Trigonelline Inhibits Inflammation and Protects ß Cells to Prevent Fetal Growth Restriction during Pregnancy in a Mouse Model of Diabetes.

BACKGROUND: As an active component from traditional Chinese medicine, trigonelline has a protective effect on diabetes. This study evaluated the protective effects of trigonelline on diabetic mice during pregnancy. METHODS: Diabetes was induced in female mice by intraperitoneal injection for continuous 5-day of 40 mg/kg/day streptozotocin. Female mice were divided into 4 groups after they were allowed to mate with normal male mice: nondiabetic, nondiabetic treated with trigonelline (70 mg/kg) for 18 days, diabetic, and diabetic treated with trigonelline (70 mg/kg). RESULTS: Diabetic pregnant mice had significantly higher levels of blood glucose, serum total cholesterol, triglyceride, insulin, and leptin but lower serum omentin-1 level and insulin sensitivity index than the nondiabetic ones. Trigonelline improved the hyperglycemia, dyslipidemia, insulin resistance, and adipocytokine of diabetic pregnant mice. Diabetic pregnant mice had significantly reduced fetus numbers, fetal weight, and fetal/placental ratio, which were reversed by trigonelline. Trigonelline prevented the increase in proinflammatory cytokines and reduced interleukin-10 level in placenta of diabetic pregnant mice. Trigonelline increased ß-cell replication and the decreased ß-cell mass, and decreased the ß-cell apoptosis of diabetic pregnant mice. CONCLUSION: These findings suggest that trigonelline protects diabetic pregnancy partly by suppressing inflammation, regulating the secretion of adipocytokines, increasing ß-cell mass, replication, and decreasing ß-cell apoptosis.

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.

Chinese consensus on early diagnosis of primary lung cancer (2014 version).

The incidence and mortality of lung cancer in China have rapidly increased. Lung cancer is the leading cause of cancer death in China, possibly because of the inadequate early diagnosis of lung cancer. Reaching a consensus on early diagnostic strategies for lung cancer in China is an unmet needed. Recently, much progress has been made in lung cancer diagnosis, such as screening in high-risk populations, the application of novel imaging technologies, and the use of minimally invasive techniques for diagnosis. However, systemic reviews of disease history, risk assessment, and patients' willingness to undergo invasive diagnostic procedures also need to be considered. A diagnostic strategy for lung cancer should be proposed and developed by a multidisciplinary group. A comprehensive evaluation of patient factors and clinical findings should be completed before treatment.

Prevention and management of lung cancer in China.

Lung cancer is the leading cause of cancer-related death worldwide. In China, the incidence of lung cancer has grown rapidly, resulting in a large social and economic burden. Several researchers have devoted their studies to lung cancer and have demonstrated that there are many risk factors for lung cancer in China, including tobacco use, environmental pollution, food, genetics, and chronic obstructive pulmonary disease. However, the lung cancer incidence is still growing rapidly in China, and there is an even higher incidence among the younger generation. One explanation may be the triple-neglect situation, in which medical policies that neglect prevention, diagnosis, and supportive care have increased patients' mortality and reduced their quality of life. Therefore, it is necessary to enhance the efficiency of prevention and early diagnosis not only by focusing more attention on treatment but also by drawing more attention to supportive care for patients with lung cancer.

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.

Role of Angptl4 in vascular permeability and inflammation.

