Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

Total infectome characterization of respiratory infections in pre-COVID-19 Wuhan, China.

At the end of 2019 Wuhan witnessed an outbreak of "atypical pneumonia" that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their "total infectome", including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen-Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016-2017.

[Value of Pediatric Early Warning Score in identifying the condition of critically ill children].

OBJECTIVE: To study the value of Pediatric Early Warning Score (PEWS) in identifying the condition of critically ill children. METHODS: A total of 120 children who were transferred to the pediatric intensive care unit (PICU) from the general ward during hospitalization or admitted to the PICU after emergency treatment in the Xiangya Hospital of Central South University from January to December, 2016 were enrolled as the PICU group. The other 120 children who were admitted to the general ward in the hospital were used as the control group. According to the disease type, the PICU group was further divided into two subgroups: respiratory/circulatory system diseases (n=55) and nervous/other system diseases (n=65). The PEWS score on admission was recorded, and the receiver operating characteristic (ROC) curve was used to analyze the value of PEWS in evaluating patients' condition. RESULTS: The PICU group had a significantly higher PEWS score than the control group (P<0.05). The respiratory/circulatory system disease subgroup had a significantly higher PEWS score than the nervous/other system disease subgroup (P<0.05). In predicting whether the child was admitted to the PICU, PEWS had a sensitivity of 85%, a specificity of 95%, and an area under the ROC curve (AUC) of 0.951 (95% confidence interval: 0.923-0.980) at the optimal cut-off value of 3.5 (PEWS score). The AUC of PEWS was 0.768 in the nervous/other system disease subgroup and 0.968 in the respiratory/circulatory system disease subgroup. The mortality rate of children with a PEWS score of >6, 4-6 and ≤3 was 40%, 21% and 0 respectively (P<0.001). CONCLUSIONS: PEWS can well identify disease severity in critically ill children, and it has different sensitivities in children with different varieties of diseases. PEWS has a good value in predicting children's prognosis.

A case of Aspergillus tracheobronchitis in a patient with chronic obstructive pulmonary disease.

Aspergillus tracheobronchitis (AT) is a unique form of invasive pulmonary aspergillosis, which is commonly found in patients with impaired immunity. Early-stage AT presents in a nonspecific way, both clinically and radiographically, thereby delaying diagnosis and resulting in a high mortality. Owing to impaired mucociliary clearance, previous nonfungal infections, and administration of corticosteroids, among other aspects, patients with chronic obstructive pulmonary disease (COPD) are predisposed to AT, although they are mostly immunocompetent. AT in COPD patients has not been well recognized and the condition is often misdiagnosed or missed. We herein report a case of AT diagnosed in a male with past COPD, with the features of pseudomembranous AT upon bronchoscopy. This contradicts the opinion that pseudomembranous AT is found in severely immunocompromised hosts with hematologic malignancies.

Circulating miR-125b but not miR-125a correlates with acute exacerbations of chronic obstructive pulmonary disease and the expressions of inflammatory cytokines.

To investigate the correlation of miR-125a/b expression with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) patients and inflammatory cytokines.Eighty-seven AECOPD patients, 93 stable chronic obstructive pulmonary disease (COPD) patients and 100 health volunteers (HCs) were recruited. Plasma samples were collected from AECOPD patients at the day 1, day 7, day 14, and day 28 of admission and from stable COPD patients as well as HCs. Total RNA was extracted from plasma, and miR-125a/b relative expressions were determined by quantitative real time-polymerase chain reaction.MiR-125b had a great capacity for distinguishing AECOPD from stable COPD (AUC = 0.926, 95% CI: 0.884-0.967) and HCs (AUC = 0.923, 95% CI: 0.880-0.966), while miR-125a did not. There were associations between miR-125b expression with TNF-α, IL-8, and LTB-4 in AECOPD patients (P = .012, P = .032, and P = .047, respectively), while no correlation of miR-125a with inflammatory cytokines was found. MiR-125b expression gradually decreased at day 7, day 14, and day 28 compared with day 1 (all P < .05) on admission, while no difference in miR-125a was discovered between each visit compared to day 1. Besides, TNF-α, IL-1ß, IL-8, and LTB-4 were elevated in AECOPD patients compared with stable COPD patients (all P < .01).MiR-125b, but not miR-125a, was positively associated with inflammatory cytokines and could be a novel biomarker for distincting AECOPD from stable COPD patients and HCs.

