Raloxifene inhibits adipose tissue inflammation and adipogenesis through Wnt regulation in ovariectomized rats and 3 T3-L1 cells.

BACKGROUND: Loss of ovarian function, as in menopause or after ovariectomy (OVX), is closely associated with obesity and white adipose tissue (WAT) inflammation. Estrogen replacement protects against postmenopausal obesity but increases the risks of carcinogenesis. In the present study, we investigated the effects of long-term treatment of raloxifene (RAL), a selective estrogen receptor modulator, on the features of estrogen deficiency-induced obesity and explored the involvement of canonical and non-canonical Wnt regulation in vivo and in vitro. METHODS: Adult female rats received bilateral OVX and divided into 5 groups: (1) Sham, (2) OVX, (3) OVX + E2: OVX rats were administered with E2 (50 µg/kg, s.c., 3 times/week), (4) OVX + RAL: OVX rats were treated with RAL (gavage, 1 mg/kg/day) suspended in 0.8% carboxymethylcellulose (CMC), (5) OVX + CMC: 0.8% CMC as vehicle control. All treatments were given for 8 weeks beginning at 1 week after OVX. In 3 T3-L1 cells, the effects of RAL on adipogenesis and lipopolysaccharide (LPS)-induced inflammation were evaluated. RESULTS: Treatment with RAL significantly decreased body weight, visceral fat pad mass, adipocyte size and plasma levels of glucose but increased plasma adiponectin. RAL reduced the elevation of HIF-1α, VEGF-A and proinflammatory cytokines (MCP-1 and TNF-α) expression by inhibition of NF-κB p65 and JNK cascades in retroperitoneal WAT. This anti-inflammatory capacity of RAL may result from upregulation of secreted frizzle-related protein 5 (SFRP5), an adipokine that repressed Wnt5a signaling. Furthermore, RAL inhibited adipogenic factors such as PPAR-γ, C/EBP-α, and FABP4, and preserved canonical Wnt10b/ß-catenin protein expression. In 3 T3-L1 adipocytes, RAL (20 µM) diminished lipid accumulation and inhibited adipogenic factors accompanied with the induction of ß-catenin, which were effectively reversed by the ß-catenin inhibitor IWR-1-endo. In addition, RAL reduced LPS-induced NF-κB p65 and p-IκB expression as well as TNF-α secretion. Suppression of SFRP5 by small interfering RNA significantly abrogated the anti-inflammatory effects of RAL. CONCLUSIONS: Distinct activation of canonical ß-catenin on inhibition of adipogenesis and non-canonical SFRP5 on suppression of WAT inflammation may contribute to the beneficial effects of RAL. Therefore, this study provides a rationale for the therapeutic potential of RAL for postmenopausal obesity.

Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif.

OBJECTIVES: An efficient bacterial surface display system based on the anchoring motif derived from Escherichia coli (E. coli) outer membrane protease OmpT was developed in this study. RESULTS: Referring to the classical Lpp-OmpA (LOA) display system, the signal peptide and nine amino acids of mature Lpp were fused to the transmembrane domain comprising five ß-strands of truncated OmpT to generate a novel Lpp-OmpT (LOT) display system. The C-terminal fusion strategy was used to fuse a small peptide (His tag) and red fluorescent protein (mCherry) to the C-terminus of LOT. Cell surface exposure of His tag and mCherry were compared between the LOA and LOT display systems. E. coli expressing LOT-His tag adsorbed more Cu2+ than E. coli expressing LOA-His tag. E. coli expressing both LOT-mCherry-His tag and LOA-mCherry-His tag adhered to Cu2+ chelating sepharose beads, and adhered cells could be dissociated from the beads after excess Cu2+ treatment. More importantly, compared with the LOA system, a higher amount of LOT-mCherry-His tag hybrid protein was demonstrated to be localized at the outer membrane by both fluorescence spectrophotometric determination of cell fractions and cell-surface immunofluorescence assay. CONCLUSIONS: These results suggest that genetically modified OmpT can be used as a potential anchoring motif to efficiently and stably display polypeptides and proteins, and that the LOT system could be used in a variety of biotechnological and industrial processes.

