Clostridium perfringens gas gangrene caused by closed abdominal injury: A case report and review of the literature.

BACKGROUND: Abdominal Clostridium perfringens (C. perfringens) gas gangrene is a rare infection that has been described in the literature as most frequently occurring in postoperative patients with open trauma. Intra-abdominal gas gangrene caused by C. perfringens infection after closed abdominal injury is extremely rare, difficult to diagnose, and progresses rapidly with high mortality risk. Here, we report a case of C. perfringens infection caused by closed abdominal injury. CASE SUMMARY: A 54-year-old male suffered multiple intestinal tears and necrosis after sustaining an injury caused by falling from a high height. These injuries and the subsequent necrosis resulted in intra-abdominal C. perfringens infection. In the first operation, we removed the necrotic intestinal segment, kept the abdomen open and covered the intestine with a Bogota bag. A vacuum sealing drainage system was used to cover the outer layer of the Bogota bag, and the drainage was flushed under negative pressure. The patient was transferred to the intensive care unit for supportive care and empirical antibiotic treatment. The antibiotics were not changed until the results of bacterial culture and drug susceptibility testing were obtained. Two consecutive operations were then performed due to secondary intestinal necrosis. After three definitive operations, the patient successfully survived the perioperative period. Unfortunately, he died of complications related to Guillain-Barre syndrome 75 d after the first surgery. This paper presents this case of intra-abdominal gas gangrene infection and analyzes the diagnosis and treatment based on a review of current literature. CONCLUSION: When the intestines rupture leading to contamination of the abdominal cavity by intestinal contents, C. perfringens bacteria normally present in the intestinal tract may proliferate in large numbers and lead to intra-abdominal infection. Prompt surgical intervention, adequate drainage, appropriate antibiotic therapy, and intensive supportive care comprise the most effective treatment strategy. If the abdominal cavity is heavily contaminated, an open abdominal approach may be a beneficial treatment.

Integrated analysis of the genomic and transcriptional profile of gliomas with isocitrate dehydrogenase-1 and tumor protein 53 mutations.

Background: The gene mutation of isocitrate dehydrogenase-1 (IDH1) is commonly found in LGG and some GBM patients and usually carries tumor protein 53 (TP53) mutations. However, the underlying mechanisms on both mutations of glioma patients in IDH1 and TP53 are still unclear. Aim: To find the potential target markers in GBM and LGG patients with IDH1 and TP53 mutation.Method: A total of 1122 glioma patients from The Cancer Genome Atlas were enrolled and divided as wild-type (without IDH1 and TP53 mutations) or both mutant (both IDH1 and TP53 mutations). The data of clinicopathological characteristics, mRNA, mutations, and copy number alteration were analyzed. Results: IDH1 and TP53 mutations, not gene expression, affect the survival probability of GBM and LGG patients, which might be related to neuron function, immune function, tumor invasion, and metastasis. The effects of the selected gene (EMILIN3, SAA1, VSTM2A, HAMP, IFT80, and CHIC2) on glioma patients could be regulated by IDH1 and TP53 mutations and had a higher survival possibility in these patients. Conclusions: The selected genes in GBM and LGG patients with IDH1 and TP53 mutations could be a potential prognosis marker in the future.

Research progress of berberine mediated photodynamic therapy.

Berberine (BBR) is a plant secondary metabolite that has been used in photodynamic therapy (PDT) in the last few decades. The present review aimed to discuss the research progress of BBR-mediated photodynamic actions. The following key words were searched in several databases: 'Berberine' combined with 'photodynamic therapy', 'sonodynamic therapy (SDT)', 'ultraviolet', 'reactive oxygen' and 'singlet oxygen'. The results demonstrated that both type I and type II reactions participated in the photodynamic progression of BBR derivatives. In addition, the photochemical characteristics of BBR derivatives were affected by the polarity, pH and O2 content of solvents. DNA binding increases the lifespan of the photoexcited BBR state and generation of singlet oxygen (1O2). The chemical properties of substituents in different positions of the BBR skeleton are pivotal for its photochemical properties, particularly the methylenedioxy group at the C-2 and C-3 positions. BBR is a promising agent for mediating both PDT- and SDT-treated diseases, particularly in tumors. However, further studies are required to validate their biological effects. In addition, the molecular mechanisms underlying the antitumor effects of BBR-PDT remain unclear and warrant further investigation. The structural modification and targeted delivery of BBR have made it possible to broaden its applications; however, experimental verification is required. Overall, BBR acts as a sensitizer for PDT and has promising development prospects.