[Inhibition of decomposing leaf litter of Cinnamomum camphora on growth of Capsicum annu- um and the alleviation effect of nitrogen application].

Effects of decomposing leaf litter of Cinnamomum camphora on growth, physiological and phenological traits of Capsicum annuum, and modification of these effects by nitrogen application were investigated using a pot experiment. C. camphora leaf litter was applied at rate of 0, 25, 50 100 g per pot, resulting into four treatments, i.e., CK (the control), L25, L50, and L100. Nitrogen application was firstly performed on the 39th d of decomposition (3.0 g urea was added to each pot six times). Leaf area, plant height, basal diameter and biomass production of C. annuum were all inhibited sharply by exposure to the leaf litter, and the inhibition effect increased with the increasing leaf litter in terms of both the intensity and the stability. Treated with L25, budding number reduced by 88.7% averagely during 55th-75th d, and the rate of fructification plant decreased by 40% on the 96th d of decomposition, while neither buds nor fruits were observed when exposed to L50 and L100 at that time. Pigment contents and net photosynthetic rate (Pn) were impacted due to leaf litter addition, and malonaldehyde (MDA) was only markedly promoted by L100. Inhibition on growth and development of C. annuum caused by leaf litter decomposition could be alleviated by nitrogen application. Leaf area treated with leaf litter recovered to the control level on the 52nd d after nitrogen application, and similar results appeared on the 83rd d after nitrogen application for other growth traits. Budding and fructification status were also visibly improved.

[Response of fine root decomposition to simulated nitrogen deposition in Pleioblastus amarus plantation, rainy area of West China].

As an important contributor to carbon (C) flux in the global C cycle, fine root litter decomposition in forests has the potential to be affected by the elevated nitrogen (N) deposition observed globally. From November 2007 to January 2013, a field experiment involving monthly simulated deposition of N in a Pleioblastus amarus plantation was conducted in the Rainy Area of West China. Four levels of nitrogen deposition were included as control (0 g N x m(-2) x a(-1)), low nitrogen (5 g N x m(-2) x a(-1)), medium nitrogen (15 g N x m(-2) x a(-1)) and high nitrogen (30 g N x m(-2) x a(-1)). After 3 years of simulated N deposition experiment (January 2011) , a two-year fine root decomposition experiment was conducted in the simulated N deposition plots using litterbag method, under monthly experimental N deposition. The decomposition rates of fine roots were fast first and then slow. Mass loss of fine roots in the first year of decomposition was up to 60%, and the change of the remaining mass was very slow in the second year. The time of 50% and 95% mass loss of fine roots was 1.20 and 5.17 years, respectively, under the conditions of no addition N input. In general, decomposition rates were underestimated using negative exponential model. Simulated N deposition significantly inhibited the decomposition of fine roots. The remaining mass in the high nitrogen treatment was 51.0% higher than that in the control, after two years of decomposition. Simulated N deposition increased C, P and K contents in the remaining mass of litter. Compared with the control, soil pH decreased significantly in the medium and high nitrogen treatments, soil organic C, total N, ammonium and nitrate contents and fine root biomass of P. amarus increased significantly in the high nitrogen treatment after simulated N deposition for 4. 5 years. Key words: nitrogen deposition; fine root decomposition; Pleioblastus amarus.

Soil biochemical responses to nitrogen addition in a bamboo forest.

Many vital ecosystem processes take place in the soils and are greatly affected by the increasing active nitrogen (N) deposition observed globally. Nitrogen deposition generally affects ecosystem processes through the changes in soil biochemical properties such as soil nutrient availability, microbial properties and enzyme activities. In order to evaluate the soil biochemical responses to elevated atmospheric N deposition in bamboo forest ecosystems, a two-year field N addition experiment in a hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis daii) plantation was conducted. Four levels of N treatment were applied: (1) control (CK, without N added), (2) low-nitrogen (LN, 50 kg N ha(-1) year(-1)), (3) medium-nitrogen (MN, 150 kg N ha(-1) year(-1)), and (4) high-nitrogen (HN, 300 kg N ha(-1) year(-1)). Results indicated that N addition significantly increased the concentrations of NH4(+), NO3(-), microbial biomass carbon, microbial biomass N, the rates of nitrification and denitrification; significantly decreased soil pH and the concentration of available phosphorus, and had no effect on the total organic carbon and total N concentration in the 0-20 cm soil depth. Nitrogen addition significantly stimulated activities of hydrolytic enzyme that acquiring N (urease) and phosphorus (acid phosphatase) and depressed the oxidative enzymes (phenol oxidase, peroxidase and catalase) activities. Results suggest that (1) this bamboo forest ecosystem is moving towards being limited by P or co-limited by P under elevated N deposition, (2) the expected progressive increases in N deposition may have a potential important effect on forest litter decomposition due to the interaction of inorganic N and oxidative enzyme activities, in such bamboo forests under high levels of ambient N deposition.

