PK/PD modeling of Ceftiofur Sodium against Haemophilus parasuis infection in pigs.

BACKGROUND: Ceftiofur Sodium is widely used in China. Our aim was to determine Ceftiofur Sodium activity and optimize dosing regimens against the pathogen Haemophilus parasuis using an in vitro and ex vivo pharmacokinetics/pharmacodynamics modeling approach. By adopting these strategies, we wanted to extend the effective life of Ceftiofur Sodium in reduce drug-resistance in pigs. RESULTS: We established an H. parasuis infection model in pigs, and assessed the pharmacokinetics of Ceftiofur Sodium in both healthy and infected animals. After Ceftiofur Sodium (10 mg/kg, i.m.) administration to healthy and H. parasuis-infected pigs, plasma based desfuroylceftiofur (a metabolite of Ceftiofur Sodium) was measured by High Performance Liquid Chromatography. The pharmacokinetics of Ceftiofur Sodium (desfuroylceftiofur) was consistent with a two-compartment open model, with first-order absorption. We observed no significant differences (P > 0.05) in pharmacokinetic parameters between healthy and infected pigs. Pharmacodynamics data showed that Ceftiofur Sodium was highly inhibitory against H. parasuis, with MIC, MBC, and MPC values of 0.003125, 0.0125 and 0.032 µg/mL, respectively. Desfuroylceftiofur in plasma also had strong bactericidal activity. Almost all H. parasuis cultured in plasma medium of Ceftiofur Sodium-inoculated healthy pigs, at each time point, were killed within 24 h. A weaker antibacterial activity was measured in infected-pig plasma medium at 18, 24, 36, and 48 h, after Ceftiofur Sodium inoculation. Pharmacokinetic parameters were combined with ex vivo pharmacodynamic parameters, and the bacteriostatic effect (36.006 h), bactericidal effect (71.637 h) and clearance (90.619 h) within 24 h, were determined using the Hill equation. Dose-calculation equations revealed the optimal dose of Ceftiofur Sodium to be 0.599-1.507 mg/kg. CONCLUSIONS: There were no significant differences in Ceftiofur Sodium pharmacokinetic parameters between healthy and infected pigs, although pharmacokinetics/pharmacodynamics fitting curves showed obviously differences. The optimal dose of Ceftiofur Sodium was lower than recommended (3 mg/kg), which may provide improved treatments for Glässers disease, with lower drug-resistance possibility.

Predictive Value of Combined LIPS and ANG-2 Level in Critically Ill Patients with ARDS Risk Factors.

To investigate the predictive value of the acute physiology and chronic health evaluation 2 (APACHE2) score and lung injury prediction score (LIPS) for acute respiratory distress syndrome (ARDS) when combined with biomarkers for this condition in patients with ARDS risk factors. In total, 158 Han Chinese patients with ARDS risk factors were recruited from the Respiratory and Emergency Intensive Care Units. The LIPS, APACHE2 score, primary diagnosis at admission, and ARDS risk factors were determined within 6 h of admission, and PaO2/FiO2 was determined on the day of admission. Blood was collected within 24 h of admission for the measurement of angiopoietin-2 (ANG-2), sE-selectin, interleukin-6 (IL-6), and interleukin-8 (IL-8) levels. ARDS was monitored for the next 7 days. Univariate and multivariate analyses and receiver operating characteristic (ROC) analyses were employed to construct a model for ARDS prediction. Forty-eight patients developed ARDS within 7 days of admission. Plasma ANG-2 level, sE-selectin level, LIPS, and APACHE2 score in ARDS patients were significantly higher than those in non-ARDS patients. ANG-2 level, LIPS, and APACHE2 score were correlated with ARDS (P < 0.001, P < 0.006, and P < 0.042, resp.). When the APACHE2 score was used in combination with the LIPS and ANG-2 level to predict ARDS, the area under the ROC curve (AUC) was not significantly increased. Compared to LIPS or ANG-2 alone, LIPS in combination with ANG-2 had significantly increased positive predictive value (PPV) and AUC for the prediction of ARDS. In conclusion, plasma ANG-2 level, LIPS, and APACHE2 score are correlated with ARDS. Combined LIPS and ANG-2 level displays favorable sensitivity, specificity, and AUC for the prediction of ARDS.