Primary culture of lung fibroblasts from hyperoxia-exposed rats and a proliferative characteristics study.

Lung fibrosis is an ultimate consequence of bronchopulmonary dysplasia (BPD) which shows the excessive proliferation of lung fibroblasts (LFs). To find a better model for studying the role of LFs in hyperoxia-induced lung fibrosis at the cellular level, we isolated LFs from the lung tissue of hyperoxia- and normoxia-exposed rat lungs on postnatal days 7, 14 and 21 for primary culture to study their proliferative behavior. In the present study, the LF predominance was > 95% in our culture method. The LFs isolated from rats exposed to hyperoxia in vivo showed significantly greater proliferation than that from normoxia-exposed rats. Flow cytometry revealed that percentage of LFs in S and G2/M stage increased, and proportion in the G0/G1 stage declined at the same time. A greater presence of myofibroblasts in LFs isolated from rats exposed to hyperoxia compared with those exposed to normoxia. In addition, elevated collagen level, transforming growth factor-ß and connective tissue growth factor protein expression in conditioned medium were also found in hyperoxia LFs. These data demonstrate that hyperoxia promotes LFs proliferation, myofibroblast transdifferentiation and collagen synthesis in a time-dependent manner. The primary culture of LFs from hyperoxia-exposed rats is a feasible method for studying the pathogenesis and treatment of lung fibrosis caused by BPD at the cellular level.

5-aza-2'-deoxycytidine, a DNA methylation inhibitor, attenuates hyperoxia-induced lung fibrosis via re-expression of P16 in neonatal rats.

BackgroundP16 methylation plays an important role in the pathogenesis of hyperoxia-induced lung fibrosis. 5-aza-2'-deoxycytidine (5-aza-CdR) is a major methyltransferase-specific inhibitor. In this study, the effects of 5-aza-CdR on a hyperoxia-induced lung fibrosis in neonatal rats were investigated.MethodsRat pups were exposed to 85% O2 for 21 days of and received intraperitoneal injections of 5-aza-CdR or normal saline (NS) once every other day. Survival rates and lung coefficients were calculated. Hematoxylin-eosin staining was performed to analyze the degree of lung fibrosis. Collagen content and TGF-ß1 levels were determined. A methylation-specific polymerase chain reaction and western blotting were performed to determine P16 methylation status and P16, cyclin D1, and E2F1 protein expression.Results5-aza-CdR treatment during hyperoxia significantly improved the survival rate and weight gain, while it decreases the degree of lung fibrosis and levels of hydroxyproline and TGF-ß1. Hyperoxia induced abnormal P16 methylation and 5-aza-CdR effectively reversed the hypermethylation of P16. Expression of the P16 protein in lung tissues was enhanced, while cyclin D1 and E2F1 protein were reduced by 5-aza-CdR treatment during hyperoxia.ConclusionThese data show that 5-aza-CdR attenuated lung fibrosis in neonatal rats exposed to hyperoxia by lowering hydroxyproline and TGF-ß1 expression and via re-expression of P16 in neonatal rats.

Autophagy regulates hyperoxia-induced intracellular accumulation of surfactant protein C in alveolar type II cells.

Surfactant protein C (SP-C) deficiency is a risk factor for hyperoxia-induced bronchopulmonary dysplasia in newborn infants. However, the role of SP-C deficiency in the process is unclear. Here, using neonatal rat BPD model and MLE-12, mouse alveolar epithelial type II cell, we examined the changes of SP-C levels during hyperoxia. Immunohistochemistry, immunofluorescence, and ELISA analysis showed SP-C accumulation in alveolar epithelial type II cells. Electron microscopy further demonstrated the accumulation of lamellar bodies and the co-localization of lamellar bodies with autophagosomes in the cytoplasm of alveolar epithelial type II cells. The inhibition of autophagy with 3-Methyladenine and knockdown of Atg7 abolished hyperoxia-induced SP-C accumulation in the cytoplasm. Furthermore, inhibition of JNK signaling with SP600125 suppressed hyperoxia-induced Atg7 expression and SP-C accumulation. These findings suggest that hyperoxia triggers autophagy via JNK signaling-mediated Atg7 expression, which promotes the accumulation of SP-C within alveolar epithelial type II cells. Our data provide a potential approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway.