Nitrogen addition significantly affects forest litter decomposition under high levels of ambient nitrogen deposition.

BACKGROUND: Forest litter decomposition is a major component of the global carbon (C) budget, and is greatly affected by the atmospheric nitrogen (N) deposition observed globally. However, the effects of N addition on forest litter decomposition, in ecosystems receiving increasingly higher levels of ambient N deposition, are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a two-year field experiment in five forests along the western edge of the Sichuan Basin in China, where atmospheric N deposition was up to 82-114 kg N ha(-1) in the study sites. Four levels of N treatments were applied: (1) control (no N added), (2) low-N (50 kg N ha(-1) year(-1)), (3) medium-N (150 kg N ha(-1) year(-1)), and (4) high-N (300 kg N ha(-1) year(-1)), N additions ranging from 40% to 370% of ambient N deposition. The decomposition processes of ten types of forest litters were then studied. Nitrogen additions significantly decreased the decomposition rates of six types of forest litters. N additions decreased forest litter decomposition, and the mass of residual litter was closely correlated to residual lignin during the decomposition process over the study period. The inhibitory effect of N addition on litter decomposition can be primarily explained by the inhibition of lignin decomposition by exogenous inorganic N. The overall decomposition rate of ten investigated substrates exhibited a significant negative linear relationship with initial tissue C/N and lignin/N, and significant positive relationships with initial tissue K and N concentrations; these relationships exhibited linear and logarithmic curves, respectively. CONCLUSIONS/SIGNIFICANCE: This study suggests that the expected progressive increases in N deposition may have a potential important impact on forest litter decomposition in the study area in the presence of high levels of ambient N deposition.

[Effects of Eucalyptus grandis leaf litter at its early stage of decomposition on the growth and photosynthetic characteristics of Cichorium intybus].

From March to May, 2010, a pot experiment was conducted to investigate the effects of Eucalyptus grandis leaf litter at its early stage of decomposition on the growth and photosynthetic characteristics of Cichorium intybus. Four treatments with different application rate of the leaf litter, i.e., 0 g x pot(-1) (CK), 30 g x pot(-1) (A1), 60 g x pot(-1) (A2), and 90 g x pot(-1) (A3), were installed. Each pot contained 12 kg soil mixed with the leaf litter, and then, C. intybus was sown. The growth indicators of the C. intybus were measured at the 30, 45, 60, and 75 d after sowing, and the photosynthetic characteristics of the C. intybus in treatment A3 were studied after the seedlings third leaf fully expanded. At each measured time, the biomass accumulation and leaf area growth of C. intybus in treatments A1, A2, and A3 were inhibited significantly. At the early stage of the leaf litter decomposition, the synthesis of photosynthetic pigments of the C. intybus seedlings was inhibited significantly, and the inhibition effect was getting stronger with the increasing amount of the leaf litter addition. The diurnal change of the seedlings photosynthetic rate in all treatments showed a bimodal curve with midday depression, the stomatal conductance and water use efficiency had the same variation trend with the net photosynthetic rate, and the total diurnal photosynthesis decreased in the order of CK > A1 > A2 > A3. The GC-MS analysis showed there were 33 kinds of small molecule compounds released gradually with the decomposition of the leaf litter, among which, allelopathic substance terpenoid dominated.

Nitrogen distribution and cycling through water flows in a subtropical bamboo forest under high level of atmospheric deposition.

