Hypermethylation of UQCRC1 is involved in cognitive impairment after neonatal sevoflurane exposure

Objective‍ ‍To investigate whether aberrant DNA methylation of ubiquinol-cytochrome C reductase core protein 1 (UQCRC1) is related to cognitive impairment caused by neonatal sevoflurane exposure. Methods‍ ‍A total of 94 SPF C57 mice of either sex, aged 6 d, and weighing 4~6 g, were randomly divided into 7 groups: control group (Con, n=6), sevoflurane-6 and -24 h exposure groups (Sev-6 and -24 h, n=6), control+DMSO group (Con+DMSO, n=19), control+5-aza-2'-deoxycytidine (5-AZA, methylation inhibitor) group (Con+5-AZA, n=19), sevoflurane+DMSO group (Sev+DMSO group, n=19), and sevoflurane+5-AZA group (Sev+5-AZA group, n=19). From 6 to 8 d after birth, the mice of the Sev-6 and -24 h exposure groups were exposed to 3% sevoflurane daily (with 97% oxygen, 2 L/min, 2 h per day), while those from the Con groups were given exposure of 100% oxygen (2 L/min, 2 h per day). For the mice of the 5-AZA and DMSO groups, 1 mg/kg of 5-AZA or an equal volume of DMSO was injected intraperitoneally 30 min before daily exposure. In 6 and 24 h after the last exposure to sevoflurane, 6 mice from the Con, Sev-6 h, and Sev-24 h groups were euthanized for biochemical analysis, and in 24 h post-exposure, 6 mice from the Con+DMSO, Con+5-AZA, Sev+DMSO, and Sev+5-AZA groups were randomly selected for biochemical analysis, while another 3 mice from above each group were also randomly selected for morphological analysis. The remaining 10 mice in these groups underwent behavioral testing (open field test, novel object test, and Y-maze test) at 30~33 d after birth to assess cognitive function, and were euthanized in 24 h after the final behavioral test. RT-qPCR and Western blotting were used to detect the hippocampal expression of UQCRC1, DNA methyltransferases (Dnmts), and methyl CpG binding protein 2 (Mecp2) at mRNA and protein levels, respectively. Immunofluorescence assay was employed to observe the distribution and expression of UQCRC1 in the hippocampus. Bisulfite sequencing PCR (BSP) was applied to measure the methylation in the UQCRC1 promoter region. Results‍ ‍Compared with the Con group, the mRNA and protein levels of UQCRC1 were down-regulated (P<0.05), and the mRNA level of Dnmts was up-regulated (P<0.05) in both the Sev-6 h and Sev-24 h exposure groups, while the methylation level in the UQCRC1 promoter region was enhanced in the Sev-24 h exposure group (P<0.05). Additionally, the Sev+5-AZA group had obviously increased mRNA and protein levels of UQCRC1 (P<0.05), and notable improvement in cognitive impairment (P<0.05) when compared with the Sev+DMSO group. Conclusion‍ ‍Hypermethylation of UQCRC1 promoter region and thus down-regulating its mRNA and protein expression might be the main mechanism by which repeated neonatal sevoflurane exposure induces cognitive impairment later in life.