The role of placenta growth factor in the hyperoxia-induced acute lung injury in an animal model.

Prolonged exposure to hyperoxia leads to acute lung injury. Alveolar type II cells are main target of hyperoxia-induced lung injury. However, the cellular and molecular mechanisms remain unknown. Here, we aimed to investigate the role of placental growth factor (PLGF) in hyperoxia-induced lung injury. Using experimental hyperoxia-induced lung injury model of neonatal rat and mouse lung epithelial type II cells (MLE-12), we examined the levels of PLGF in bronchoalveolar lavage fluid and in the supernatants of MLE-12 cells. Our results revealed that exogenous PLGF induced hyperoxia-induced lung injury. Furthermore, PLGF triggered a shift of vinculin from insoluble to soluble cell fraction, similar to the observation under hyperoxia stimulation. Moreover, we observed significantly reduced phosphorylation of focal adhesion kinase and increased permeability in MLE-12 cells treated with PLGF. These results suggest that PLGF triggers focal adhesion disassembly in alveolar type II cells via inhibiting the activation of focal adhesion kinase. Our findings reveal a novel role of PLGF in hyperoxia-induced lung injury and provide a potential target for the management of hyperoxia-induced acute lung injury.

Specific oligopeptides in fermented soybean extract inhibit NF-κB-dependent iNOS and cytokine induction by toll-like receptor ligands.

The ethanol extract of fermented soybean from Glycine max (chungkookjang, CHU) has been claimed to have chemopreventive and cytoprotective effects. In the present study, we examined the inhibitory effect of CHU on inducible nitric oxide synthase (iNOS) and cytokine induction by toll-like receptor (TLR) ligands treatment and attempted to identify the responsible active components. Nitric oxide (NO) content and iNOS levels in the media or RAW264.7 cells were measured using the Griess reagent and real-time polymerase chain reaction assays. CHU treatment inhibited NO production and iNOS induction elicited by lipopolysaccharide (LPS, TLR4L) in a concentration-dependent manner. Tumor necrosis factor-α and interleukin-6 productions were also diminished. Peptidoglycans (TLR2/6L) and CpG-oligodeoxynucleotides (TLR9L) from CHU inhibited iNOS induction, but not poly I:C (TLR3L) or loxoribine (TLF7L). The anti-inflammatory effect resulted from the inhibition of nuclear factor-kappa B (NF-κB) through the inhibition of inhibitory-κB degradation. Of the representative components in CHU, specific oligopeptides (AFPG and GVAWWMY) had the ability to inhibit iNOS induction by LPS, whereas others failed to do so. Daidzein, an isoflavone used for comparative purposes, was active at a relatively higher concentration. In an animal model, oral administration of CHU to rats significantly diminished carrageenan-induced paw edema and iNOS induction. Our results demonstrate that CHU has anti-inflammatory effects against TLR ligands by inhibiting NF-κB activation, which may result from specific oligopeptide components in CHU. Since CHU is orally effective, dietary applications of CHU and/or the identified oligopeptides may be of use in the prevention of inflammatory diseases.

Mitigation of carbon tetrachloride-induced hepatic injury by methylene blue, a repurposed drug, is mediated by dual inhibition of GSK3ß downstream of PKA.