BACKGROUND: The hydrological cycle is an important way of transportation and reallocation of reactive nitrogen (N) in forest ecosystems. However, under a high level of atmospheric N deposition, the N distribution and cycling through water flows in forest ecosystems especially in bamboo ecosystems are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate N fluxes through water flows in a Pleioblastus amarus bamboo forest, event rainfall/snowfall (precipitation, PP), throughfall (TF), stemflow (SF), surface runoff (SR), forest floor leachate (FFL), soil water at the depth of 40 cm (SW1) and 100 cm (SW2) were collected and measured through the whole year of 2009. Nitrogen distribution in different pools in this ecosystem was also measured. Mean N pools in vegetation and soil (0-1 m) were 351.7 and 7752.8 kg ha(-1). Open field nitrogen deposition at the study site was 113.8 kg N ha(-1) yr(-1), which was one of the highest in the world. N-NH4(+), N-NO3(-) and dissolved organic N (DON) accounted for 54%, 22% and 24% of total wet N deposition. Net canopy accumulated of N occurred with N-NO3(-) and DON but not N-NH4(+). The flux of total dissolved N (TDN) to the forest floor was greater than that in open field precipitation by 17.7 kg N ha(-1) yr(-1), due to capture of dry and cloudwater deposition net of canopy uptake. There were significant negative exponential relationships between monthly water flow depths and monthly mean TDN concentrations in PP, TF, SR, FFL and SW1. CONCLUSIONS/SIGNIFICANCE: The open field nitrogen deposition through precipitation is very high over the world, which is the main way of reactive N input in this bamboo ecosystem. The water exchange and N consume mainly occurred in the litter floor layer and topsoil layer, where most of fine roots of bamboo distributed.

[Effects of simulated nitrogen deposition on soil enzyme activities in a Betula luminifera plantation in Rainy Area of West China].

From January 2008 to January 2009, a field experiment was conducted to investigate the effects of simulated nitrogen (N) deposition (0, 5, 15, and 30 g N x m(-2) x a(-1)) on the soil enzyme activities in a Betula luminifera plantation in Rainy Area of West China. As compared with the control (0 g N x m(-2) x a(-1)), simulated N deposition stimulated the activities of soil hydrolases (beta-fructofuranosidase, cellulase, acid phosphatase, and urease) significantly, but depressed the activities of soil oxidases (polyphenol oxidase and peroxidase). These results suggested that the increased exogenous inorganic N could stimulate soil microbial activity and increase the demands of both B. luminifera and soil microbes for C and P, whereas the depress of soil polyphenol oxidase and peroxidase activities under N addition could inhibit the degradation of litter and promote its accumulation in soil, leading to the increase of soil C storage in the B. luminifera plantation ecosystem.

[Effects of Eucalyptus grandis leaf litter decomposition on the growth and photosynthetic characteristics of Cichorium intybus].

A pot experiment was conducted to study the effects of Eucalyptus grandis leaf litter during its early stage decomposition on the growth and the photosynthesis of Cichorium intybus. Each pot contained 12 kg soil mixed with different amounts of E. grandis leaf litter (30 g x pot(-1), A1; 60 g x pot(-1), A2; 90 g x pot(-1), A3; and 0 g x pot(-1), CK), and sowed with C. intybus. The growth indicators and the photosynthetic characteristics of C. intybus were measured after the third leaf of C. intybus seedlings fully expanded in treatment A3. At the early stage of leaf litter decomposition, the C. intybus biomass accumulation, leaf area growth, and synthesis of photosynthetic pigments were inhibited significantly, and the inhibition effect was getting stronger with the increasing amount of the leaf litter addition. The intercellular CO2 concentration of C. intybus was increased by litter addition, while the net photosynthetic rate, stomatal conductance, and transpiration rate were significantly lower than those of the control. With the increase of leaf litter addition, all the parameters of C. intybus light response and CO2 response except CO2 compensation point showed an obvious downward trend, and there existed significant differences between the treatments of litter additions and the control. It was suggested that during the decomposition of E. grandis leaf litter, its allelopathic substances released gradually and acted on receptor plants, inhibited the synthesis of photosynthetic pigments and the photosynthesis of the receptors, decreased the receptors environmental adaptation ability, and accordingly, inhibited the growth of C. intybus.

Effects of mitochondrial potassium channel and membrane potential on hypoxic human pulmonary artery smooth muscle cells.