BACKGROUND: Angptl4 is a secreted protein involved in the regulation of vascular permeability, angiogenesis, and inflammatory responses in different kinds of tissues. Increases of vascular permeability and abnormality changes in angiogenesis contribute to the pathogenesis of tumor metastasis, ischemic-reperfusion injury. Inflammatory response associated with Angptl4 also leads to minimal change glomerulonephritis, wound healing. However, the role of Angptl4 in vascular permeability, angiogenesis, and inflammation is controversy. Hence, an underlying mechanism of Angptl4 in different kind of tissues needs to be further clarified. METHODS: Keywords such as angptl4, vascular permeability, angiogenesis, inflammation, and endothelial cells were used in search tool of PUBMED, and then the literatures associated with Angptl4 were founded and read. RESULTS: Data have established Angptl4 as the key modulator of both vascular permeability and angiogenesis; furthermore, it may also be related to the progression of metastatic tumors, cardiovascular events, and inflammatory diseases. This view focuses on the recent advances in our understanding of the role of Angptl4 in vascular permeability, angiogenesis, inflammatory signaling and the link between Angptl4 and multiple diseases such as cancer, cardiovascular diseases, diabetic retinopathy, and kidney diseases. CONCLUSIONS: Taken together, Angptl4 modulates vascular permeability, angiogenesis, inflammatory signaling, and associated diseases. The use of Angptl4-modulating agents such as certain drugs, food constituents (such as fatty acids), nuclear factor (such as PPARα), and bacteria may treat associated diseases such as tumor metastasis, ischemic-reperfusion injury, inflammation, and chronic low-grade inflammation. However, the diverse physiological functions of Angptl4 in different tissues can lead to potentially deleterious side effects when used as a therapeutic target. In this regard, a better understanding of the underlying mechanisms for Angptl4 in different tissues is necessary.

Pretreatment with anti-flagellin serum delays acute lung injury in rats with sepsis.

OBJECTIVE: This study examined the reduction of sepsis-induced ALI by inhibition of flagellin-stimulated TLR5 signaling. METHODS: Rats were randomly divided into three groups: one group served as the sham-operated group (control group), and the other two groups received the induction of sepsis (sepsis and treatment groups). The treatment group was injected with anti-flagellin serum before induction of sepsis. At 2, 4, 6, 12, 24, and 48 h following induction of sepsis (six time-point subgroups, n = 10 per subgroup), arterial PaO(2), wet/dry (W/D) lung weight ratios, levels of serum and BALF flagellin and TNF-α, pulmonary pathological alterations, and TLR5 mRNA expression in the lungs were examined. RESULTS: Compared to sham-operated rats, septic rats had: increased levels of serum and BALF flagellin at 6, 12, 24, and 48 h; reduced arterial PaO(2); elevated W/D lung weight ratio; increased serum and BALF TNF-α levels; and up-regulated TLR5 mRNA expression at 12, 24, and 48 h (P < 0.01). Pretreatment with anti-flagellin serum, however, significantly inhibited sepsis-associated declines in arterial PaO(2), increased W/D lung weight ratios, elevated serum and BALF TNF-α levels, and up-regulated TLR5 mRNA expression at 24 and 48 h (P < 0.01). CONCLUSION: Neutralizing the actions of circulating flagellin with anti-flagellin serum delayed the development of ALI in rats with sepsis.

[The negative enrichment by immunomagnetic beads for tumor cells from malignant pleural effusions].

