NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.     

Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28 days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p < 0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47 ± 1.57% vs. 33.47 ± 1.34%, p < 0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.     

Tauroursodeoxycholate (TUDCA), chemical chaperone, enhances function of islets by reducing ER stress

The exposure to acute or chronic endoplasmic reticulum (ER) stress has been known to induce dysfunction of islets, leading to apoptosis. The reduction of ER stress in islet isolation for transplantation is critical for islet protection. In this study, we investigated whether tauroursodeoxycholate (TUDCA) could inhibit ER stress induced by thapsigargin, and restore the decreased glucose stimulation index of islets. In pig islets, thapsigargin decreased the insulin secretion by high glucose stimulation in a time-dependent manner (1 h, 1.35 ± 0.16; 2 h, 1.21 ± 0.13; 4 h, 1.17 ± 0.16 vs. 0 h, 1.81 ± 0.15, n = 4, p < 0.05, respectively). However, the treatment of TUDCA restored the decreased insulin secretion index induced by thapsigargin (thapsigargin, 1.25 ± 0.12 vs. thapsigargin + TUDCA, 2.13 ± 0.19, n = 5, p < 0.05). Furthermore, the culture of isolated islets for 24 h with TUDCA significantly reduced the rate of islet regression (37.4 ± 5.8% vs. 14.5 ± 6.4%, n = 12, p < 0.05). The treatment of TUDCA enhanced ATP contents in islets (27.2 ± 3.2 pmol/20IEQs vs. 21.7 ± 2.8 pmol/20IEQs, n = 9, p < 0.05). The insulin secretion index by high glucose stimulation is also increased by treatment of TUDCA (2.42 ± 0.15 vs. 1.92 ± 0.12, n = 12, p < 0.05). Taken together, we suggest that TUDCA could be a useful agent for islet protection in islet isolation for transplantation.     

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.     

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14/CD16 circulating monocytes

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4 μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63 ± 3 vs 13 ± 1%, p < 0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63 ± 3 vs 41 ± 2%, p < 0.001), with less distribution in caveolae (13 ± 1 vs 34 ± 2%, p < 0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41 ± 2 vs 66 ± 3%, p < 0.001) and caveolae (34 ± 2 vs 18 ± 1%, p < 0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins.     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Exposure to Biomass Smoke Extract Enhances Fibronectin Release from Fibroblasts

COPD induced following biomass smoke exposure has been reported to be associated with a more fibrotic phenotype than cigarette smoke induced COPD. This study aimed to investigate if biomass smoke induced extracellular matrix (ECM) protein production from primary human lung fibroblasts in vitro. Primary human lung fibroblasts (n = 5–10) were stimulated in vitro for up to 72 hours with increasing concentrations of biomass smoke extract (BME) or cigarette smoke extract (CSE) prior to being assessed for deposition of ECM proteins, cytokine release, and activation of intracellular signalling molecules. Deposition of the ECM proteins perlecan and fibronectin was upregulated by both CSE (p<0.05) and BME (p<0.05). The release of the neutrophilic chemokine IL-8 was also enhanced by BME. ERK1/2 phosphorylation was significantly upregulated by BME (p<0.05). Chemical inhibition of ERK signalling molecules partially attenuated these effects (p<0.05). Stimulation with endotoxin had no effect. This study demonstrated that BME had similar effects to CSE in vitro and had the capacity to directly induce fibrosis by upregulating production of ECM proteins. The mechanisms by which both biomass and cigarette smoke exposure cause lung damage may be similar.     

Resistance exercise acutely enhances mesenteric artery insulin-induced relaxation in healthy rats

Aims

We evaluated the mechanisms involved in insulin-induced vasodilatation after acute resistance exercise in healthy rats.

Main methods

Wistar rats were divided into 3 groups: control (CT), electrically stimulated (ES) and resistance exercise (RE). Immediately after acute RE (15 sets with 10 repetitions at 70% of maximal intensity), the animals were sacrificed and rings of mesenteric artery were mounted in an isometric system. After this, concentration–response curves to insulin were performed in control condition and in the presence of LY294002 (PI3K inhibitor), L-NAME (NOS inhibitor), L-NAME + TEA (K⁺ channels inhibitor), LY294002 + BQ123 (ET-A antagonist) or ouabain (Na⁺/K⁺ ATPase inhibitor).

