Transferase-mediated dUTP Nick End-labeling Method Research Articles

Aspirin attenuates morphine antinociceptive tolerance in rats with diabetic neuropathy by inhibiting apoptosis in the dorsal root ganglia.

Morphine is a drug used in chronic pain such as diabetic neuropathy, but the development of tolerance to its antinociceptive effect is an important clinical problem. Aspirin is an analgesic and antiapoptotic drug used in combination with morphine as an adjuvant in diabetic neuropathy. Our aim in this study was to investigate the effects of aspirin on morphine-induced neuronal apoptosis and analgesic tolerance in rats with diabetic neuropathy. The antinociceptive effects of aspirin (50mg/kg) and morphine (5mg/kg) were evaluated by thermal pain tests. Streptozotocin (65mg/kg) was injected intraperitoneally to induce diabetic neuropathy. To evaluate apoptosis, ELISA kits were used to measure caspase-3, Bax and Bcl-2 levels. Apoptotic cells were detected histologically by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. Study results indicate that prior administration of aspirin to diabetic rats significantly increased the antinociceptive efficacy of morphine compared to morphine alone. Thermal pain tests showed that aspirin significantly reduced morphine tolerance in rats with diabetic neuropathy. Biochemical analysis revealed that aspirin significantly decreased the levels of pro-apoptotic proteins, caspase-3 and Bax, while increasing the anti-apoptotic Bcl-2 in DRG neurons. Semiquantitative scoring demonstrated that aspirin provided a significant reduction in apoptotic cell counts in diabetic rats. In conclusion, these data suggested that aspirin attenuated morphine antinociceptive tolerance through anti-apoptotic activity in diabetic rat DRG neurons.

Retinoic acid inhibits the pyroptosis of degenerated nucleus pulposus cells by activating Sirt1-SOD2 signaling

ABSTRACT Aim Intervertebral disc (IVD) degeneration is a common disease initiated by the degeneration of the nucleus pulposus (NP). The pyroptosis of degenerated NP cells (dNPCs) plays an important role in NP degeneration. The purpose of this study is to identify a feasible solution that can inhibit NP cell pyroptosis to therapy the degeneration of the intervertebral disc. Methods Cell viability and proliferation were quantified by Cell Counting Kit-8 assay. The measurement of cellular reactive oxygen species (ROS) was detected by 2,7-Dichlorodi-hydrofluorescein diacetate. The death of cells was analyzed by the Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) method of fluorescence analysis. The pyroptosis of cells was assessed by flow cytometry analyses. The contents of sulfate glycosaminoglycans were detected by a blyscan assay kit. Result In this study, we determined the effects of retinoic acid (RA) on dNPCs and investigated the underlying mechanism of RA-mediated pyroptosis in dNPCs. We also verified the effects of RA on IVD degeneration in vivo. Our results demonstrated that RA significantly increased the proliferation and the protein expression of sox9, aggrecan, and collagen II of dNPCs. Pyroptosis-related proteins and the pyroptosis rate of dNPCs were significantly decreased by RA. We found that Sirt1-SOD2 signaling was activated, while ROS generation and TXNIP/NLRP3 signaling in dNPCs were inhibited after the addition of RA. Furthermore, RA also recovered the structure of NP and increased the contents of sulfated glycosaminoglycans and collagen in vivo. Conclusion Our study demonstrated that RA could inhibit the pyroptosis and increase the extracellular matrix synthesis function of dNPCs and verified that RA has a protective effect on IVD degeneration.

Metformin prevents morphine-induced apoptosis in rats with diabetic neuropathy: a possible mechanism for attenuating morphine tolerance.

