Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids.

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the µ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCß3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion-Induced Acute Lung Injury.

Diverse clinical factors, including intestinal ischemia, contribute to acute lung injury (ALI), which has up to a 40% mortality rate. During the development of lung injury an immune response is elicited that exacerbates the lung insult. Neutrophils have been well studied in mediating the pulmonary insults through an assortment of mechanisms, such as release of granule contents and production of proinflammatory cytokines due to the overactivation of complement and cytokines. In this study, we found that enhanced endoplasmic reticulum (ER) stress was observed in infiltrated neutrophils in the early stage of an ALI mice model. In neutrophils, complement 5a (C5a) inspires strong ER stress through inositol-requiring kinase 1a and, to a less extent, the protein kinase R-like ER kinase signaling pathway. The granule release induced by C5a was ER stress mediated. Knowkdown of X-box-binding protein 1, a downstream signaling molecule of inositol-requiring kinase 1a, impaired granule release, based on myeloperoxidase production. Further analysis revealed that C5a induced ER stress by binding to C5a receptor in neutrophils. Using xbp(f/f) MRP8-cre mice in which X-box-binding protein 1 is deficient specifically in neutrophils and ER stress is deprived, we confirmed that ER stress in neutrophils was required for granule release in vivo and led to ALI, whereas dampening ER stress in neutrophils substantially alleviated ALI. Taken together, our results demonstrated that C5a receptor-mediated ER stress induced granule release in neutrophils, contributing to the development of ALI. This novel mechanism suggests a new potential therapeutic target in autophagy regulation for ALI.

Isoflurane Preconditioning Promotes the Survival and Migration of Bone Marrow Stromal Cells.

BACKGROUND: Preconditioning with the volatile anesthetic isoflurane exerts protective effects in animal models of ischemia. The cytoprotective effects of isoflurane are dependent on the expression of hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor that mediates cellular responses to hypoxia. METHODS: We investigated the effect of isoflurane preconditioning on bone marrow stromal cell (BMSC) survival and function. RESULTS: Short exposures to low isoflurane concentrations promoted in vitro survival and migration of BMSCs, whereas long exposures and high doses had the opposite effect. At specific doses and times, isoflurane upregulated the expression of HIF-1α and the stromal-derived factor-1 receptor CXCR4, and induced the activation of Akt, similar to hypoxia, and the effect of isoflurane was abrogated by silencing of HIF-1α or inhibition of PI3K/Akt signaling. In vivo experiments showed that isoflurane preconditioning increased the engraftment of BMSCs into the ischemic brain and improved functional recovery in a mouse model of stroke. CONCLUSION: Isoflurane preconditioning at specific doses and times improves the survival and function of BMSCs through the upregulation of CXCR4 via a mechanism involving HIF-1α expression and the PI3K/Akt pathway, suggesting that anesthetic preconditioning could be developed as a strategy to improve the efficiency of cell therapy.

Isoflurane preconditioning increases survival of rat skin random-pattern flaps by induction of HIF-1α expression.

BACKGROUND: Survival of random-pattern skin flaps is important for the success of plastic and reconstructive surgeries. This study investigates isoflurane-induced protection against ischemia of skin flap and the underlying molecular mechanism in this process. METHODS: Human umbilical vein endothelial cells (HUVECs) and human skin fibroblast cells were exposed to isoflurane for 4 h. Expression of hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were analyzed up to 24 h post isoflurane exposure using qRT-PCR and western blot, or ELISA analyses. PI3K inhibitors--LY 294002 and wortmannin, mTOR inhibitor--rapamycin, and GSK3ß inhibitor--SB 216763 were used respectively to assess the effects of isoflurane treatment and HIF-1α expression. Furthermore, 40 rats were randomly divided into 5 groups (control, isoflurane, scrambled siRNA plus isoflurane, HIF-1α siRNA plus isoflurane, and DMOG) and subjected to random-pattern skin flaps operation. Rats were prepared for evaluation of flap survival and full-feld laser perfusion imager (FLPI) (at 7 day) and microvessel density evaluation (at 10 day). RESULTS: Isoflurane exposure induced expression of HIF-1α protein, HO-1 and VEGF mRNA and proteins in a time-dependent manner. Both LY 294002 and wortmannin inhibited phospho-Akt, phospho-mTOR, phospho-GSK 3ß and HIF-1α expression after isoflurane exposure. Both wortmannin and rapamycin inhibited isoflurane-induced phospho-4E-BP1 (Ser 65) and phospho-P70(s6k) (Thr 389) and HIF-1α expression. SB 216763 pre-treatment could further enhance isoflurane-induced expression of phospho-GSK 3ß (Ser 9) and HIF-1α protein compared to the isoflurane-alone cells. In animal experiments, isoflurane alone, scrambled siRNA plus isoflurane, or DMOG groups had significantly upregulated vascularity and increased survival of the skin flaps compared to the controls. However, HIF-1α knockdown abrogated the protective effect of isoflurane preconditioning in rats. CONCLUSIONS: Isoflurane preconditioning improves survival of skin flaps by up the regulation of HIF-1α expression via Akt-mTOR and Akt-GSK 3ß signaling pathways.