Key findings

Acute RE increased insulin-induced vasorelaxation as compared to control (CT: R_max = 7.3 ± 0.4% and RE: R_max = 15.8 ± 0.8%; p < 0.001). NOS inhibition reduced (p < 0.001) this vasorelaxation from both groups (CT: R_max = 2.0 ± 0.3%, and RE: R_max = − 1.2 ± 0.1%), while PI3K inhibition abolished the vasorelaxation in CT (R_max = − 0.1 ± 0.3%, p < 0.001), and caused vasoconstriction in RE (R_max = − 6.5 ± 0.6%). That insulin-induced vasoconstriction on PI3K inhibition was abolished (p < 0.001) by the ET-A antagonist (R_max = 2.9 ± 0.4%). Additionally, acute RE enhanced (p < 0.001) the functional activity of the ouabain-sensitive Na⁺/K⁺ ATPase activity (R_max = 10.7 ± 0.4%) and of the K⁺ channels (R_max = − 6.1 ± 0.5%; p < 0.001) in the insulin-induced vasorelaxation as compared to CT.

Significance

Such results suggest that acute RE promotes enhanced insulin-induced vasodilatation, which could act as a fine tuning to vascular tone.     

Is there any association of apolipoprotein E gene polymorphism with obesity status and lipid profiles? Tehran Lipid and Glucose Study (TLGS)

Aims

Considering the key role played by the apolipoprotein E (Apo E) gene in the regulation of lipid metabolism and obesity, the current study has evaluate the association between abdominal obesity and Apo E gene polymorphism in a population of Tehran.

Materials and methods

A cross-sectional study was performed on 345 men and 498 women, aged 19–86 years, selected from among participants of the Tehran Lipid and Glucose Study. The RFLP-PCR technique was employed to investigate polymorphism in the gene fragments. Based on the national survey of risk factors for non-communicable diseases of Iran, waist circumference (WC) cut off was set at 89 cm for men and 91 cm for women. The risk effect of obesity related variables and lipid profiles in two groups of WC were examined by logistic regression. For body mass index (BMI), waist to hip ratio (WHR), high-density lipoprotein-cholesterol (HDL-C), triglyceride (TG), fasting blood sugar (FBS), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and blood pressure (BP), the standard risk cut-offs were applied.

Results

Frequencies of E2, E3, and E4 alleles were 9.7, 73, and 14.6%, respectively. The presence of the E3 allele was significantly associated with higher TG level in subjects with high WC, while, the presence of E4 allele decreased the plasma HDL-C (E2:52.1 ± 13.1 vs., E3:48.9 ± 11.2 vs., E4:44.6 ± 10.6 mg/dl, p < 0.05), HDL-C2 (E2:20.4 ± 9.2 vs., E3:19.1 ± 8.8 vs., E4:16.3 ± 7.9 mg/dl, p < 0.05), and HDL-C3 (E2:32.1 ± 7.4 vs., E3:30.3 ± 6.2 vs., E4:28.3 ± 6.1 mg/dl, p < 0.05) in normal WC subjects. The presence of the E3 carrier increased the risk of having higher plasma TG, compared with the E2 carrier (95% CI OR = 1.91, 1.02–3.57; p = 0.04).

Conclusion

According to the results of this study, the E3 carrier, caused an approximately 90% increase in the levels of TG in the group with abdominal obesity.     