Chronic hypoxia induces proliferation of human pulmonary artery smooth muscle cells (hPASMCs), leading to remodeling and pulmonary hypertension, but the mechanism remains unclear. The present study tested the roles of mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) and mitochondrial membrane potential (DeltaPsi(m)) on hPASMCs under normoxic or hypoxic conditions. Our results demonstrated that diazoxide or hypoxia, alone or in combination, could depolarize DeltaPsi(m) through opening mitoK(ATP), release of cytochrome C, and overproduction of hydrogen peroxide by mitochondria, resulting in increased proliferation and decreased apoptosis of hPASMCs. Five-hydroxydecanoate could partly reduce these hypoxia-dependent responses. These results suggest that the opening of mitoK(ATP) followed by a depolarization of DeltaPsi(m) might play an important role in hypoxic proliferation of hPASMCs through cytochrome C accumulation within the mitochondria or mitochondrial overproduction of hydrogen peroxide.

[Regulation of rat airway smooth muscle cell proliferation by mitochondrial ATP-sensitive K(+) channel in asthmic rats.].

The objective of this paper was to investigate the effect and mechanism of mitochondrial ATP-sensitive K(+) (MitoK(ATP)) channel on the proliferation of airway smooth muscle cells (ASMCs) in asthmic rats. Thirty-six Sprague-Dawley (SD) rats were randomly assigned into 2 groups (18 in each): (1) Asthma group: the asthmic rat model was established by ovalbumin (OVA) sensitization and excitation; (2) Normal group: rats were subjected to inhalation of equal amount of normal saline. The rat ASMCs were isolated from fresh lung tissues and cultured respectively as follows: (1) CONTROL GROUP: normal ASMCs were cultured under normoxia for 24 h; (2) Diazoxide group: normal ASMCs were cultured under normoxia for 24 h with diazoxide (an opener of MitoK(ATP) channel); (3) 5-HD group: normal ASMCs were cultured under normoxia for 24 h with 5-hydroxydecanoate (5-HD) (an antagonist of MitoK(ATP) channel); (4) Asthma group: Asthmic ASMCs were cultured under normoxia for 24 h; (5) Asthma + diazoxide group: Asthmic ASMCs were cultured under normoxia with diazoxide for 24 h; (6) Asthma + 5-HD group: Asthmic ASMCs were cultured under normoxia with 5-HD for 24 h. The mitochondrial membrane potential (ΔΨm) was detected using Rhodamine 123 (R-123). The level of reactive oxygen species (ROS) was detected by DCF fluorescence. The expression of nuclear factor-kappa B (NF-κB) mRNA was examined by RT-PCR. The proliferation and apoptosis of rat ASMCs were examined respectively by MTT colorimetric assay and cell cycle analysis. The results were as follows. (1) After exposure to diazoxide for 24 h, the R-123 fluorescence intensity, the ROS level, NF-κB mRNA expression and the MTT absorbance value (A value) in normal ASMCs were significantly increased, and the apoptosis of rat ASMCs was significantly decreased compared to the control group (P<0.05). However, there was no significant changes in those indices after the normal ASMCs had been exposed to 5-HD for 24 h. (2) In Asthma and Asthma + diazoxide groups, the R-123 fluorescence intensity, ROS level and the MTT A value were markedly increased, and the apoptosis was markedly decreased compared to control group (P<0.05). These changes were more obvious in Asthma + diazoxide group than those in Asthma group (P<0.05). 5-HD partly weakened the effect of asthma on the R-123 fluorescence intensity, ROS level and the MTT A value and the apoptosis of rat ASMCs (P<0.05). R-123 fluorescence intensity and NF-κB mRNA expression were positively correlated with ROS level. NF-κB mRNA expression was positively correlated with the MTT A value and negatively correlated with the apoptosis of rat ASMCs. All the results suggest that the opening of MitoK(ATP) channel followed by a depolarization of ΔΨm contributes to the increase in ROS level and NF-κB mRNA expression in rat ASMCs and to the unbalance between cell proliferation and apoptosis of ASMCs induced by asthma. This might be a mechanism of the development of airway remodeling in asthma.

The relationships among reactive oxygen species, hypoxia-inducible factor 1alpha and cell proliferation in rat pulmonary arterial smooth muscle cells under hypoxia.

