It is noticed that MDL

It is noticed that MDL-28170 only partly reversed isoflurane-induced AIF release and nuclear translocation. In addition to calpain mechanism, activation of the DNA repair enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is also essential for AIF release (Culmsee et al., 2005; Moubarak et al., 2007), and results in the production of high levels of neurotoxic PAR polymer, which has been directly linked to the induction of AIF translocation (Andrabi et al., 2006). Moreover, PARP-1-mediated AIF release is sequentially linked to calpain dependent AIF release (Vosler et al., 2009). The increase of [Ca2+]m is responsible for mitochondria-induced ROS production to produce severe DNA damage (Hara and Snyder, 2007). DNA damage activates PARP-1 to produce PAR polymer leading to severe NAD depletion (Islam et al., 2017) and secondary mitochondrial Ca2+ dysregulation with subsequent calpain activation and AIF release (Lu et al., 2013). Isoflurane is also demonstrated to cause extensive ROS upregulation and lipid peroxidation (Wei et al., 2008; Yang et al., 2008), enhance activation of PARP in the Amrubicin mg of developing mice (Liang et al., 2010), suggesting isoflurane-induced AIF release and nuclear translocation may be also linked to activation of PARP-1. Whether there is a parallel or additive relationship between PARP-1 and calpain activation in the regulation of isoflurane-induced AIF release needs further investigation. JNK is one of the major members in the MAPK family. JNK signaling pathway is activated by [Ca2+]i increase and plays crucial cellular roles on nervous system development (Kuan et al., 1999; Mousa and Bakhiet, 2013) and neurodegeneration (Harper and Wilkie, 2003). Our previous study has identified that isoflurane-induced caspase-3 activation is by JNK pathway in the neonatal rats (Li et al., 2013). An increasing amount of studies have reported that JNK signaling pathway is also involved in AIF-dependent apoptosis in the injuries induced by glutamate (Yang et al., 2013), cadmium (Jiang et al., 2014) and cambogin (Shen et al., 2015). Our present results showed that the JNK inhibitor SP600125 not only reduced isoflurane-induced increase of mitochondrial Bax expression, cytochrome c release to cytosol, activation of caspase-3, but also mitigated isoflurane-induced AIF release to cytosol and nuclear translocation, illustrating that JNK is involved in isoflurane-induced caspase-dependent and AIF-dependent apoptosis pathway in the brains of neonatal rat. Additionally, SP600125 attenuated isoflurane-induced memory impairment in the developing rats evidenced by increasing the percentage of freezing time in the contextual fear-conditioning experiment, suggesting that the activation of JNK is also an important mechanism of isoflurane-induced cognitive function impairment. Since the activation of both JNK and calpain was required for isoflurane-induced AIF translocation and neurotoxicity, it is possible there is a cross-talk between these two pathways. It was noted that SP600125 did not influence isoflurane-induced increase of μ-calpain and AIF truncation in the mitochondria; whereas SP600125 significantly attenuated isoflurane-induced increase of m-calpain and AIF release to cytosol. These results illustrated that JNK pathway-induced AIF-dependent apoptosis was related with activating m-calpain and subsequently promoting AIF release. How the activation of JNK and calpain induces AIF release is not completely clarified. One potential mechanism of AIF release is by mPTP. This is supported by the finding that the mPTP inhibitor cyclosporine A blocks calpain-mediated calcium-induced AIF release in isolated mitochondria (Baburina et al., 2015). Mitochondrial Bax can activate the mPTP in addition to forming transmembrane pores, which is an important mechanism of cytochrome c and AIF release (Favreau et al., 2012). mPTP opening is also regulated by ROS/JNK pathway (Favreau et al., 2012). Our present results found that the JNK inhibitor, which is different from the calpain inhibitor, attenuated isoflurane-induced increase of mitochondrial Bax expression and Bax/Bcl-2 ratio, indicating that JNK activation may promote AIF and cytochrome c release by mPTP, although we have not directly measured the opening of mPTP. It is also possible that JNK itself can induce mitochondrial–nuclear AIF translocation in addition to activation of m-calpain since calpain inhibitor only partly reversed isoflurane-induced AIF nuclear translocation and neuroapoptosis at 24 h after exposure. The study of Douglas and Baines has shown that the activation of JNK, but not Ca2+/calpain, induces mitochondrial–nuclear AIF translocation in the β-Lapachone and MNNG-induced PARP1-dependent necrosis model (Douglas and Baines, 2014). Further experiments are needed to testify the additional mechanism of JNK-induced AIF nuclear translocation in the developing rats after isoflurane exposure.