Neuregulin-1ß Protects the Rat Diaphragm during Sepsis against Oxidative Stress and Inflammation by Activating the PI3K/Akt Pathway.

Sepsis-induced diaphragm dysfunction (SIDD) which is mainly characterized by decrease in diaphragmatic contractility has been identified to cause great harms to patients. Therefore, there is an important and pressing need to find effective treatments for improving SIDD. In addition, acetylcholinesterase (AChE) activity is a vital property of the diaphragm, so we evaluated both diaphragmatic contractility and AChE activity. Though neuregulin-1ß (NRG-1ß) is known to exert organ-protective effects in some inflammatory diseases, little is known about the potential of NRG-1ß therapy in the diaphragm during sepsis. Our study was aimed at exploring the effects of NRG-1ß application on diaphragmatic contractility and AChE activity during sepsis. Proinflammatory cytokines, muscle injury biomarkers in serum, contractile force, AChE activity, proinflammatory cytokines, oxidative parameters, histological condition, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling proteins in the diaphragm were measured and compared between nonseptic and septic groups with or without NRG-1ß treatment. In vitro, the effects of NRG-1ß on reactive oxygen species (ROS) production in the lipopolysaccharide- (LPS-) stimulated L6 rat muscle skeletal cells with or without the Akt inhibitor MK-2206 were detected. NRG-1ß inhibited proinflammatory cytokine release and muscle injury biomarkers soaring in serum and improved the sepsis-induced diaphragm dysfunction and AChE activity decrease significantly during sepsis. Meanwhile, the inflammatory response, oxidative stress, pathological impairment, and cell apoptosis in the diaphragm were mitigated by NRG-1ß. And NRG-1ß activated the PI3K/Akt signaling in the diaphragm of septic rats. Elevated ROS production in the LPS-stimulated L6 rat skeletal muscle cells was reduced after treatment with NRG-1ß, while MK-2206 blocked these effects of NRG-1ß. In conclusion, our findings underlined that NRG-1ß could reduce circulating levels of proinflammatory cytokines in rats with sepsis, adjust diaphragmatic proinflammatory cytokine level, mitigate diaphragmatic oxidative injury, and lessen diaphragm cell apoptosis, thereby improving diaphragmatic function, and play a role in diaphragmatic protection by activating PI3K/Akt signaling.

The Role of Oxidative Stress in Decreased Acetylcholinesterase Activity at the Neuromuscular Junction of the Diaphragm during Sepsis.

Our recent study demonstrated that acetylcholinesterase (AChE) activity at the neuromuscular junction (NMJ) of the diaphragm decreased during sepsis. However, the mechanisms were not clearly identified. In this study, we aimed to investigate whether the decreased AChE activity was related to oxidative stress by observing AChE activity in different grades of sepsis induced by caecal ligation and puncture (CLP). At 24 h after surgery, an assay of thiobarbituric acid reactive species (TBARS) and protein carbonyls, as well as the myeloperoxidase (MPO), superoxide dismutase (SOD), and catalase (CAT) activity, was conducted. AChE activity was measured by biochemical and histological detection. AChE and CAT activity in the diaphragm decreased, while the contents of TBARS and protein carbonyls, the activity of MPO and SOD, and the SOD/CAT ratios increased. The above changes were much more significant in the mid-grade septic group than in the low-grade septic group. The colour of the AChE activity staining at the NMJ gradually lightened from the sham surgery group to the mid-grade septic group. AChE activity was significantly negatively correlated with the levels of TBARS and protein carbonyls. We consider that oxidative stress might be responsible for decreased AChE activity in the diaphragms of rats induced with sepsis.

Effects of propofol on ischemia-induced ventricular arrhythmias and mitochondrial ATP-sensitive potassium channels.

AIM: To investigate the potential of propofol in suppressing ventricular arrhythmias and to examine whether mitochondrial ATP-sensitive potassium channels are involved. METHODS: Male Sprague-Dawley rats were pretreated with intravenous infusion of propofol (Prop), a selective mitochondrial KATP channel inhibitor 5-hydroxydecanoate (5-HD), propofol plus 5-HD (Prop+5-HD), a potent mitochondrial K(ATP) channel opener diazoxide (DZ) or NS, respectively. The dosage of each drug was 10 mg/kg. The animals then underwent a 30 min-ligation of the left anterior descending artery. The severity of arrhythmias, the incidence of ventricular fibrillation (VF), and the time of the first run of ventricular arrhythmias were documented using an arrhythmia scoring system. Mitochondrial membrane potential (ΔΨm) was measured in freshly isolated rat cardiomyocytes with a fluorescence microscope. RESULTS: The arrhythmia scores in the Prop and DZ group were 2.6(0-5) and 2.4(0-5), respectively, which were significantly lower than that in the control group [4.9(2-8)]. VF was not observed in both Prop and DZ groups. The first run of ventricular arrhythmias was significantly postponed in the Prop group (10.5±2.2 vs 7.3±1.9 min). Bracketing of propofol with 5-HD eliminated the anti-arrhythmic effect of propofol. In isolated rat cardiomyocytes, propofol (50 µmol/L) significantly decreased ΔΨm, but when propofol was co-administered with 5-HD, the effect on ΔΨm was reversed. CONCLUSION: Propofol preconditioning suppresses ischemia-induced ventricular arrhythmias in the rat heart, which are proposed to be caused by opening of mitochondrial K(ATP) channels.

