Association between myocardial cell apoptosis and calpain-1/caspase-3 expression in rats with hypoxic-ischemic brain damage.

The present study aimed to investigate the association between myocardial cell apoptosis and calpain-1/caspase-3 expression in a rat model of hypoxic-ischemic brain damage (HIBD). A total of 64 newborn rats were divided into control (n=8; sacrificed on day 7) and HIBD groups (n=56). HIBD group rats were sacrificed 2, 12 or 24 h, or 2, 3, 5 or 7 days following HIBD (n=8/group). A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to detect myocardial apoptotic cells and calculate the apoptosis index (AI), reverse transcription-polymerase chain reaction was performed to detect myocardial calpain-1/caspase-3 mRNA expression levels and a western blot analysis was conducted to detect calpain­1 protein expression levels. The correlations between calpain­1 and caspase­3 expression levels and AI were analyzed. The results demonstrated that apoptotic myocardial cells in the HIBD groups were markedly increased compared with the control group, with AI peaking in the day 3 group. Caspase­3 and calpain­1 mRNA expression levels were increased from 2 and 12 h following HIBD, respectively, with the most elevated levels in the day 2 group. Compared with the control group, calpain­1 protein expression levels were increased from 2 h, with the greatest expression levels in the day 3 group (P<0.05). Calpain­1 mRNA and protein (76/80 kDa) expression levels demonstrated positive linear correlations with AI (r=0.786, P=0.001; and r=0.853, P=0.001, respectively) Caspase-3 mRNA expression levels were positively correlated with AI (r=0.894; P=0.001). In conclusion, the present study demonstrated that in rats with HIBD, there is a positive correlation between increased apoptosis of myocardial cells and expression levels of calpain-1 and caspase-3.

PKCε regulates contraction-stimulated GLUT4 traffic in skeletal muscle cells.

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc-epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol-induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF-109203X, Gö6983, and Ro-31-8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated-EAVSLKPT), but not one of PKCδ (myristoylated-SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol-induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA-mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction-stimulated GLUT4 traffic in muscle cells.

Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4myc skeletal muscle cells.

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C(2)C(12) myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 beta-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.

[Protective and curative effects of prophylactic administration of pulmonary surfactant on neonatal respiratory distress syndrome].

OBJECTIVE: To evaluate the protective and curative effects of prophylactic administration of pulmonary surfactant (PS) on neonatal respiratory distress syndrome (NRDS). METHODS: One hundred neonates aged 0.5 h after birth, with the gestational age < 32 w, birth weight < 1500 g, and number of gastric, stable microbubble < or = 7/mm(2) by gastric stable microbubble test (SMT), but without clinical or radiological manifestations of RDS at the admission, were randomly divided into 2 equal groups: prophylactic group (PG), receiving curosurf, a product of PS, immediately after admission; and non-prophylactic group (N-PG), receiving curosurf only after development of RDS. RESULTS: One hour after the administration of PS, the PaO(2), a/APO(2), pH, and PaO(2) of the PG group were 86.2 mm Hg +/- 8.1 mm Hg, 0.30 +/- 0.04, 7.38 +/- 0.06, and 178 +/- 37, all significantly higher than those of the non-PG group (all P < 0.01), and the PaCO(2) of the PG group was 37.3 mm Hg +/- 9.8 mm Hg, significantly lower than that of the non-PG group (53.6 mm Hg +/- 11.1 mm Hg, P < 0.01). In comparison with the level before the administration of PS (0.75 +/- 0.06), the level of FiO(2) of the 47 pediatric patients receiving mechanical ventilation decreased after the administration of curosurf time-dependently, e.g., was 0.50 +/- 0.09, 0.34 +/- 0.06, and 0.25 +/- 0.07 8, 48, and 96 hours after the administration. In comparison with the level before the administration of curosurf 9.0 +/- 1.0 cm H2O, the level of mean airway pressure (MAP) decreased time-dependently after the administration, e.g., were 7.5 +/- 0.8 and 6.0 +/- 0.3 48 and 96 hours after the administration (all P < 0.01). Compared with that before the administration of curosurf (3.02 +/- 0.2), the X-ray chest score decreased time-dependently after the administration of curosurf, e.g., were 1.89 +/- 0.34, 1.82 +/- 0.33, and 1.17 +/- 0.42 6, 12, and 72 hours after the administration (all P < 0.01). The RDS rate of the PG group was 30%, significantly lower than that of the non-PG group (P < 0.01). The severe case rate of the PG group was 20%, significantly lower than that of the N-PG group (53%, P = 0.01). The mortally of the PG group was 0, significantly lower than that of the non-PG group (P < 0.05). The total times of supplemental oxygen administration, assisted ventilation and hospitalization of the 47 patients with RDS in the PG group were significantly shortened compared with the RDS patients in the N-PG group [(3.6 +/- 1.7) d vs. (5.9 +/- 3.6) d, P < 0.05; (8.6 +/- 5.5 d vs. (14.1 +/- 6.2) d, P < 0.01; and (20.5 +/- 10.0) d vs. (32.8 +/- 17.8) d, P < 0.05). CONCLUSION: Prophylactic administration of PS to the preterm neonates with high risk of RDS effectively decreases the incidence of RDS, development of severe cases and mortality, shorten the disease course, the duration of supplemental oxygen administration and assisted ventilation, thus decreasing the potential morbidity associated with long-term oxygen supplement and assisted ventilation.

