Somatostatin Reduces the Acute Lung Injury of Mice via Increasing the Affinity of Glucocorticoid Receptor.

BACKGROUND/AIMS: Although it has been reported that somatostatin (SOM) upregulated the level of 90-kD heat shock protein (Hsp90), which participates in the inflammatory regulation by its client proteins, such as glucocorticoid receptor (GR), it remains unclear if it has a protective role against acute lung injury (ALI). METHODS: ALI model was established by the injection of oleic acid (OA) into the tail vein of mice. Lung injury was assessed by histological analysis, lung water content and arterial blood gases. The levels of Hsp90 and GR, the binding capacity and the affinity of GR were examined. RESULTS: It was showed that pretreatment with SOM significantly increased Hsp90 levels and alleviated lung injuries in OA-injected mice. Furthermore, SOM increased the GR expression and improved the affinity of the GR in animals with lung injury. However, little alteration was found in the maximum binding capacity of the GR in mice with or without SOM. CONCLUSION: The data indicate SOM exerts a protective effect by increasing Hsp90 abundant and further enhancing the affinity of the GR. The beneficial effects of SOM treatment provide a new strategy for modulation of GR efficiency and alleviation of acute lung injury.

Chronic caffeine exposure attenuates blast-induced memory deficit in mice.

OBJECTIVE: To investigate the effects of three different ways of chronic caffeine administration on blast- induced memory dysfunction and to explore the underlying mechanisms. METHODS: Adult male C57BL/6 mice were used and randomly divided into five groups: control: without blast exposure, con-water: administrated with water continuously before and after blast-induced traumatic brain injury (bTBI), con-caffeine: administrated with caffeine continuously for 1 month before and after bTBI, pre-caffeine: chronically administrated with caffeine for 1 month before bTBI and withdrawal after bTBI, post-caffeine: chronically administrated with caffeine after bTBI. After being subjected to moderate intensity of blast injury, mice were recorded for learning and memory performance using Morris water maze (MWM) paradigms at 1, 4, and 8 weeks post-blast injury. Neurological deficit scoring, glutamate concentration, proinflammatory cytokines production, and neuropathological changes at 24 h, 1, 4, and 8 weeks post-bTBI were examined to evaluate the brain injury in early and prolonged stages. Adenosine A1 receptor expression was detected using qPCR. RESULTS: All of the three ways of chronic caffeine exposure ameliorated blast-induced memory deficit, which is correlated with the neuroprotective effects against excitotoxicity, inflammation, astrogliosis and neuronal loss at different stages of injury. Continuous caffeine treatment played positive roles in both early and prolonged stages of bTBI; pre-bTBI and post-bTBI treatment of caffeine tended to exert neuroprotective effects at early and prolonged stages of bTBI respectively. Up-regulation of adenosine A1 receptor expression might contribute to the favorable effects of chronic caffeine consumption. CONCLUSION: Since caffeinated beverages are widely consumed in both civilian and military personnel and are convenient to get, the results may provide a promising prophylactic strategy for blast-induced neurotrauma and the consequent cognitive impairment.

Adenosine A2A receptor deficiency alleviates blast-induced cognitive dysfunction.

Traumatic brain injury (TBI), particularly explosive blast-induced TBI (bTBI), has become the most prevalent injury among military personnel. The disruption of cognitive function is one of the most serious consequences of bTBI because its long-lasting effects prevent survivors fulfilling their active duty and resuming normal civilian life. However, the mechanisms are poorly understood and there is no treatment available. This study investigated the effects of adenosine A2A receptor (A2AR) on bTBI-induced cognitive deficit, and explored the underlying mechanisms. After being subjected to moderate whole-body blast injury, mice lacking the A2AR (A2AR knockout (KO)) showed less severity and shorter duration of impaired spatial reference memory and working memory than wild-type mice did. In addition, bTBI-induced cortical and hippocampal lesions, as well as proinflammatory cytokine expression, glutamate release, edema, cell loss, and gliosis in both early and prolonged phases of the injury, were significantly attenuated in A2AR KO mice. The results suggest that early injury and chronic neuropathological damages are important mechanisms of bTBI-induced cognitive impairment, and that the impairment can be attenuated by preventing A2AR activation. These findings suggest that A2AR antagonism is a potential therapeutic strategy for mild-to-moderate bTBI and consequent cognitive impairment.