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling.

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

Requirement of miR-9-dependent regulation of Myocd in PASMCs phenotypic modulation and proliferation induced by hepatopulmonary syndrome rat serum.

Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3'-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS.

MiR-206 controls the phenotypic modulation of pulmonary arterial smooth muscle cells induced by serum from rats with hepatopulmonary syndrome by regulating the target gene, annexin A2.

BACKGROUND: Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease that is characterised by intrapulmonary vascular dilatation (IPVD) and arterial hypoxemia. Pulmonary vascular remodelling (PVR) is an important pathological feature of HPS, but the potential mechanisms underlying PVR remain undefined. Recent findings have established the essential role of changes in Annexin A2 (ANXA2) in controlling the phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS. However, the mechanism by which upstream signalling regulates ANXA2 is unclear. METHODS: In the present study, computational analysis was used to predict which miRNA might target the 3´-untranslated region (3´-UTR) of the ANXA2 mRNA. Real-time PCR and western blotting were performed to study the level of correlation between ANXA2 and the differentiation marker with the predicted miRNAs in PASMCs stimulated with serum from normal rats or those with HPS. Functional analysis of the miRNA and a luciferase reporter assay were performed to demonstrate that the predicted miRNA suppressed ANXA2 expression by directly targeting the predicted 3´-UTR site of the ANXA2 mRNA. RESULTS: Computational analysis predicted that miR-206 would target the 3´-UTR of ANXA2 mRNA. In HPS rat serum-stimulated PASMCs, the expression of miR-206 displayed an inverse correlation with ANXA2, while a positive correlation was observed with the phenotypic marker smooth muscle α-actin (SM α-actin). The miRNA functional analysis and luciferase reporter assay demonstrated that miR-206 effectively downregulated the expression of ANXA2 by binding to the 3´-UTR of the ANXA2 mRNA. Consistently, miR-206 effectively inhibited the HPS rat serum-induced phenotypic modulation and proliferation, while these effects were reversed in ANXA2-overexpressing PASMCs. CONCLUSION: This study demonstrates that miR-206 inhibits the HPS rat serum-induced phenotypic modulation and proliferation in PASMCs by down-regulating ANXA2 gene expression.

Metagenomic next-generation sequencing for pleural effusions induced by viral pleurisy: A case report.

BACKGROUND: Viral pleurisy is a viral infected disease with exudative pleural effusions. It is one of the causes for pleural effusions. Because of the difficult etiology diagnosis, clinically pleural effusions tend to be misdiagnosed as tuberculous pleurisy or idiopathic pleural effusion. Here, we report a case of pleural effusion secondary to viral pleurisy which is driven by infection with epstein-barr virus. Viral infection was identified by metagenomic next-generation sequencing (mNGS). CASE SUMMARY: A 40-year-old male with a history of dermatomyositis, rheumatoid arthritis, and secondary interstitial pneumonia was administered with long-term oral prednisone. He presented with fever and chest pain after exposure to cold, accompanied by generalized sore and weakness, night sweat, occasional cough, and few sputums. The computed tomography scan showed bilateral pleural effusions and atelectasis of the partial right lower lobe was revealed. The pleural fluids were found to be yellow and slightly turbid after pleural catheterization. Thoracoscopy showed fibrous adhesion and auto-pleurodesis. Combining the results in pleural fluid analysis and mNGS, the patient was diagnosed as viral pleuritis. After receiving Aciclovir, the symptoms and signs of the patient were relieved. CONCLUSION: Viral infection should be considered in cases of idiopathic pleural effusion unexplained by routine examination. mNGS is helpful for diagnosis.

Relationship between the anti-inflammatory properties of salmeterol/fluticasone and the expression of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in COPD.