[Expression of and relationship between caspase-1 and interleukin-18 mRNA in brain tissue of hypoxic-ischemic neonatal rats].

OBJECTIVE: Caspase-1 is a member of cysteinyl aspartate specific protease family and it plays important roles in the pathophysiology of many diseases for its proinflammatory and proapoptotic peculiarity. Interleukin (IL)-18, one of its substrates, is a pleiotropic cytokine and processed by caspase-1 to become fully bioactive. The aim of this study was to investigate the relationship and effect of caspase-1 and IL-18 mRNA expressions after hypoxic-ischemic brain damage (HIBD) in neonatal rats. METHODS: Totally 112 seven-day-old Wistar rats were assigned to control group, HIBD 3 h, 8 h, 24 h, 3 d, 6 d and 14 d groups via complete randomization (n = 16 per group), and the model of HIBD was induced by unilateral carotid ligation followed by timed exposure to 8% oxygen. In each group, 8 rats were used for measuring the mRNA for caspase-1 and IL-18 in the cerebral cortex by semi-quantitative RT-PCR, and the brains of another 8 rats were observed for light microscopic changes by HE staining. RESULTS: The expression of caspase-1 mRNA was low in control group (0.2918 +/- 0.0809). After HIBD, the level of caspase-1 mRNA in ischemic cortex began to increase at 24 h (0.5222 +/- 0.0941, P < 0.01 vs. control), peaked at 6 d (0.7886 +/- 0.0480, P < 0.01 vs. the other groups) and decreased at 14 d (0.5314 +/- 0.1272). The level of IL-18 mRNA was 0.3218 +/- 0.0466 in control group. After HIBD, the expression of mRNA for IL-18 increased progressively at 24 h to 6 d (24 h: 0.5823 +/- 0.0740; 3 d: 0.6976 +/- 0.1073; 6 d: 0.9110 +/- 0.0647, P < 0.01 vs. control), reached its greatest level at 6 d (P < 0.01 vs. other groups). The expression of IL-18 mRNA after HIBD showed a close correlation to caspase-1 in time (r = 0.871, P < 0.01). Histological study showed that the degenerated and necrotic neurons increased progressively at 1 d to 6 d after HIBD, at the same time the proliferation of glial cells appeared. CONCLUSION: The increased expression of caspase-1 and IL-18 mRNA after HIBD, regularity of which was consistent with the time frame for development of brain injury, implied that they may play important roles in the pathogenesis of HIBD in neonatal rats.

[Expression of caspase - 1 after hypoxic-ischemic brain damage].

OBJECTIVE: To study the expression of caspase-1 mRNA in the cerebral cortex after hypoxic-ischemic brain damage(HIBD) in neonatal rats. METHODS: One hundred and twelve 7-day-old Wistar rats were randomly assigned to control group, HIBD 3 hours, 8 hours, 24 hours, 3 days, 6 days and 14 days groups (n=16 in each group), and standardized HIBD was given. In each group, 8 rats were used to assess the mRNA expression of caspase-1 in cerebral cortex by semi-quantitative reverse transcription-polymerase reaction, and another 8 rats were used to study histological changes with hematoxylin-eosin(HE) staining. RESULTS: The expression of caspase-1 mRNA was observed in the cerebral cortex in both control and HIBD groups. After HIBD, the level of caspase-1 mRNA in ischemic cortex began to increase at 24 hours (P<0.01 vs. controls), peaked at 6 days (P<0.01 vs. other groups) and decreased at 14 days. Histological study showed that the degenerated and necrotic neurons were increased progressively from 1 day to 6 days after HIBD, together with proliferation of glial cells. CONCLUSION: The increased expression of caspase-1 mRNA after HIBD, which was consistent with the time frame of the development of brain injury, indicates that it might play an important role in pathogenesis of HIBD in neonatal rats.

[Expression of neuronal nitric oxide synthase in brain tissue of hypoxic-ischemic neonatal rat and the cerebral protective effect of neuronal nitric oxide synthase inhibitor 7-nitroindazole].

OBJECTIVE: To explore the effect of nNOS and its selective inhibitor 7-NI in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS: The model of HIBD in neonatal rat was made and the expression of nNOS mRNA was measured at different time points and compared with the control group. The levels of NOS, NO, MDA, SOD were also assayed at different time points after aderministering 7-NI, and compared with the HIBD group and the control group. In addition, we observed the degree of apoptosis after 24 h of HIBD. RESULTS: Expression of nNOS mRNA was higher at 2 h and 6 h after hypoxia than those in the control group (P < 0.05) and reached more at 6 h (P < 0.05). Concentrations of NOS, NO, MDA increased after HIBD, but concentration of SOD decreased (P < 0.01); 7-NI reduced the levels of NOS, NO, MDA, and level of SOD was higher than those in the HIBD group (P < 0.05). There was no apoptosis in the control group, 7-NI could effectively inhibit the degree of apoptosis after HIBD (P < 0.001). CONCLUSION: nNOS has an important role in the pathogenesis of neonatal rats with HIBD, its specific inhibitor 7-NI can protect brains from hypoxic-ischemic damage through anti-oxidation and prohibiting apoptosis.