Morphine is a drug of choice for the treatment of severe and chronic pain, but tolerance to the antinociceptive effect limits its use. The development of tolerance to morphine has recently been associated with neuronal apoptosis. In this study, our aim was to investigate the effects of metformin on morphine-induced neuronal apoptosis and antinociceptive tolerance in diabetic rats. Three days of cumulative dosing were administered to establish morphine tolerance in rats. The antinociceptive effects of metformin (50mg/kg) and test dose of morphine (5mg/kg) were considered at 30-min intervals by thermal antinociceptive tests. To induce diabetic neuropathy, streptozotocin (STZ, 65mg/kg) was injected intraperitoneally. ELISA kits were used to measure caspase-3, bax, and bcl-2 levels from dorsal root ganglion (DRG) tissue. Semi-quantitative scoring system was used to evaluate apoptotic cells with the the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. The findings suggest that co-administration of metformin with morphine to diabetic rats showed a significant increase in antinociceptive effect compared to morphine alone. The antinociceptive tests indicated that metformin significantly attenuated morphine antinociceptive tolerance in diabetic rats. In addition, metformin decreased the levels of apoptotic proteins caspase 3 and Bax in DRG neurons, while significantly increased the levels of antiapoptotic Bcl-2. Semi-quantitative scoring showed that metformin provided a significant reduction in apoptotic cell counts in diabetic rats. These data revealed that metformin demonstrated antiapoptotic activity in diabetic rat DRG neurons and attenuated morphine tolerance. The antiapoptotic activity of metformin probably plays a significant role in reducing morphine tolerance.

Shenjing Guben Wan promotes sperm development by increasing the activity of seminiferous epithelium Sertoli cells.

BackgroundInfertility is an important social problem. Asthenozoospermia (AZS) is a common pathological cause of male infertility, but its pathogenesis is unclear. Shenjing Guben Wan (SJGBW), a traditional Chinese medicine, has shown remarkable effects during the clinical treatment of oligozoospermia or AZS.MethodsIn this study, clinical evaluations were carried out on 184 AZS patients receiving SJGBW treatment, including sperm count, sperm quality, and pregnancy rate. Also, ornidazole was used to build an AZS mouse model, and SJGBW treatment was administered. The sperm quantity and fertility of mice in different groups were evaluated; a cholecystokinin octapeptide-8 (CCK-8) experiment was carried out to test the activity of seminiferous epithelium Sertoli cells, and immunohistochemistry and the Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) method were employed to test the pathological information and expression of the Sertoli cell surface marker in the testicular tissues of mice in each group.ResultsThe sperm vitality, progressive sperm motility, and sperm morphology of patients who received SJGBW treatment were all improved (P<0.05). In the AZS group, the average sperm count, sperm vitality, pregnancy rate, and female mouse litters were all lower relative to mice in the control group. Following SJGBW treatment, the average sperm count, sperm vitality, pregnancy rate, and female mouse litters of mice in the AZS group were all significantly improved. The cytobiological experimental results showed that compared with the serum of normal male mice in the control group, the drug serum containing SJGBW could improve the cell vitality and proliferative ability of seminiferous epithelium Sertoli cells in AZS mice. Furthermore, the TUNEL results showed that the seminiferous tubule Sertoli cells and mesenchymal cells of the AZS mice exhibited the most significant apoptosis, which was alleviated following SJGBW treatment. Moreover, the levels of Sertoli cell marker, SOX9, and anti-apoptosis protein, Bcl2, in SJGBW-treated mice were both higher than that in AZS mice.ConclusionsSJGBW can promote the development and maturation of germ cells by facilitating the proliferation of Sertoli cells in AZS patients, thereby improving the fertility of these patients.

Protection of curcumin to intestinal mucosal barrier by inhibiting enterocyte apoptosis in septic rats