Dexamethasone inhibits U937 cell adhesion via the down-regulation of ROCK1 activity.

OBJECTIVE: To explore the effect of dexamethasone (DEX) on monocyte adhesion function and its underlying mechanism. METHODS: The effects of DEX and fasudil on adhesion of cultured U937 monocytes to human umbilical vein endothelial cells (HUVEC) following stimulation with phorbol myristate acetate (PMA) were studied; Changes in the Rho-associated coiled-coil protein kinase 1 (ROCK1) protein content and activity were evaluated. RESULTS: DEX and fasudil significantly inhibited U937 cell adhesion rates under PMA stimulation and inhibited ROCK1 activity. Mifepristone (RU-486) and cycloheximide (CHX) did not alter these effects of DEX. CONCLUSIONS: DEX interferes with the adhesion function of U937 cells through the inhibition of ROCK1 activity.

Roles of Rho-associated coiled-coil protein kinase in multiple cell behaviors.

Rho-associated coiled-coil protein kinase (ROCK) is a serine/threonine kinase that belongs to AGC family of kinases. By inducing the formation of stress fibers and reorganizing the cytoskeleton, it is involved in many biological behaviors of cells including cell contraction, cell migration, cell division, and morphological changes, and thus exerts important roles in regulating the multiple functions of cells.

Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear.

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-ß-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.

[Effect of (S)-4C3HPG on brain damage in the acute stage of moderate traumatic brain injury model of mice and underlying mechanism].

The aim of this study is to investigate the effect of (S)-4-carboxy-3-hydroxy-phenylglycine [(S)-4C3HPG], a mixed group I glutamate metabotropic receptor antagonist and a group II agonist, on impairment in a cortical impact model of traumatic brain injury (TBI) in mice and to elucidate the possible mechanisms. Mice were injected (i.p.) with saline, 1 mg/kg (S)-4C3HPG, 5 mg/kg (S)-4C3HPG and 10 mg/kg (S)-4C3HPG (n=10 per group), respectively, at 30 min before moderate TBI. Neurological deficit scores, water content in injured brain and glutamate concentration in cerebral spinal fluid (CSF) were detected at 24 h after TBI. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) mRNA in injured cortex were also detected by real-time RT-PCR. The results showed that the neurological deficits and cerebral edema were significantly attenuated in mice pretreated with (S)-4C3HPG (5 and 10 mg/kg respectively) compared with those in mice pretreated with saline. Furthermore, (S)-4C3HPG treatment also decreased the glutamate concentration in CSF and the expressions of TNF-α and IL-1ß mRNA remarkably in a dose-dependent manner. These results suggest that (S)-4C3HPG treatment attenuates cortical impact-induced brain injury possibly via suppression of glutamate release and inhibition of excessive inflammatory cytokine production. These findings highlight the potential benefit of glutamate metabotropic receptor ligand for preventing TBI.

[Effect of small interfering RNA-mediated Smad3 gene silencing on transforming growth factor-beta1-induced bi-directional effects on skin fibroblast proliferation].

OBJECTIVE: To study the role of Smad3 in transforming growth factor-beta1 (TGF-beta1)-induced bi-directional effects on skin fibroblast proliferation. METHODS: The Smad3 small interfering (siRNA) plasmid was constructed using a pSUPER vector. The efficiency of cell transfection was detected by fluorescence microscopy, and the inhibitory effect of the plasmid was assessed by real-time quantitative RT-PCR and immunohistochemistry. The effect of the plasmid on the fibroblast proliferation and Smad3 binding activity was analyzed by BRDU ELISA and EMSA, respectively. RESULTS: The transfection efficiency of the plasmid into the cells was 41.2%. The Smad3 siRNA plasmid produced efficient and specific inhibition of the expression of Smad3, and promoted the cell proliferation in a dose-dependent manner and abrogated the bi-directional effect of TGF-beta1 on the cell proliferation and Smad3 binding activity. CONCLUSION: The siRNA targeting Smad3 gene can inhibit the protein expression and RNA transcription of Smad3, and TGF-beta1 exerts bi-directional regulation on fibroblast proliferation by modulating Smad3 activity.

Local glutamate level dictates adenosine A2A receptor regulation of neuroinflammation and traumatic brain injury.