OBJECTIVE: To establish a method (negative enrichment by immunomagnetic beads) for detection of tumor cells in pleural effusions and to evaluate the sensitivity and specificity of the method for clinical application. METHODS: Five, 10, 20, 50 and 100 A549 (lung adenocarcinoma) cells were labeled with DAPI and added into 20 ml pleural effusions [containing (1 - 10)×10(6)cells] from heart failure patients, followed by immunomagnetic negative enrichment method. Recovered cancer cells were enumerated using a fluorescent microscope. Tumor cells were enriched from pleural effusion samples by means of density gradient centrifugation and negative enrichment by immunomagnetic beads method, followed by identification with cytology analysis (Wright's Giemsa's staining), immunofluorescence staining (IF) and fluorescence in situ hybridization (FISH) using centromere DNA probes of chromosome 7 and 8. Cytology, IF and FISH evaluations were performed in 53 pleural effusion samples, including 36 cases of malignant disease (25 male and 11 female patients aging 40 to 78 years, mean age (63 ± 9) and 17 cases of benign disease (8 male and 9 female patients aging 25 to 81 years, mean age (53 ± 18). RESULTS: After DAPI staining and mixing with pleural effusions from heart failure patients, the cell recovery rates of A549 cells evaluated under fluorescence microscope were 75%, 78%, 82%, 85%, 88%, and the average recovery rate was 81.6%. Using negative enrichment method and density gradient centrifugation combined with cytology analysis, the positive rates of tumor cells in 36 malignant pleural effusion samples were 81% (29/36) and 61% (22/36), respectively (χ(2) = 4.00, P = 0.039). Using negative enrichment method combined with IF, the positive rate of CK18(+), DAPI(+), CD(45)(-) cells was 100%. Moreover, using negative enrichment method combined with FISH analysis, the positive rate of tumor cells was 86% (31/36), much higher than that using density gradient centrifugation combined with cytology analysis (χ(2) = 5.818, P = 0.012). In 17 cases of benign pleural effusions, using negative enrichment method combined with IF, the positive rate was 100%. But other methods didn't find cancer cells from benign pleural effusions. CONCLUSIONS: It was applicable to enrich tumor cells from pleural effusions using negative enrichment method by immunomagnetic beads. This method combined with cytology analysis or FISH significantly enhanced the sensitivity and specificity of tumor cell detection in pleural effusions. But it was difficult to distinguish cancer cells from mesothelial cells using immunofluorescence staining with CK18, DAPI and CD(45) label. More specific markers were needed to recognize tumor cells from pleural effusions.

Mitochondrial and energy metabolism-related properties as novel indicators of lung cancer stem cells.

Energy metabolism is the foundation of survival for all organisms, and mitochondria are the most important energy-supplying organelles in eukaryotic cells. However, the mitochondrial and energy/metabolism-related properties of cancer stem cells (CSCs), the stem cell-like subpopulation in tumor masses, remain unknown. In our study, we compared the masses of mitochondria and mitochondrial DNA (mtDNA), the mitochondrial membrane potential (Δψm), oxygen/glucose consumption, and the concentration of reactive oxygen species (ROS) and ATP between lung CSCs (LCSCs) and non-LCSCs. In addition, the change in features during differentiation was examined. Some mitochondrial and energy metabolism-related properties, such as perinuclear mitochondrial distribution, a lower quantity of mtDNA, higher Δψm, lower oxygen/glucose consumption, and lower intracellular concentrations of ROS and ATP, can be used as indicators of LCSCs.

Over-expression of caveolin-1 aggravate LPS-induced inflammatory response in AT-1 cells via up-regulation of cPLA2/p38 MAPK.

OBJECTIVE AND DESIGN: The aim of this study was to study the effect of caveolin-1 on the cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB (NF-kappaB) in mouse lung alveolar type-1 cells' (AT-1 cells) inflammatory response induced by LPS. MATERIALS AND METHODS: Gene clone technique was used to over-express caveolin-1 in AT-1 cells by lentivirus vector. The level of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), cPLA2, p38 MAPK and NF-kappaB was measured by ELISA, western blotting and EMSA. TREATMENT: AT-1 cells were treated with LPS. RESULTS: Over-expression of caveolin-1 not only increased the production of pro-inflammatory cytokine TNF-alpha and IL-6, but also enhanced the expression of the cPLA2, p38 MAPK, and NF-kappaB. CONCLUSIONS: Our data demonstrated that over-expression of caveolin-1 aggravates the AT-1 injury induced by LPS, involving in modulation of the cPLA2 mediated by the cPLA2/p38 MAPK pathway.

[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.

[Small interfering RNA (siRNA) targeting vascular endothelial growth factor inhibits vascular leakage in brain of rats exposed to high altitude].