Hypoxia inducible factor-1α is involved in the neurodegeneration induced by isoflurane in the brain of neonatal rats.

More and more data show isoflurane, a commonly used volatile anesthetic has dual effects on neuron fate. However, the underlying mechanisms that can explain the apparent paradox are poorly understood. Hypoxia inducible factor (HIF)-1α, a transcription factor, has been found regulating both prosurvival and prodeath pathways in the CNS. Previously, we found that isoflurane can activate HIF-1α under normoxic conditions in vitro and HIF-1α has been found to be involved in the pre-conditioning effect of isoflurane in various organs. Here, we investigated whether HIF-1α is a contributing factor in the neurodegenration in rodent primary cultured neurons and in developing rat brain. Isoflurane dose-dependently induced apoptotic neurodegeneration in neonatal rats as assessed by S100ß, cleaved caspase 3 and poly-(ADP-ribose) polymerase (PARP), respectively. Notably, isoflurane up-regulates HIF-1α protein levels in vivo and in vitro during induction of neurodegeneration. Likewise, isoflurane resulted in a significant elevation of cytosonic calcium levels in neuron cultures. Furthermore, knockdown of HIF-1α expression in cultured neurons attenuated isoflurane-induced neurotoxicity. Finally, Morris water maze (MWM) test showed neonatal exposure to isoflurane impaired juvenile learning and memory ability in rats. These findings indicate that HIF-1α is involved in the neurodegeneration induced by isoflurane in the brain of neonatal rats, suggesting HIF-1α may be a candidate for the dual effects of isoflurane on neuron fate.

Inhibition of hypoxia inducible factor-1α ameliorates lung injury induced by trauma and hemorrhagic shock in rats.

AIM: Ischemia/reperfusion is an initial triggering event that leads to gut-induced acute lung injury (ALI). In this study, we investigated whether hypoxia inducible factor-1α (HIF-1α) played a role in the pathogenesis of lung injury induced by trauma and hemorrhagic shock (T/HS). METHODS: Male Wistar rats underwent laparotomy and hemorrhagic shock for 60 min. Sham-shock animals underwent laparotomy but without hemorrhagic shock. After resuscitation for 3 hr, the rats were sacrificed. Morphologic changes of the lungs and intestines were examined. Bronchoalveolar lavage fluid (BALF) was collected. Lung water content, pulmonary myeloperoxidase (MPO) activity and the levels of malondialdehyde (MDA), nitrite/nitrate, TNF-α, IL-1ß, and IL-6 in the lungs were measured. The gene expression of pulmonary HIF-1α and iNOS, and HIF-1α transcriptional activity in the lungs were also assessed. The apoptosis in the lungs was determined using TUNEL assay and cleaved caspase-3 expression. RESULTS: Lung and intestinal injuries induced by T/HS were characterized by histological damages and a significant increase in lung water content. Compared to the sham-shock group, the BALF cell counts, the pulmonary MPO activity and the MDA, nitrite/nitrate, TNF-α, IL-1ß, and IL-6 levels in the T/HS group were significantly increased. Acute lung injury was associated with a higher degree of pulmonary HIF-1α and iNOS expression as well as apoptosis in the lungs. Intratracheal delivery of HIF-1α inhibitor YC-1 (1 mg/kg) significantly attenuated lung injury, and reduced pulmonary HIF-1α and iNOS expression and HIF-1α transcriptional activity in the T/HS group. CONCLUSION: Local inhibition of HIF-1α by YC-1 alleviates the lung injury induced by T/HS. Our results provide novel insight into the pathogenesis of T/HS-induced ALI and a potential therapeutic application.