During brain injury, extracellular adenosine and glutamate levels increase rapidly and dramatically. We hypothesized that local glutamate levels in the brain dictates the adenosine-adenosine A(2A) receptor (A(2A)R) effects on neuroinflammation and brain damage outcome. Here, we showed that, in the presence of low concentrations of glutamate, the A(2A)R agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) inhibited lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) activity of cultured microglial cells, an effect that was dependent on the protein kinase A (PKA) pathway. However, in high concentrations of glutamate, CGS21680 increased LPS-induced NOS activity in a protein kinase C (PKC)-dependent manner. Thus, increasing the local level of glutamate redirects A(2A)R signaling from the PKA to the PKC pathway, resulting in a switch in A(2A)R effects from antiinflammatory to proinflammatory. In a cortical impact model of traumatic brain injury (TBI) in mice, brain water contents, behavioral deficits, and expression of tumor necrosis factor-alpha, interleukin-1 mRNAs, and inducible NOS were attenuated by administering CGS21680 at post-TBI time when brain glutamate levels were low, or by administering the A(2A)R antagonist ZM241385 [4-(2-{[5-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-yl]amino}ethyl)phenol] at post-TBI time when brain glutamate levels were elevated. Furthermore, pre-TBI treatment with the glutamate release inhibitor (S)-4C3HPG [(S)-4-carboxy-3-hydroxyphenylglycine] converted the debilitating effect of CGS21680 administered at post-TBI time with high glutamate level to a neuroprotective effect. This further indicates that the switch in the effect of A(2A)R activation in intact animals from antiinflammatory to proinflammatory is dependent on glutamate concentration. These findings identify a novel role for glutamate in modulation of neuroinflammation and brain injury via the adenosine-A(2A)R system.

Adenosine A2A receptors in both bone marrow cells and non-bone marrow cells contribute to traumatic brain injury.

Adenosine A2A receptors (A(2A)Rs) in bone marrow-derived cells (BMDCs) are involved in regulation of inflammation and outcome in several CNS injuries; however their relative contribution to traumatic brain injury (TBI) is unknown. In this study, we created a mouse cortical impact model, and BMDC A(2A)Rs were selectively inactivated in wild-type (WT) mice or reconstituted in global A(2A)R knockout (KO) mice (i.e. inactivation of non-BMDC A(2A)Rs) by bone marrow transplantation. When compared with WT mice, selective inactivation of BMDC A(2A)Rs significantly attenuated the neurological deficits, brain water content and cell apoptosis at 24 h post-TBI as global A(2A)R KO did. However, compared with the A(2A)R KO mice, selective reconstitution of BMDC A(2A)Rs failed to reinstate brain injury, indicating the contribution of the non-BMDC A(2A)R to TBI. Furthermore, the protective outcome by selective inactivation of BMDC A(2A)R or broad inactivation of non-BMDC A(2A)Rs was accompanied with reduced CSF glutamate level and suppression of the inflammatory cytokines interleukin-1, or interleukin-1 and tumor necrosis factor-alpha. These findings demonstrate that inactivation of A(2A)Rs in either BMDCs or non-BMDCs is sufficient to confer the protective effect as global A(2A)R KO against TBI, indicating the A(2A)R involvement in TBI by multiple cellular mechanisms of A(2A)R involvement including inhibition of glutamate release and inflammatory cytokine expressions.

Genetic variations of heat shock protein 84 in mice mediate cellular glucocorticoid response.

Heat shock protein 90 (Hsp90), encoded by hsp84 and hsp86 in mice, has been confirmed to modulate glucocorticoid receptor (GR) function; however, the contribution of Hsp90 in glucocorticoid (GC) sensibility/resistance has received less attention. Previously, we found that genetic variations of Hsp84 are related to differences in the in vivo GC-GR responses between BALB/c and C57BL/6 mice suffering from traumatic injury. To evaluate the modulation of Hsp84 polymorphisms on the GC response, we used a cellular heat-stress injury (HSI) model combined with a transgene-plasmid infection approach and assessed HSI-induced cellular damage and GR nuclear translocation, with or without dexamethasone pretreatment. We demonstrated that after HSI, fibroblasts from the C57BL/6 line exhibit higher cellular survival, higher nuclear GR levels and lower lactate dehydrogenase activity compared to those from the BALB/c line. We showed that dexamethasone-rescued HSI-induced damage is accompanied by increasing nuclear GR levels in both lines. Importantly, this protection against HSI was greater in C57BL/6 fibroblasts and was resistant to geldanamycin, a selective inhibitor of Hsp90. Importantly, transfection of the hsp84-transgene from C57BL/6 mice increased the nuclear GR levels and lessened HSI-induced damage in BALB/c fibroblasts. Our data thereby demonstrate that Hsp84 from C57BL/6 mice modulates higher cellular GC-GR responsiveness.