The objectives of this paper were to observe the changes of reactive oxygen species (ROS) in rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxic condition and to test if hypoxia-induced proliferation of PASMCs was mediated by ROS. PASMCs were divided into three groups: normal group, hypoxia group and hypoxia + Mn-TBAP (a ROS scavenger) group. The level of ROS was determined by a laser scanning confocal microscope. The expression of hypoxia-inducible factor 1alpha (HIF-1alpha) mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). HIF-1alpha protein was detected using immunohistochemical staining, and the proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows: (1) The level of ROS in hypoxia group was significantly increased as compared with that in the normal group (P<0.05). The level of ROS in hypoxia + Mn-TBAP group was significantly decreased as compared with that in hypoxia group (P<0.05), but was increased as compared with that in the normal group (P<0.05). (2) The expressions of HIF-1alpha mRNA and protein in hypoxia group and hypoxia + Mn-TBAP group were increased as compared with those in the normal group (P<0.05), and these changes were more significant in hypoxia group than those in hypoxia + Mn-TBAP group. (3) The proliferation of PASMCs in hypoxia group was more obvious than that in the normal group and hypoxia + Mn-TBAP group (P<0.05), and the proliferation of PASMCs in hypoxia + Mn-TBAP group was increased more significantly than that in the normal group (P<0.05). The results indicate that ROS is significantly increased in rat PASMCs under hypoxia, and that ROS affects the expression of HIF-1alpha and the proliferation of PASMCs under hypoxia. Therefore, ROS may play an important role in the pathogenesis of pulmonary hypertension and hypoxic signal transductions.

[Effect of opening of mitochondrial ATP-sensitive K(+) channels on the expression of hypoxia inducible factor-1alpha and cell proliferation in pulmonary arterial smooth muscle cells of rats].

The objective of this paper was to investigate the effect of mitochondrial ATP-sensitive K(+) (MitoK(ATP)) channels on the expression of hypoxia inducible factor-1alpha (HIF-1alpha) and cell proliferation in pulmonary arterial smooth muscle cells (PASMCs) of rats. Cultured PASMCs were divided into six groups as follows: (1) normoxia group: cultured under normoxia for 24 h; (2) normoxia + diazoxide group: cultured under normoxia with diazoxide, an opener of MitoK(ATP) channel, for 24 h; (3) normoxia + 5-HD group: cultured under normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK(ATP) channel, for 24 h; (4) hypoxia group: cultured under hypoxia (37 degrees C, 5% O(2), 5% CO(2), 90% N(2)) for 24 h; (5) hypoxia + diazoxide group: cultured under hypoxia (37 degrees C, 5% O(2), 5% CO(2), 90% N(2)) with diazoxide for 24 h; (6) hypoxia + 5-HD group: cultured under hypoxia (37 degrees C, 5% O(2), 5% CO(2), 90% N(2)) with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with Rhodamine 123 (R-123) fluorescence technique. Immunohistochemical method was used to trace the expression of HIF-1alpha. The proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows: The intensity of R-123 fluorescence in normoxia + diazoxide group was significantly increased as compared with that in normoxia group (P<0.05), and the intensity of R-123 fluorescence in hypoxia + diazoxide group was also significantly increased as compared with that in hypoxia group (P<0.05). 24-hour hypoxia or 24-hour hypoxia + diazoxide markedly increased the intensity of R-123 fluorescence in PASMCs as compared with normoxia (P<0.05), and the change was more prominant in hypoxia + diazoxide group than that in hypoxia group (P<0.05). There was no significant difference in the intensity of R-123 fluorescence between normoxia group and normoxia + 5-HD group (P>0.05). However, 5-HD weakened the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. The intensity of R-123 fluorescence in hypoxia + 5-HD group was significantly decreased as compared with that in hypoxia group (P<0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the expression of HIF-1alpha and the proliferation of PASMCs were significantly increased as compared with that in normoxia or normoxia + diazoxide group (P<0.05), and the change was more significant in hypoxia + diazoxide group than that in hypoxia group (P<0.05). There was no significant difference in the expression of HIF-1alpha and the proliferation of PASMCs between normoxia group and normoxia + 5-HD group (P>0.05). However, the expression of HIF-1alpha and the proliferation of PASMCs in hypoxia + 5-HD group were significantly decreased as compared with that in hypoxia group (P<0.05). All these results suggest that the opening of MitoK(ATP) channels followed by a depolarization of mitochondrial membrane might contribute to the increase of the expression of HIF-1alpha and the proliferation of PASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.

[Effect of opening of mitochondrial ATP-sensitive K⁺ channel on the distribution of cytochrome C and on proliferation of human pulmonary arterial smooth muscle cells in hypoxia].