BACKGROUND: Salmeterol and fluticasone combination (SFC) has anti-inflammatory effects and improves clinical symptoms in patients with chronic obstructive pulmonary disease (COPD). However, the anti-inflammatory mechanism of SFC remains unclear. In this study, we investigated the inflammatory responses of COPD, as well as the relationship of the inflammatory factors with the levels of CD4+CD25+Foxp3+ regulatory T cells (Foxp3+Tregs) after SFC therapy. METHODS: Twenty-one patients with moderate or severe COPD received treatment with 50/500 µg of SFC twice a day for 12 weeks. Before and after treatment, the patients were evaluated using the Modified Medical Research Council (MMRC) dyspnea scale and by conducting a 6-min walk test. The number of neutrophils, monocytes and lymphocytes in induced sputum were counted. Levels of cytokines, including pre-inflammatory IL-8, TNF-α, IL-17A and cytokine IL-10, in the sputum supernatant and peripheral blood were measured by ELISA. The proportion of Foxp3+Tregs in the total CD4+ T cell of the peripheral blood was determined by flow cytometry. The relationship between IL-17A levels and the percentage of Foxp3+Tregs was analyzed by statistical analysis. RESULTS: After treatment with SFC, the forced expiratory volume in 1 s as a percentage of predicted values (FEV1%) and the 6-min walk distance in the COPD patients significantly increased, while dyspnea scores decreased. The total number of cells, neutrophils, and the percentage of neutrophils in induced sputum reduced notably, while the proportion of monocytes was significantly increased. Levels of the inflammatory cytokines IL-8, TNF-α, and IL-17A in the sputum supernatant and in the blood were markedly lowered, while IL-10 levels were unchanged. The proportion of Foxp3+Tregs in the total CD4+T cell population in the peripheral blood was drastically higher than that before treatment. The level of IL-17A was negatively correlated with the proportion of Foxp3+Tregs in CD4+T cells. CONCLUSION: SFC can reduce the levels of inflammatory factors and improve symptoms of COPD. The levels of inflammatory factors are associated with the variation of Foxp3+Tregs in COPD. TRIAL REGISTRATION: This study was registered with http://www.chictr.org (Chinese Clinical Trial Register) as follows: ChiCTR-TNC-10001270.

[Effects of methyl ß cyclodextrin on the proliferation and transdifferentiation of type II alveolar epithelial cells].

OBJECTIVE: To study the destructive effects of the membrane lipid microdomain with methyl ß cyclodextrin (MßCD) on the proliferation, transdifferentiation and cell cycle of type II alveolar epithelial cell (AEC II). METHODS: The membrane lipid microdomain of AEC II was destroyed by MßCD (MßCD interference group) in vitro, and then cultured with DMEM as control. Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT); flow cytometry was used to assay the cell cycle. The expressions of AEC II specific surfactant protein-C (SP-C) and AECI specific aquaporin 5 (AQP5) were detected by immunofluorescence and Western blotting analyses. RESULTS: Compared with control group, cell number and the cell proliferation was decreased in MßCD interference group at 24, 48 and 72 hours after interaction [cell numbers (×10(6)/ml): 2.74±0.56 vs. 8.05±0.92, 4.45±0.68 vs. 10.52±0.81, 7.82±0.59 vs. 11.39±0.81; MTT results (A value): 0.25±0.20 vs. 0.45±0.02, 0.35±0.03 vs. 0.54±0.28, 0.48±0.04 vs. 0.59±0.05, all P<0.01]. MßCD could increase the percentage of cells in G0/G1 phases and decreased the percentage in S phases at 24 hours [G0/G1 phases: (60.06±1.65)% vs. (43.43±3.59)%; S phases: (16.20±2.17)% vs. (34.07±2.63)%, both P<0.05 ]. Incubation of AEC II with MßCD resulted in up regulation of the expression of SP-C (0.54±0.04 vs. 0.47±0.03, 0.19±0.03 vs. 0.06±0.02) and down regulation of AQP5 (0.30±0.04 vs. 0.43±0.06, 0.39±0.04 vs. 0.59±0.04) at 48 hours and 72 hours after interaction (P<0.05 or P<0.01). CONCLUSION: The destruction of membrane lipid microdomain by the MßCD can inhibit proliferation and transdifferentiation of AEC II, and induce cell cycle arrest in G1 phase.

[Adenovirus-mediated protein-kinase-GIalpha suppresses the hypoxia-induced proliferation and phenotype-switching of pulmonary arterial smooth muscle cell].