To study the effect of curcumin on enterocyte apoptosis and its protective effect on intestinal mucosal barrier in septic rats. Eighty-seven 3-month male Sprague-Dawley (SD) rats were divided into Sham group, model group and curcumin group by random number table method, with 29 rats in each group. The septic rat model was reproduced by cecal ligation and puncture (CLP). 4 mL dimethyl sulfoxide solution were intraperitoneally injected in both Sham group and model group, 200 mg/kg curcumin dissolved by 4 mL dimethyl sulfoxide solution were intraperitoneally injected in curcumin group 10 minutes after operation. The blood samples (15 rats in each group) were collected 2, 12, 24 hours after operation, and the levels of serum procalcitonin (PCT), tumor necrosis factor-α (TNF-α), D-lactic acid and diamine oxidas (DAO) were tested by enzyme linked immunosorbent assay (ELISA). The ileum tissues were collected 12 hours, 24 hours after operation in three groups, water content was tested by weighting, pathologic structure was observed by light microscope, the enterocyte apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling method (TUNEL). The 7-day survival rate was observed in three groups (14 rats in each group). The serum levels of PCT, TNF-α, D-lactic acid and DAO were higher in model group at 2, 12, 24 hours after operation than those in Sham group, PCT, TNF-α levels were significantly higher in model group than those in Sham group 2 hours after operation [PCT (μg/L): 1.89±0.17 vs. 0.10±0.02, TNF-α (ng/L): 216.51±1.47 vs. 85.25±8.20, both P < 0.01], D-lactic acid, DAO levels were significantly higher in model group than those in Sham group 12 hours after operation [D-lactic acid (mg/L): 40.53±7.76 vs. 11.29±1.28, DAO (ng/L): 1 120.40±302.35 vs. 330.02±81.28, both P < 0.01]. Compared with model group, the levels of serum PCT, TNF-α, D-lactic acid and DAO were lower in curcumin group 2, 12, 24 hours after operation, the statistical difference appeared from 12 hours after operation [PCT (μg/L): 5.37±0.44 vs. 8.67±0.64, TNF-α (ng/L): 211.12±4.31 vs. 313.30±18.46, D-lactic acid (mg/L): 29.74±1.41 vs. 40.53±7.76, DAO (ng/L): 810.71±201.41 vs. 1 120.40±302.35, all P < 0.05], curcumin group had lower water content in ileum tissues 12 hours, 24 hours after operation [(68.34±0.68)% vs. (70.55±0.87)%, (69.41±0.59)% vs. (71.69±0.87)%, both P < 0.05]. The pathologic structures of intestinal villus were normal in Sham group, however, in model group intestinal villus were atrophic, edematous and shorten 12 hours after operation, it was further exacerbated 24 hours after operation. Compared with model group, the pathologic structures of intestinal villus in curcumin group were relived 12 hours, 24 hours after operation. The number of apoptotic enterocytes were significantly increased in model group compared with Sham group 24 hours after operation (cells: 25.48±6.10 vs. 4.00±2.04, P < 0.05), and the number of apoptotic enterocytes was lower in curcumin group than that in model group at the same time (cells: 15.48±3.75 vs. 25.48±6.10), the difference was statistically significant (both P < 0.05). Seven-day survival rate was significantly lower in curcumin than that in model group [42.9% (6/14) vs. 50.0% (7/14)], however, the difference was not statistically significant (P > 0.05). Curcumin can protect the intestinal mucosal barrier by inhibiting enterocyte apoptosis in septic rats.

Effects of Cyclosporine Therapy on Liver and Kidney Retrieval in Poisoned Male Rats by Mesobuthus eupeus Scorpion Venom

