Improvement of mitochondrial function mediated the neuroprotective effect of 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone in rats with cerebral ischemia-reperfusion injuries

Abstract

Deficits in mitochondrial function is a critical inducement in the major pathways that drive neuronal cell death in ischemic process particularly. Drugs target to improve the mitochondrial function may be a feasible therapeutic choice in treatment with ischemic diseases. In the present study, we investigated whether 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone (RD-1), a compound derived from rhodanine, could protect against ischemic neuronal damage via improving mitochondrial function. We tested the neuroprotective effect of RD-1 both in rats modeled by middle cerebral artery occlusion reperfusion in vivo and in primary cortical neurons subjected to hypoxia/reperfusion injury in vitro. Results showed that treatment with RD-1 for 14 days remarkably reduced infarct size, decreased neurological deficit score and accelerated the recovery of somatosensory function in vivo. Meanwhile, RD-1 also increased the cellular viability after 48 h treatment in vitro. In addition, RD-1 protected the primary cortical neurons against mitochondrial damage as evidenced by stabilizing the mitochondrial membrane potential and reducing the overproduction of reactive oxygen species. Furthermore, hypoxia/reperfusion injury induced damaged mitochondrial axonal transport and consequently neurotransmitter release disorder, which were ameliorated by RD-1 treatment. Besides, RD-1 inhibited the downregulation of proteins related with mitochondrial transport and neurotransmitter release induced by ischemic injury both in vivo and in vitro. The obtained data demonstrated the neuroprotective effect of RD-1 and the involved mechanisms were partially attributed to the improvement in mitochondrial function and the synaptic activity. Our study indicated that RD-1 may be a potential therapeutic drug for the ischemic stroke therapy.

Keywords: 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone; cerebral ischemia reperfusion; mitochondrial function; neuroprotection; synaptic activity.

Figure 1. Protective effect of RD-1 against brain injury after cerebral ischemia reperfusion in MCAO rats

The NSS scores were tested at 1, 3, 7 and 14 days after reperfusion. Adhesive-removal test was evaluated at 14 days after reperfusion. (A) Representative infarct volume measured by HE staining at 14 days after reperfusion. (B) Quantitative evaluation of infract volume in experimental groups. (C) NSS scores. (D) Adhesive-removal for somatosensory test. Data were expressed as mean ± SD, *P < 0.001 vs. Sham; ^#P < 0.05, ^##P < 0.01 vs. Model; n = 8-10.

Figure 2. Protective effect of RD-1 against hypoxia-induced damage in PCNs subjected to OGD/R injury

Cells were post-treated with serial concentrations of RD-1 (0, 0.1, 1, 5 and 10 μmol/L). Cell viability was measured by CCK-8 assay after 48 h treatment. The values were represented as mean ± SD, *P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.001 vs. Model; n = 3.

Figure 3. RD-1 reduced the ROS generation in PCNs subjected to OGD/R injury

ROS generation was measured by fluorescent probe DCFH-DA after 12 h treatment in the presence or absence of 5 μmol/L RD-1. The concentration of RD-1 was selected based on the cell viability analysis. (A) Representative image of ROS fluorescence in each group (magnification 40×, Scale bar = 100 μm). (B) Quantitative analysis of relative DCFH-DA fluorescence intensity. The values were represented as mean ± SD, ^*P < 0.001 vs. Control; ^#P < 0.001 vs. Model, n = 3.

Figure 4. RD-1 stabilized MMP in PCNs subjected to OGD/R injury

MMP was measured by JC-1, an indicator of mitochondrial function. Red fluorescence represents the mitochondrial aggregate JC-1 and green fluorescence indicates the monomeric JC-1. The concentration of RD-1 was selected based on the cell viability analysis. (A) Representative image of JC-1 labeled MMP staining in each group (magnification 40×, Scale bar = 100 μm). (B) Quantitative analysis of MMP. The values were represented as mean ± SD, *P < 0.001 vs. Control; ^#P < 0.001 vs. Model, n = 3.

Figure 5. Effects of RD-1 on the impaired axonal mitochondrial transport

(A) Representative view frame of mitochondrial movement along the axon (Scale bar = 33 μm), mitochondria move along the axon shown in a rectangle area; the arrow pointed to the track of an retrograde moving mitochondrion. (B) Mean speed of motile mitochondria in each group. (C) and (D) Expression of KIF1B and Miro2 in PCNs and ischemic cortex, the intensity of each band was normalized to that of β-actin. The values were represented as mean ± SD, *P < 0.05, **P < 0.01 vs Control or Sham; ^#P < 0.05, ^##P < 0.01 vs Model, n = 3.

Figure 6. Effects of RD-1 on the synaptic vesicle release

FM1-43 dye was used to image synaptic vesicle release. (A) Representative time-relapse destaining of synaptic boutons of different groups (magnification 20×, Scale bar = 50 μm). (B) Analytical results of synaptic vesicle release efficacy as indicated by percentage of remaining fluorescence intensity. (C) and (D) Expression of SNAP25 and Complexin-1/2 in PCNs and ischemic cortex, the intensity of each band was normalized to that of β-actin. The values were represented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 vs Control or Sham; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs Model, n = 3.