OBJECTIVE: To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell (PASMC) induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling (HPVR). METHODS: To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle alpha actin (SM-alpha-actin) determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and ³H-TdR incorporated way. RESULTS: Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-alpha-actin protein (44.25 ± 5.34 in normoxia, 32.18 ± 4.19 in 12 h hypoxia condition, 21.90 ± 2.44 in 24 h hypoxia condition, P < 0.05), that could be blocked by the transfection of Ad-PKGIα. Hypoxia could push PASMC mitosis and proliferating (³H-TdR incorporated way: 7570 ± 371 in normoxia, 12,020 ± 831 in 12 h hypoxia condition, 14,924 ± 1491 in 24 h hypoxia condition, P < 0.05), that could be blocked by the transfection of Ad-PKGIα, too. CONCLUSIONS: The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.

Restoration of SOCS3 suppresses human lung adenocarcinoma cell growth by downregulating activation of Erk1/2, Akt apart from STAT3.

SOCS3 is regarded as a major negative regulator of STAT3. Recent evidence indicates that SOCS3 regulates strength and duration of other signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in non-malignant cells. The repression or silence of SOCS3 expression in a few tumor types has led to speculation that loss of SOCS3 gene is closely related to deregulation of multiple signal pathways during tumorigenesis. However, apart from STAT3, little is known in malignant cells about the mechanism by which SOCS3 modulates other intracellular signal cascades such as Erk1/2 and Akt, whose aberrant activation has been implicated in many human tumors. Expression of SOCS3 proved deficient in human lung adenocarcinoma A549 cells, and forced expression of SOCS3 resulted in growth inhibition. Growth suppression due to SOCS3 was associated with attenuated activation of Erk1/2, Akt as well as STAT3. The results suggested that SOCS3, as negative regulators of cytokine signaling, might maintain homeostasis by regulating multiple signaling pathways and reverse cell malignant behavior.

[Study on expression of liver X receptor-alpha in rat lung tissue in acute lung injury].

OBJECTIVE: To observe changes in liver X receptor-alpha (LXR alpha) in acute lung injury (ALI) in rats induced by lipopolysaccharide (LPS) to explore mechanism of LXR alpha in pathogenesis of ALI. METHODS: Forty-eight Wistar rats were randomly divided into two groups. ALI model was reproduced by intravenous injection of LPS (5 mg/kg), and control group was injected with normal saline (2.5 ml/kg). At 1, 2, 4, 8 hours after ALI, artery blood gas analysis, lung tissue wet/dry weight (W/D) ratio, myeloperoxidase (MPO) activity, lung histopathologic changes were observed. The expressions of LXR alpha and tumor necrosis factor-alpha (TNF-alpha) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). TNF-alpha content was measured with enzyme linked immunosorbent assay (ELISA). LXR alpha protein in lung tissues was assessed by immunohistochemistry. RESULTS: Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood (PaO(2)) decreased significantly, lung W/D weight ratio and MPO activity increased significantly (all P<0.05), histopathology of lung revealed signs of injury. After injury, expression of LXR alpha mRNA in lung tissue decreased markedly, and expression of TNF-alpha mRNA in lung tissue increased markedly (all P<0.05). TNF-alpha increased markedly in lung homogenate and blood serum at the same period, and TNF-alpha reached peak value at 4 hours. Immunohistochemical staining of LXR alpha showed that lung tissues of normal rats express LXR alpha significantly, however, after injury, expression of LXR alpha in lung tissue decreased markedly (all P<0.05). CONCLUSION: Lung tissues of normal rats express LXR alpha. The decreased LXR alpha mRNA and protein expressions in the lung tissue of rats with ALI caused by LPS may be associated with the occurrence of ALI.

[Effect of nuclear factor-kappaB on peroxisome proliferator activated receptor gamma expression in Ana-1 cells].