Fungal community enhanced humification and influenced by heavy metals in industrial-scale hyperthermophilic composting of municipal sludge.

The succession of fungal community and effects of heavy metals on fungi during industrial-scale hyperthermophilic composting of municipal sludge remain unclear. Results showed hyperthermophilic composting enhanced decomposition and humification of municipal sludge in the short terms, while heavy metal concentrations and speciation had no significant change with high copper and zinc levels (101-122 and 292-337 mg/kg, respectively) in compost samples. The fungal community and its ecological assembly displayed dynamic change during hyperthermophilic composting. Some thermophilic-resistant fungi, such as phylum Ascomycota and genera Candida, Aspergillus, Thermomyces and Petriella dominated in hyperthermophilic phase. Heavy metals served important effects on fungal community structure and functions during composting. Some fungal drivers (e.g., Thermomyces, Petriella and Schizophyllum) and keystone fungi (e.g., Candida and Pichia) might be thermophilic- and heavy metal-resistant fungi which played important roles in decomposition and humification of municipal sludge. This study reveals fungal community accelerating humification and its influencing factors during composting.

Inflammatory stimulation and hypoxia cooperatively activate HIF-1{alpha} in bronchial epithelial cells: involvement of PI3K and NF-{kappa}B.

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1α. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1α in a cell-specific manner. Here, we demonstrate that stimulation of BEAS-2B cells and primary human bronchial epithelial cells by proinflammatory cytokines TNFα/IL-4 strongly induced expression and transcriptional activity of HIF-1α under normoxic conditions and amplified hypoxic HIF-1α activation. TNFα/IL-4 stimulated de novo HIF-1α gene transcription and translation rather than affected HIF-1α protein degradation and mRNA decay process. The activation of HIF-1α by TNFα/IL-4 was countered by the phosphoinositol 3-kinase (PI3K) inhibitor LY-294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the MAPK pathway. In line, TNFα/IL-4 also activated NF-κB, whereas blocking of NF-κB by an inhibitor or silencing NF-κB subunit p65 attenuated HIF-1α activation by TNFα/IL-4. We also found the collaborative induction of VEGF, a potent angiogenic factor required for airway remodeling, by TNFα/IL-4 and hypoxia partially via HIF-1α pathway in BEAS-2B cells. This study reports the previously unsuspected collaborative regulation of HIF-1α by TNFα/IL-4 and hypoxia in bronchial epithelial cells partially via PI3K-mTOR and NF-κB pathway, and thereby will lead to the elucidation of the importance of HIF-1 in integrating inflammatory and hypoxic response in the pathogenesis of airway diseases.

Heme oxygenase-1 mediates the anti-inflammatory effect of isoflurane preconditioning in LPS-stimulated macrophages.

AIM: The aim of this study was to investigate the anti-inflammatory action of isoflurane preconditioning in a model of lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and examine the role of heme oxygenase (HO)-1 in this process. METHODS: Murine 264.7 macrophages were pretreated with or without 1%-3% isoflurane for 1 h. Thirty minutes later, the cells were incubated with or without LPS for 24 h. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell injury was assessed by measuring the release of lactate dehydrogenase (LDH). HO-1 and inducible nitric oxide synthase (iNOS) protein expression was analyzed by Western blotting. Tumor necrosis factor (TNF)-alpha levels, nitrite production and HO activity were also determined. RESULTS: Pretreatment with the nontoxic and clinically approved anesthetic isoflurane potently attenuated the cell injury and the decrease in cell viability that was induced by LPS. Treatment or pretreatment with 2% isoflurane induced HO-1 protein expression and caused an induction of HO activity. This result correlated with a decrease in iNOS expression, a decrease in the production of nitric oxide (NO) and impaired release of TNF-alpha in LPS-stimulated macrophages. Blockade of HO activity with tin protoporphyrin (SnPP) reversed these effects. CONCLUSION: Isoflurane preconditioning exerts its anti-inflammatory activity through the HO-1 pathway in an in vitro inflammation model.

Isoflurane preconditioning ameliorates endotoxin-induced acute lung injury and mortality in rats.