Background: Mesobuthus eupeus venom is a member of Buthidae family, which can enter the blood circulation exerting detrimental effects on body organs, such as the liver and kidney through inflammation. Cyclosporine, known as an anti-inflammatory drug, is used to treat many inflammation-associated diseases. Objectives: In this study, cyclosporine was selected to inhibit the scorpion toxin effects on rat organs. Methods: This experimental study was conducted in the Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization, Karaj, Iran, from June to November 2019. Fifty male rats were randomly divided into five groups of 10, including the control (10 mg/kg olive oil i.p), M. eupeus venom (10 mg/kg i.p.), cyclosporine 10 mg/kg (venom 10 mg/kg for 30 min i.p followed by cyclosporine 10/kg mg for 7 day i.p.), cyclosporine 20 mg/kg (venom 10 mg/kg for 30 min i.p followed by cyclosporine 20 mg/kg for 7 day i.p.), and cyclosporine 30 mg/kg (venom 10 mg/kg for 30 min i.p followed by cyclosporine 30 mg/kg for 7 day i.p.). After treatment with cyclosporine, the liver and kidney function was analyzed by calculating some biochemical enzymes, including serum glutamate-pyruvate transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), nitric oxide (NO), interleukin-2 (IL-2), malondialdehyde (MDA), creatinine, and urea via ELISA and spectrophotometry. Then, to determine the rate of apoptosis in tissue, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method was done. Results: At the end of the study, the results showed a significant elevation in SGPT (164.5 ± 10 vs. 126.2 ± 7, P &lt; 0.0001), SGOT (190.37 ± 11 vs. 148 ± 10, P &lt; 0.0001), NO (24.4 ± 1.17 vs. 17.4 ± 1.4, P = 0.02), and MDA (0.42 ± 0.05 vs. 0.22 ± 0.04, P &lt; 0.0001) in the venom group compared with the control group. There were no significant differences in the urea, IL-2, and creatinine between the venom and control groups. However, the group receiving cyclosporine (30 mg/kg) showed a significant decline in SGPT (96.42 ± 5.7 vs. 164.5 ± 10, P &lt; 0.0001), SGOT (144.57 ± 9.24 vs. 190.37 ± 11, P &lt; 0.0001), urea (28.83 ± 1.32 vs. 38.83 ± 1.6, P = 0.00), creatinine (0.023 ± 0.01vs. 0.29 ± 0.005, P &lt; 0.0001), and MDA (0.10 ± 0.01 vs. 0.42 ± 0.05, P &lt; 0.0001), as well as increased apoptosis rate (P &lt; 0.05), compared with the venom group. No significant difference was observed between the cyclosporine and venom groups in NO and IL-2. Conclusions: Cyclosporine at a dose of 30 mg was able to decrease inflammatory responses and induce apoptosis rate. Therefore, it could be a suitable drug for patients bitten by a scorpion sting.

Effects of chronic treatment with botulinum toxin type A in salivary glands of rats: Histological and immunohistochemical analyses.

The intraglandular application of botulinum neurotoxin type A (BoNT-A) is used in patients with neuromotor disorders to control the escape of saliva. The aim of this study was to analyze the impact of repeated treatment with BoNT-A on the submandibular-sublingual complex of rats. A total of 35 Wistar rats were divided into three groups: control group (C), in which animals were not treated; group B, treated with 2.5 U intraglandular injections of BoNT-A (Prosigne® ) and group G, that received bovine gelatine (Prosigne stabilizer). Three applications were performed in intervals of 35days. Twelve and 35days after ending the treatment, submandibular-sublingual complex was collected for histological analysis. Immunohistochemical reactions for calponin and specific muscle actin were also performed, besides detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. A decrease in mucosal and serous acini diameter was observed, with increased interstitial space after 12days of treatment with BoNT-A, which was observed in a lesser degree on the 35th day. At 12days, immunohistochemical analysis revealed a decrease in myoepithelial cells of serous acini in group B. TUNEL methods evidenced apoptosis in animals from group B. BoNT-A caused histological and cellular changes in submandibular-sublingual complex, followed by a tendency toward reversal after 35days. The reversal characteristic of cellular changes in the submandibular-sublingual complex suggests that this BoNT-A formulation may be safely used for sialorrhea treatment.

GTS-21 Protected Against LPS-Induced Sepsis Myocardial Injury in Mice Through α7nAChR.

Sepsis-induced myocardial injury is a well-known cause of mortality. The cholinergic anti-inflammatory pathway (CHAIP) is a physiological mechanism by which the central nervous system regulates immune response through the vagus nerve and acetylcholine; the α7-nicotinic acetylcholine receptor (α7nAChR) is the main component of CHAIP; GTS-21, a synthetic α7nAChR selective agonist, has repeatedly shown its powerful anti-inflammatory effect. However, little is known about its effect on LPS-induced myocardial injury. We investigated the protective effects of GTS-21 on lipopolysaccharide (LPS)-induced cardiomyopathy via the cholinergic anti-inflammatory pathway in a mouse sepsis model. We constructed the model of myocardial injury in sepsis mice by C57BL/6 using LPS and determined the time of LPS treatment by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). C57BL/6 mice were randomized into five groups: blank control group, model group, α-bungarotoxin + LPS group, GTS-21 + LPS group, and α-bungarotoxin + GTS-21 + LPS group. The pathological results of myocardial tissue were detected by the HE method; the apoptosis rate was detected by the TUNEL method; the relative expressions of NF-κB p65, Caspase-3, Caspase-8, Bcl-2, Bax, p53, and a7nAChR were detected by real-time quantitative PCR (RT-PCR); and the protein expressions of IL-6, IL-1 β, TNF-α, and pSTAT3 were detected by western blot. The results showed that LPS-induced myocardial pathological and apoptosis changes were significant compared with the blank group, which was reversed by GTS-21; however, pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21. NF-κB p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1β, TNF-α, and pSTAT3 were significantly increased in the model group, while a7nAChR and Bcl-2 were significantly decreased; GTS-21 treatment reversed that result, while pretreatment with α-bungarotoxin strengthened the result in the model. And pretreatment with α-bungarotoxin blocked the protective effect of GTS-21. GTS-21 can alleviate the LPS-induced damage in the heart via a7nAChR, and pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21 on sepsis in mice.