OBJECTIVE: To determine the effects of nuclear factor-alpha B (NF-alpha B) on peroxisome proliferator activated receptor gamma (PPAR gamma) expression in the murine macrophage cell line Ana-1, based on the investigation of the PPAR gamma expression stimulated by lipopolysaccharide (LPS). METHODS: Ana-1 cells were divided randomly into seven groups: control group, LPS groups (cells were activated by 0.1 mg/L LPS for 1, 2, 4, 8 hours respectively), SN50 group (cells were stimulated by 50 mg/L SN50 for 4 hours) and NF-alpha B high expression plasmid transfected group. PPAR gamma and tumor necrosis factor-alpha(TNF-alpha) mRNA levels were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and the TNF-alpha protein was measured by enzyme linked immunosorbent assay (ELISA) after being activated by LPS. The eukaryotic expression vector of murine NF-alpha B p65 gene was constructed and stably transfected into Ana-1 cells with DOTAP liposome . The PPAR gamma expression and NF-alpha B p65 translocation as stimulated by LPS and SN50 were assayed by Western blotting. RESULTS: Expressions of both PPAR gamma mRNA and protein were downregulated when cells were stimulated by LPS, which were accompanied with the activity of NF-alpha B and TNF-alpha in Ana-1 cells (all P<0.05). The eukaryotic expression vectors containing murine p65 gene were successfully constructed and stably transfected into Ana-1 cells. LPS stimulation and NF-alpha B p65 gene overexpression resulting in upregulation of p65 could downregulate PPAR gamma expression in Ana-1 cells (r=-0.913, P<0.05).But downregulation of NF-alpha B by SN50 could upregulate PPAR gamma expression in the nucleus (r=-0.959, P<0.05). CONCLUSION: LPS can markedly decrease the expression of PPAR gamma in Ana-1 cells, which may be related to its activity of enhancing NF-alpha B. NF-alpha B p65 gene can control PPAR gamma expression in the reverse direction in Ana-1 cells.

[The effect of seawater on expression of protease-activated receptor 2 in adenocarcinoma of lung cell line A549 cells].

OBJECTIVE: To investigate the effect of seawater on expression of protease-activated receptor 2 (PAR-2) in adenocarcinoma of lung cell line A549 cells, and the inflammatory injury on A549 cells induced by seawater. METHODS: A549 cells were randomly divided into four groups: control group, 2 hours group, 4 hours group, 8 hours group, in which cells were treated with seawater for 2, 4 and 8 hours respectively. After seawater treatment, cells were collected for real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis to determine the expression of PAR-2 mRNA and its protein. Supernatant were collected for tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) which were determined by enzyme-linked immunosorbent essay (ELISA). RESULTS: The expression of PAR-2 mRNA and protein of A549 cells increased in a time-dependent manner after seawater treatment, significantly so after 2 hours in all groups (both P<0.05), and peaked at 4 hours after seawater treatment (1.8-fold and 2.2-fold respectively, both P<0.01), followed by a decrease though still higher than those of control group significantly (both P<0.01). TNF-alpha and IL-8 in supernatant increased significantly after seawater treatment, peaking at 2 hours after seawater treatment [(214.35+/-20.85) ng/L, (55.86+/- 5.65) ng/L ] and then followed by a slight decrease though still significantly higher than those of control group [(25.86+/-3.85) ng/L, (6.97+/-1.77) ng/L, all P<0.01]. CONCLUSION: Seawater can induce significant inflammation of A549 cells and up-regulate the expression of PAR-2 on A549 cells.

The involvement of aquaporin 1 in the hepatopulmonary syndrome rat serum-induced migration of pulmonary arterial smooth muscle cells via the p38-MAPK pathway.

Hepatopulmonary syndrome (HPS) is characterized by arterial oxygenation defects induced by intrapulmonary vascular dilation (IPVD). Pulmonary vascular remodeling (PVR) is an important pathological feature of IPVD; however, the details regarding the underlying mechanisms of this process remain undefined. Recent studies have determined that the abnormal migration of pulmonary arterial smooth muscle cells (PASMCs) plays a role in the pathogenesis of the PVR associated with HPS. Additionally, aquaporin 1 (AQP1) not only functions as a water channel molecule but also promotes cell migration by facilitating water transport in the lamellipodia of migrating cells. Common bile duct ligation (CBDL) rat is a well-accepted HPS model; we determined that the immunoperoxidase labeling of AQP1 was enhanced in the media of the pulmonary vessels in CBDL rats. HPS rat serum mediated the overexpression of AQP1 in PASMCs, and also upregulated PASMC migration. Small interfering RNAs (siRNAs) that targeted rat AQP1 caused significant downregulation of AQP1, which resulted in decreased PASMC migration. Furthermore, the inhibition of the p38-MAPK pathway abolished AQP1-dependent PASMC migration. In conclusion, this study demonstrated that AQP1 enhanced PASMC migration via the p38-MAPK pathway in rat with HPS and may represent a potential therapeutic strategy in the setting of pulmonary vascular remodeling associated with HPS.