BACKGROUND AND PURPOSE: Methylene blue (MB) has recently been considered for new therapeutic applications. In this study, we investigated whether MB has antioxidant and mitochondria-protecting effects and can prevent the development of toxicant-induced hepatitis. In addition, we explored the underlying basis of its effects. EXPERIMENTAL APPROACH: Blood biochemistry and histopathology were assessed in mice injected with CCl4 (0.5 mL·kg(-1)) following MB administration (3 mg·kg(-1) ·day(-1), 3 days). Immunoblottings were performed to measure protein levels. Cell survival, H2 O2 , and mitochondrial superoxide and membrane permeability transition were determined in HepG2 cells. KEY RESULTS: MB protected cells from oxidative stress induced by arachidonic acid plus iron; it restored GSH content and decreased the production of H2 O2 . It consistently attenuated mitochondria dysfunction, as indicated by inhibition of superoxide production and mitochondrial permeability transition. MB inhibited glycogen synthase kinase-3ß (GSK3ß) and protected the liver against CCl4. Using siRNA, the inhibition of GSK3ß was shown to depend on AMPK. MB increased the activation of AMPK in vitro (3-24 h) and in vivo. MB also increased the phosphorylation of liver kinase B1 (LKB1) via cAMP-dependent PKA. SiRNA knockdown of LKB1 eliminated phosphorylation of AMPK and inhibited MB activation of AMPK. In addition, MB treatment (≤1 h) facilitated PKA-mediated GSK3ß serine phosphorylation independently of AMPK. CONCLUSIONS AND IMPLICATIONS: MB has antioxidant and mitochondria-protecting effects and protects the liver from toxicants, which results from the dual inhibition of GSK3ß by AMPK downstream of PKA-activated LKB1, and PKA itself. Our findings reveal a novel pharmacological effect of MB and its molecular basis.

Z-palatopharyngoplasty plus genioglossus advancement and hyoid suspension for obstructive sleep apnea hypopnea syndrome.

OBJECTIVE: To explore the feasibility and efficiency of Z-palato-pharyngoplasty (ZPPP) plus genioglossus advancement and hyoid suspension (GAHM) for severe obstructive sleep apnea hypopnea syndrome (OSAHS). STUDY DESIGN: Case series with planned data collection. SETTING: A university medical center. SUBJECTS AND METHODS: Twenty-six patients who had OSAHS with Friedman obstructive sleep apnea stage II/III and posterior airway space <11 mm received GAHM and ZPPP. All patients were reevaluated 6 months after surgery using the preoperative methods. RESULTS: Based on success criteria, defined as an apnea-hypopnea index (AHI) of <20 and a decrease >50%, the success rate was 46.2% at 6 months postoperatively. The AHI showed a significant reduction from 65.6 ± 17.6 preoperatively to 30.1 ± 23.1 postoperatively. The percentage of time with oxyhemoglobin saturation below 90% (CT(90)) decreased from 30.9% ± 28.1% preoperatively to 15.5% ± 25.6% postoperatively (P < .01). Sleep architecture was effectively changed. The S3 + S4 percentage of total sleep time increased from 3.6% ± 4.4% to 8.7% ± 5.0% (P < .05). The success rates were 100% (8/8) and 22.2% (4/18) in patients with Friedman obstructive sleep apnea stage II and III, respectively. CONCLUSION: The success rate of ZPPP plus GAHM for patients with severe OSAHS who suffer from oropharyngeal and hypopharyngeal obstruction was limited. Friedman stage was a predictor of ZPPP plus GAHM surgical success.

[Maxillomandibular advancement for obstructive sleep apnea/hypopnea syndrome].

OBJECTIVE: To explore effectiveness of maxillomandibular advancement (MMA) in the treatment of obstructive sleep apnea/hypopnea syndrome (OSAHS). METHODS: MMA was performed in 10 OSAHS patients with mandibular dysplasia diagnosed by mandibular protrusion angle (SNB) < 75 degrees and a posterior airway space (PAS) < 11 mm. Six patients had uvulopalatopharyngoplasty (UPPP) also. Six patients had over 6 months postoperative follow up. RESULTS: The blood loss was about 250-600 ml in the operation, and the serious complications didn't happen. The patients were satisfied with the postoperative facial change. Based on success criteria of 2009, of 5 patients showed highly responsive result and 1 patient was responsive (valid). rate was 83% and the responsive rate 100%. The snoring loudness score and Epworth sleepy score were reduced from preoperative 8 (6-10) and 15 (11-24) to postoperative 2 (0-4) and 5 (1-8). AHI was reduced from preoperative 52.2 (23.7-83.8) to postoperative 12.6 (7.6-31.8), lowest mean oxygen saturation increased from 0.64 (0.57-0.83) to 0.82 (0.78-0.93). Percentage of time with oxyhemoglobin saturation below 0.90 (CT90) reduced from 21.0% (12.0%-37.2%) to 2.0% (0%-8.0%). CONCLUSIONS: MMA is effective for the OSAHS patients with mandibular dysplasia.