BACKGROUND: The effects of isoflurane pretreatment on pulmonary proinflammatory cytokines and survival in severe endotoxin-induced acute lung injury (ALI) have not been studied systemically. We investigated the effect of preadministration of isoflurane on ALI induced by lipopolysaccharide (LPS) in rats. METHODS: Male Sprague-Dawley rats weighing 250-300 g were randomly assigned to 1 of 4 groups: sham rats (injected intraperitoneally [IP] with saline) pretreated with vehicle (100% O(2)) (sham-vehicle); sham rats pretreated with isoflurane (sham-ISO); LPS rats (injected IP with LPS) pretreated with vehicle (vehicle-LPS); and LPS rats pretreated with isoflurane (ISO-LPS). Endotoxemia was induced by IP injection of LPS. Isoflurane 1.4% was administered 30 min before LPS injection. The animals were then observed for 6 h. We monitored arterial blood pressure, heart rate, and blood gas. The extent of ALI was evaluated by lung wet/dry ratio, Evans blue dye extravasation, and histologic examination. We also measured pulmonary nitric oxide (NO), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 levels. In addition, survival statistics and pulmonary inducible NO synthase (iNOS) gene expression were also determined. RESULTS: LPS caused systemic hypotension and severe ALI, as evidenced by the increases in the extent of ALI, impairment of pulmonary functions, and increases in pulmonary NO, TNF-alpha, IL-1beta, and IL-6. Isoflurane preconditioning mitigated systemic hypotension and the development of ALI. Isoflurane preconditioning also attenuated the LPS-induced increases in pulmonary nitrate/nitrite and proinflammatory cytokine release and improved survival of rats with severe sepsis. The expression of iNOS was upregulated by LPS and reduced by isoflurane pretreatment. CONCLUSIONS: Isoflurane preconditioning can attenuate pulmonary proinflammatory cytokine release and decrease the mortality induced by severe sepsis. Early protection seems to be mediated partly through inhibition of iNOS-NO pathway activation.

Hypoxia upregulates hypoxia inducible factor (HIF)-3alpha expression in lung epithelial cells: characterization and comparison with HIF-1alpha.

The role of the hypoxia-inducible factor (HIF) subunits 1alpha and 2alpha in response to hypoxia is well established in lung epithelial cells, whereas little is known about HIF-3alpha with respect to transcriptional and translational regulation by hypoxia. HIF-3alpha and HIF-1alpha are two similar but distinct basic helix-loop-helix-PAS proteins, which have been postulated to activate hypoxia responsive genes in response to hypoxia. Here, we used quantitative real time RT-PCR and immunoblotting to determine the activation of HIF-3alpha vs. HIF-1alpha by hypoxia. HIF-3alpha was strongly induced by hypoxia (1% O2) both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation, whereas HIF-1alpha protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stability in A549 cells, as measured on mRNA and protein levels. Interestingly, HIF-3alpha and HIF-1alpha exhibited strikingly similar responses to a variety of activating or inhibitory pharmacological agents. These results demonstrate that HIF-3alpha is expressed abundantly in lung epithelial cells, and that the transcriptional induction of HIF-3alpha plays an important role in the response to hypoxia in vitro. Our findings suggest that HIF-3alpha, as a member of the HIF system, is complementary rather than redundant to HIF-1alpha induction in protection against hypoxic damage in alveolar epithelial cells.

[The expression of capillary hypoxia-induced factor-alpha and pulmonary artery remodeling in chronic obstructive pulmonary disease].

OBJECTIVE: To observe the expression of hypoxia-inducible factor-alpha (HIF-1alpha, HIF-2alpha, HIF-3alpha) and pulmonary vascular remodeling in pulmonary arteries of patients with chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, of whom 12 had concurrent COPD and 10 without COPD. Pulmonary vascular remodeling was observed with optical microscope, and HIF-alpha mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively. RESULTS: Vascular remodeling parameters (WT%, WA%) of COPD patients [(24 +/- 3)%, (48 +/- 6)%, respectively] were statistically different from those of the control subjects [(15 +/- 2)%, (39 +/- 4)%, respectively]. Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-1alpha in COPD group (0.172 +/- 0.011, 0.089 +/- 0.013, respectively) were statistically higher than those of the control subjects (0.103 +/- 0.010, 0.042 +/- 0.010, P < 0.01). Furthermore, they correlated positively with the parameters for vascular remodeling. The mRNA and protein levels of HIF-2alpha in COPD group (0.038 +/- 0.010, 0.077 +/- 0.010, respectively) were significantly lower than those of the control subjects (0.133 +/- 0.017, 0.169 +/- 0.010, respectively, P < 0.01), and correlated negatively with the parameters for vascular remodeling. Regarding HIF-3alpha, only the mRNA level in COPD group (0.077 +/- 0.010) was statistically lower than that of the control subjects (0.088 +/- 0.010) (P < 0.05), and no correlation with vascular remodeling parameters was observed. CONCLUSIONS: Pulmonary vascular remodeling in COPD patients was accompanied by the differential expression of HIF-alpha gene, which may be involved in the process of hypoxic pulmonary vascular remodeling in COPD.

