Phospholipase A2 of Peroxiredoxin 6 Plays a Critical Role in Cerebral Ischemia/Reperfusion Inflammatory Injury

Abstract

Microglia-mediated inflammation is an important step in the progression of cerebral ischemia/reperfusion injury and the associated production of receptors of immunomoudulation, including Toll-like receptors (TLRs). Peroxiredoxin 6 (Prdx6) has been demonstrated as the endogenous antioxidant protein for its peroxidase properties. However, the role of the independent phospholipase A2 (iPLA2) activity of Prdx6 in stroke has not been well studied. In this study, we evaluated whether blocking the calcium-iPLA2 activity of Prdx6 using siRNA and inhibitors (1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol, MJ33) would have a critical effect on inflammatory brain damage. We conducted oxygen-glucose deprivation (OGD)/recovery (R) in vitro and middle cerebral artery occlusion (MCAO) in vivo in a microglia/neuron co-culture system and in rats. In vitro, we found that Prdx6-iPLA2 activity was associated with the secretion of neurotoxic inflammatory mediators interleukin1β (IL-1β), interleukin-17 (IL-17) and interleukin-23 (IL-23) and elevated expression of Toll-like receptor 2/4 (TLR2/4), leading to the formation of nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in microglial cells. In vivo, combined treatment with Prdx6-iPLA2 activity inhibitor MJ33 showed a greater diminution in neurologic deficits, cerebral infarction, brain water content and inflammatory molecules than Prdx6-siRNA treatment alone. Our findings provide new insight into Prdx6-iPLA2 function in the brain. Inhibition of Prdx6-iPLA2 activity by gene therapy and/or pharmacology may constitute a promising new therapeutic approach to the treatment of stroke.

Keywords: MJ33; OGD/R; Prdx6-iPLA2 activity; cerebral ischemia/reperfusion; microglia/neuron co-culture system.

Figure 1

The efficiency of phospholipase A2 of peroxiredoxin 6 (Prdx6-iPLA2) siRNA. Effects of siRNA on mRNA (A) and protein expression (B) of Prdx6. (C) An independent phospholipase A2 (iPLA2) enzyme-linked immunosorbent assay (ELISA) kit was used to measure the PLA2 activity in microglia. (D) Both Prdx6-iPLA2 siRNA and 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33) had no effect on GSH activity. Values are expressed as mean ± SEM of three independent experiments, *p < 0.05 vs. Control; **p < 0.01 vs. Control; ^#p < 0.05 vs. Scramble, ^##p < 0.01 vs. Scramble.

Figure 2

Effects of Prdx6-iPLA2 on neuron viability and cell damage in response to oxygen glucose deprivation and regeneration (OGD/R). (A) Neuron viability was measured by MTS assay. (B) Neuron damage was measured by Lactate dehydrogenase (LDH) assay. Number of experiments: 9. Values are mean ± SEM, **p < 0.01 vs. Control; ^#p < 0.05 vs. Scramble.

Figure 3

Effect of Prdx6-iPLA2 on the release of interleukin1β (IL-1β), interleukin-17 (IL-17) and interleukin-23 (IL-23) in culture medium in response to OGD/R (A–C). Number of experiments: 9. Values are mean ± SEM, *p < 0.05 vs. Control; **p < 0.01 vs. Control; ^#p < 0.05 vs. Scramble; ^##p < 0.01 vs. Scramble.

Figure 4

Effect of Prdx6-iPLA2 on Toll-like receptor 2 (TLR2) and TLR4 expression in microglia in response to OGD/R. TLR2 and TLR4 were activated during OGD/R exposure. Prdx6-iPLA2 siRNA or MJ33 treatment could inhibit TLR2 and TLR4 mRNA (A,B) and protein expression (C–E). Results are expressed as the mean ± SEM of three independent experiments. **p < 0.01 vs. Control; ^#p < 0.05 vs. Scramble; ^##p < 0.01 vs. Scramble.

Figure 5

Effect of Prdx6-iPLA2 on nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in microglia in response to OGD/R. (A–C) mRNA levels analyzed by quantitative RT-PCR. Representative Western blot bands and semi-quantitative analyses of NF-κB (D,E), iNOS (D,F) and COX-2 (D,G) expression in astrocytes under OGD/R. Results are expressed as mean ± SEM of three independent experiments. *p < 0.05 vs. Control; **p < 0.01 vs. Control; ^#p < 0.05 vs. Scramble; ^##p < 0.01 vs. Scramble.

Figure 6

Effect of Prdx6-siRNA and MJ33 on the expression of Prdx6 and iPLA2 activity following middle cerebral artery occlusion (MCAO). qPCR (A) and Western blot (B) results showed that the expression of Prdx6 was reduced in the Prdx6-siRNA group. ELISA (C) results also showed that a significant decrease of iPLA2 activity was found in the Prdx6 + MJ33 group. Results are expressed as mean ± SEM of three independent experiments. ^&p < 0.01 vs. Sham; **p < 0.01 vs. MCAO; ^##p < 0.01 vs. Prdx6-siRNA.

Figure 7

Effect of Prdx6 siRNA and MJ33 on cerebral infarct volume, neurologic deficit scores and brain water content following transient MCAO. (A) Representative images of TTC-stained sections 24 h after MCAO. (B) Prdx6 siRNA significantly increased cerebral infarct volume compared with the MCAO group. Combine treatment with Prdx6 siRNA and MJ33 attenuated the injury. Neurological function (C) and brain water content (D) were tested after 24 h of MCAO in rats. Values are mean ± SEM of nine animals in each group. ^&p < 0.01 vs. Sham; **p < 0.01 vs. Scramble; ^#p < 0.05, vs. Prdx6 siRNA; ^##p < 0.01 vs. Prdx6 siRNA.

Figure 8

The influence of Prdx6-siRNA and MJ33 on the production of inflammatory cytokines IL-1β, IL-17 and IL-23. The ELISA results indicated that IL-1β (A), IL-17 (B) and IL-23 (C) were upregulated by Prdx6 siRNA after MCAO. These increase were attenuated by additional treatment of rats with MJ33. The results are expressed as the mean ± SEM of nine animals in each group. ^&p < 0.01 vs. Sham; **p < 0.01 vs. Scramble; ^#p < 0.05, vs. Prdx6 siRNA.

Figure 9

Expression of TLR2 and TLR4 in response to Prdx6 siRNA and MJ33. Real-time PCR showed that TLR2 (A) and TLR4 (B) expression increased after MCAO or giving Prdx6-siRNA. Combined treatment with Prdx6-siRNA and MJ33 downregulated TLR2 and TLR4 levels. Representative Western blot (C) and semi-quantitative analyses of the levels of TLR2 (D) and TLR4 (E) in the cortex after MCAO. Results are expressed as the mean ± SEM of three independent experiments. ^&p < 0.01 vs. Sham; **p < 0.01 vs. Scramble; ^#p < 0.05, vs. Prdx6 siRNA; ^##p < 0.01 vs. Prdx6 siRNA.

Figure 10

Expression of NF-κB, iNOS and COX-2 in response to Prdx6 siRNA and MJ33. Real-time PCR results, representative Western blot bands, and semi-quantitative analyses of NF-κB (A,D,E), iNOS (B,D,F) and COX-2 (C,D,G) in the cortex following MCAO. Results are expressed as the mean ± SEM of three independent experiments. ^&p < 0.01 vs. Sham; **p < 0.01 vs. Scramble; ^#p < 0.05, vs. Prdx6 siRNA; ^##p < 0.01 vs. Prdx6 siRNA.