Human urinary kallidinogenase suppresses cerebral inflammation in experimental stroke and downregulates nuclear factor-kappaB

Abstract

The purpose of this study is to investigate the possible mechanism and the neuroprotective effect of human urinary kallidinogenase (HUK) in cerebral ischemia. The mouse middle cerebral artery occlusion (MCAO) model was used. Mice were treated with HUK (20 PNAU/g per day, intravenous) or saline as control, from the beginning of reperfusion to 72 h. Neurological deficits, infarct size, and BWC were measured at 6, 24, 48, and 72 h after MCAO, respectively. Pathological changes of brain were observed by TUNEL assay. Inflammatory factors were measured by real-time PCR and western blotting. Activation of MAPKs, Akt, and nuclear factor-kappaB (NF-kappaB) was detected by western blotting. Our results indicated that HUK significantly improved neurofunction, decreased infarct size, and suppressed edema, as well as inflammatory mediators as compared with the vehicle group. Furthermore, HUK inhibited the NF-kappaB pathway and activated the MAPK/ERK pathway in this neuroprotection.

Figure 1

NSS and BWC at different time points. (A) NSS increased at different time points after MCAO versus that in the sham group, and HUK reverse it at 24, 48, and 72 h; ^*P<0.05 versus the MCAO group. (B) BWC as a measure of brain edema of the ischemic hemisphere. HUK could significantly reduce the ischemic brain edema at 6, 24, 48, and 72 h caused by MCAO; ^*P<0.05 versus the sham group and ^#P<0.05 versus the MCAO group. Values are mean±s.e.m., n=10.

Figure 2

Infarct size was measured by TTC. (A) Infarct images obtained by TTC staining at 48 h after MCAO. The normal tissue was stained deep red and the infarct area was stained pale gray. (B) Infarct size was expressed by bar graphs at 6, 24, 48, and 72 h after MCAO with HUK or vehicle, respectively; ^*P<0.05 versus MCAO with vehicle. Values are mean±s.e.m., n=10.

Figure 3

Apoptosis determined by TUNEL. TUNEL-positive cells significantly increased at 24 h after MCAO (B) versus that in the sham group (A), and HUK could reverse it (C). The percent of TUNEL-positive was 8.39±1.81% in the MCAO group and decreased to 3.11±0.68% after treatment with HUK (D). Six representative microscopic fields were analyzed for each group; ^*P<0.05 versus the sham group and ^#P<0.05 versus the HUK group.

Figure 4

Expression of mice mRNA for IL-1β (A), IL-6 (B), ELAM-1 (C), iNOS (D), TNF-α (E), COX-2 (F), and MCP-1 (G) at 6, 24, 48, and 72 h after MCAO or treated with HUK. Six independent samples were used in the experiments per group; ^*P<0.05, ^**P<0.01 versus the sham group and ^#P<0.05, ^##P<0.01 versus the MCAO group.

Figure 5

Expression of protein level for iNOS and COX-2 at 6, 24, 48, and 72 h after MCAO in the treated or untreated group. Three independent experiments were performed. ^*P<0.05 versus the sham group and ^#P<0.05, ^##P<0.01 versus the MCAO group.

Figure 6

(A) Effect of HUK on activation of the NF-κB pathway as analyzed by western blotting. (B) A graphical representation of the densitometric measurements showing the ratio of p-IκBα and IκBα in the cytoplasm and (C, D) graphical representation of the densitometric measurements showing the protein level of p65 and p50 in the nucleus at 6, 24, 48, and 72 h after MCAO in the treated or untreated group. Three independent experiments were performed. ^**P<0.01 versus the sham group and ^#P<0.05, ^##P<0.01 versus the MCAO group.

Figure 7

Effect of HUK on activation of the MAPK pathway and the Akt pathway as analyzed by western blotting. (A–C) A graphical representation of the densitometric measurements showing the ratio of p-JNK and JNK; p-p38 and p38; and pERK and ERK; and (D) graphical representation of the densitometric measurements showing the ratio of p-AKT and AKT at 6, 24, 48, and 72 h after MCAO in the treated or untreated group. Three independent experiments were performed. ^*P<0.05 versus the sham group and ^#P<0.05 versus the MCAO group.