Sinoporphyrin sodium is a promising sensitizer for photodynamic and sonodynamic therapy in glioma.

The aim of the present study was to explore the antitumor effects of sinoporphyrin sodium (DVDMS)­mediated photodynamic therapy (PDT) and sonodynamic therapy (SDT) in glioma, and to reveal the underlying mechanisms. The uptake of DVDMS by U­118 MG cells was detected by flow cytometry (FCM). A 630­nm semiconductor laser and 1­MHz ultrasound were used to perform PDT and SDT, respectively. Cell proliferation and apoptosis were evaluated using the Cell Counting Kit­8 assay, FCM and Hoechst 33258 staining, respectively. Western blot analysis was used to detect protein expression and phosphorylation levels. BALB/c nude mice were used to establish a xenograft model of U­118 MG cells. DVDMS was injected intravenously and PDT and SDT were performed 24 h later. An in vivo imaging system was used to evaluate the fluorescence of DVDMS, to measure tumor sizes, and to evaluate the therapeutic effects. The uptake of DVDMS by U­118 MG cells was optimal after 4 h. PDT and SDT following DVDMS injection significantly inhibited the proliferation and increased apoptosis of glioma cells in vitro (P<0.05, P<0.01) respectively. In vivo, the fluorescence intensity of DVDMS was lower in the PDT and SDT groups compared with the DVDMS group, while tumor cell proliferation and weight were lower in the PDT and SDT groups than in the control group (P<0.05, P<0.01). However, there was no significant difference when laser, ultrasound or DVDMS were applied individually, compared with the control group. Hematoxylin and eosin staining suggested that both PDT and SDT induced significant apoptosis and vascular obstruction in cancer tissues. DVDMS­mediated PDT and SDT inhibited the expression levels of proliferating cell nuclear antigen (PCNA) and Bcl­xL, increased cleaved ­caspase 3 levels, and decreased the protein phosphorylation of the PI3K/AKT/mTOR signaling pathway. Changes in the expression of PCNA, and Bcl­xL and in the levels of cleaved­caspase 3 were partly reversed by N­acetyl­L­cysteine, a reactive oxygen species (ROS) scavenger. Similar results were obtained with FCM. DVDMS­mediated PDT and SDT inhibited glioma cell proliferation and induced cell apoptosis in vitro and in vivo, potentially by increasing the generation of ROS and affecting protein expression and phosphorylation levels.

FBW7 protects against spinal cord injury by mitigating inflammation-associated neuronal apoptosis in mice.

Spinal cord injury (SCI) leads to severe and long-lasting neurological disability. Presently, the lack of effective therapies for SCI is largely attributable to an incomplete understanding of its pathogenesis. F-box and WD repeat domain-containing protein 7 (FBW7, also known as FBXW7) is a type of E3 ubiquitin ligase complex, and plays essential roles in regulating different pathological and physiological processes. In this study, we attempted to explore the effects of FBW7 on SCI progression by the in vivo and in vitro experiments. SCI mice showed significantly reduced expression of FBW7 in spinal cord tissues. Promoting FBW7 expression via intrathecal injection of AAV9/FBW7 effectively improved locomotor function in SCI mice. Neuronal death in spinal cords of SCI mice was obviously ameliorated by FBW7 over-expression, along with greatly decreased expression of cleaved Caspase-3. In addition, microglial activation in spinal cord specimens was detected in SCI mice through increasing Iba-1 expression levels, which was, however, attenuated in SCI mice injected with AAV9/FBW7. Additionally, FBW7 over-expression dramatically restrained inflammatory response in spinal cord tissues of SCI mice, as evidenced by the down-regulated expression of tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß) through blocking the activation of nuclear factor-κB (NF-κB) signaling. These anti-inflammatory effects of FBW7 were confirmed in LPS-stimulated mouse microglial BV2 cells. Finally, our in vitro studies showed that conditional medium (CM) collected from LPS-incubated BV2 cells markedly induced apoptosis in the isolated primary spinal neurons; However, this effect was overtly ameliorated by CM from LPS-exposed BV2 cells over-expressing FBW7. Thus, FBW7-regulated inflammation in microglial cells was involved in the amelioration of neuronal apoptosis during SCI development. Collectively, these findings illustrated that FBW7 expression was down-regulated in spinal cords of SCI mice, and promoting its expression could effectively mitigate SCI progression by repressing microglial inflammation and neuronal death.

