Sevoflurane preconditioning alleviates myocardial ischemia reperfusion injury through mitochondrial NAD+-SIRT3 pathway in rats. / 七氟醚预处理通过线粒体NAD+-SIRT3é€šè·¯å‡è½»å¤§é¼ å¿ƒè‚Œç¼ºè¡€å†çŒæ³¨æŸä¼¤.

OBJECTIVES: Myocardial ischemia reperfusion injury (IRI) occurs occasionally in the process of ischemic heart disease. Sevoflurane preconditioning has an effect on attenuating IRI. Preserving the structural and functional integrity of mitochondria is the key to reduce myocardial IRI. Silent information regulator 3 (SIRT3), a class of nicotinamide adenine dinucleotide (NAD+) dependent deacetylases, is an important signal-regulating molecule in mitochondria. This study aims to explore the role of mitochondrial NAD+-SIRT3 pathway in attenuating myocardial IRI in rats by sevoflurane preconditioning. METHODS: A total of 60 male Sprague Dawley (SD) rats were randomly divided into 5 groups (n=12): A sham group (Sham group), an ischemia reperfusion group (IR group), a sevoflurane preconditioning group (Sev group, inhaled 2.5% sevoflurane for 30 min), a sevoflurane preconditioning+SIRT3 inhibitor 3-TYP group (Sev+3-TYP group, inhaled 2.5% sevoflurane for 30 min and received 5 mg/kg 3-TYP), and a 3-TYP group (5 mg/kg 3-TYP). Except for the Sham group, the IR model in the other 4 groups was established by ligating the left anterior descending coronary artery. The size of myocardial infarction was determined by double staining. Serum cardiac troponin I (cTnI) level was measured. The contents of NAD+ and ATP, the activities of mitochondrial complexes I, II, and IV, the content of MDA, the activity of SOD, and the changes of mitochondrial permeability were measured. The protein expression levels of SIRT3, SOD2, catalase (CAT), and voltage dependent anion channel 1 (VDAC1) were detected by Western blotting. The ultrastructure of myocardium was observed under transmission electron microscope. MAP and HR were recorded immediately before ischemia (T0), 30 min after ischemia (T1), 30 min after reperfusion (T2), 60 min after reperfusion (T3), and 120 min after reperfusion (T4). RESULTS: After ischemia reperfusion, the content of NAD+ in cardiac tissues and the expression level of SIRT3 protein were decreased (both P<0.01), and an obvious myocardial injury occurred, including the increase of myocardial infarction size and serum cTnI level (both P<0.01). Correspondingly, the mitochondria also showed obvious damage on energy metabolism, antioxidant function, and structural integrity, which was manifested as: the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level and mitochondrial permeability were increased (all P<0.01). Compared with the IR group, the content of NAD+ in cardiac tissues and the expression level of SIRT3 protein were increased in the Sev group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were decreased in the Sev group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were increased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were decreased in the Sev group (all P<0.01). Compared with the Sev group, the content of NAD+ in cardiac tissues and the expression level of SIRT3 protein were decreased in the Sev+3-TYP group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were increased in the Sev+3-TYP group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were increased in the Sev+3-TYP group (all P<0.01). CONCLUSIONS: Sevoflurane preconditioning attenuates myocardial IRI through activating the mitochondrial NAD+-SIRT3 pathway to preserve the mitochondrial function.

Detergent-insoluble inclusion constitutes the first pathology in PFN1 transgenic rats.

Mutation of profilin 1 (PFN1) can cause amyotrophic lateral sclerosis (ALS). To assess how PFN1 mutation causes the disease, we created transgenic rats with human genomic DNA that harbors both the coding and the regulatory sequences of the human PFN1 gene. Selected transgenic lines expressed human PFN1 with or without the pathogenic mutation C71G at a moderate and a comparable level and in the similar pattern of spatial and temporal expression to rat endogenous PFN1. The artificial effects of arbitrary transgene expression commonly observed in cDNA transgenic animals were minimized in PFN1 transgenic rats. Expression of the mutant, but not the wild type, human PFN1 in rats recapitulated the cardinal features of ALS including the progressive loss of motor neurons and the subsequent denervation atrophy of skeletal muscles. Detergent-insoluble PFN1 inclusions were detected as the first pathology in otherwise asymptomatic transgenic rats expressing mutant human PFN1. The findings suggest that protein aggregation is involved in the neurodegeneration of ALS associated with PFN1 mutation. The resulting rat model is useful to mechanistic study on the ALS.