[Effectiveness and related factors of Z-palatopharyngoplasty for treating obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: To investigate the efficacy and related factors of Z-palatopharyngoplasty for treating severe obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: Thirty four OSAHS patients with graded 1-3 tonsil, posterior airway space (PAS) > or = mm , Friedman II and III oropharyngeal airway were included in this study, all cases had Z-palatopharyngoplasty. The follow up was at least 6 months postoperatively. Measurement parameters of responders and nonresponders were analyzed. RESULTS: According to related criterion of China, cure rate was 35.3%, accumulative total excellence rate 64.7% and accumulative valid rate 70. 6%. The cured and excellence patients were considered as responders, the other as nonresponders. The lowest oxygen saturation (LSaO2), percentage of time with oxyhemoglobin saturation below 0.90 (CT90), mandibular plane angle (MPA), mandibular body length, position of tongue and Friedman clinical stage are statistically significant between responder and nonresponder. The best cut points of LSaO2, CT90 and MPA were 0.72, 22.80% and 29.40 degrees respectively. The logistic regression showed that Friedman stage and MPA entered into equation, which was Y = ln [P/(1-P)] = -122.85 + 31.57X1 + 1.01X2, if setting X1 as Friedman stage, and X2 as MPA. CONCLUSIONS: Z-palatopharyngoplasty is effective surgical approach for OSAHS patients with posterior airway space (PAS) > or = 11 mm. The affective factors of Z-palatopharyngoplasty included LSaO2, CT90, MPA, mandibular body length, position of tongue and Friedman clinical stage. Among them, the mandibular plane angle and Friedman clinical stage were predominant factors.

Genioglossus advancement and hyoid suspension plus uvulopalatopharyngoplasty for severe OSAHS.

OBJECTIVE: To evaluate the outcome of a comprehensive surgical approach on the treatment of severe obstructive sleep apnea hypopnea syndrome (OSAHS) and find out possible predictors to the effectiveness of this approach. STUDY DESIGN AND SETTING: Eighteen patients received genioglossus advancement with hyoid suspension (GAHM) and uvulopalatopharyngoplasty (UPPP). The multiple logistic regression was used to analyze predictors for the outcome of treatment. RESULTS: Apnea hypopnea index (AHI) showed a reduction in the preoperative vs postoperative polysomnography (63.83 +/- 16.34 vs 21.43 +/- 20.34). With success defined as a final postoperative AHI of less than 20 events per hour, the success rate was 67%. The main differences between responders and nonresponders include age, posterior airway space (PAS), time of oxyhemoglobin saturation below 90% (CT90), and body mass index (BMI). Age and BMI were key predictors for therapeutic effect. CONCLUSION: GAHM plus UPPP may benefit severe OSAHS patients with oropharyngeal and hypopharyngeal obstruction. The success was best predicted by low BMI and younger age. SIGNIFICANCE: This paper provides reference for patient selection of UPPP plus GAHM, and considers that older or morbidly obese patients with OSAHS should be excluded from this operation.

Anatomic and spiral computed tomographic study of the genial tubercles for genioglossus advancement.

