry (a-l) and RT-PCR (m-n) were carried out at 2 days post-MCAO. (a-j) Coronal brain sections were obtained 2 days after surgery in the sham (a, f), MCAO (b, g), MCAO + DIPOPA (c, h), MCAO + DEOPA (d, i), and MCAO + EP (e, j) groups and stained using anti-Iba-1 (a-e) or anti-Mac2 (f-j) antibodies. The images in the lower boxes (a-j) are high-magnification photographs, and insets in each image are high-magnification photographs of the indicated regions (white box). Photographs are representative of 3 independent experiments. Scale bars in a-j represent 1 mm and those in high-magnification photographs represent 100 μm or 25 μm. (k-l) Numbers of Iba-1⁺ and Mac2⁺ cells in indicated regions in a (*, 0.1 mm²) were counted and are presented as means ± SEMs (n = 12 from 3 animals). (m-n) RT-PCR samples were prepared from the indicated region (the black box) at 2 days post-MCAO and RNA levels of iNOS, IL-6, TNF-α, and IL-1β are presented as means ± SEMs (n = 3). Sham, sham-operated controls (n = 6); MCAO + PBS, PBS-treated MCAO control rats (n = 6); MCAO + DIPOPA, DIPOPA-administered MCAO rats (n = 6); MCAO + DEOPA, DEOPA-administered MCAO rats (n = 6); MCAO + EP, EP-administered MCAO rats (n = 6). **p < 0.01 versus the MCAO group; #p < 0.05, ##p < 0.01 versus the MCAO + DIPOPA group

Fig. 5

Suppression of vascular neutrophil activation after MCAO by DIPOPA. (a) DIPOPA, DEOPA, or EP (5 mg/kg) was administered 6 h post-MCAO, and neutrophils were purified from peripheral blood for immunoblot analysis at 12 h post-MCAO and immunohistochemistry was carried out at 48 h post-MCAO. (b-g) Coronal brain sections were obtained 48 h after surgery from the sham (b), MCAO (c), MCAO + DIPOPA (d), MCAO + DEOPA (e), and MCAO + EP (f) groups. Neutrophils were stained using anti-MPO antibody, and insets are high-magnification photographs of the indicated regions (*) in b. Photographs are representative of 3 independent experiments. Scale bars in a-f represent 1 mm and those in insets represent 50 μm. (g) Numbers of MPO⁺ cells in indicated regions in b (*, 0.1 mm²) were counted and are presented as means ± SEMs (n = 12 from 3 animals). (h-i) Vascular neutrophils were extracted from blood samples at 12 h post MCAO, and the expressions of PSGL-1 and LFA-1 in neutrophils were measured by western blot. Representative immunoblots and protein levels presented as means ± SEMs (n = 3). Sham, sham-operated rats (n = 5); MCAO + PBS, PBS-treated MCAO control rats (n = 5); MCAO + DIPOPA, DIPOPA-administered MCAO rats (n = 5); MCAO + DEOPA, DEOPA-administered MCAO rats (n = 5); MCAO + EP, EP-administered MCAO rats (n = 5). **p < 0.01 versus the MCAO group; #p < 0.05, ## p < 0.01 versus the MCAO + DIPOPA group

Fig. 6

Suppression of the LPS-induced activation of BV2 cells by DIPOPA. (a) The cytotoxicities of DIPOPA, DEOPA, and EP were measured by cell viability assay using CCK-8. (b-f) Nitrite production (b) and expressions of proinflammatory markers (c-f) were assessed using a Griess assay and by RT-PCR, respectively. BV2 cells (0.7 × 10⁴ cells/well in 24-well culture dishes) were pretreated with DIPOPA, DEOPA, or EP (1, 5, or 10 mM) for 1 h, washed, and then incubated with LPS (100 ng/ml) for 24 h. Changes in nitrite and proinflammatory cytokine mRNA levels are presented as means ± SEMs. &p < 0.05 versus vehicle controls; *p < 0.05, **p < 0.01 versus LPS treatment alone; #p < 0.05, ##p < 0.01 versus 1 mM of DIPOPA-treated cells; †p < 0.05, ††p < 0.01 versus 5 mM of DIPOPA-treated cells; $p < 0.05, $$p < 0.01 versus 10 mM of DIPOPA-treated cells

Fig. 7

Suppressions of neutrophil to endothelial adhesion, transendothelial neutrophil migration, and chemokine and cell adhesion molecule inductions by DIPOPA. (a) HL-60 cells were activated by 1 μM of ATRA for 3 days, and HUVEC cells were plated on gelatin-coated plates 1 day before assays. Both cell types were treated with DIPOPA, DEOPA, or EP for 1 h and activated with TNF-α (10 U/ml) for 12 h. HL-60 cells and HUVECs were labeled with CellTracker™ Red CMFDA or CellTracker Green CMTPX Dye, respectively. (b, c) After TNF-α treatment, adhesion molecule expressions were determined in both cell lines by western blot and beta-actin was used as the loading control. (d, e) For the adhesion assay, HL-60 cells and HUVECs were cocultured for 30 min, washed with chilled PBS 3 times, fixed, and the numbers of attached dHL-60 cells were counted. (f, g) Labeled dHL-60 cells were seeded on HUVEC monolayers in upper Boyden chambers, cocultured for 4 h, and fixed. Numbers of dHL-60 cells that measured were determined using a MTT assay, and results are presented as means ± SEMs (n = 3). Scale bars in d and f represent 200 μm or 10 μm. **p < 0.01 versus TNF-α-treated control cells; ^##p < 0.01 versus 10 mM of DIPOPA-treated group

Fig. 8

Suppression of NF-kB activity by DIPOPA in LPS-treated BV2 cells. (a-d) Effects of DIPOPA, DEOPA, or EP (all at 5 mM) on cytoplasmic IκB levels (a, b) and on nuclear p65 levels (c, d) in BV2 cells were examined by immunoblotting after treating cells with LPS (100 ng/ml) for 15 min or 1 h, respectively. Alpha-Tubulin and lamin B were used as loading controls. (e) BV2 cells were transfected with NF-κB-Luc reporter plasmid 1 day before assay, and NF-κB activities induced by LPS (100 ng/ml) in the presence or absence of DIPOPA, DEOPA, or of EP (5 or 10 mM) were examined. NF-κB activities are presented as means ± SEMs (n = 3). (f) Endogenous NF-κB binding was assessed using the TransAM p65 assay kit in the presence or absence of DIPOPA, DEOPA, or EP (all at 5 mM). *p < 0.05, **p < 0.01 versus LPS treatment alone; ##p < 0.01 versus DIPOPA-treated cells