[Anti-tumor effect of brucine-loaded chitosan nanoparticles in vitro].

We designed two novel polymer materials N-glycyrrhetinic acid-polyethylene glycol-chitosan derivatives (NGPC) and N-quaternary ammonium-chitosan derivatives (NQC). We prepared three kinds of drug loaded chitosan nanoparticles (brucine/NGPC-NPs, brucine/NQC-NPs, brucine/MNPs) by ionic crosslinking method with brucine as a model drug and chitosan nanoparticles（brucine/NGPC-NPs, brucine/NQC-NPs） as the reference formulation. Using high content analysis, flow cytometry, immunofluorescence, transmission electron microscopy and other advanced technology, we tested the effect of 20 µg·mL(-1) concentration of brucine solution and brucine/ chitosan nanoparticles（brucine/CTS-NPs） in hepatocarcinoma (HEpG2) cells and evaluated the apoptosis induced by the treatment. The results suggested that brucine-CTS/NPs had a strongest activity in killing tumor cells, and increased the total cell apoptosis rate with a significant formation of "crescent-shaped" body, swelling mitochondria, mitochondria cristae missing, decreased mitochondrial membrane potential and release of cytochrome C. The activity was enhanced by multifunctional nanocomposite particles that increased the cumulative amount of drug in the mitochondria for the anti-tumor effect.

Controllable organization and high throughput production of recoverable 3D tumors using pneumatic microfluidics.

Three-dimensional tumor culture methods offer a high degree of biological and clinical relevance to in vitro models as well as cancer therapy. However, a straightforward, dynamic, and high-throughput method for micro-manipulation of 3D tumors is not yet well established. In this study, we present a novel and simple strategy for producing biomimetic 3D tumors in a controllable, high throughput manner based on an integrated microfluidic system with well-established pneumatic microstructures. Serial manipulations, including one-step cell localization, array-like self-assembly, and real-time analysis of 3D tumors, are accomplished smoothly in the microfluidic device. The recovery of tumor products from the chip is performed by dynamic off-switch of the pneumatic microstructures. In addition, this microfluidic platform is demonstrated to be capable of producing multiple types of 3D tumors and performing the evaluation of tumor targeting by nanomedicine. The pneumatic microfluidic-based 3D tumor production shows potential for research on tumor biology, tissue engineering, and drug delivery.

High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PµSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PµS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PµSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PµS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PµS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PµS groups. We demonstrate that PµS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays.

High-throughput rare cell separation from blood samples using steric hindrance and inertial microfluidics.

The presence and quantity of rare cells in the bloodstream of cancer patients provide a potentially accessible source for the early detection of invasive cancer and for monitoring the treatment of advanced diseases. The separation of rare cells from peripheral blood, as a "virtual and real-time liquid biopsy", is expected to replace conventional tissue biopsies of metastatic tumors for therapy guidance. However, technical obstacles, similar to looking for a needle in a haystack, have hindered the broad clinical utility of this method. In this study, we developed a multistage microfluidic device for continuous label-free separation and enrichment of rare cells from blood samples based on cell size and deformability. We successfully separated tumor cells (MCF-7 and HeLa cells) and leukemic (K562) cells spiked in diluted whole blood using a unique complementary combination of inertial microfluidics and steric hindrance in a microfluidic system. The processing parameters of the inertial focusing and steric hindrance regions were optimized to achieve high-throughput and high-efficiency separation, significant advantages compared with existing rare cell isolation technologies. The results from experiments with rare cells spiked in 1% hematocrit blood indicated >90% cell recovery at a throughput of 2.24 × 10(7) cells min(-1). The enrichment of rare cells was >2.02 × 10(5)-fold. Thus, this microfluidic system driven by purely hydrodynamic forces has practical potential to be applied either alone or as a sample preparation platform for fundamental studies and clinical applications.