Lyophilized somatic cells direct embryonic development after whole cell intracytoplasmic injection into pig oocytes

The study investigated the feasibility of lyophilization for long-term preservation of somatic cells and embryonic development after whole cell intracytoplasmic injection (WCICI) into enucleated pig oocyte. Confluent cultured porcine fetal fibroblast (pFF) cells were lyophilized and stored at 4 °C for at least 6 months. Results showed that compared to non-lyophilized control cells, lyophilized cells had drastically reduced cellular viability (P < 0.01). WCICI of reconstituted lyophilized cells could support complete embryonic development. However, the rates of cleavage (64.7 ± 2.7 vs. 43.5 ± 4.7%) and blastocyst formation (18.2 ± 0.6 vs. 10.2 ± 1.6%) were lower than that of control (P < 0.05). Total nuclei number per blastocyst (30.4 ± 4.5 vs. 25.2 ± 4.7) and intensity of acetylation at histone H3 (AcH3) protein (55.9 ± 3.5 vs. 53.3 ± 3.8) did not differ (P > 0.05). The development ability of embryos, produced from lyophilized somatic cells, was further increased (19.5 ± 2.4 vs. 10.2 ± 1.6%; P < 0.05) by treatment with trichostatin A (TSA) for 24 h post-activation. These TSA-treated embryos also had AcH3 level comparable with in vitro fertilized embryos (63.1 ± 3.2 vs. 69.9 ± 1.3). In conclusion, our results suggest that lyophilized somatic cells can direct embryonic development up to blastocyst stage after WCICI into pig oocytes. Treatment of embryos, produced from lyophilized somatic cells, with TSA can further increase their in vitro developmental potential.     

The effect of TSPP-mediated photodynamic therapy and Parecoxib in experimental tumours

Aims

The study investigated the effects of the combined treatment Parecoxib (Pcox) and 5,10,15,20-tetra-sulphonato-phenyl-porphyrin(TSPP)-mediated photodynamic therapy on Walker 256 carcinosarcoma.

Main methods

Five groups of male Wistar rats were used: the control group, treated with TSPP, group 2, irradiated 24 h thereafter, group 3, treated with Pcox and irradiated 24 h thereafter, groups 4 and 5 treated with combined therapies, TSPP and Pcox before irradiation, and Pcox 24 h after TSPP and irradiation respectively. Tumour inflammation, growth and non-growth factors, apoptosis/necrosis rate and oxidative/nitrosative stress markers were investigated.

Key findings

Malondialdehyde levels and cyclooxygenase (COX)-2 expression increased significantly in the group treated with Pcox after TSPP-PDT when compared with TSPP + IR group (p < 0.05, p < 0.001 respectively), in correlation with a decrease in glutathione levels (p < 0.05). The quantification of apoptosis, based on the TUNEL-assay, and necrosis rate revealed an increase of apoptotic/necrotic index in the same group (p < 0.05). On the other hand, Pcox administered before irradiation showed a significant increase in both vascular endothelial growth factor (VEGF) and COX-2 levels (p < 0.05) and in nitric oxide production (p < 0.01), when compared with the control group.

Significance

The administration of Pcox after TSPP-mediated PDT showed promising antitumoural effects, leading to an increase in oxidative and nitrosative stress as well as apoptosis/necrosis rate in tumour tissue. These results show that combined regimens that involve selective COX-2 inhibitors administration after irradiation may improve the therapeutic effectiveness of PDT.     

Clinical value of plasma pentraxin 3 levels for predicting cardiac troponin elevation after percutaneous coronary intervention

Aims

Post-procedural myocardial necrosis manifested by elevated cardiac troponin T (cTnT) often complicates percutaneous coronary intervention (PCI). Plasma pentraxin 3 (PTX3) levels are increased in patients with arterial inflammation and especially unstable angina pectoris (UAP). This study tested whether plasma PTX3 levels can predict post-PCI cTnT elevation.

Main methods

We evaluated 94 consecutive patients with AP and normal pre-PCI cTnT levels who underwent PCI. Pre-PCI virtual histology-intravascular ultrasound was performed to assess culprit plaque composition. Plasma PTX3 and serum hs-CRP levels were measured pre-PCI. Patients were divided into 2 groups according to presence (Group I, n = 34) or absence (Group II, n = 60) of post-PCI cTnT elevation > 3 × the upper limit of normal at 24 h after PCI.