Expression of histone deacetylase 1 gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor

Objective To investigate the expression of histone deacetylase 1 (HDAC1) gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor. Methods HDAC1 small interfering RNA (siRNA) and recombinant expression vector pcDNA3.1-HDAC1 were transfected with A549 lung cancer cells, respectively. Stable transfected cell lines were screened by G418 selection. reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression levels of HDAC1 mRNA and HDAC1 protein in the cell; cell proliferation was measured by cell counting kit-8 (CCK-8). Clone formation experiment was used to observe the effect of up-regulation and down-regulation of HDAC1 expression on the radiation sensitivity of A549 cells, cell apoptosis was detected by transferase-mediated dUTP nick-end labelling (TUNEL) method. Nude mice experiment of transplantable tumor was applied for in vivo detection of the influence of up-regulated and down-regulation of HDAC1 expression combined with X-ray irradiation in the inhibition of lung cancer growth. Results The expression of HDAC1 gene and protein were significantly lower to 1.86±0.09 and 2.41±0.10 (P=0.015, 0.006) in HDAC1 cell line A549, and the HDAC1 up-regulation group were significantly higher to 3.65±0.14 and 5.41±0.12 (P=0.012, 0.008); The radiation sensitivity of lung cancer A549 cells was increased in the down-regulation group of HDAC1 (P=0.025), and the radiation sensitivity of lung cancer A549 cells was decreased (P=0.009)in the HDAC1 up-regulation group. Down-regulation of HDAC1 inhibited the proliferation of A549 lung cancer, increased the rate of apoptosis to (42.17±3.19)% (P=0.008), proliferation of A549 cells was significantly enhanced in HDAC1 up-regulated group, associated with reduced percentage of apoptotic cells (9.11±1.46)% (P=0.006); Average volume in the nude mice of HDAC1 down-regulated group (547.37±89.68) mm3 were significantly smaller than the control group (1 238.44±212.36) mm3 (P=0.008); furthermore, mean volume of tumor was greater in the HDAC1 up-regulated group than in the control group (1 681.27±195.37) mm3 (P=0.006). After 20 Gy γ irradiation, the average volume of HDAC1 down-regulated group were significantly decreased (214.15±54.26) mm3 (P=0.011). Compared with that before irradiation, the average volume of the tumor in the nude mice was not significantly changed after irradiation (P=0.264). Conclusion Down-regulated expression of HDAC1 can inhibit the growth of human lung cancer A549 cells in nude mice, enhance the radiation sensitivity of tumor cells, whereas up-regulated expression of HDAC1 can produce tumor radiation resistance. Key words: Lung cancer; A549 cell; Histone deacetylase 1; Transplantable tumor; Nude mice

Ocular Lesions in Red-Tailed Hawks ( Buteo jamaicensis) With Naturally Acquired West Nile Disease.