Effects of poly(L-lysine)-modified Fe3O4 nanoparticles on endogenous reactive oxygen species in cancer stem cells.

Intracellular reactive oxygen species (ROS) have been extensively shown to play an important role in the regulation of cell proliferation and cell cycle progression. The effects of endogenous ROS on the proliferation and differentiation of cancer stem cells (CSCs) have received increasing attention because of the unique properties of these cells that allow them to drive tumor growth and evade conventional cancer therapies. In this study, poly(L-Lysine) (PLL)-modified Fe(3)O(4) nanoparticles were synthesized to label CSCs derived from U251 glioblastoma multiform. A featured peroxidase-like activity within PLL-modified Fe(3)O(4) nanoparticles that could greatly reduce intracellular H(2)O(2) activity was identified. We also found that PLL-modified Fe(3)O(4) nanoparticles could accelerate the progression of CSC cell cycle, probably due to the impaired activity of endogenous ROS in CSCs. These results show that growth and proliferation of CSCs could be promoted by Fe(3)O(4) nanocarriers in an ROS-dependent manner, and Fe(3)O(4) nanocarriers may be suitable for certain tumor therapies as a drug delivery system.

Synthesis of polyethylene glycol- and sulfobetaine-conjugated zwitterionic poly(L-lactide) and assay of its antifouling properties.

A new antifouling polyester monomethoxy-poly(ethylene glycol)-b-poly(L-lactide)-b-poly(sulfobetaine methacrylate) (MPEG-PLA-PSBMA) was obtained by ring-opening polymerization of L-lactide, and subsequent click chemistry to graft the azide end-functionalized poly(sulfobetaine methacrylate) (polySBMA) moieties onto the alkyne end-functionalized MPEG-PLA (MPEG-PLA-alkyne). The chemical structure of the polymer was characterized using (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and its physical properties (including molecular weight, glass transition temperature, and melting point) were determined using gel permeation chromatography and differential scanning calorimetry. To investigate its hydrophilicity and stability, as well as its antifouling properties, the polymer was also prepared as a surface coating on glass substrates. The wettability and stability of this polyester was examined by contact angle measurements. Furthermore, its antifouling properties were investigated via protein adsorption, cell adhesion studies, and bacterial attachment assays. The results suggest that the prepared zwitterionic polyester exhibits durable wettability and stability, as well as significant antifouling properties. The new zwitterionic polyester MPEG-PLA-PSBMA could be developed as a promising antifouling material with extensive biomedical applications.

Construction of oxygen and chemical concentration gradients in a single microfluidic device for studying tumor cell-drug interactions in a dynamic hypoxia microenvironment.

Recent microfluidic advancements in oxygen gradients have greatly promoted controllable oxygen-sensitive cellular investigations at microscale resolution. However, multi-gradient integration in a single microfluidic device for tissue-mimicking cell investigation is not yet well established. In this study, we describe a method that can generate oxygen and chemical concentration gradients in a single microfluidic device via the formation of an oxygen gradient in a chamber and a chemical concentration gradient between adjacent chambers. The oxygen gradient dynamics were systematically investigated, and were quantitatively controlled using simple exchange between the aerial oxygen and the oxygen-free conditions in the gas-permeable polydimethylsiloxane channel. Meanwhile, the chemical gradient dynamics was generated using a special channel-branched device. For potential medical applications of the established oxygen and chemical concentration gradients, a tumor cell therapy assessment was performed using two antitumor drugs (tirapazamine and bleomycin) and two tumor cell lines (human lung adenocarcinoma A549 cells and human cervical carcinoma HeLa cells). The results of the proof-of-concept experiment indicate the dose-dependent antitumor effect of the drugs and hypoxia-induced cytotoxicity of tirapazamine. We demonstrate that the integration of oxygen and chemical concentration gradients in a single device can be applied to investigating oxygen- and chemical-sensitive cell events, which can also be valuable in the development of multi-gradient generating procedures and specific drug screening.