Protective effect of ginsenoside Rg1 on lidocaine-induced apoptosis.

Lidocaine, as an anesthetic substance, is often used for surface and spinal anesthesia. However, studies have shown that lidocaine may induce transient neurological symptoms and cauda equina syndrome. In the present study the effects of the ginsenoside Rg1 (Rg1) on lidocaine­induced apoptosis were assessed in Jurkat cells using flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The data showed that incubation with Rg1 provides protection against lidocaine­induced apoptosis in cultured Jurkat cells. In order to investigate the effect of Rg1 on the apoptosis pathway, caspase 3 gene expression was determined. The results suggested that the protective effect of Rg1 on lidocaine­induced apoptosis is mediated by altering the level of B­cell lymphoma­2 (BCL­2) family proteins and downregulating caspase­3 expression. In conclusion, the present study demonstrated that incubation with Rg1 provides protection against lidocaine­induced apoptosis in cultured Jurkat cells. In addition, the study demonstrated that Rg1 is a notable antiapoptotic molecule that is capable of blocking the caspase­dependent signaling cascade in Jurkat cells, and that the protective effect of Rg1 on lidocaine­induced apoptosis is mediated by altering levels of BCL­2 family proteins and downregulating caspase­3 expression. The present study provides the basis for understanding and evaluating the effect of Rg1 in the in vivo treatment of lidocaine-induced transient neurological symptoms and cauda equina syndrome by lidocaine.

[Construction of RNAi vector of dopamine D1 receptor and identification of its silencing effects].

OBJECTIVE: To construct dopamine D1 receptor (DRD1) expression interference vectors to study the role of DRD1 in nerve cells and lay a foundation for drug development in anti-convulsion. METHODS: Based on DRD1 gene sequence in GenBank, 10 interfere vectors of DRD1 were designed. Liposomal was used to transfect NG-108-15 and the transfect effect was assayed by GFP. With realtime PCR and Western blot, the DRD1 expression was detected. RESULTS: The 10 constructed interfere vectors transfected into NG-108-15 cells by liposomal method and inhibited DRD1 mRNA and protein expression. DRD1 mRNA expression in NG-108-15 cells transfected with pGPU6-GFP-Neo-si-DRD1-5 was the lowest whereas DRD1 protein expression in NG-108-15 cells transfected with pGPU6-GFP-Neo-si-DRD1-1, -2, -6, -7 was the lowest. CONCLUSION: DRD1 expression interference vector is successfully constructed.

[Limb ischemic preconditioning reduces rabbit hepatic ischemia-reperfusion injury through inhibition the phosphorylation of proteins in the MAPK signal pathway in the late phase].

OBJECTIVE: To investigate the liver protection mechanisms of MAPK signaling pathway of limb ischemia preconditioning in the late phase. METHODS: Thirty-six adult male New Zealand white rabbits, weighing 1.8-2.0 kg, were randomly divided equally into 3 groups: group C (sham operation), group L (liver ischemia-reperfusion 24 h after limb ischemia preconditioning), group IR (liver ischemia-reperfusion without limb ischemia preconditioning). Serum alanine transaminase (ALT) was measured during ischemia reperfusion. The tissue and cell injury of liver were examined by optical and electron microscopy. Activation of P38MAPK, P44/P42MAPK, and JNK in hepatic tissue was assessed by western blot after 30 min of reperfusion. RESULTS: Serum ALT and cell injury in the liver as examined by optical and electron microscopy was decreased in group L as compared with the group IR. Phosphorylation of P38MAPK, P44/ P42MAPK, and JNK were all increased significantly after 30 min of reperfusion. Phosphorylation of P38MAPK and JNK was reduced by limb ischemia pre-treatment. CONCLUSION: Limb ischemia pre-treatment can induce the late phase of preconditioning in rabbit liver through the inhibition of the phosphorylation of P38MAPK and JNK.