Ocular lesions are common in red-tailed hawks with West Nile (WN) disease. These lesions consist of pectenitis, choroidal or retinal inflammation, or retinal necrosis, but detailed investigation of the ocular lesions is lacking. Postmortem examination of the eyes of 16 red-tailed hawks with naturally acquired WN disease and 3 red-tailed hawks without WN disease was performed using histopathology, immunohistochemistry for West Nile virus (WNV) antigen, glial fibrillary acid protein, cleaved caspase-3, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Retinal lesions were classified as type I or type II lesions. Type I lesions were characterized by lymphoplasmacytic infiltrates in the subjacent choroid with degeneration limited to the outer retina (type Ia lesion) or with degeneration and necrosis of the outer retina or outer and inner retina (type Ib lesion) while retinal collapse, atrophy, and scarring were hallmarks of type II lesions. Type II retinal lesions were associated with a more pronounced choroiditis. Although not statistically significant, WNV antigen tended to be present in larger quantity in type Ib lesions. Type I lesions are considered acute while type II lesions are chronic. The development of retinal lesions was associated with the presence of an inflammatory infiltrate in the choroid. A breakdown of the blood-retina barrier is suspected to be the main route of infection of the retina. Within the retina, virus appeared to spread via both neuronal and Müller cell processes.

Automated TUNEL assay on tissue sections via robotics

The manual TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) method for detecting deoxyribonucleic acid (DNA) fragmentation during apoptosis is widely used to determine cell death in tissue sections. However, manually staining large numbers of slides is cumbersome and may result in inconsistent signal intensities. We have developed a simple automated TUNEL assay using a robotic platform and a popular apoptosis detection kit. Comparing manual and automatic staining methods of serially sectioned murine tissue blocks showed no differences in quality or quantity of signal. The automated TUNEL assay allows larger throughput and provides consistent results across many tissue types.

Grape seed proanthocyanidin extract protects the retina against early diabetic injury by activating the Nrf2 pathway.

The present study aimed to investigate whether grape seed proanthocyanidin extract (GSPE) has a protective effect on diabetic retinal function. A total of 30 Wistar rats were randomly divided into three equal groups, including the control, diabetic and GSPE-treated diabetic groups. Retinal tissue was harvested and subsequently stained with hematoxylin and eosin. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and methane dicarboxylic aldehyde (MDA) levels were evaluated using respective assay kits; whereas nuclear erythroid 2-related factor 2 (Nrf2) and heme oxygenase (HO)-1 expression levels were assessed by immunohistochemical and western blot analysis. Cell apoptosis in the retina was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. The results showed that the structure of the retina was damaged in diabetic rats, as compared with the control rats. Notably, the structure of the retina improved in the GSPE-treated diabetic group, as compared with the diabetic group. SOD and GSH-Px activities were significantly increased in the retina of rats in the GSPE-treated diabetic group, as compared with the diabetic group (P=0.011 and P=0.001, respectively). Furthermore, a significant reduction in MDA was detected (P=0.013) and the expression levels of Nrf2 and HO-1 in the bladders of rats in the GSPE-treated diabetic group were significantly increased, as compared with the diabetic group (P=0.038 and P=0.043, respectively). Apoptosis of retinal cells was significantly increased in the diabetic group, as compared with the control group (P<0.001); a significant reduction was also detected in the GSPE-treated diabetic group, as compared with the diabetic group (P=0.014). These results demonstrate that GSPE administration may protect the retina against hyperglycemic damage, possibly by ameliorating oxidative stress-mediated injury via the activation of the Nrf2 pathway.

Effect of Maxing Shigan Tang on H1N1 Influenza A Virus-Associated Acute Lung Injury in Mice

Objective: This study is aimed at examining the effects of Maxing Shigan Tang (MST) treatment on H1N1-associated acute lung injury (ALI) and exploring the possible mechanism. Material and Methods: Mice were randomly divided into a control group, model group, peroxisomal proliferator activator receptor γ (PPARγ) inhibition group (PPARγ-), PPARγ activation group (PPARγ+), and MST group. Influenza A (H1N1) virus of the Fort Monmouth 1 (FM1) strain was used to induce an ALI mice model. Hematoxylin and eosin staining was performed to investigate the effect of MST treatment on H1N1-associated ALI. Cell apoptosis of lung tissues of each group were conducted through transferase-mediated dUTP nick end-labeling methods. Moreover, the expression level of caspase 3, activity of caspase 3, and serum level of tumor necrosis factor (TNF)-α of each group were also analyzed. Finally, quantitative real-time polymerase chain reaction and Western blotting analysis were carried out to detect angiopoietin-like 4 (ANGPTL4) expression level. Results: We found that mice infected with the FM1 strain of H1N1 influenza A virus developed severe ALI, and MST could improve H1N1-induced ALI. Moreover, MST decreased lung cell apoptosis and reduced the serum content of TNF-α. In addition, MST significantly induced the ANGPTL4 expression in H1N1-induced ALI. Conclusion: MST improves H1N1-associated ALI maybe through targeting ANGPTL4 in mice.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

BackgroundCigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).MethodsPNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.ResultsCSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.ConclusionsAcrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms.