Dynamic trapping and high-throughput patterning of cells using pneumatic microstructures in an integrated microfluidic device.

Microfluidic trapping methods create significant opportunities to establish highly controlled cell positioning and arrangement for the microscale study of numerous cellular physiological and pathological activities. However, a simple, straightforward, dynamic, and high-throughput method for cell trapping is not yet well established. In the present paper, we report a direct active trapping method using an integrated microfluidic device with pneumatic microstructures (PµSs) for both operationally and quantitatively dynamic localization of cells, as well as for high-throughput cell patterning. We designed and fabricated U-shape PµS arrays to replace the conventional fixed microstructures for reversible trapping. Multidimensional dynamics and spatial consistency of the PµSs were optically characterized and quantitatively demonstrated. Furthermore, we performed a systematic trapping investigation of the PµSs actuated at a pressure range of 0 psi to 20 psi using three types of popularly applied mammalian cells, namely, human lung adenocarcinoma A549 cells, human hepatocellular liver carcinoma HepG2 cells, and human breast adenocarcinoma MCF-7 cells. The cells were quantitatively trapped and controlled by the U-shape PµSs in a programmatic and parallel manner, and could be opportunely released. The trapped cells with high viability were hydrodynamically protected by the real-time actuation of specifically designed umbrella-like PµSs. We demonstrate that PµSs can be applied as an active microfluidic component for large-scale cell patterning and manipulation, which could be useful in many cell-based tissue organization, immunosensor, and high-throughput imaging and screening.

Cancer stem cell labeling using poly(L-lysine)-modified iron oxide nanoparticles.

Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(L-lysine) (PLL)-modified γ-Fe(2)O(3) nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.

[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.

[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 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.

Enhancement of antiangiogenic effects of human canstatin with a hypoxia-regulated transgene vector in lung cancer model.

UNLABELLED: Canstatin, a newly identified antiangiogenesis protein, has a potent inhibitory effect on the proliferation and growth of endothelial cells. To enhance the expression and antiangiogenic effects of canstatin in solid tumors, we constructed a eukaryotic expression vector that encodes human canstatin cDNA downstream from nine copies of the hypoxia-response element. METHODS: Canstatin complementary DNA from adult liver tissues was cloned into the mammalian expression vector pCMV-Script. Nine copies of the hypoxia-response element were ligated upstream from the canstatin gene near the cytomegalovirus promoter. The recombinant vector, pCMV9Cans, was transformed into A549 cells by cationic liposomes. The transformed cells were cultured under oxic and anoxic conditions. We detected canstatin messenger ribonucleic acid and protein expression in transformed cells by TaqMan polymerase chain reaction and Western blot analysis, respectively. Human umbilical vein endothelial cells were cocultured with recombinant vector transformed A549 cells using Transwell plates under oxic and anoxic conditions. The proliferation and apoptosis of the cocultured endothelial cells were evaluated with 3H-thymidine incorporation and terminal deoxynucleotidyl-mediated biotinylated deoxyuridine triphosphate nick end-labeling methods (TUNEL), respectively. A canstatin-encoding vector with no hypoxia-response element, pCMVCans, was used as the positive control, and naked plasmid-transformed and singly cultured parental cells were used as negative controls. The biologic activity of the vector in tumor tissues of lung cancer-bearing nude mice was evaluated by microvessel counts. Canstatin protein expression was assessed by Western blot analysis in tumor tissues. pCMVCans and empty vector were used as controls in the in vivo assays. RESULTS: Canstatin messenger RNA and protein were detected in both pCMV9Cans- and pCMVCans-transformed A549 clones. Under oxic conditions, canstatin expression was not significantly different in clones stably transformed with pCMV9Cans or pCMVCans. However, under anoxic conditions canstatin expression was significantly higher in pCMV9Cans-transformed cells than in pCMVCans-transformed cells. Moreover, the 3H-thymidine uptake rate of the human umbilical vein endothelial cells was markedly lower than that of the pCMVCans-transformed cells, and many endothelial cells underwent apoptosis when cocultured with pCMV9Cans-transformed A549 cells, especially under anoxic conditions. We detected canstatin expression in tumor tissues; the expression level in pCMV9Cans-transformed tumors was significantly higher than that in pCMVCans-transformed tumors. An in vivo assay showed that tumors transformed with pCMV9Cans remained small, and microvessels in those tumors were much fewer than those in pCMVCans-transformed tumors. CONCLUSION: The hypoxia-regulated vector pCMV9Cans increases the expression of canstatin, thereby enhancing its biological effects. We believe that the hypoxia-inducible canstatin-expressing vector is a promising gene therapy tool for antiangiogenesis research.

