Objective:To evaluate the effect of sevoflurane on mitochondrial-associated endoplasmic reticulum membranes (MAMs) in mouse microglia and the relationship with silent information regulator-related enzyme 3 (SIRT3)/adenosine triphosphatase family protein 3A (ATAD3A) signaling pathway.Methods:After normal culture of mouse microglia (BV-2 cell line), the cells were divided into 4 groups ( n=33 each) by a random number table method: empty adenovirus-control group (group C), empty adenovirus-sevoflurane exposure group (group Sev), SIRT3 overexpression adenovirus-control group (group SIRT3 + C) and SIRT3 overexpression adenovirus-sevoflurane exposure group (group SIRT3 + Sev). The cells were infected with SIRT3 overexpression adenovirus 100 pmol/well in SIRT3+ C group and SIRT3+ Sev group and with empty adenovirus 100 pmol/well in C group and Sev group. After 48 h of infection, the cells were routinely cultured for 48 h in C group and SIRT3+ C group, the cells were incubated with 3% sevoflurane for 2 h, once a day for 3 consecutive days, followed by routine culture for 48 h in Sev group and SIRT3+ Sev group. The contents of mitochondrial Ca 2+ and reactive oxygen species (mtROS) were measured by flow cytometry. The mitochondrial membrane potential (MMP) was measured by JC-1 probe. The mitochondrial ATP content was measured by luciferase luminescence method. The expression of SIRT3 was detected by Western blot. The expression of acetylated ATAD3A was detected by immunoprecipitation. The co-localization of endoplasmic reticulum and mitochondria was determined by confocal fluorescence microscopy, and the Manders co-localization coefficient was calculated to evaluate the development of MAMs. Results:Compared with group C, the contents of mitochondrial Ca 2+ and mtROS were significantly increased, the contents of MMP and mitochondrial ATP were decreased, the expression of SIRT3 was down-regulated, the expression of acetylated ATAD3A was up-regulated, and the development of MAMs was increased in group Sev ( P<0.05). Compared with Sev group, the contents of mitochondrial Ca 2+ and mtROS were significantly decreased, the contents of MMP and mitochondrial ATP were increased, the expression of SIRT3 was up-regulated, the expression of acetylated ATAD3A was down-regulated, and the development of MAMs was decreased in SIRT3 + Sev group ( P<0.05). Compared with SIRT3+ C group, the contents of mitochondrial Ca 2+ and mtROS were significantly increased, the contents of MMP and mitochondrial ATP were decreased, the expression of SIRT3 was down-regulated, the expression of acetylated ATAD3A was up-regulated, and the development of MAMs was increased in SIRT3+ Sev group ( P<0.05). Conclusions:The mechanism by which sevoflurane induces mitochondrial dysfunction in mouse microglia may be related to the inhibition of SIRT3/ATAD3A signaling pathway, leading to excessive development of MAMs.

Objective:To evaluate the effect of sevoflurane on mitochondrial-associated endoplasmic reticulum membranes (MAMs) in mouse microglia and the relationship with silent information regulator-related enzyme 3 (SIRT3)/adenosine triphosphatase family protein 3A (ATAD3A) signaling pathway.Methods:After normal culture of mouse microglia (BV-2 cell line), the cells were divided into 4 groups ( n=33 each) by a random number table method: empty adenovirus-control group (group C), empty adenovirus-sevoflurane exposure group (group Sev), SIRT3 overexpression adenovirus-control group (group SIRT3 + C) and SIRT3 overexpression adenovirus-sevoflurane exposure group (group SIRT3 + Sev). The cells were infected with SIRT3 overexpression adenovirus 100 pmol/well in SIRT3+ C group and SIRT3+ Sev group and with empty adenovirus 100 pmol/well in C group and Sev group. After 48 h of infection, the cells were routinely cultured for 48 h in C group and SIRT3+ C group, the cells were incubated with 3% sevoflurane for 2 h, once a day for 3 consecutive days, followed by routine culture for 48 h in Sev group and SIRT3+ Sev group. The contents of mitochondrial Ca 2+ and reactive oxygen species (mtROS) were measured by flow cytometry. The mitochondrial membrane potential (MMP) was measured by JC-1 probe. The mitochondrial ATP content was measured by luciferase luminescence