OBJECTIVE: To measure and compare Chinese mandibular genial tubercles measured anatomically and with computed tomography (CT). STUDY DESIGN AND SETTING: Spiral CT scans were taken of 40 adult human skulls; the superior genial spines were measured using anatomic and CT methods. RESULTS: The height and width of the superior genial spines, mandible thickness, and distance from the menton to the inferior and superior margins of the superior genial spines were 5.82 +/- 0.71, 6.98 +/- 1.35, 11.95 +/- 1.59, 11.08 +/- 2.05, and 16.91 +/- 2.30 mm from anatomic measurements and 6.17 +/- 0.71, 7.01 +/- 1.13, 12.19 +/- 1.64, 10.41 +/- 1.55, and 15.73 +/- 2.12 mm using spiral CT, respectively. The anatomic and CT measurements were correlated. CONCLUSION: Spiral CT of the genial tubercles can help locate the osteotomy in genioglossus advancement. SIGNIFICANCE: This study acquired reference data on Chinese genial tubercles demonstrating that CT measurements of the genial tubercles reflect their anatomy, which should allow accurately locate the osteotomy.

[Effectiveness of combined surgery for treating severe obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: To explore the efficiency of a comprehensive surgical approach of genioglossus advancement and hyoid suspension (GAHM) plus uvulopalatopharyngoplasty (UPPP) for treating severe obstructive sleep apnea hypopnea syndrome (OSAHS) and to evaluate related factors on surgery outcomes. METHODS: Eighteen patients with severe OSAHS (apnea hypopnea index, AHI > 40/h) confirmed with polysomnography received genioglossus advancement and hyoid suspension plus uvulopalatopharyngoplasty. The obstruction in both the oropharynx and the hypopharynx were evaluated by preoperative physical examination, fiberoptic pharyngolaryngoscopy, cephalometry, and computed tomography of the upper airway. The follow up was at least 6 months postoperatively. The Wilcoxon signed rank test was used to compare the preoperative and postoperative results by SPSS 11.0 for windows. The Mann-Whitney test was used to analyze the difference between responders and nonresponders. RESULTS: The follow up time ranges from 6 to 24 months, there were statistically significance in all but body mass index (BMI) between preoperative and postoperative measurements. Mean AHI was reduced from preoperative (x +/- s, 63.8 +/- 16.3)/h to postoperative (23.6 +/- 19.5)/h, lowest mean oxygen saturation increased from 0.72 +/- 0.07 to 0.81 +/- 0.13(x +/- s). According to criterion at home, the 6-month rate of responder is 83%, if AHI <20/h and decreased by at least 50% as success, the rate of success is 67%. The age, posterior airway space (PAS) and percentage of time with oxyhemoglobin saturation below 0.90 (CT90) were (39.1 +/- 7.4) years, (8.3 +/- 0.9) mm, (18.5 +/- 10.9)% in responder, while (52.5 +/- 9.4) years, (6.8 +/- 1.3) mm, (37.7 +/- 23.6) % in nonresponder, and there are statistically significant between responder and nonresponder. CONCLUSIONS: GAHM plus UPPP is effective surgical approach for patients with severe OSAHS who suffer from oropharyngeal and hypopharyngeal obstruction. Age, PAS and CT90 were possible affective factors on surgical outcomes.

[Etiology of laryngeal stridor in neonates: analysis of 16 cases].

OBJECTIVE: To reduce misdiagnosis and explore etiology of laryngeal stridor in neonates. METHODS: Sixteen neonates with laryngeal stridor were reviewed. All the patients were examined with CT scan for larynx and lungs and some were examined with laryngoscopy. Laboratory examination and chromosome analysis were also done in all or some patients. RESULTS: All the 16 neonates were diagnosed before admission as congenital simple laryngeal stridor in other hospitals. Of the 16 neonates with laryngeal stridor, 5 (31%) had thyroglossal ductal cyst, 1 (6%) neonate with cri du chat (5p(-)) syndrome, 2 (13%) neonates with Pierre-Robin syndrome and the rest 8 (50%) were suspected of congenital simple laryngeal stridor. CONCLUSION: Neonates with laryngeal stridor are easily misdiagnosed; neonatal thyroglossal duct cyst is one of common causes. The diagnosis for a congenital simple laryngeal stridor should be carefully made excluding the possibility of related diseases. The authors recommend that laryngoscopy and CT scan for larynx should be performed in the neonates with laryngeal stridor.