[Enhanced expression of signal transducers and activators of transcription in lung tissue of hypoxic pulmonary hypertension rat models].

OBJECTIVE: To investigate the expression levels of signal transducers and activators of transcription (STATs) in the lung tissue of hypoxic pulmonary hypertension (HPH) rat models. METHODS: The Wister rat HPH models were divided into 4 groups: 1 week group (H1), 2 week group (H3), 3 week group (H3), and 4 week group (H4) (n = 12 in each group). The levels of STATsmRNA expression of the lung tissue were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot. The protein expression of STATs and cellular morphologic changes were observed by immunohistochemistry and Tiger image analysis. RESULTS: The RT-PCR showed that the levels of STAT1mRNA expression of the lung tissue in H1 1.25 +/- 0.12, H2 2.28 +/- 0.15 and H3 1.27 +/- 0.12 were significantly higher than that in the healthy control group 0.61 +/- 0.07 (P < 0.01); the levels of STAT2mRNA expression in H1 0.54 +/- 0.06, H2 1.01 +/- 0.08 and H3 1.36 +/- 0.09 were significantly higher than that in control group 0.30 +/- 0.03 (P < 0.01); the mRNA expressions of STAT3 in H1 0.74 +/- 0.11, H2 1.19 +/- 0.13 and H3 0.80 +/- 0.08 were significantly higher than that in control group 0.26 +/- 0.10 (P < 0.01); and the mRNA expressions of STAT5 in H1 0.92 +/- 0.10, H2 1.23 +/- 0.10 and H3 1.03 +/- 0.11 were significantly higher than that in control group 0.60 +/- 0.11 (P < 0.01). The Northern blot assay demonstrated that the expressions of STAT1mRNA in H1 0.49 +/- 0.10 and H3 0.67 +/- 0.07 were significantly different from that in H2 0.91 +/- 0.07 (P < 0.01); the expressions of STAT3mRNA in H1 2.10 +/- 0.21 and H3 2.58 +/- 0.17 were significantly different from that in H1 3.56 +/- 0.29 (P < 0.01); the expressions of STAT5mRNA in H1 0.99 +/- 0.10 and H3 1.45 +/- 0.12 were significantly different from that in H2 1.79 +/- 0.15 (P < 0.01). It is obvious that the mRNA expressions of STATs in the lung tissue of rat HPH models were increased in the 1st week, to the highest in the 2nd week, and decreased in the 3st week, and all higher than that of control group. The granules of STAT3 and STAT5, positively stained, were observed in the cytoplasm of pulmonary alveolar wall, intrapulmonary vascular and bronchial wall in H3. The protein expressions of STAT3 8.16 +/- 0.49 and STAT5 6.03 +/- 0.37 in H3 were markedly higher than those in H1, H4, and control group. CONCLUSION: The up-regulation of the STATs expression in lung tissue of HPH rat models suggested that the STATs were involved in the pathogenesis of HPH formation.

[Changes of interleukin-6 and Janus kinases in rats with hypoxic pulmonary hypertension].