OBJECTIVE: To observe the change of expression of vascular endothelial growth factor (VEGF) and vascular leakage in the brain of rats exposed to high altitude with siRNA targeting vascular endothelial growth factor and explore the pathological mechanism and preventive approach of high altitude cerebral edema (HACE). METHODS: Fifty male Wistar rats were divided randomly into normal control group (Ncon), high altitude control group (Hcon), intraventricular normal saline control group (Scon), intraventricular siRNA group (CVI) and intravenous siRNA group (IVI). Rats in Ncon were raised normally. Rats in Hcon, Scon, CVI and IVI pretreated with intravenous injection of normal saline, intraventricular injection of normal saline, intraventricular injection of siRNA and intravenous injection of siRNA respectively were exposed to a low-pressure cabin mimicking a high altitude of 7000 m for 24 h. The ratio of dry and wet brain weight was calculated and the sodium fluorescein leakage calculated to evaluate the cerebral edema and the blood brain barrier permeability. Also the real-time quantitative RT-PCR was employed to detect the expression of VEGF mRNA and the Western blot the expression of VEGF. RESULTS: Compared with rats in NC, high altitude exposure led to a significant increase in the levels of VEGF mRNA (from 21.6 + or - 3.5 K copies/microg to 36.3 + or - 3.9 K copies/microg, P < 0.01) and protein (from 48 + or - 0.09 to 0.77 + or - 0.12, P < 0.01) in rat brain and fluorescence intensity of sodium fluorescein increased significantly (from 548 + or - 48 rfu/mg to 674 + or - 32 rfu/mg, P < 0.01). Intravenous injection of siRNA targeting to VEGF caused no significant change of expression VEGF mRNA and protein and fluorescence intensity of sodium fluorescein in rat brain (P > 0.05, respectively). While compared with rats in HC, intraventricular injection of siRNA targeting to VEGF caused the significant reduction of expression of VEGF mRNA (from 36.3 + or - 3.9 to 19.9 + or - 4.3, P < 0.01) and protein (from 0.77 + or - 0.12 to 0.44 + or - 0.13, P < 0.01) and fluorescence intensity of sodium fluorescein (from 674 + or - 32 rfu/mg to 542 + or - 77 rfu/mg, P < 0.05) in rat brain. There were no significant change in the ratio of dry and wet brain weight among five groups. CONCLUSION: VEGF may play a key role in the pathologic process of HACE. Intraventricular injection of siRNA targeting to VEGF inhibits the expression of VEGF and prevent the high altitude-induced vascular leakage. These findings might provide a basis for new preventive approaches of cerebral edema.

[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.

[Transfection of human beta defensin 2 gene into the lung by aerosol inhalation: experiment with rats].

OBJECTIVE: To investigate the feasibility to transfect human beta defensin 2 (hBD2) gene into the lung so as to enhance the endogenous hBD2 expression to defend infectious diseases. METHODS: Recombinant plasmid containing hBD2 gene, pLXSN-hBD2, was mixed with a new cationic liposome prepared by film at different weight ratios. Sixteen Wistar rats were divided into 2 groups: experimental group (n = 11), undergoing aerosol inhalation of liposome/pLXSN-hBD2 complex, and control group (n = 5), undergoing aerosol inhalation of blank vector pLXSN. The tracheae were taken out from 5 rats of the experiment and control groups each 2 days later, and from 2 rats of the experimental group 6, 15, and 21 days later respectively so as to obtain the epithelial cells of trachea. DNA was extracted from the tracheal epithelial cells and PCR was used to examine the transfection and integration of hBD2. Western blotting was used to detect the protein expression of hBD2. RESULTS: Aerosolization impacted obviously the microcapsule structure of liposome/plasmid complexes, and there was the least structural destruction of complex at a ratio of 10:1 that suited for aerosolization best. After the inhalation the relevant plasmids were all successfully integrated into the epithelial cells in both groups. Protein expression of hBD2 was not detected in the control group and the hBD2 protein expression level 2 days after transfection of the experimental group was 4866.9 +/- 148.2, and then decreased gradually, and reached 3.2 +/- 1.5 twenty-one days after the transfection. CONCLUSION: The recombinant plasmid pLXSN-hBD2 can be transfect into the airway epithelial cells via aerosol inhalation and the expression of hBD2 sustains for a period of time.