Key findings

Plasma PTX3 (4.06 ± 2.05 ng/ml vs 2.17 ± 1.02 ng/ml, p < 0.001), serum hs-CRP levels (0.25 ± 0.03 vs 0.16 ± 0.03 mg/dl, p = 0.048), plaque burden (80.9 ± 5.3 vs 75.4 ± 10.6%, p = 0.047), presence of positive remodeling (59 vs 25%, p = 0.034), and percent necrotic core area (19.0 ± 7.4 vs 14.0 ± 5.9%, p = 0.046) were significantly higher in Group I than in Group II. Receiver-operating characteristic curve analysis showed that with a best cut-off value of 2.83 ng/ml, plasma PTX3 level (AUC 0.823) predicted post-PCI cardiac TnT elevation better than did serum hs-CRP level (AUC 0.618). Multiple logistic regression analysis showed that plasma PTX3 level was the most independent predictor of post-PCI cardiac cTnT elevation (OR: 2.65; 95% CI: 1.56–10.1; p = 0.003).

Significance

Plasma PTX3 level may be a useful marker for predicting post-PCI cardiac cTnT elevation, which is associated with inflammatory status of culprit lesions.     

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.     

The bed nucleus of the stria terminalis has developmental and adult forms in mice,with the male bias in the developmental form being dependent on testicular AMH

Canonically, the sexual dimorphism in the brain develops perinatally, with adult sexuality emerging due to the activating effects of pubescent sexual hormones. This concept does not readily explain why children have a gender identity and exhibit sex-stereotypic behaviours. These phenomena could be explained if some aspects of the sexual brain networks have childhood forms, which are transformed at puberty to generate adult sexuality. The bed nucleus of stria terminalis (BNST) is a dimorphic nucleus that is sex-reversed in transsexuals but not homosexuals. We report here that the principal nucleus of the BNST (BNSTp) of mice has developmental and adult forms that are differentially regulated. In 20-day-old prepubescent mice, the male bias in the principal nucleus of the BNST (BNSTp) was moderate (360 ± 6 vs 288 ± 12 calbindin^+ ve neurons, p < 0.0001), and absent in mice that lacked a gonadal hormone, AMH. After 20 days, the number of BNSTp neurons increased in the male mice by 25% (p < 0.0001) and decreased in female mice by 15% (p = 0.0012), independent of AMH. Adult male AMH-deficient mice had a normal preference for sniffing female pheromones (soiled bedding), but exhibited a relative disinterest in both male and female pheromones. This suggests that male mice require AMH to undergo normal social development. The reported observations provide a rationale for examining AMH levels in children with gender identity disorders and disorders of socialization that involve a male bias.     

Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.     

Novel vasoactive intestinal peptide derivatives with improved stability protect rat alveolar L2 cells from cigarette smoke-induced cytotoxicity and apoptosis

Vasoactive intestinal peptide (VIP) has been thought to be a promising candidate for asthma/chronic obstructive pulmonary disease (COPD), and our group previously developed several long-lasting VIP derivatives. The objective of the present study was to clarify the therapeutic potential of new VIP derivatives with improved chemical and metabolic stability. Exposure of rat alveolar L2 cells to cigarette smoke extract (CSE) for 1 h led to release of lactate dehydrogenase (LDH) and decreased viability in a CSE concentration-dependent manner. There appeared to be marked induction of apoptosis after CSE exposure, as demonstrated by 59% elevation of caspase-3 activity and TUNEL staining. In contrast, a stabilized VIP derivative, [R^15,20,21, L¹⁷]-VIP-GRR (IK312532), at a concentration of 10⁻⁷ M, exhibited 71% attenuation of LDH release and 85% decrease of the number of apoptotic cells. In addition to IK312532, new VIP derivatives also showed anti-apoptotic effects against CSE toxicity and marked reduction of nitric oxide production. In terms of cytoprotective effects, [R^15,20,21, L¹⁷, A^24,25, des-N²⁸]-VIP-GRR was more effective than VIP and IK312532, possibly due to the improved stability. Thus, the present study is the first to demonstrate that novel stabilized VIP derivatives exert anti-apoptotic and cytoprotective effects on CSE-induced cytotoxicity.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

Antihypertensive and endothelium-dependent vasodilator effects of aqueous extract of Cistus ladaniferus