Differential alteration of heat shock protein 90 in mice modifies glucocorticoid receptor function and susceptibility to trauma.

Heat shock protein 90 (Hsp90), encoded by the murine hsp84 and hsp86 genes in mice, is a pivotal regulator of glucocorticoid receptor (GR) function in the hypothalamus-pituitary-adrenal axis and affords stress protection. To explore the underlying molecular mechanisms of strain susceptibility to traumatic stress, we investigated the alteration by Hsp90 of the function of the glucocorticoid-glucocorticoid receptor (GC-GR) pathway in attenuating stress responses in C57BL/6 and BALB/c mice using the whole-body blast injury (WBBI) model. We found that C57BL/6 mice had a lower WBBI-induced mortality, higher nuclear GR level, and higher glucocorticoid-response element (GRE) binding activity than BALB/c mice. This study is the first report identifying four genetic variations of the murine hsp84 gene: 226A>C, 996G>C, 1483G>C, and 2000G>T. These nucleotide changes occur in the functional domains associated with the nuclear/cytosolic translocation of GR, GR-Hsp90 interaction, ATP binding, and self-dimerization of Hsp90, respectively. Further, we used a specific Hsp90 inhibitor, geldanamycin (GA), to assess the role of Hsp90 in the discriminative traumatic response in C57BL/6 mice. Pretreatment with GA reduced nuclear GR levels and GRE binding activity, and enhanced WBBI-induced mortality. These findings suggest that Hsp90 may underlie the strain-selective (C57BL/6 versus BALB/c) susceptibility to WBBI by mediating the nuclear translocation of GRs and GRE binding. Thus, pharmacological manipulation of Hsp90 may represent a therapeutic strategy to modify the function of the GC-GR pathway and traumatic stress response.

[H19 expression in placenta with pre-eclampsia].

OBJECTIVE: To explore the role of H19 imprinting in etiology of pre-eclampsia. METHODS: Placentas of 24 women with pre-eclampsia (3 with mild pre-eclampsia and 21 with severe pre-eclampsia) and 50 healthy pregnant women at full term (control) were collected during selected cesarean delivery between August 2007 and March 2008. The statuses of H19 imprinting with placental tissues from normal pregnancy and patients with pre-eclampsia were identified upon polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The systolic and diastolic pressure were analyzed in H19 heterozygotic women. RESULTS: (1) There were 20 (40%) heterozygotes in 50 cases placenta tissues of the third trimesters, 11 (45%) heterozygotes in 24 cases placenta tissues of pre-eclampsia. There were no significant difference between two groups (P > 0.05). (2) All 20 heterozygotes in placenta tissues of the third trimesters are exclusively monoallelically expressed, while 5 cases (45%) in 11 heterozygotes of pre-eclampsia are biallelically expressed (loss of imprinting, LOI). There were significant difference between two groups (P < 0.01). (3) The values of systolic and diastolic pressure of patients with monoallelic expression of H19 were (171 +/- 9) mm Hg (1 mm Hg = 0.133 kPa) and (104 +/- 8) mm Hg, the values of systolic and diastolic pressure with biallelic expression were (194 +/- 21) mm Hg and (124 +/- 18) mm Hg. There were significant difference between two groups (P < 0.05). CONCLUSION: LOI of H19 can be identified in pre-eclamptic placentas and is associated with maternal blood pressures, which implies the involvement of H19 gene LOI in the pathogenesis of pre-eclampsia and its potential relationship with the severity of the disease.