[The changes of PTEN/Akt1 expression and cell proliferation in pulmonary arterial smooth muscle cells induced by hypoxia in rats].

OBJECTIVE: To observe the changes of PTEN/Akt1 mRNA and protein expression level in pulmonary arterial smooth muscle cells (PASMC) induced by hypoxia in rats, and to investigate the role of PTEN/Akt1 signaling pathway in hypoxic pulmonary hypertension (HPH). METHODS: Pure PASMCs were grown and cultured from rat pulmonary arterial tissues. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) were used to examine PTEN and Akt1 mRNA expression after PASMCs were exposed to hypoxia for 2 h, 8 h, 12 h and 24 h respectively. PTEN/Akt1 protein expressions were determined by Western blotting. The changes of PASMC proliferation were determined by MTT and (3)H-TdR incorporation. Results were expressed as (-x) +/- s. Statistical comparisons of results were performed by t test of Excel 2003. Differences were considered significant if P < 0.05. RESULTS: PASMCs were induced to keep proliferating by hypoxia. The value of (3)H-TdR was 0.37 +/- 0.06 under normoxia, but was increased with prolonged exposure to hypoxia, reaching 0.70 +/- 0.10 at 12 h (t = 14.29, P < 0.01), being significantly different compared to the normoxia group. The value of MTT was 8374 +/- 545 under normoxia, but was increased by exposure to hypoxia, reaching 11 208 +/- 679 at 24 h (t = 19.56, P < 0.01), being significantly different compared to the normoxia group. The mRNA and protein of PTEN/Akt1 were detectable in all the groups. The values of mRNA, total protein and phosphorylated protein of Akt1 were 0.76 +/- 0.09, 25 +/- 6, 48 +/- 8 respectively in the normoxia group. After exposure to hypoxia, the values were 1.05 +/- 0.09, 41 +/- 7, 79 +/- 14 respectively at 8 h, being significantly different compared to the normoxia group (t = 168.00, 58.54, 8.31, P < 0.01 and P < 0.05), but the values decreased thereafter and returned to the level comparable to the normoxia group at 24 h. The values of mRNA, phosphorylated protein of PTEN were 0.25 +/- 0.06, and 98 +/- 8 respectively in the normoxia group, but were increased after exposure to hypoxia, reaching 0.38 +/- 0.05, and 232 +/- 12 respectively at 24 h, being significantly different compared to the normoxia group (t = 22.04, 50.46, all P < 0.01). CONCLUSION: The results suggested that PTEN/Akt1 signaling pathway might play an important role in hypoxic pulmonary hypertension.

[The protective effects and mechanisms of peroxisome proliferator-activated receptor-gamma agonist in rats with acute lung injury].

