Proportion Of TUNEL-positive Cells Research Articles

Lacosamide alleviates bilateral cavernous nerve injury-induced erectile dysfunction in the rat model by ameliorating pathological changes in the corpus cavernosum.

Bilateral cavernous nerve injury-related erectile dysfunction (BCNI-ED) shows a limited response to type 5 phosphodiesterase inhibitors. Furthermore, lacosamide (LCM) can alleviate peripheral neuropathy. To explore whether LCM can improve the erectile response after BCNI, we randomly divided 30 young Sprague-Dawley rats into three groups (n = 10 per group), namely, the sham operation, 0.9% normal saline-treated (BCNI + 0.9% NS), and LCM-treated BCNI (BCNI + LCM) groups. LCM was injected intraperitoneally at a dose of 90 mg/kg/day for 7 consecutive days. Erectile function was assessed by measuring the ratio of peak intracavernous pressure (ICP) to mean arterial pressure (MAP), and tissues were harvested for transmission electron microscopy, immunofluorescence, Masson's trichrome staining, TUNEL staining, and Western blot analysis. The BCNI + 0.9% NS group showed reduced ICP/MAP ratio (0.93 ± 0.04 vs. 0.44 ± 0.05, P < 0.0001). An increased proportion of TUNEL-positive cells (0.04 ± 0.01 vs 0.87 ± 0.03, P < 0.0001) and a decreased smooth muscle/collagen ratio (0.44 ± 0.01 vs. 0.33 ± 0.01, P < 0.001) were observed in the BCNI + 0.9% NS compared with the sham group. Administration of LCM significantly restored the ICP/MAP ratio (0.44 ± 0.05 vs. 0.74 ± 0.05, P < 0.001) and decreased the proportion of TUNEL positive cells (0.87 ± 0.03 vs. 0.60 ± 0.04, P < 0.0001) in the corpus cavernosum following BCNI. The ratio of smooth muscle to collagen (0.43 ± 0.01vs. 0.33 ± 0.01, P < 0.01) and expression of α-SMA (P < 0.0001) in the BCNI + LCM group significantly increased compared with BCNI + 0.9% NS group, indicating alleviation of fibrosis. Apoptotic markers, including Bax/Bcl-2 (P < 0.01) and Caspase-3 (P < 0.0001) in the BCNI + LCM group was significantly lower than that in the BCNI + 0.9% NS group. LCM treatment partially upregulated the expression of vWF and eNOS in cavernous tissue in rats subjected to BCNI (P < 0.05). Increases in S100-β and nNOS expression in the major pelvic ganglion (MPG) were observed after LCM administration. In summary, LCM can recover erectile function in BCNI-ED rat model by suppressing corporal apoptosis and fibrosis, and protecting the cavernous nerve.

Effects of Iron on Efficacy of Photodynamic Therapy Using Photolon in a Mouse Model of CT26 Colon Cancer.

Photodynamic therapy (PDT) -a minimally invasive anti-cancer therapy-is undergoing experimental studies to increase its anti-cancer effects. This study investigated the influence of iron on the anti-cancer effects of PDT. PDT was performed in a cancer-bearing mouse model, which was created by using a murine colon carcinoma (CT26) cell line after administration of Photolon and iron. Tumor volume and the results of TdT-mediated dUTP-biotin nick end labeling (TUNEL), 8-OHdG, and TBARS assays were used to measure anti-cancer effect. On day 14, tumor volume had increased by 49% in the PDT group and decreased by 72% in the iron+PDT group. The percentage of TUNEL-positive cells in tumor tissues was 45% in the PDT group and 69% in the iron+PDT group, suggesting that the proportion of TUNEL-positive cells had increased in the iron+PDT group. The 8-OHdG content in tumor tissues was 33% higher in the iron+PDT group than in the PDT group. The TBARS content in tumor tissues was 46% higher in the iron+PDT group than in the PDT group. Iron enhances the anti-cancer effect of PDT using Photolon, most likely by increasing oxidative damage.