Cistus ladaniferus L. (Cistaceae) is a medicinal plant originated from the Mediterranean region which exerts different pharmacological effects. In the present study, our goal was to examine whether the plant possessed antihypertensive properties. Aqueous extract of Cistus leaves (AEC, 500 mg/kg/day) reduced systemic blood pressure (SBP) in two animal models of hypertension, the l-NAME and renovascular two kidney-one clip (2K-1C) hypertensive rats. In the former, AEC prevented the increase in SBP when co-administered with l-NAME during four weeks (164 ± 3 mm Hg in l-NAME vs. 146 ± 1 mm Hg in l-NAME + AEC, p < 0.001). In the latter, AEC reversed the increase in SBP when administered during four weeks after installation of the hypertension (146 ± 5 mm Hg with AEC vs. 179 ± 6 mm Hg without, p < 0.05). AEC treatment also reversed the endothelial dysfunction observed in both animal models of hypertension. A direct effect on cardiac and vascular tissue was also tested by examining the contractile effects of AEC in rat isolated aortic rings and Langendorff perfused hearts. AEC (10 mg/L) had no effect on left ventricular developed pressure and heart rate in isolated perfused heart. However, AEC produced a strong relaxation of pre-contracted rat aortic rings (80 ± 2% relaxation, n = 25). When the rings were denuded from endothelium or were incubated with 1 mM Nω-nitro-l-arginine (l-NNA), the relaxant effect of AEC was lost. We conclude that C. ladaniferus possesses antihypertensive properties which are mainly due to an endothelium-dependent vasodilatory action.     

MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis

MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-ß1 plus cytomix (a mixture of IL-1ß, IFN-γ and TNF-α). TGF-ß1 plus cytomix (TCM) induced apoptosis in HBECs (3.4 ± 0.6% of control vs 83.1 ± 4.0% of TCM treated cells, p < 0.01), and this was significantly blocked by the miRNA-146a mimic (8.8 ± 1.5%, p < 0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.     

Factors affecting the incidence of postpartum oestrus, ovarian activity and reproductive performance in Thoroughbred mares bred at foal heat under Indian subtropical conditions

Decreased reproductive performance due to summer stress is a well known phenomenon in farm livestock. Whether this occurs in the mare and specifically how this might affect postpartum reproductive activity and performance, especially at Foal Heat (FH), is unknown. This study, therefore, aims to investigate this and the factors that might affect postpartum reproductive activity. Reproductive records of 228 Thoroughbred mares (694 mare years) bred in subtropical north-western India were retrospectively analysed. Overt oestrous activity occurred within 21 d postpartum in 92.94% (645/694) of mares. Significantly (p < 0.001) more April foaling mares (97.37%, 185/190) expressed postpartum oestrous activity than those foaling in January (83.61%; 51/61) and February (88.49; 123/139). Similarly significantly (p < 0.01) fewer multiparous mares failed to demonstrate oestrous activity than primiparous mares (6.12% vs.15.07%; 38/621 vs. 11/73, respectively). 190 of these 694 mares were additionally monitored to confirm ovulation; in these mares onset of FH (oestrus plus confirmed ovulation) occurred 8.42 ± 0.17 d and first ovulation 13.64 ± 0.20 d postpartum. Month, stud farm, year, and parity did not affect interval from parturition to FH onset or to first ovulation; or FH onset to ovulation. In FH bred mares Day 16 pregnancy rate and overall foaling rate were 53.76% (100/186) and 46.24% (86/186) respectively and were similar to those of mares bred later postpartum. FH pregnancy rates were not affected by stud, season, month, year, number of matings, or day of ovulation but were significantly (p < 0.008) lowered by increasing mare age. Significantly (p < 0.01) lower Day 16 pregnancy rates were observed in uterine treated mares compared to untreated mares (31.09% vs. 57.96%; 9/29 vs. 91/157, respectively), this difference was not evident during the rest of pregnancy. In conclusion, postpartum reproductive and ovarian activity appears to be affected by environment, i.e., delayed in subtropical kept Thoroughbred mares compared to those kept in temperate climates. However, resulting reproductive performance at FH and the factors affecting postpartum reproductive activity are similar.