Early inhibition of HIF-1α with small interfering RNA reduces ischemic–reperfused brain injury in rats

Abstract

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1α siRNA can protect the brain from ischemic damage by inhibiting HIF-1α induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1α siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1α mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1α, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1α positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1α siRNA may protect the ischemic–reperfused neurons in vivo via inhibition of HIF-1α, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke.

Introduction

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcriptional complex composed of inducible HIF-1α subunit and constitutive HIF-1β subunit. Whereas HIF-1β is a common subunit for ARNT Sim (PAS) protein, HIF-1α is the specific and oxygen-regulated subunit of HIF-1. In severe hypoxic cases, HIF-1α is accumulated and leads to cell death by activating different target genes (Semenza et al., 2000). It may bind to proapoptotic members of the Bcl-2 family such as BNIP3 (Bruick, 2000), Nix (Sowter et al., 2001), and p53 as well as caspases (Li et al., 2005), which contribute to cell death or apoptosis.

RNA interference (RNAi) has been established as a powerful tool to characterize gene function in various species, notably in mammals (Fire et al., 1998, Elbashir et al., 2001). RNAi is triggered by the presence of double-stranded RNA (dsRNA) in the cell and results in rapid destruction of the mRNA containing the identical sequence. SiRNAs exert their gene-silencing activity through the RNA interfering silencing complex (RISC) that degrades cognate mRNA (Sharp, 2001). The demonstration that siRNA can effectively inhibit gene expression in mammals opens up many possibilities for exploring the use of siRNA in rats or mice for dissecting gene function and drug target validation studies in tissue specific manners in vivo. Our previous study has demonstrated that hyperbaric oxygen can protect the brain from global ischemia damage partly by suppressing the level of HIF-1α (Li et al., 2005). Additionally, protein inhibitors of HIF-1α, such as 2ME2 and D609, can protect the brain from ischemic damage in a focal ischemia rat model (Chen et al., 2007).

In the present study, we tested the feasibility of using small inducible RNAi in vivo to obtain an unbiased evaluation of the potential therapeutic effect of inhibiting HIF-1α in middle cerebral artery occlusion induced focal ischemic rat model. We also attempted to clarify the mechanisms of HIF-1α in the neuronal cell death after cerebral ischemia. Our hypothesis is that HIF-1α is one of the important proapoptotic factors in the development of brain infarcts and neuronal death, and HIF-1α siRNA can protect the brain from ischemic damage by inhibiting HIF-1α induced apoptotic pathway at the RNA level. We have examined the RNA and protein expression of HIF-1α, the apoptotic genes it regulates in the infarct tissues and the effect of HIF-1α siRNA on the cerebral ischemic neuronal death.