Effects of postnatal hyperoxia exposure on the rat dentate gyrus and subventricular zone

Premature newborns may be exposed to hyperoxia in the first postnatal period, but clinical and experimental works have raised the question of oxygen toxicity for the developing brain. However, specific analysis of hyperoxia exposure on neurogenesis is still lacking. Thus, the aim of the present study was to evaluate possible changes in the morphometric parameters of the main neurogenic sites in newborn rats exposed to 60 or 95 % oxygen for the first 14 postnatal days. The optical disector, a morphometric method based upon unbiased sampling principles of stereology, was applied to analyse cell densities, total volumes, and total cell numbers of the dentate gyrus (DG) and subventricular zone (SVZ). Apoptosis and proliferation were also studied by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling method and anti-ki67 immunohistochemistry, respectively. Severe hyperoxia increased the percentage of apoptotic cells in the DG. Moderate and severe hyperoxia induced a proliferative response both in the DG and SVZ, but the two neurogenic sites showed different changes in their morphometric parameters. The DG of both the hyperoxic groups showed lower volume and total cell number than that of the normoxic one. Conversely, the SVZ of newborn rats exposed to 95 % hyperoxia showed statistically significant higher volume and total cell number than SVZ of rats raised in normoxia. Our findings indicate that hyperoxia exposure in the first postnatal period affects both the neurogenic areas, although in different ways, i.e. reduction of DG and expansion of SVZ.

Effects of Morphine Postconditioning on Myocardial Ischemia-Reperfusion Injury in Rats In Vivo

Objective.To investigate the effects of morphine postconditioning on Myocardial ischemia reperfusion injury in rats in vivo. Methods. To randomly divide 40 male SD rats equally into 4 groups, including Sham group in which the chest was opened without ligating the left coronary artery, ischemia-reperfusion group(Group I/R ), ischemic preconditioning group(Group IPC ) and morphine postconditioning group(GroupMOR) in which 0. 3 mg/kg morphine was given intravenously 5 min before reperfusion. The left anterior descending coronary arterys(LAD) of rats in five groups are ligated for 30 minutes and are re-perfused for 90 minutes. Cardiac Apoptosis was determined quantitatively by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） methods． To calculate the concentration of the serum malondialdehyde(MDA) with Thiobarbituric acid (TBA) reaction method and the activity of the superoxide dismutase (SOD) with xanthine oxidase reaction method. Result. Comparing with Group S, the quantity of the cardiac apoptosis in Group I/R, IPC and MOR rised in different levels. Comparing with Group 1/R, the quantity of the cardiac apoptosis in Group IPC and MOR reduced obviously. Comparing with Group 1/R, the concentration of the serum malondialdehyde (MDA) in the other four groups all reduced and the activity of the superoxide dismutase increased. Conclusion. Morphine postconditioning can significantly reduce myocardial apoptosis induced by ischemia-reperfusion injury，reduce myocardial infarct size, decrease the concentration of MDA, and increase the activity of SOD. Therefore, morphine postconditioning has protective effects on myocardial ischemia-reperfusion injury in rats in vivo. Morphine is a potent kind of opioid analgesics, which is widely used in clinical anesthesia. However, further studies are needed on effects of morphine postconditioning on myocardial ischemia reperfusion injury. In order to provide the foundations for clinical application, the authors investigate the effects of morphine postconditioning on myocardial ischemia reperfusion injury through comparison and analysis of the cardiac apoptosis, the concentration of the serum malondialdehyde (MDA) and the activity of the superoxide dismutase (SOD) in Group S, I/R, IPC and MOR.