OBJECTIVE: To investigate the expressions of interleukin 6 (IL-6) and Janus kinases (JAKs) in rats with hypoxia induced pulmonary hypertension (HPH). METHODS: Sixty male Wistar rats were randomized into 5 groups with 12 animals in each: a one-week hypoxic group (H(1) group), a two-week hypoxic group (H(2) group), a three-week hypoxic group (H(3) group), a four-week hypoxic group (H(4) group), a normal oxygen group (N group). The rat model of HPH was replicated in normal baric hypoxic cabin. The levels of IL-6 and JAKs mRNA were measured by using reverse transcription-polymerase chain reaction (RT-PCR); The expression of JAKs protein and cell morphologic changes were observed by immunohistochemistry. RESULTS: (1) The levels of IL-6 mRNA in H(1) (1.67 +/- 0.09), H(2) (2.26 +/- 0.12) and H(3) (1.55 +/- 0.11) groups were significantly higher than that in N group (1.20 +/- 0.11, all P < 0.01). The levels of JAK1 mRNA in H(1) (2.11 +/- 0.09), H(2) (2.85 +/- 0.12) and H(3) (2.36 +/- 0.13) groups were significantly higher than that in N group (1.62 +/- 0.10, all P < 0.01); The levels of JAK2 mRNA in H(1) (1.41 +/- 0.07), H(2) (2.02 +/- 0.13) and H(3) (1.36 +/- 0.09) groups were significantly higher than that in N group (1.01 +/- 0.09, all P < 0.01); The levels of JAK3 mRNA in H(1) (0.86 +/- 0.11), H(2) (1.45 +/- 0.10) and H(3) (0.91 +/- 0.13) groups were significantly higher than that in N group (0.55 +/- 0.08, all P < 0.01). The levels of TYK2 mRNA in H(1) (1.36 +/- 0.10), H(2) (1.76 +/- 0.11) groups were significantly higher than that in N group (0.57 +/- 0.07, all P < 0.01). The levels of IL-6 and JAKs mRNA in H(2) group were significantly higher than those in H(1) and H(3) groups (all P < 0.01). (2) Histochemical staining of JAK1 and JAK3 in H(3) group showed yellow cytoplasm of alveolar wall cells, bronchial wall cells and small blood wall cells; The active protein contents of JAK1 (5.36 +/- 0.32) and JAK3 (4.88 +/- 0.29) were markedly increased as compared with N group (1.52 +/- 0.18, 1.22 +/- 0.09, respectively; all P < 0.01) by TIGER image analysis method. CONCLUSIONS: The levels of IL-6, JAKs mRNA and protein can increase in pulmonary tissues of rats with HPH. It suggests that IL-6/JAKs pathway may take part in the pathogenesis of HPH.

Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.

BACKGROUND: LPS-binding protein (LBP) and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. Blocking LBP and CD14 binding might prevent LPS-induced inflammation. In previous studies, we obtained a peptide analog (MP12) for the LBP/CD14 binding site and showed that this peptide analog had anti-endotoxin activity. In this study, we used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity. METHODS: We used error-prone PCR (ep-PCR) and induced mutations in the C-terminus of LBP and attached the PCR products to T7 phages to establish a mutant phage display library. The positive clones that competed with LBP for CD14 binding was obtained by screening. We used both in vivo and in vitro experiments to compare the anti-endotoxin activities of a polypeptide designated P1 contained in a positive clone and MP12. RESULTS: 11 positive clones were obtained from among target phages. Sequencing showed that 9 positive clones had a threonine (T) to methionine (M) mutation in amino acid 287 of LBP. Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-α expression and NF-κB activity in U937 cells (P<0.05). Compared to MP12, P1 significantly improved arterial oxygen pressure, an oxygenation index, and lung pathology scores in LPS-induced ARDS rats (P<0.05). CONCLUSION: By in vitro directed evolution of peptide analogs for the LBP/CD14 binding site, we established a new polypeptide (P1) with a threonine (T)-to-methionine (M) mutation in amino acid 287 of LBP. This polypeptide had high anti-endotoxin activity in vitro and in vivo, which suggested that amino acid 287 in the C-terminus of LBP may play an important role in LBP binding with CD14.

Over-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation, and phenotype modulation of human PASMCs: the role of phenotype modulation of PASMCs in pulmonary vascular remodeling.

The proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a role in pulmonary vascular remodeling (PVR). Recently, it was shown that vascular smooth muscular cell phenotype modulation is important for their proliferation in other diseases. However, little is known about the role of human PASMC phenotype modulation in the proliferation induced by hypoxia and its molecular mechanism during PVR. In this study, we found using primary cultured human PASMCs that hypoxia suppressed the expression of endogenous PKGIα, which was reversed by transfection with a recombinant adenovirus containing the full-length cDNA of PKGIα (Ad-PKGIα). Ad-PKGIα transfection significantly attenuated the hypoxia-induced downregulation of the expression of smooth muscle α-actin (SM-α-actin), myosin heavy chain (MHC) and calponin in PASMCs, indicating that hypoxia-induced phenotype modulation was blocked. Furthermore, flow cytometry and (3)H-TdR incorporation demonstrated that hypoxia-induced PASMC proliferation was suppressed by upregulation of PKGIα. These results suggest that enhanced PKGIα expression inhibited hypoxia-induced PASMC phenotype modulation and that it could reverse the proliferation of PASMCs significantly. Moreover, our previous work has demonstrated that Akt protein is activated in the process of hypoxia-induced proliferation of human PASMCs. Interestingly, we found that Akt was not activated by hypoxia when PASMC phenotype modulation was blocked by Ad-PKGIα. This result suggests that blocking phenotype modulation might be a key up-stream regulatory target.

Over-starvation aggravates intestinal injury and promotes bacterial and endotoxin translocation under high-altitude hypoxic environment.

AIM: To study whether over-starvation aggravates intestinal mucosal injury and promotes bacterial and endotoxin translocation in a high-altitude hypoxic environment. METHODS: Sprague-Dawley rats were exposed to hypobaric hypoxia at a simulated altitude of 7000 m for 72 h. Lanthanum nitrate was used as a tracer to detect intestinal injury. Epithelial apoptosis was observed with terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Serum levels of diamino oxidase (DAO), malondialdehyde (MDA), glutamine (Gln), superoxide dismutase (SOD) and endotoxin were measured in intestinal mucosa. Bacterial translocation was detected in blood culture and intestinal homogenates. In addition, rats were given Gln intragastrically to observe its protective effect on intestinal injury. RESULTS: Apoptotic epithelial cells, exfoliated villi and inflammatory cells in intestine were increased with edema in the lamina propria accompanying effusion of red blood cells. Lanthanum particles were found in the intercellular space and intracellular compartment. Bacterial translocation to mesenteric lymph nodes (MLN) and spleen was evident. The serum endotoxin, DAO and MDA levels were significantly higher while the serum SOD, DAO and Gln levels were lower in intestine (P < 0.05). The bacterial translocation number was lower in the high altitude hypoxic group than in the high altitude starvation group (0.47 ± 0.83 vs 2.38 ± 1.45, P < 0.05). The bacterial translocation was found in each organ, especially in MLN and spleen but not in peripheral blood. The bacterial and endotoxin translocations were both markedly improved in rats after treatment with Gln. CONCLUSION: High-altitude hypoxia and starvation cause severe intestinal mucosal injury and increase bacterial and endotoxin translocation, which can be treated with Gln.

JAK-STAT signaling pathway in pulmonary arterial smooth muscle cells is activated by hypoxia.

Pulmonary arterial smooth muscle cells (PASMC) were divided into a normoxic group (N), 2, 8 and 12 h hypoxic groups (H2, H8 and H12) and an AG490 plus 8 h hypoxic group (AG490). The expression of JAK1, JAK2, JAK3 and TYK2 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). STAT1 and STAT3 protein expressions were determined by Western blotting. The results showed that the levels of JAK1, JAK2 and JAK3 mRNA did not change significantly in the N group but were increased after 2 h exposure to hypoxia. JAK1, JAK2 and JAK3 mRNA expressions peaked at 8 h. It decreased at 12 h but remained above those in the N group. TYK2 mRNA was not found in either hypoxic or normal PASMC. The phospho-STAT1 and -STAT3 protein levels increased after 2 h exposure to hypoxia. They were about 2.8 and 4.1 times the N group, respectively, after 8 h. They decreased at 12 h but remained above those in the N group. AG490 inhibited phospho-STAT3 protein expression by about 25.5% at 8 h but did not block the expression of phospho-STAT1 protein. These findings suggest that hypoxia induces the expression of JAK1, JAK2, JAK3 and phospho-STAT1 and -STAT3 in PASMC. Hypoxia activates the JAKs-STATs signaling pathway, which may participate in the pathogenesis of hypoxic PASMC injury.