By up-regulating μ- and δ-opioid receptors, neuron-restrictive silencer factor knockdown promotes neurological recovery after ischemia

Abstract

We investigated the effects of neuron-restrictive silencer factor (NRSF) on proliferation of endogenous nerve stem cells (NSCs) and on μ- and δ-opioid receptor (MOR/DOR) expression in rats after cerebral ischemia. Among 100 rats subjected to cerebral ischemia, 20 rats were transfected with NRSF shRNA, and the remaining 80 were randomly assigned to normal, sham, model, and negative control (NC) groups. On days 7, 14, and 28 after ischemia and reperfusion, neurological function scores were assigned and a step-down passive avoidance test was conducted. Nerve function scores, step-down reaction periods, error times and apoptosis rates, as well as levels of B-cell CLL/lymphoma 2 (Bcl-2), BCL2-associated X protein (Bax), and NRSF expression were lower in the NRSF shRNA group than in the model and NC groups. By contrast, step-down latency, numbers of bromodeoxyuridine-positive cells, MOR/DOR expression, and phosphorylation of extracellular signal regulated protein kinase (ERK) and cAMP response element binding protein (CREB) were higher in the NRSF shRNA group than in the model and NC groups. These results suggest that by up-regulating MOR/DOR expression, NRSF knockdown accelerates recovery of neurological function after cerebral ischemia, at least in part by promoting NSC proliferation and inhibiting apoptosis.

Keywords: apoptosis; cerebral ischemia; neuron-restrictive silencer factor; proliferation; μ- and δ-opioid receptors.

Figure 1. Changes in local CBF in the parietal cortex of model group rats

CBF: cerebral blood flow.

Figure 2

(A) NRSF mRNA expression in each group. (B) protein expressions of NRSF and β-actin in each group detected by western blot. (C) NRSF protein expression in each group. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NC, negative control; NRSF, neuron-restrictive silencer factor.

Figure 3

BrdU-positive cell morphology (A), BrdU-positive cell rates (B), numbers of apoptotic nerve cells (C), and histogram of apoptosis rate (D) for brain tissues from each group. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NC, negative control; NRSF, neuron-restrictive silencer factor.

Figure 4

Expression of apoptosis-related proteins in brain tissues (A) and a histogram showing apoptosis-related protein expression in each group (B) based on Western blots. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NC, negative control; NRSF, neuron-restrictive silencer factor.

Figure 5

Histogram of NRSF and μ- and δ-opioid receptor mRNA expression (A), Western blot images of MOR, DOR, and NRSF expression (B), and histogram of NRSF, MOR, and DOR protein expression (C) for brain tissues from each group. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NRSF, neuron-restrictive silencer factor; NC, negative control; MOR, μ-opioid receptors; DOR, δ-opioid receptors.

Figure 6

p-ERK and p-CREB immunofluorescence in cortical ischemia and hippocampal CA1 regions (A), comparison of p-ERK and p-CREB expression intensity (B, C), Western blot images of t-ERK, p-ERK, t-CREB, and p-CREB proteins (D), and histogram of t-ERK, p-ERK, t-CREB, and p-CREB protein expression (E) in each. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NC, negative control; NRSF, neuron-restrictive silencer factor; ERK, extracellular signal-regulated kinase; CREB, response element binding protein.

Figure 7

Western blot images of t-ERK, p-ERK, t-CREB, and p-CREB proteins (A) and histogram of t-ERK, p-ERK, t-CREB, and p-CREB protein expression (B) in each group. ^*P < 0.05 compared to the normal group; ^#P < 0.05 compared to the model group; NC, negative control; NRSF, neuron-restrictive silencer factor; ERK, extracellular signal-regulated kinase; CREB, response element binding protein.

Figure 8. NRSF binds to MOR/DOR promoter genes

Paraformaldehyde cross-linking was performed after chromatin from rat brain neural stem cells was cut into fragments of BP 200~2000 using sonication (A); Chromatin and ChIP antibodies were separated by shear after immunoprecipitation, and purified DNA introns were amplified using PCR (B). NRSF, neuron-restrictive silencer factor; MOR/DOR, μ- and δ-opioid receptors; ChIP, chromatin immunoprecipitation assay.