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by tannic acid and resveratrol

Tannic acid (TA), a plant polyphenol, has been described as having anticarcinogenic, antioxidant, antimutagenic, antimicrobial, antiallergic, antiinflammatory and astringent properties. Resveratrol (RES), a phytoalexin phenolic compound, has been known as an antioxidant, anticarcinogenic, antiinflammatory, antimutagenic, antiproliferative, antiviral, antibacterial, estrogenic and vasodilator agent. Although molecular mechanisms of TA and RES responsible for anticancer activity are yet to be known, there are in vitro studies indicating that these substances contribute to anticancer activity by inducing apoptosis. In this study, the effects of TA and RES in MCF-7 breast cancer cells on apoptotic index, FAS-associated death domain (FADD) and Bak proteins were investigated. Terminal transferase-mediated dUTP nick end-labeling (TUNEL) method was applied for apoptosis and immunohistochemical staining for FADD and Bak proteins. Our study results show that TA and RES increased apoptotic index, percentage ratios of apoptotic proteins Bak and FADD in MCF-7 cell line. Keywords : Bak, Fas-associated death domain (FADD), MCF-7, resveratrol, tannic acid African Journal of Biotechnology Vol. 12(12), pp. 1431-1437

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine‐treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H(1)- and H(2)-receptors. Cimetidine is a H(2)-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100 mg kg(-1) of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.

P251 The Role of Reactive Oxidative Species Within Cigarette Smoke Extract on Apoptosis and Inflammation in Primary Nasal Epithelial Cells: Abstract P251 Table 1

<h3>Introduction</h3> The responses of the bronchial epithelium to cigarette smoke (CS) are well characterized, but effects on the nasal epithelium, also important in respiratory disease, are not as well defined. The mechanism of cell death due to cigarette smoke extract (CSE) exposure is controversial. As there is convincing evidence that cigarette smoke decreases levels of protective antioxidants, we hypothesised that reactive oxidative species contained within CSE contribute to its immunomodulatory and cytotoxic properties. <h3>Methods</h3> Nasal brushings were obtained from 16 healthy volunteers from the medial aspect of the inferior turbinate as previously described. CSE was prepared by combusting 1 Marlboro cigarette through 25 ml of media. Cell viability was determined after primary nasal epithelial cells (PNECs) were stimulated with 5% CSE for 24 h (caspase 3 levels determined after 4 h), in the presence or absence of 20 mM N-acetylcysteine (NAC). In separate experiments, cultures were stimulated with <i>Pseudomonas aeruginosa</i> lipopolysaccharide (PA LPS) for 24 h (0 – 30 µg/ml), and the effects of pre-incubation with CSE±20 mM NAC for 4 h evaluated in terms of cytokine release. Phospho-NF-κB activity was determined after 1 h PA LPS exposure. Apoptosis was evaluated using annexin-V staining and the terminal transferase-mediated dUTP nick end-labelling (TUNEL) method. <h3>Results</h3> 5% CSE (4 h) exposure was immunosuppressive in PNEC cultures for both IL-8 and IL-6 release (0.53 fold reduction in IL-8 and 0.49 fold reduction in IL-6 release after stimulation with 30 µg/ml PA LPS for 24 h). 4 h exposure to CSE heightened active caspase 3 levels, and a 24 h exposure induced both early and late apoptosis established by annexin-V staining (Table 1). Apoptosis was confirmed using the TUNEL assay. All of these effects were mitigated with the addition of 20 mM NAC to the CSE (0.85 fold reduction in IL-8 and 0.73 fold reduction in IL-6 release after stimulation with 30µg/ml PA LPS for 24 h). <h3>Conclusions</h3> A 4 h CSE exposure was immunosuppressive in PNEC cultures and induced apoptosis. Reactive oxidative species are at least partially responsible for these observations. Research funded by NI RDO.

S87 Differential Inflammatory Responses of Primary Bronchial Epithelial Cells from Subjects with COPD, Healthy Smokers and Never Smokers

IntroductionWe investigated the responses of primary bronchial epithelial cells (PBECs) obtained from subjects with COPD, healthy smokers (HS), and non-smokers (NS) to cigarette smoke extract (CSE) treatment. We hypothesised that...