[Differential expression of the three hypoxia-inducible factor alpha subunits in pulmonary artery of rats with hypoxia-induced pulmonary hypertension].

OBJECTIVE: To differentiate the expression patterns of all hypoxia-inducible factor alpha (HIF-alpha) subunits (HIF-1alpha, HIF-2alpha and HIF-3alpha) in pulmonary artery of rats undergoing systemic hypoxia. METHODS: Forty male healthy wistar rats were assigned randomly to 5 groups, 8 rats per group, then exposed to hypoxia [O2, (10.0 +/- 0.5)%] for 0 d (H(0)), 3 d (H(3)), 7 d (H(7)), 14 d (H(14)) and 21 d (H(21)) respectively, 8 h per day intermittently. Mean pulmonary arterial pressure (mPAP), arterial wall area (WA, microm), pulmonary artery medium thickness (PAMT%) and right ventricle hypertrophy index (RVHI) were measured. HIF-1alpha, HIF-2alpha and HIF-3alpha gene expression were determined by immunohistochemistry, in situ hybridization and Western blot. RESULTS: mPAPs in H(7), H(0) and H(14) groups were [(18.40 +/- 0.40) mm Hg, 1 mm Hg = 0.133 kPa], [(14.40 +/- 0.40) mm Hg] and [(21.20 +/- 0.20) mm Hg], respectively, statistically different when H(7) group was compared with H(0) and H(14) groups (all P < 0.05). Arterial morphology showed that WA%, PAMT and RVHI% in H(7) group were (47.8 +/- 0.8)%, (12.3 +/- 0.5) microm, (24.0 +/- 1.0)%, respectively; in H(0) group were (35.5 +/- 1.3)%, (11.9 +/- 0.6)%, (23.6 +/- 0.5) microm, respectively; in H(21) group were (65.0 +/- 0.7)%, (23.0 +/- 0.8) microm, (27.7 +/- 1.0)%, respectively. When H(7) group was compared with H(0) group, only WA% was statistically different; when H(14) group was compared with H(0) group, all the three parameters were statistically different (P < 0.05). In situ hybridization demonstrated that the mRNA levels (absorbance, A) of HIF-1alpha, HIF-2alpha, and HIF-3alpha in H(14) group were 0.200 +/- 0.020, 0.080 +/- 0.010, 0.170 +/- 0.010, respectively; in H(7) group were 0.050 +/- 0.020, 0.160 +/- 0.020, 0.160 +/- 0.020, respectively; in H(0) group were 0.050 +/- 0.010, 0.140 +/- 0.020, 0.060 +/- 0.010, respectively. When H(7) group was compared with H(0) group, only HIF-3alpha was statistically different; when H(14) group was compared with H(0) group, all the three genes were significantly different (P < 0.05). Immunohistochemistry showed that HIF-1alpha, HIF-2alpha and HIF-3alpha protein levels in H(3) group were 0.200 +/- 0.020, 0.020 +/- 0.010, 0.050 +/- 0.010, respectively; in H(14) group were 0.160 +/- 0.010, 0.100 +/- 0.020, 0.160 +/- 0.010, respectively; in H(7) group were 0.220 +/- 0.020, 0.030 +/- 0.010, 0.180 +/- 0.020, respectively; in H(0) group were 0.050 +/- 0.010, 0.020 +/- 0.010, 0.040 +/- 0.010, respectively. HIF-1alpha in H(3) group, HIF-2alpha in H(14) group, HIF-3alpha in H(7) and H(3) group were statistically different with that of H(0) group (P < 0.05). Protein bands of HIF-alpha subunits in lung tissue, measured by Western blot, showed that HIF-3alpha decreased in H(7) group as compared to H(0) group, but the other two proteins showed a marked increase in H(3) group as compared to H(0) group, and increased further corresponding to the duration of hypoxia and peaked in H(14) group as compared to H(7) group. CONCLUSION: The differential expression of the three HIF-alpha subunits may play a role in the development of hypoxia-induced pulmonary hypertension.

