Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats.

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

[Protective effects of microRNA-181b on aged rats with sepsis-induced hippocampus injury in vivo].

OBJECTIVE: To investigate the protective effect of microRNA-181b (miR-181b) on aged rats with sepsis-induced hippocampus injury in vivo. METHODS: Seventy-five male healthy old Sprague-Dawley (SD) rats were randomly divided into five groups (n = 15) using a random number table: sham operation group (Sham group), sepsis group [cecal ligation and puncture (CLP) group], miR-181b Agomir+CLP group (Ag+CLP group), miR-181b Antagomir+CLP group (An+CLP group) and normal saline (NS) control group (NS+CLP group). Rats sepsis model was reproduced by CLP, and in Sham group, the cecum of rats was separated only after abdominal operation without ligation or perforation. The rats in Ag+CLP group were given miR-181b Agomir 10 µL via lateral ventricle at 24 hours before CLP, the rats in An+CLP group were given 10 µL miR-181b Antagomir, and those in NS+CLP group were given 10 µL NS. At 6, 12, 24 hours after CLP, 5 rats of each group were sacrificed randomly, and hippocampus were harvested. The expression of miR-181b in hippocampus was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression of nuclear factor-ΚB p65 (NF-ΚB p65) was determined by Western Blot. The contents of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with Sham group, the expression of miR-181b in hippocampus of CLP group was significantly decreased at 6 hours after CLP (2-ΔΔCT: 0.70±0.12 vs. 0.98±0.06, P < 0.05), and the expressions of NF-ΚB p65, IL-1ß and TNF-α were significantly increased [NF-ΚB p65/Histone H3: 0.30±0.03 vs. 0.07±0.01, IL-1ß (ng/L): 120.39±8.02 vs. 50.55±11.12, TNF-α (ng/L): 59.48±4.60 vs. 40.31±3.96, all P < 0.05], this trend was continued till 24 hours, and these results indicated that there was obvious inflammation in hippocampus of sepsis rats. There was no statistical difference in the expression of miR-181b, NF-ΚB p65, IL-1ß or TNF-α in hippocampus between NS+CLP group and CLP group, which indicated that injection of NS into the rat lateral ventricle, had not aggravated the damage degree of hippocampus. Compared with CLP group, the expression of miR-181b in hippocampus of Ag+CLP group was significantly increased at 6 hours after CLP (2-ΔΔCT: 1.87±0.25 vs. 0.70±0.12, P < 0.05), and the expressions of NF-ΚB p65, IL-1ß and TNF-α were significantly lowered [NF-ΚB p65/Histone H3: 0.16±0.03 vs. 0.30±0.03, IL-1ß (ng/L): 73.76±8.17 vs. 120.39±8.02, TNF-α (ng/L): 49.52±4.77 vs. 59.48±4.60, all P < 0.05]. There was no statistical difference in the expression of miR-181b in hippocampus between An+CLP group and CLP group (2-ΔΔCT: 0.80±0.08 vs. 0.70±0.12 at 6 hours, 0.48±0.03 vs. 0.46±0.05 at 12 hours, 0.61±0.09 vs. 0.63±0.07 at 24 hours, all P > 0.05), but the expressions of NF-ΚB p65, IL-1ß and TNF-α in hippocampus at 6 hours after CLP of An+CLP group were significantly higher than those of CLP group [NF-ΚB p65/Histone H3: 0.44±0.02 vs. 0.30±0.03, IL-1ß (ng/L): 134.21±5.78 vs. 120.39±8.02, TNF-α (ng/L): 67.62±5.86 vs. 59.48±4.60, all P < 0.05], this trend was continued till 24 hours after CLP. The above results showed that overexpression of miR-181b might attenuate the inflammation of hippocampus through down-regulation of NF-ΚB, IL-1ß and TNF-α. CONCLUSIONS: The expression of hippocampal miR-181b was significantly decreased in septic rats. Up-regulation of miR-181b could inhibit the activation of NF-ΚB signal pathway and the release of the inflammatory cytokine IL-1ß and TNF-α stimulated by sepsis, and alleviate the inflammatory reaction and hippocampus injury in rat with sepsis.

Simple simultaneous determination of butylated hydroquinone (TBHQ) and butylated hydroxyanisole (BHA) antioxidants in oil using high-performance liquid chromatography with chemiluminescence detection.

A highly sensitive and convenient high-performance liquid chromatography technique coupled with chemiluminescence detection for the simultaneous determination butylated hydroquinone (TBHQ) and butylated hydroxyanisole (BHA) in oil is established. The detection is based on the inhibitory effect on the CL reaction between luminol and potassium ferricyanide in an alkaline medium. Samples were separated through a reverse-phase C18 column using a mobile phase of methanol and water (80: 20, v/v) at a flow rate of 0.5 mL/min. The effects of various parameters including mobile phase, flow rate and chemiluminescence regent were studied. Under optimum conditions, both TBHQ and BHA showed good linear relationships in the range 1 × 10(-7) -1 × 10(-5) g/mL with detection limits of 24 and 33 ng/mL, respectively. The proposed method is simple and sensitive, with low costs. The method was successfully applied for the quantification of TBHQ and BHA in sesame oil. The possible inhibition mechanism is also discussed briefly.