method. The expression of SIRT3 was detected by Western blot. The expression of acetylated ATAD3A was detected by immunoprecipitation. The co-localization of endoplasmic reticulum and mitochondria was determined by confocal fluorescence microscopy, and the Manders co-localization coefficient was calculated to evaluate the development of MAMs. Results:Compared with group C, the contents of mitochondrial Ca 2+ and mtROS were significantly increased, the contents of MMP and mitochondrial ATP were decreased, the expression of SIRT3 was down-regulated, the expression of acetylated ATAD3A was up-regulated, and the development of MAMs was increased in group Sev ( P<0.05). Compared with Sev group, the contents of mitochondrial Ca 2+ and mtROS were significantly decreased, the contents of MMP and mitochondrial ATP were increased, the expression of SIRT3 was up-regulated, the expression of acetylated ATAD3A was down-regulated, and the development of MAMs was decreased in SIRT3 + Sev group ( P<0.05). Compared with SIRT3+ C group, the contents of mitochondrial Ca 2+ and mtROS were significantly increased, the contents of MMP and mitochondrial ATP were decreased, the expression of SIRT3 was down-regulated, the expression of acetylated ATAD3A was up-regulated, and the development of MAMs was increased in SIRT3+ Sev group ( P<0.05). Conclusions:The mechanism by which sevoflurane induces mitochondrial dysfunction in mouse microglia may be related to the inhibition of SIRT3/ATAD3A signaling pathway, leading to excessive development of MAMs.

Objective To investigate the effect of esmolol pretreatment on Toll like receptor-4 (TLR4)/nuclear factor-kappa-B (NF-кB) pathway in rats with repeated cerebral ischemia/reperfusion (IR) injury. Methods Forty-eight adult male Sprague-Dawley rats were randomly allocated into sham-operated group, IR group and esmolol group (n=16). Rats in the IR group and esmolol group were injected intravenously with esmolol at a dose of 200 g/(kg?min) or normal saline for one h before surgery, and then, bilateral common carotid arteries were clipped to establish the repeated IR injury models. Bilateral common carotid arteries in rats of sham-operated group were only isolated but not clipped, and injected intravenously with normovolemic normal saline for one h. Learning and memory abilities of rats were measured by Morris water maze test before, and one, 3 and 7 d after surgery. Rats were euthanized and hippocampus tissues were excised. The wet to dry (W/D) ratio of the hippocampus was tested. The permeability of blood-brain barrier was determined by Evans blue (EB) method. The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1β in the hippocampus were tested by enzyme linked immunosorbent assay (ELISA). The NF-кB p65 and TLR4 mRNA expressions in the hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The NF-кB p65 and TLR4 protein expressions in hippocampus were detected by Western blotting. Results As compared with those in the sham-operated group, the escape latency was significantly prolonged and swimming distance was signficantly longer in rats of IR group one, 3 and 7 d after surgery (P<0.05), the W/D ratio of the hippocampus and the content of EB in brain tissues were significantly increased in the IR group (P<0.05), the levels of TNF-α, IL-6 and IL-1β in hippocampus were significantly increased in the IR group (P<0.05), and the NF-кB p65 or TLR4 mRNA and protein expressions in the hippocampus of IR group were statistically higher (P<0.05). As compared with IR group, the escape latency and swimming distance of rats in the esmolol group were significantly shortened one, 3 and 7 d after surgery (P<0.05), the W/D ratio of the hippocampus and content of EB in brain tissues of esmolol group were significantly decreased (P<0.05), the levels of TNF-α, IL-6 and IL-1β in the hippocampus of esmolol group were signficantly lower(P<0.05), and the NF-кB p65 or TLR4 mRNA and protein expressions in hippocampus of esmolol group were statistically lower in the esmolol group (P<0.05). Conclusion Esmolol preconditioning can alleviate cerebral injury and improve learning and memory abilities of rats with repeated cerebral IR injury, which may be involved in alleviating inflammation and suppressing TLR4/NF-кB pathway.