Protective effects and mechanism of insulin on brain in septic rats

Abstract

Objective To investigate the protective effects and mechanism of insulin(INS) on brain in septic rats, and explore the possible role of uncoupling protein 2 (UCP2) in these effects. Methods Fifty male specific pathogen free(SPF) Sprague-Dawley rats were randomly divided into normal control (CN) group(n=10), lipopolysaccharide(LPS) group(n=20) and INS group (n=20) according to random number table.The septic rat model was established through an intraperitoneal injection of 15 mg/kg LPS of gram- negative bacteria.The rats in the INS group received a 1 U/kg INS injection subcutaneously 30 minutes before the injection of LPS, and the rats in the CN group were given equivalent 9 g/L saline in the same way.Eight rats in each group were killed, and their cerebral cortex were collected after the injection of LPS for 24 h. Pathological change of cerebral cortex was detected by Hematoxylin-Eosin(HE) staining.The cerebral cortex mitochondia were extracted for detecting the levels of reactive oxygen species(ROS), malondialdehyde (MDA) and the activity of superoxide dismutase(SOD). Neuronal apoptosis was detected by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling staining.UCP2 mRNA expression was detected by quantitative real-time(RT)-PCR.Apoptosis-associated protein B lymphocyte tumor-2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved cysteinyl aspartate specific protease(cleaved Caspase-9) and UCP2 protein expression were determined by Western blot. Results (1)Compared with the CN group, obvious abnormal pathological change was revealed by HE staining in cerebral cortex of rats in the LPS group and the INS group, but the pathological change was attenuated in the INS group compared with the LPS group.(2)Compared with the CN group, the levels of mitochondrial ROS[(210.01±14.09) RFU vs.(49.06±7.28) RFU] and MDA[(2.19±0.18) nmol/mg pro vs.(1.25±0.11)nmol/mg pro]in the LPS group significantly increased, whereas SOD activity significantly decreased [(238.49±35.60) U/g pro vs.(446.66±24.90)U/g pro], and the differences were significant(all P<0.05). Compared with the LPS group, the levels of ROS [(152.69±15.83) RFU vs.(210.01±14.09) RFU] and MDA[(1.55±0.14) nmol/mg pro vs.(2.19±0.18) nmol/mg pro] in the INS group decreased, while SOD activity increased[(327.8±23.26) U/g pro vs.(238.49± 35.60) U/g pro], and the differences were significant(all P<0.05). (3)Compared with the CN group, the neuronal apoptosis index of cortex in the LPS group was elevated[(54.16±6.84)% vs.(5.45±1.43)%], while the expression of Bcl-2 decreased (627±0.018 vs.0.739±0.020), but the expressions of Bax(0.768±0.019 vs.0.520±0.010) and cleaved Caspase-9(0.739±0.016 vs.0.467±0.030) increased, and the differences were significant(all P<0.05). Compared with the LPS group, the neuronal apoptosis index of cortex in the INS group decreased [(33.30±3.07)% vs.(54.16±6.84)%], but the Bcl-2 expression increased (0.743±0.022 vs.0.627±0.018), and Bax (0.687±0.034 vs.0.768±0.019) and cleaved Caspase-9(0.551±0.013 vs.0.739±0.016) were reduced, and the differences were significant (all P<0.05). (4)Compared with the CN group, the mRNA (2.248±0.155 vs.1.000±0.100) and protein expression of UCP2 (0.659±0.016 vs.0.599±0.018) were elevated in the LPS group.Compared with the LPS group, the UCP2 mRNA (2.944±0.117 vs.2.248±0.155) and UCP2 protein (0.719±0.018 vs.0.659±0.016) increased, and the differences were significant(all P<0.05). Conclusions INS can protect the brain of septic rats through alleviating mitochondrial oxidative stress and inhibiting the mitochondrial-initiated apoptotic pathway to reduce neuronal apoptosis.INS upregulates UCP2 expression in the brain of septic rats, which may play a role in the protective effects mentioned above. Key words: Sepsis; Brain; Insulin; Reactive oxygen species; Oxidative stress; Apoptosis; Uncoupling protein 2