OBJECTIVE: To observe if peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist (troglitazone) was able to alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats, and explore the underlying mechanisms. METHODS: Seventy-two wistar rats were randomized into the the following groups, the LPS groups (32 rats), and the troglitazone intervention groups (T group, 32 rats) and a control group (8 rats). T groups and LPS groups were divided into 1, 2, 4, 8 h subgroups (n = 8 each) according to the experimental protocol. LPS (5 mg/kg) was administered through the vein in the LPS groups. In the T groups, 15 min after LPS injecting, troglitazone was administrated (3 mg/kg) through the vein. PaO2, myeloperoxidase activity (MPO), lung tissue histopathological changes were observed. Expressions of PPAR-gamma mRNA and TNF-alpha mRNA were assayed by RT-PCR, TNF-alpha levels measured with ELISA, expression of PPAR-gamma protein in lung tissue detected by immunohistochemistry method, and expression of NF-kappaB P65 protein assayed by Western Blot. The data were expressed as mean +/- SD and analyzed with SPSS 10.0 software. RESULTS: PaO2 in 1, 2, 4, and 8 h groups were (85 +/- 10), (80 +/- 10), (81 +/- 10), (82 +/- 13) mm Hg (1 mmHg =0.133 kPa) in the T groups, (75 +/- 11), (69 +/- 12), (63 +/- 11), (71 +/- 13) mm Hg in the LPS groups, respectively, the difference being significant between groups (F = 4.32, P < 0.05). MPO activity in 2, 4 and 8 h groups were (10.6 +/- 1.2), (14.1 +/- 2.1), (11.1 +/- 1.8) U/g in the LPS groups, (8.2 +/- 0.8), (9.2 +/- 0.9), (8.8 +/- 0.7) U/g in the T groups, and comparison between groups showed statistical significance (F = 14.99, P <0.05). TNF-alpha mRNA expression (A) in 1 h group and 2 h group were 0.68 +/- 0.07, 0.92 +/- 0.05 in the LPS groups and 0.39 +/- 0.07, 0.50 +/- 0.09 in the T groups, and comparison between groups showed statistical significance (q = 3.09, 3.99, P <0.05). TNF-alpha levels in 1 h group and 2 h group in lung homogenate and plasma were (340 +/- 33), (757 +/- 47), (12.3 +/- 1.8), (54.7 +/- 6.6) ng/L in LPS groups, (306 +/- 30), (685 +/- 47), (10.0 +/- 1.7), (46.8 +/- 5.9) ng/L in T groups, the difference between groups being significant(q =3.92, 4.71, 4.81, 5.17, all P<0.05). PPAR-gamma mRNA expression (A) in 1 h, 2 h and 4 h groups were 0.36 +/- 0.05, 0.25 +/- 0.04, 0.30 +/- 0.05 in the LPS groups, 0.39 +/- 0.02, 0.44 +/- 0.05, 0.46 +/- 0.04 in the T groups, the difference between groups being significant (q =6.13, 5.69, 3.72, all P <0.05). NF-kappaB P65 translocated from plasma to nucleus in 1 h and 8 h group; the A values were 0.81 +/- 0.14, 1.91 +/- 0.16, 0.33 +/- 0.06, 2.01 +/- 0.18 in the LPS groups and 1.14 +/- 0.15, 1.06 +/- 0.21, 0.81 +/- 0.14, 1.03 +/- 0.18 in the T groups, comparison between groups showed statistical difference (q = 3.29, 6.25, 5.59, 6.81, all P <0.05). CONCLUSION: PPAR-gamma agonist (troglitazone) decreased the expression levels of inflammatory mediators such as TNF-alpha, reduced infiltration and activation of inflammatory cells in lung tissues, and alleviated LPS-induced through PPAR-gamma upregulation and inhibition of NF-kappaB activity.

[The expression of scavenger receptor class A in the lung in mice with acute lung injury induced by lipopolysaccharide].

OBJECTIVE: To investigate the expression of scavenger receptor class A (SR-A) in lung tissue and alveolar macrophage (AM) in mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: The model of ALI was reproduced by intraperitoneal (ip) injection of LPS (5 mg/kg), and the expression of SR-A was observed in lung tissue and AM, and the expression and distribution of J774 A.1 were also studied by using mouse macrophage line. RESULTS: Partial pressure of oxygen in artery (PaO(2)) of lung tissue in LPS groups was significantly lower while wet/dry weight (W/D) ratio was higher than those in normal saline control group (both P<0.01). SR-A was widely expressed in murine lung, including AM, pulmonary vascular endothelial cells, vascular smooth muscle cells, lung epithelial cells and polymorphonuclear neutrophils. Immunohistochemical staining showed that, during the course of development of ALI in mice, the expression of SR-A was gradually down-regulated with the elapse of time after ip injection of LPS, The same result was seen in the isolated AM, and especially marked in group 4 hours and group 8 hours. In vitro, the expression of SR-A on J774 A.1 cells decreased was also down-regulated when treated with LPS. CONCLUSION: SR-A was widely expressed in murine lung. During the course of ALI, the expression of SR-A in the murine lung and AM is down-regulated, which may be induced by LPS.