[Experimental study on the expression and function of aquaporin-1 and aquaporin-5 in rats with acute lung injury induced by lipopolysaccharide].

OBJECTIVE: To investigate whether the expression and function of aquaporin-1 (AQP-1) is altered by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in primary rat lung microvessel endothelial cells (LMECs) after exposure to lipopolysaccharide (LPS), and to study the expressions of AQP-1 and AQP-5 in lung tissue of rats with acute lung injury (ALI) induced by LPS. The aim is to further clarify the pathogenesis of ALI/acute respiratory distress syndrome (ARDS). METHODS: (1) In vitro: The third passage LMECs were randomly divided into LPS group, TNF-alpha group, IL-1beta group and DMEM control group, and the experimental groups were exposed to LPS, TNF-alpha and IL-1beta respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantify AQP-1 mRNA changes and an immunocytochemistry method was used for determining AQP-1 protein changes in cultured rat LMECs. Isotope tracer technique was applied for the assay of the intra-cellular tritium water ((3)H2O) signal intensity in rat LMECs. (2) In vivo: Forty male Wistar rats were randomly divided into five groups: LPS 2 h group, LPS 4 h group, LPS 6 h group, LPS 8 h group and a control group, eight rats per group; The LPS treated groups served as the ALI models. RT-PCR was used to observe the changes of AQP-1 and AQP-5 mRNA and the immunohistochemistry method was used for determining AQP-1 and AQP-5 protein changes in ALI rats. RESULTS: (1) In vitro: The expression of AQP-1 mRNA and protein in LMECs were decreased significantly in the LPS group (0.428 +/- 0.026, 0.366 +/- 0.009), the TNF-alpha group (0.446 +/- 0.029, 0.374 +/- 0.014) and IL-1beta group (0.454 +/- 0.023, 0.377 +/- 0.007) as compared to the DMEM control group (0.793 +/- 0.035, 0.660 +/- 0.013, respectively; all P < 0.01). The quantities of tritium water's permeability in the LPS group, the TNF-alpha group and the IL-1beta group [(726 +/- 58), (738 +/- 45), (774 +/- 44) counts per minute] were significantly less than that in the DMEM control group [(1 148 +/- 70) counts per minute, P < 0.01]. (2) In vivo: The expression levels of AQP-1 and AQP-5 mRNA in ALI rats (LPS 2 h group 0.409 +/- 0.018, 0.421 +/- 0.020; LPS 4 h group 0.421 +/- 0.023, 0.412 +/- 0.023; LPS 6 h group 0.435 +/- 0.020, 0.388 +/- 0.031; LPS 8 h group 0.438 +/- 0.016, 0.386 +/- 0.019, respectively) were significantly lower than that in the control group (0.794 +/- 0.015, 0.787 +/- 0.022; all P < 0.01). CONCLUSION: AQP-1 and AQP-5 may play a role in abnormal fluid transportation and probably involve in the formation of pulmonary edema in ALI/ARDS.