[Propofol provides ischemic postconditioning on myocardial ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate whether the infusion of propofol during early reperfusion provides ischemic postconditioning (I-postC) on myocardial ischemia-reperfusion injury in rats. METHODS: Sixty adult rats were randomly divided into 5 groups (n = 12 each): sham operation (group S); normal saline (group C); propofol 1 mg/kg (group P1); propofol 2 mg/kg (group P2); propofol 5 mg/kg (group P3). The left anterior descending coronary artery (LAD) was occluded for 60 min and reperfused for 120 min. Normal saline, propofol 1 mg/kg, 2 mg/kg or 5 mg/kg (propofol diluted to 2.5 ml with normal saline equally) were intravenously infused 3 min before reperfusion until 5 min after reperfusion. The heart were obtained for determination of (1) the size of area at risk and infarct size (Evans Blue and TTC staining); (2) expression of Caspase-3 (immunohistochemistry staining); (3) percentage of apoptotic cardiomyocytes (flow cytometry); (4) levels of phosphorylated Akt (Western blot). RESULTS: Compared with group C [size of area at risk (41.5 +/- 1.0)%, infarct size (45.5 +/- 1.0)%, expression of caspase-3 (5.87 +/- 0.29), percentage of apoptotic cardiomyocytes (26.8 +/- 1.3)%, level of phosphorylated Akt (10.8 +/- 1.9)%], propofol 1 mg/kg and 2 mg/kg significantly reduced the size of area at risk and infarct size [size of area at risk (38.3 +/- 1.0)% and (37.3 +/- 1.2)%; infarct size (33.8 +/- 1.2)% and (30.2 +/- 1.7)%, P < 0.05], inhibited the expression of caspase-3 (1.50 +/- 0.36 and 1.48 +/- 0.30, P < 0.05), decreased the percentage of apoptotic cardiomyocytes [(16.3 +/- 1.2)% and (16.5 +/- 1.0)%, P < 0.05] and promoted the phosphorylation of Akt [(68.7 +/- 4.0)% and (58.3 +/- 2.8)%, P < 0.05]. CONCLUSION: Propofol 1 mg/kg and 2 mg/kg can provide I-postC to myocardial ischemia-reperfusion injury in rats by activation of Akt pathway.

[Association of glucocoid receptor binding activity and heat shock protein expressions after acute lung injury in mice].

OBJECTIVE: To study the association between decreased ligand binding activity of glucocoid receptor (GR) and heat shock protein 90 (Hsp90), the molecular chaperone of GR, after acute lung injury (ALI) in mice. METHODS: In mouse models of oleic acid-induced ALI, the levels of GR, Hsp90 and Hsp70 were dynamically observed using Western blotting, and the binding capacity and binding affinity of GR assessed with radioligand binding assay. RESULTS: After ALI, pulmonary edema was significantly aggravated in the mice with significantly increased lung body index and lung water ratio. GR increased within 1 h after the injury, but then decreased significantly to the lowest level at 12 h after the injury, and the levels of Hsp90 and Hsp70 was increased obviously and reached the highest at 12 h. Radioligand binding assay showed that the Bmax decreased gradually and Kd value increased, and these changes were consistent with the changes of Hsp90/GR. CONCLUSION: The ligand binding activity of GR is related to the changes of Hsp90 after ALI.

[Construction and verification of bait protein RGS4-fusion expression plasmid in yeast two-hybrid system].

OBJECTIVE: To research the interaction between glucocorticosteroid receptor domains and regulator of G protein signaling 4 (RGS4) protein. METHODS: The RGS4 gene was used as the bait protein gene to construct the fusion bait expression plasmid of yeast two-hybrid. The whole encoding sequence of RGS4 gene was amplified by RT-PCR method. With confirmed by electrophoresis, the RGS4 gene was cloned into the MCS of the plasmid pGBKT7 to form a recombinant plasmid pGBKT7-RGS4 and the sequence of the recombinant plasmid was sequenced. According to the protocol of yeast two-hybrid system gold III, the competent Y187 yeast was prepared and transformed with recombinant plasmid pGBKT7-RGS4. Following that, the toxicity and autonomous activation of this recombinant plasmid pGBKT7-RGS4 in Y187 yeast were tested. In the end, we verified the normal expression of the RGS4 fusion protein in vitro by TNT system and in vivo by Weston blot. RESULTS: The sequence of the recombinant plasmid was verified to be correct, as compared with the sequence provided by GenBank. The protein could be correctly synthesized in vivo, and no autonomous activation and toxicity was observed in Y187 yeast. CONCLUSIONS: The recombinant plasmid may be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombinant RGS4 fusion protein can be used as the bait protein successfully.