[Protective effect of hypothermic propofol on ischemic spinal cords].

OBJECTIVE: To evaluate the protective effect of hypothermic propofol infused via the aorta against ischemia/reperfusion injury of spinal cords in rabbits. METHODS: Sixty New Zealand white rabbits were randomly divided into six groups (n=10 in each group). The infrarenal circum-aortic clamping model was used in this study. During the 30-clamping time, 5 mL/kg of normal saline, 10% intralipid, propofol, 4 degrees C saline, 4 degrees C intralipid and 4 degrees C propofol were infused into the left femoral arteries of the rabbits in group SN, IPN, PN, SH, PH and IPH, respectively, at a rate 10 mL/(kg x h). The heart rates, blood pressures, respiratory rates and SPO2 were measured during the ischemic-reperfusion processes. The neurological status (Tarlov Scale system) were assessed 6 h, 24 h, and 48 h after the reperfusions. The spinal cords were harvested 48 h after the reperfusions for histological analysis. The concentrations of excitatory amino (EAA, aspartate and glutamate), malondialdehyde (MDA) and superoxide dismutas (SOD) in the harvested spinal cords were determined. RESULTS: Group PN, SH, PH and IPH had better neurological outcomes and less severe pathological changes than group SN and IPN (P<0.05). There were no significant differences between group SN and IPN (P>0.05). Group PN, SH, PH and IPH had lower concentrations of EAA in spinal cords than group SN and IPN (P<0.05). Group PH had the best neurological outcome, the least histopathological changes of spinal cords, and the lowest concentrations of EAA (P<0.05). Groups PN and PH had lower concentrations of malondialdehyde than group SN, IPN, SH and IPH. Groups PN and PH had higher concentrations of superoxide dismutas than group SN, IPN, SH and IPH (P<0.05). CONCLUSION: Both propofol and hypothermic liquids can protect spinal cords against ischemia/reperfusion injuries. Combined use of propofol and hypothermia results in significant recovery of spinal cord functions.

[The reperfusion injury model improvement and the tolerance time investigation of rabbit spinal cord ischemia under normothermia].

OBJECTIVE: This study is designed to improve the rabbit model of ischemic- reperfusion injury and determine the safe clamping duration relevant to the spinal cord tolerance to ischemia at normothermia. METHODS: 50 New Zealand white rabbits were assigned randomly to 5 groups (Group C20, C25, C30, C40 and C60, 10 rabbits in each group) according to different clamping durations, ranging from 20 min to 60 min. The rabbits were endotracheally intubated for ventilation, and their left ear arteries were catheterized for monitoring the mean artery pressure. The spinal cord ischemia was induced by infrarenal aorta occlusion. A catheter was inserted into the aorta distal clamped site for monitoring the distal artery pressure. The neurological functional status of animal was assessed with the Tarlov scale system (0 or 1 meaning the rabbit paraplegia), at the moment of revival, 6 h, 24 h, and 48 h after the reperfusion. After last scoring, the lumbar segments of spinal cord (L4-L6) were removed for pathological examination, and the normal motor neurons of anterior horn were counted. RESULTS: Forty-eight hours after the infusion, the severe neurological impairments were not detected in the rabbits whose aorta were only clamped for 20 min (Group C20). However, the rabbits in Group CSO became totally paraplegic, and the rabbits in Group C25 C30 or C40 developed the paraplegia at 30% , 80% or 90% respectively. The median number of normal motor neuron was 12. 5, 10 or 2 respectively in Group C20, C25 or C30, and 0 median number resulted in Group C40 and C60. CONCLUSION: The rabbit model of ischemic-reperfusion injury is successfully improved, of which the safe clamping duration without spinal cord injury is not more than 20 min at normothermia.

Mutation analysis of the Smad3 gene in human osteoarthritis.

Osteoarthritis (OA) is the most common joint disease worldwide. Recent studies have shown that targeted disruption of Smad3 in mouse results in OA. To reveal the possible association between the Smad3 gene mutation and human OA, we employed polymerase chain reaction-single strand conformation polymorphism and sequencing to screen mutations in all nine exons of the Smad3 gene in 32 patients with knee OA and 50 patients with only bone fracture. A missense mutation of the Smad3 gene was found in one patient. The single base mutation located in the linker region of the SMAD3 protein was A --> T change in the position 2 of codon 197 and resulted in an asparagine to isoleucine amino-acid substitution. The expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 in sera of the patient carrying the mutation were higher than other OA patients and controls. This is the first report showing that the Smad3 gene mutations could be associated with the pathogenesis of human OA.

[Techniques for producing transgenic animals and the recent development].

In this article the author reviews the currently prevailing techniques in producing transgenic animals with recount of those employing microinjection, retrovirus infection, embryonic stem cells, sperm vectors and nuclei transfer of somatic cells. The prospective of these techniques are also dealt with in brief.