Assisted exercise improves bone strength in very low birthweight infants by bone quantitative ultrasound.

AIM: To evaluate whether assisted exercise could prevent the development of osteopenia of prematurity, we performed assisted exercise in the study group of very low birthweight (VLBW) premature infants. METHODS: Sixteen premature infants with birthweight below 1500 g were enrolled in this study and randomly assigned into the exercise (n = 8) and control (n = 8) groups. Assisted exercise involved full extension and flexion range of motion of the upper and lower extremities by a trained nurse with a schedule of 5 days a week for a total of 4 weeks. Bone strength was determined by quantitative ultrasound measurement of tibial bone speed of sound every 2 weeks during the study period. RESULTS: No difference in gender, birthweight, and gestation age between the exercise and control groups was noted. There was statistically significant less tibial bone speed of sound decrease in the exercise group on the sixth and eighth week of life. During the study period, there were no statistically significant differences in blood biochemistry data, including calcium, phosphorus, magnesium, alkaline phosphatase, osteocalcin and osteoprotegerin, between the two groups. CONCLUSIONS: This study revealed that early assisted exercise could improve bone strength in very low birthweight infants. The biochemical markers of bone metabolism, osteocalcin and osteoprotegerin, could not be the indicators for early diagnosis of osteopenia of prematurity.

Heme oxygenase-1 suppresses the infiltration of neutrophils in rat liver during sepsis through inactivation of p38 MAPK.

To investigate the molecular mechanism underlying heme oxygenase-1 (HO-1)-modulated infiltration of neutrophils, the sepsis model of cecal ligation and puncture in Sprague-Dawley rats was used. In vivo induction and suppression of HO-1 were performed by pretreatment with cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX, respectively. Tricarbonyldichlororuthenium(II) dimer, [Ru(CO)3Cl2]2 (a carbon monoxide [CO] releaser), and hemoglobin (a CO scavenger) were used to examine the participation of HO-1/CO in the effect of CoPP pretreatment on formylated peptide (fMLP)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Anisomycin (a p38 MAPK activator) and SB203580 (a p38 MAPK inhibitor) were used to examine p38 MAPK mediation in the attenuation of fMLP-attracted migration by HO-1. The results demonstrated that zinc protoporphyrin IX and CoPP pretreatment conferred enhancing and inhibitory effects, respectively, on hepatic neutrophil infiltration. Pretreatment with CoPP inhibited fMLP-induced migration and p38 MAPK phosphorylation in neutrophils ex vivo. The [Ru(CO)3Cl2]2 stimulated whereas hemoglobin diminished the suppression of fMLP-induced p38 MAPK phosphorylation by CoPP. Moreover, anisomycin diminished the suppressive effects of CoPP pretreatment on fMLP-induced migration, actin polymerization, polarization, and migration speed of neutrophils. These results suggest that HO-1 in neutrophil attenuates its infiltration during sepsis via the inactivation of p38 MAPK. Understanding the mechanism that diminishes neutrophil infiltration by HO-1 may help prevent hepatic failure during sepsis.

Role of cAMP-response element-binding protein phosphorylation in hepatic apoptosis under protein kinase C alpha suppression during sepsis.

Previous studies have shown that a decrease in protein kinase C (PKC) alpha levels contributes to hepatic failure and/or apoptosis during sepsis, and suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which can modulate expression of Bcl-xL. However, the underlying molecular mechanism remains uncertain. In the present study, we examined whether a decrease in the nuclear PKCalpha levels causes hepatic apoptosis via modulation of cAMP-response element-binding protein (CREB) or nuclear factor-kappaB (NFkappaB), the crucial factors regulating the expression of prosurvival Bcl-xL. For polymicrobial sepsis induction, a cecal ligation and puncture model was used; at 9 or 18 h after CLP, experiments were terminated, referring as early or late sepsis, respectively. Additionally, PKCalpha was suppressed by stable transfection of antisense PKCalpha plasmid into a Clone-9 rat hepatic epithelial cell. The results showed that the nuclear PKCalpha was significantly decreased in the liver during sepsis, which was accompanied by decreases in phospho-CREB content, DNA-binding activity of CREB, and Bcl-xL expression. Likewise, the binding activity of NFkappaB increased significantly, which was associated with a decrease in cytosolic inhibitory-kappaBalpha content. The in vitro suppression of PKCalpha also resulted in decreases in the phospho-CREB content and DNA-binding activity, which were accompanied by down-regulation of Bcl-xL and apoptosis, but no significant alteration in NFkappaB-binding activity. The in vivo and in vitro results suggest that the suppression of PKCalpha results in a decreased CREB phosphorylation and subsequent down-regulation of Bcl-xL, which may contribute to the hepatic apoptosis during sepsis.

Early expression of heme oxygenase-1 in leukocytes correlates negatively with oxidative stress and predicts hepatic and renal dysfunction at late stage of sepsis.

Oxidative stress triggered by septic insult may be the major cause of multiple organ dysfunction syndrome (MODS) in intensive unit care patients. The inducible form of heme oxygenase-1 (HO-1) can be induced by cytokines, lipopolysaccharide, and reactive oxygen species during sepsis. These facts raise the question of whether the expression of HO-1 in leukocytes can indicate the level of oxidative stress of multiple organs in sepsis. Clinical peritonitis was simulated in an animal model by cecal ligation and puncture (CLP). The level of oxidative stress was examined by plasma lipid peroxidation (LPO). Liver function was analyzed by plasma aspartate aminotransferase, alanine aminotransferase, total bilirubin, and direct bilirubin. Lung function was evaluated by severity of edema. Renal function was measured by blood urea nitrogen and creatinine. The correlation between early HO-1 induction and LPO level or organ functional indicators of the same rat at late sepsis was analyzed by linear regression. The results showed that the protein content of HO-1 increased at 9 h after CLP, whereas expression of HO-1 mRNA in leukocytes was significantly increased (P < 0.01) at 6 h after CLP. Plasma level of LPO and the indices of hepatic, pulmonary, and renal function were significantly increased at 18 h after CLP. Moreover, highly negative correlations were observed between HO-1 mRNA expression at 6 h after CLP and level of LPO or severity of hepatic/renal dysfunction at 18 h after CLP. These results suggest that early HO-1 mRNA expression in leukocytes may represent oxidative stress and may predict the severity of liver and renal dysfunction during sepsis.

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line.

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.

Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line.

Inactivation of protein kinase C (PKC)alpha plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKCalpha inactivation mediates the apoptosis, PKCalpha was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKCalpha and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKCalpha and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKCalpha were decreased by 54.5% and 41.4%, respectively, after PKCalpha antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKCalpha-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKCalpha and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKCalpha declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.