ROCK2 knockdown alleviates LPS-induced inflammatory injury and apoptosis of renal tubular epithelial cells via the NF-κB/NLRP3 signaling pathway.

Rho-associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis-associated acute kidney injury (S-AKI). HK-2 cells were stimulated with lipopolysaccharide (LPS) to simulate S-AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS-induced HK-2 cells was knocked down using short hairpin RNA-ROCK2, in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription-quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK-2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF-α, IL-6 and IL-1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL-positive cells, decreased Bcl-2 protein expression levels and increased cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS-induced HK-2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro-inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS-induced expression of phosphorylated-NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD and caspase-1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS-treated HK-2 cells. Therefore, ROCK2 knockdown may alleviate LPS-induced HK-2 cell injury via the inactivation of the NF-κB/NLRP3 signaling pathway.

Plasmodium infection suppresses colon cancer growth by inhibiting proliferation and promoting apoptosis associated with disrupting mitochondrial biogenesis and mitophagy in mice

BackgroundColon cancer is a common gastrointestinal tumor with a poor prognosis, and thus new therapeutic strategies are urgently needed. The antitumor effect of Plasmodium infection has been reported in some murine models, but it is not clear whether it has an anti-colon cancer effect. In this study, we investigated the anti-colon cancer effect of Plasmodium infection and its related mechanisms using a mouse model of colon cancer.MethodsAn experimental model was established by intraperitoneal injection of Plasmodium yoelii 17XNL-infected erythrocytes into mice with colon cancer. The size of tumors was observed dynamically in mice, and the expression of Ki67 detected by immunohistochemistry was used to analyze tumor cell proliferation. Apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, and the expression of apoptosis-related proteins including Bax, Bcl-2, caspase-9, and cleaved caspase-3 was detected by western blot and immunohistochemistry, respectively. Transmission electron microscopy (TEM) was used to observe the ultrastructural change in colon cancer cells, and the expression of mitochondrial biogenesis correlative central protein, PGC-1α, and mitophagy relevant crucial proteins, PINK1/Parkin, were detected by western blot.ResultsWe found that Plasmodium infection reduced the weight and size of tumors and decreased the expression of Ki67 in colon cancer-bearing mice. Furthermore, Plasmodium infection promoted mitochondria-mediated apoptosis in colon cancer cells, as evidenced by the increased proportion of TUNEL-positive cells, the upregulated expression of Bax, caspase-9, and cleaved caspase-3 proteins, and the downregulated expression of Bcl-2 protein. In colon cancer cells, we found destroyed cell nuclei, swollen mitochondria, missing cristae, and a decreased number of autolysosomes. In addition, Plasmodium infection disturbed mitochondrial biogenesis and mitophagy through the reduced expression of PGC-1α, PINK1, and Parkin proteins in colon cancer cells.ConclusionsPlasmodium infection can play an anti-colon cancer role in mice by inhibiting proliferation and promoting mitochondria-mediated apoptosis in colon cancer cells, which may relate to mitochondrial biogenesis and mitophagy.Graphical

Dietary resveratrol attenuation of intestinal inflammation and oxidative damage is linked to the alteration of gut microbiota and butyrate in piglets challenged with deoxynivalenol