Expression of bone morphogenetic proteins and their receptors in the normal adult rat spinal cord.

OBJECTIVE: To observe the expression distribution of bone morphogenetic proteins (BMP) in the spinal cord of normal adult rats. METHODS: Expression of BMP2, BMP4, and BMP7, and their receptors BMPR Ia, BMPR Ib, and BMP II were detected by immunochemistry analysis in the spinal cord of normal adult rats. RESULTS: Expression of BMPR Ia or BMPR Ib was observed in the motor neurons of the anterior horn, sensory neurons of the dorsal horn, oligodentrocytes, some microglia, and some astrocytes. Expression of receptor BMPR II was found in the oligodentrocytes and motor neurons in the gray matter of anterior horn. It was also expressed in some glial fibrillary acidic protein (GFAP)-positive astrocytes in the white matter but not in the gray matter. BMP2 and BMP4 were not expressed in the spinal cord of normal adult rats by immunohistochemistry. BMP7 was expressed in all the APC-positive oligodentrocytes, all the NeuN-positive motor neurons in the anterior horn, and some astrocytes in the normal spinal cord. Phosphated pSmad 1/5/8 protein was expressed in all the oligodentrocytes, all the neurons, and some astrocytes, especially in the GFAP-positive astrocytes which were RC2-positive radial glia in the subventricular zone. CONCLUSION: BMP7, BMP receptors, and phosphated pSmad 1/5/8 are expressed in many types of cells whereas BMP2 and BMP4 are not expressed in the spinal cord of normal adult rats, which suggests an important function of BMP signal pathway in the neuron and glia of spinal cord.

[Effects of ulinastatin on cerebral inflammatory response during cardiopulmonary bypass].

OBJECTIVE: To investigate the effects of ulinastatin (UTI) on cerebral inflammatory response during cardiopulmonary bypass (CPB). METHODS: Twenty-four NYHA II-III patients (13 males and 11 females) aged 23-45 years, undergoing elective cardiac valve replacement under hypothermic CPB were randomly divided into 2 groups: ulinastatin group (Group U, n=12) and control group (Group C, n=12). In group U, UTI (1.2 x 10(4) U/kg) was given intravenously after the induction of anesthesia, 0.6 x 10(4) U/kg UTI was added to the priming solution, and 0.6 x 10(4) U/kg UTI was given about 5 min before the aortic decamping. In Group C, normal saline was given instead of UTI. Internal jugular vein was cannulated and the catheter was advanced retrogradely till jugular bulb. Blood samples were taken simultaneously from artery and jugular bulb after induction of anesthesia (T1), 60 min (T2) and 6 h (T3) after discontinuation of CPB for determination of TNFalpha, IL-6, IL-8 and IL-10. The juguloarterial gradients of these cytokines (deltaTNFalpha, deltaIL-6, deltaIL-8, and deltaIL-10) were calculated. RESULTS: In Group C, arterial levels of TNFalpha, IL-6, IL-8, IL-10 at T2 and T3, deltaTNFalpha, deltaIL-8 and deltaIL-10 at T2, deltaTNFalpha, deltaIL-6 and deltaIL-10 at T3 significantly increased (P < 0.01). deltaIL-8 increased at T3 (P < 0.05). In Group U, arterial levels of IL-6, IL-8, IL-10 at T2, arterial levels of IL-6, IL-8,IL-L-10 and deltaTNFalpha, deltaIL-8 at T3 significantly increased (P < 0.01). Arterial levels of TNFalpha at T2 and T3, deltaTNFalpha, deltaIL-10 at T2, deltaIL-6 at T3 increased (P < 0.05). Arterial levels of TNFalpha, IL-6 and deltaTNFalpha, deltaIL-8 at T2, arterial levels of TNFalpha and deltaIL-6 at T3 in Group U were lower than those in Group C (P < 0.05). Arterial levels of IL-6 at T3, IL-8 at T2 and T3 in Group U were significantly lower than those in Group C (P < 0.01). Arterial levels of IL-10 and deltaIL-10 at T3 in Group U were higher than those in Group C (P < 0.05). CONCLUSION: Systemic and cerebral activation of inflammatory response during CPB can be alleviated by ulinastatin.