The objective of this paper was to investigate the contribution of mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) and mitochondrial membrane potential (Deltapsim) to the distribution of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to the proliferation of HPASMCs induced by hypoxia. HPASMCs were divided into 6 groups, as following: (1) control group: cultured under normoxia; (2) diazoxide group: cultured in normoxia with diazoxide, an opener of mitoK(ATP); (3) 5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of mitoK(ATP); (4) 24-hour hypoxia group: cultured in hypoxia for 24 h; (5) 24-hour hypoxia + diazoxide group: cultured in hypoxia with diazoxide for 24 h; (6) 24-hour hypoxia + 5-HD group: cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. Western blot was used to detect the expression of cytochrome C protein in cell plasma and mitochondria,respectively. The expression of cell caspase-9 protein was determined with Western blot. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay. The results were as following: after exposure to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased compared with that in the control group (P<0.05), but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24-hour hypoxia or 24-hour hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMCs compared with normoxia (P<0.05), and the change was more significant in 24-hour hypoxia + diazoxide group than that in 24-hour hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased compared with that in the control group (P<0.05), and the expression of caspase-9 protein was significantly decreased compared with that in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). But there were no significant changes in these tests after HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein and the expression of caspase-9 protein were significantly decreased compared with those in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). These changes were more significant in 24-hour hypoxia + diazoxide group than those in 24-hour hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of the distribution of cytochrome C, the expression of caspase-9 in HPASMCs and the proliferation of HPASMCs induced by hypoxia (P<0.05). All these results suggest that the opening of mitoK(ATP) followed by a depolarization of Deltapsim induced by hypoxia might contribute to the inhibition of the release of cytochrome C from mitochondria to plasma in HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. The signal transduction pathway of mitochondria might play an important role in the relationship between Deltapsim and apoptosis of HPASMCs.

[The effect of mitochondrial membrane potential on changes of reactive oxygen species and on proliferation of hypoxic human pulmonary arterial smooth muscle cells].

OBJECTIVE: To investigate the contribution of the opening of mitochondrial ATP-sensitive K+ channel (MitoK(ATP)) and mitochondrial membrane potential (delta psi m) to changes of reactive oxygen species (ROS) and to the imbalance of proliferation/apoptosis of human pulmonary arterial smooth muscle cells (HPASMC) induced by hypoxia. METHODS: HPASMC were divided into the following groups: (1) control group (A group): cultured under normoxia; (2) 5-HD group (B group): cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK(ATP), for 24 h; (3) diazoxide group (C group): cultured in normoxia with diazoxide, an opener of MitoK(ATP), for 24 h; (4) chronic hypoxia group (D group): cultured under hypoxia for 24 h; (5) chronic hypoxia + diazoxide group (E group): cultured in hypoxia with diazoxide for 24 h; (6) chronic hypoxia + 5-HD group (F group): cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. The level of ROS in HPASMC was tested with chemiluminescence method. The proliferation of HPASMC was examined by the expression of PCNA, c-fos and c-jun proteins, and by MTT colorimetric assay. RESULTS: The mitochondrial membrane potentials (expressed by intensity of R-123) of C group (105 +/- 4), D group (95 +/- 13) and E group (126 +/- 8) were significantly depolarized as compared with A group (75 +/- 7, q = 5.474, 3.659, 9.213, all P < 0.05). The levels of ROS of C group (3045 +/- 126), D group (3116 +/- 34) and E group (3236 +/- 31) were significant increased than that of A group (2772 +/- 49) (q = 6.882, 7.448, 16.289, all P < 0.05). The cell viability of C group (0.305 +/- 0.022), D group (0.328 +/- 0.078) and E group (0.440 +/- 0.023) were significant increased than that of A group (0.237 +/- 0.013) in HPASMC (q = 2.993, 4.017, 8.919, all P < 0.05). The mitochondrial membrane potential, the level of ROS and the cell viability in HPASMC of E group were significantly different as compared with D group (q = 5.554, 8.841, 4.902, all P < 0.05). The mitochondrial membrane potential (71 +/- 4), the level of ROS (2863 +/- 132) and the cell viability (0.264 +/- 0.045) of F group were significantly decreased than those of D group (95 +/- 13, 3 116 +/- 34, 0.328 +/- 0.078, q = 4.367, 5.907, 2.832, all P < 0.05). CONCLUSIONS: The results suggested that the opening of MitoK(ATP) followed by a depolarization of delta psi m might contribute to the increasing of the level of ROS in HPASMC, and play an important role in the proliferation of HPASMC. This might be a mechanism for the development of hypoxic pulmonary hypertension.