[Transforming growth factor beta1 correlates inducible nitric oxide synthase in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension].

OBJECTIVE: To investigate the dynamic expression of transforming growth factor beta(1)(TGF-beta(1)) and inducible nitric oxide synthase (iNOS) in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension (HPH). METHODS: Forty adult male Wistar rats were randomly divided into five groups: a control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H(3), H(7), H(14), H(21) group), eight rats per group. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were measured. Lungs were inflation fixed for in situ hybridization and immunohistochemistry. RESULTS: mPAP increased significantly in H(7) group [(18.41 +/- 0.37) mm Hg, 1 mm Hg = 0.133 kPa, P < 0.05], reaching its peak in H(14) group [(21.17 +/- 0.23) mm Hg], then remained at the high level. Hypoxia induced pulmonary artery remodeling and right ventricle hypertrophy index became evident in H(14) group. Expression of iNOS protein in control group (0.109 +/- 0.021) was weakly positive in pulmonary arterial tunica media, while the level of iNOS protein was markedly up-regulated in H(3) group (0.225 +/- 0.030, P < 0.01), reaching its peak in H(7) group (0.312 +/- 0.036), then remained at the high level. Expression of iNOS mRNA in C group (0.112 +/- 0.030) was weakly positive in pulmonary arterial wall, while the level of iNOS mRNA was markedly up-regulated in H(3) group (0.245 +/- 0.036), reaching its peak in H(7) group (0.318 +/- 0.034, P < 0.01), then remained at the high level. Expression of TGF-beta(1) protein in C group (0.042 +/- 0.012) was weakly positive, but the level of TGF-beta(1) protein was markedly up-regulated in H(3) group (0.198 +/- 0.031), reaching its peak in H(7) group (0.267 +/- 0.035, P < 0.01), and then tended to decline in H(14) and H(21) group. TGF-beta(1) mRNA staining was weakly positive in C group (0.145 +/- 0.018), H(3) group (0.163 +/- 0.021) and H(7) group (0.176 +/- 0.026), but began to increase significantly in H(14) group (0.385 +/- 0.028, P < 0.01), and then remained stable. TGF-beta(1) protein and mRNA were located predominantly in tunica adventitia and tunica media. Linear correlation analysis showed that TGF-beta(1) mRNA, iNOS mRNA and protein were positively correlated with mPAP, vessel morphometry and RVHI (r = 0.843 - 0.937, all P < 0.01). TGF-beta(1) protein (tunica adventitia) was negatively correlated with iNOS mRNA and protein (r = -0.856, -0.835, all P < 0.01). CONCLUSIONS: Interaction of TGF-beta(1) and iNOS plays a role in the pathogenesis of HPH in rats. iNOS can regulate the expression of TGF-beta(1) gene by NO. TGF-beta(1) can regulate the expression of iNOS gene by decreased stability and translation of iNOS mRNA and increased degradation of iNOS protein.

[Preventive effect of dexmedetomidine on postoperative delirium in elderly patients with oral cancer].

PURPOSE: To observe and analyze the preventive effect of dexmedetomidine on postoperative delirium in elderly patients with oral cancer. METHODS: One hundred and fifty-six elderly patients with oral cancer who received radical surgery under general anesthesia were studied. They were randomly divided into 2 groups: experimental group (n=78) and control group (n=78). All patients stayed in PACU for 2 hours after surgery, and then were transferred to SICU when they had waken up. Subsequently, patients in experimental group were assigned to take intravenous dexmedetomidine at a dose of 0.2 µg/kg.h for 12 hours while patients in control group were assigned to take intravenous normal saline for 12 hours. All patients were given compound analgesia consisted of tramadol and tropisetron in the same dose. During the first three postoperative days, patients were evaluated with the Confusion Assessment Method for the Intensive Care Unit (CAM-ICU), Visual Analogue Scale and Richmond Agitation Sedation Scale twice a day(8:00 am and 8:00 pm). Statistical analysis was performed using SPSS16.0 software package. RESULTS: There was no significant difference on postoperative VAS, the incidence of postoperative bradycardia and hypotension between 2 groups. In addition, no postoperative respiratory depression was found in 2 groups. Richmond Agitation Sedation Scale on the first and second day after surgery in the experimental group was better than in the control group. The incidence of postoperative delirium, nausea and vomiting in the experimental group was lower than in the control group. CONCLUSIONS: Intravenous dexmedetomidine at a dose of 0.2 µg/kg.h for 12 hours after operation in elderly patients with oral cancer can ameliorate postoperative sedation status, reduce the incidence of postoperative delirium, and will not cause postoperative bradycardia, hypotension and other complications.

