Abstract
Neuronal apoptosis is an important factor accounting for the poor outcomes of intracerebral hemorrhage (ICH). This study first showed that inhibition of activating transcription factor 6 (ATF6) could alleviate secondary brain injury through anti-apoptosis after ICH in rats. Melatonin, ATF6 and CCAAT/enhancer-binding protein homologous protein (CHOP) siRNAs were applied in this study. Brain edema, neurological functions, blood-brain barrier (BBB) integrity were evaluated at 24 h after ICH. Western blot analysis was used to evaluate the protein level of target proteins (ATF6, CHOP, Bip, Bcl-2, Bax, and cleaved caspase-3). Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA level of ATF6, CHOP and cleaved caspase-3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 immunofluorescence staining were applied to evaluate the neuronal cell death. The results suggested that the levels of ATF6 and its downstream protein, CHOP, were upregulated and reached the peak at 24 h after ICH. ATF6 was highly expressed in neurons. The administration of melatonin significantly decreased the mRNA and protein levels of ATF6, and its downstream targets, CHOP and cleaved caspase-3, but increased the Bcl-2/Bax ratio, which ameliorated the neurological functions. The CHOP siRNA significantly reversed the pro-apoptotic effect induced by the increased ATF6 level after ICH. Melatonin could protect against neuronal apoptosis via suppression of ATF6/CHOP arm of ER-stress-response pathway.
Highlights
MATERIALS AND METHODSIntracerebral hemorrhage is one of the most common types of stroke (Feigin et al, 2009; Steiner et al, 2014), which is featured by a high rate of death and disability (van Asch et al, 2010; Krishnamurthi et al, 2014)
The Evans Blue (EB) extravasation was increased in ipsilateral hemisphere of intracerebral hemorrhage (ICH) compared with the rats in sham group (p < 0.05 vs. sham), while the rats receiving the higher dose of melatonin displayed a lower level of EB leakage (p < 0.05 vs. ICH + vehicle, Figure 2C)
The results showed that the administration of activating transcription factor 6 (ATF6) siRNA could significantly reduce the level of CCAAT/enhancer-binding protein homologous protein (CHOP) expression in both protein and mRNA levels, while CHOP siRNA had no effects on the expression of ATF6, which was elevated at 24 h after ICH (p < 0.05, vs. sham)
Summary
Intracerebral hemorrhage is one of the most common types of stroke (Feigin et al, 2009; Steiner et al, 2014), which is featured by a high rate of death and disability (van Asch et al, 2010; Krishnamurthi et al, 2014). The rats received trans-cardiac perfusion with 0.1M PBS after being anesthetized, we collected the brain tissues around hematoma (Figure 2A), which were further processed as previously reported (Nakka et al, 2010), and 40 μg of protein from each sample was used for electrophoresis (100 V, 1 h) and transferred to the polyvinylidene fluoride membranes at 250 V for 1 h. Afterward, following the manufacturer’s instructions, a total volume of 5 μl (500 pmol) of rat ATF6 siRNA or CHOP siRNA (Thermo Fisher Scientific, United States) dissolved in nuclease-free water was injected into the right ventricle (3.5 mm depth below the skull) with a pump at the rate of 0.5 μl/min at FIGURE 2 | Effects of melatonin on neurological functions, brain edema and BBB leakage. We used non-parametric statistics to analyze data regarding the time-course of ATF6 as it did not pass the normality test
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