Hypoxia-inducible factor-1 alpha regulates the role of vascular endothelial growth factor on pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

BACKGROUND: Hypoxia-inducible factor-1alpha (HIF-1alpha) is one of the pivotal mediators in the response of lungs to decreased oxygen availability, and increasingly has been implicated in the pathogenesis of pulmonary hypertension. Vascular endothelial growth factor (VEGF), a downstream target gene of HIF-1alpha, plays an important role in the pathogenesis of hypoxic pulmonary hypertension and hypoxic pulmonary artery remodelling. In this study, we investigated the dynamic expression of HIF-1alpha and VEGF in pulmonary artery of rats with hypoxia-induced pulmonary hypertension. METHODS: Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were estimated. Lungs were inflated and fixed for in situ hybridisation and immunohistochemistry. RESULTS: mPAP values were significantly higher than the control values after 7days of hypoxia [(18.4 +/- 0.4) mmHg, P < 0.05]. RVHI developed significantly after 14 days of hypoxia. Expression of HIF-1alpha protein increased in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, HIF-1alpha protein was markedly increased by day 3 (0.20 +/- 0.02, P < 0.05), reached the peak by day 7, then declined after day 14 of hypoxia. HIF-1alpha mRNA increased significantly after day 14 of hypoxia (0.20 +/- 0.02, P < 0.05). VEGF protein began to increase markedly after day 7 of hypoxia, reaching its peak around day 14 of hypoxia (0.15 +/- 0.02, P < 0.05). VEGF mRNA began to increase after day 7 of hypoxia, then remained more or less stable from day 7 onwards. VEGF mRNA is located mainly in tunica intima and tunica media, whereas VEGF protein is located predominantly in tunica intima. Linear analysis showed that HIF-1alpha mRNA, VEGF and mPAP were correlated with hypoxic pulmonary artery remodelling. HIF-1alpha mRNA was positively correlated with VEGF mRNA and protein (P < 0.01). CONCLUSION: HIF-1alpha and VEGF are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.

[Hypoxia-inducible factor 1alpha regulates vascular endothelial growth factor's roles on pulmonary arteries of rats with hypoxia-induced pulmonary hypertension].

OBJECTIVE: To investigate the dynamic expression of hypoxia inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. METHODS: Forty male adult Wistar rats were randomly divided into five groups: a control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H(3), H(7), H(14) and H(21) group), eight rats per group. Mean pulmonary pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were measured. Lungs were inflation fixed for in situ hybridization and immunohistochemistry. RESULTS: mPAP increased significantly after 7-day of hypoxia [(18.41 +/- 0.37) mm Hg, P < 0.05], reaching its peak after 14-day of hypoxia, then remained on the high level. Pulmonary artery remodeling index (outer diameter 100 - 150 micro m) and RVHI became evident after 14-day of hypoxia. Expression of HIF-1alpha protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1alpha protein was markedly up-regulated after 3-day (0.178 +/- 0.017, P < 0.05), reaching its peak after 7-day of hypoxia (0.221 +/- 0.021, P < 0.05), then tended to decline after 14-day and 21-day of hypoxia. HIF-1alpha mRNA staining was poorly positive in control, hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly after 14-day of hypoxia (0.203 +/- 0.024, P < 0.05), then remained stable. Expression of VEGF protein began to increase after 7-day of hypoxia (0.074 +/- 0.022, P < 0.05), reaching its peak after 14-day of hypoxia (0.147 +/- 0.017, P < 0.05), then remained on the high level. VEGF mRNA in control and hypoxia for 3 days was poorly positive, but began to increase after 7-day of hypoxia (0.138 +/- 0.010, P < 0.05), and remained on the high level thereafter. VEGF mRNA located mainly in tunica intima and tunica media, whereas VEGF protein located predominantly in tunica intima. Linear correlation analysis showed that HIF-1alpha mRNA, VEGF, and mPAP were correlated with vessel morphometry and RVHI (P < 0.01). HIF-1alpha protein (tunica intima) was positively correlated with VEGF mRNA and protein (P < 0.01). CONCLUSIONS: HIF-1alpha and VEGF are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats. HIF-1alpha protein may regulate the expression of VEGF gene by transcriptional activation, resulting in the occurrence and development of hypoxic pulmonary hypertension.

Isoflurane preconditioning activates HIF-1alpha, iNOS and Erk1/2 and protects against oxygen-glucose deprivation neuronal injury.

Preconditioning neurons with isoflurane, a commonly used volatile anesthetic in clinical practice, improves tolerance of subsequent ischemia in both intact animal models and in vitro preparations. To investigate the mechanisms of this protection, we primarily cultured rat hippocampal neurons and simulated ischemia in vitro by oxygen-glucose deprivation (OGD). Neuron viability was measured. Neuron injury was observed by inverted phase contrast microscope and assessed by lactate dehydrogenase (LDH) release. Gene expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Isoflurane exposure for 2 h at 24 h before a 2 h OGD dose-dependently reduced cell injury. Isoflurane accumulated phosphorylation/activation of extracellular signal-related kinases 1 and 2 (Erk1/2) and hypoxia inducible factor (HIF)-1alpha, a transcription factor involved in cell survival. Inhibition of the phospho-Erk1/2 partially abolished the isoflurane preconditioning-induced HIF-1alpha protein content accumulation and neuroprotection. Isoflurane also increased inducible nitric oxide synthase (iNOS) mRNA levels, a downstream gene of HIF-1alpha. Thus, the current results indicate that isoflurane preconditioning activates HIF-1alpha during protection against OGD neuronal injury and the activation might be partly mediated by the Erk1/2 pathway.

Transforming growth factor-beta1 induces transdifferentiation of fibroblasts into myofibroblasts in hypoxic pulmonary vascular remodeling.

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-beta1 (TGF-beta1) can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of a-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-beta1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-beta1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-beta1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.

Up-regulation of hypoxia inducible factor 1alpha by isoflurane in Hep3B cells.

BACKGROUND: The volatile anesthetic isoflurane induces hypoxia inducible factor (HIF)-1-responsive genes heme oxygenase 1, inducible nitric oxide synthase, and vascular endothelial growth factor (VEGF) expression. Little is known about the extent to which induction of HIF-1alpha is affected by isoflurane. METHODS: Hep3B cells were exposed to isoflurane at various concentrations (0.5-4%) or for different time periods (2-8 h) at 37 degrees C. HIF-1alpha gene expression and transcriptional activity, heme oxygenase 1, inducible nitric oxide synthase, and VEGF gene expression were quantified. RESULTS: Isoflurane induced a time- and concentration-dependent increase in HIF-1alpha protein but not for HIF-1alpha messenger RNA (mRNA) in Hep3B cells. The maximal increase was induced by 2% isoflurane, and the cells incubated with 2% isoflurane for 4-8 h expressed the highest protein. Similarly, HIF-1alpha transcriptional activity was higher in Hep3B cells exposed to 2% isoflurane for 16 h than that in control cells. The combination of 2% isoflurane and desferrioxamine, a hypoxia mimetic, caused a higher level of HIF-1alpha protein than that induced by 2% isoflurane alone. Reoxygenation and inhibitor of proteasome pathway MG132 did not affect the isoflurane-induced HIF-1alpha protein accumulation. Cycloheximide, an inhibitor for protein synthesis, completely abrogated the induction of HIF-1alpha protein by isoflurane. Isoflurane stimulated heme oxygenase 1, inducible nitric oxide synthase, and VEGF mRNA expression in a concentration-dependent manner, and inactivation of HIF-1alpha attenuated the induction of VEGF mRNA by isoflurane. CONCLUSION: Isoflurane can up-regulate HIF-1alpha and enhance HIF-1-responsive genes heme oxygenase 1, inducible nitric oxide synthase, and VEGF mRNA expression in Hep3B cells. The induction of HIF-1alpha by isoflurane does not involve protein degradation but depends on translation pathway.