BackgroundDeoxynivalenol (DON) is a widespread mycotoxin that induces intestinal inflammation and oxidative stress in humans and animals. Resveratrol (RES) effectively exerts anti-inflammatory and antioxidant effects. However, the protective effects of RES on alleviating DON toxicity in piglets and the underlying mechanism remain unclear. Therefore, this study aimed to investigate the effect of RES on growth performance, gut health and the gut microbiota in DON-challenged piglets. A total of 64 weaned piglets [Duroc × (Landrace × Yorkshire), 21-d-old, 6.97 ± 0.10 kg body weight (BW)] were randomly allocated to 4 treatment groups (8 replicate pens per treatment, each pen containing 2 males; n = 16 per treatment) for 28 d. The piglets were fed a control diet (CON) or the CON diet supplemented with 300 mg RES/kg diet (RES group), 3.8 mg DON/kg diet (DON) or both (DON+RES) in a 2 × 2 factorial design.ResultsDON-challenged piglets fed the RES-supplemented diet had significantly decreased D-lactate concentrations and tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) mRNA and protein expression, and increased zonula occludens-1 (ZO-1) mRNA and protein expression compared with those of DON-challenged piglets fed the unsupplemented diet (P < 0.05). Compared with unsupplemented DON-challenged piglets, infected piglets fed a diet with RES showed significantly decreased malondialdehyde (MDA) levelsand increased mRNA expression of antioxidant enzymes and antioxidant genes (i.e., GCLC, GCLM, HO-1, SOD1 and NQO-1) and glutamate-cysteine-ligase modulatory subunit (GCLM) protein expression (P < 0.05). Moreover, RES supplementation significantly abrogated the increase in the proportion of TUNEL-positive cells and the protein expression of caspase3 in DON-challenged piglets (P < 0.05). Finally, RES supplementation significantly increased the abundance of Roseburia and butyrate concentrations, while decreasing the abundances of Bacteroides and unidentified-Enterobacteriaceae in DON-challenged piglets compared with DON-challenged piglets alone (P < 0.05).ConclusionsRES supplementation improved gut health in DON-challenged piglets by strengthening intestinal barrier function, alleviating intestinal inflammation and oxidative damage, and positively modulating the gut microbiota. The protective effects of RES on gut health may be linked to increased Roseburia and butyrate concentrations, and decreased levels of Bacteroides and unidentified-Enterobacteriaceae.

Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

Amelioration of dry eye syndrome by oral administration of cultivated wild ginseng extract

The present study investigates the effect of ludartin on spinal cord injury in rat model. Ludartin treatment decreased the expression of myeloperoxidase and malondialdehyde in spinal cord tissues. The expression of glutathione and superoxide dismutase was enhanced. It also exhibited inhibitory effect on reduction of NeuN-positive cells. The proportion of TUNEL positive cells was also regulated. In addition, ludartin treatment prevented the onset of apoptosis which was evident by decrease in caspase-3 and Bax level and increase of Bcl‑2 level. Up-regulation of tumor necrosis factor‑α, inter-leukin‑1β and I interleukin‑6 by spinal cord injury was suppressed by ludartin treatment. There was improvement in locomotion of rats by treatment with ludartin. Ludartin treatment of spinal cord injury rats improves locomotion by inhibiting inflammatory cytokine expression and preventing cell apoptosis. Thus, ludartin has therapeutic importance in spinal cord injury treatment.

Ludartin exhibits therapeutic effect on spinal cord injury through inhibition of apoptosis and inflammation

The present study investigates the effect of ludartin on spinal cord injury in rat model. Ludartin treatment decreased the expression of myeloperoxidase and malondialdehyde in spinal cord tissues. The expression of glutathione and superoxide dismutase was enhanced. It also exhibited inhibitory effect on reduction of NeuN-positive cells. The proportion of TUNEL positive cells was also regulated. In addition, ludartin treatment prevented the onset of apoptosis which was evident by decrease in caspase-3 and Bax level and increase of Bcl‑2 level. Up-regulation of tumor necrosis factor‑α, inter-leukin‑1β and I interleukin‑6 by spinal cord injury was suppressed by ludartin treatment. There was improvement in locomotion of rats by treatment with ludartin. Ludartin treatment of spinal cord injury rats improves locomotion by inhibiting inflammatory cytokine expression and preventing cell apoptosis. Thus, ludartin has therapeutic importance in spinal cord injury treatment.&#x0D; Video Clip of Methodology:&#x0D; 3 min 35 sec: Full Screen Alternate

Mito-TEMPO improves development competence by reducing superoxide in preimplantation porcine embryos

Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p < 0.05) compared to those from G1 zygotes. In G2 embryos, the proportion of TUNEL-positive cells was also higher than that of G1 embryos. Superoxide in G2 embryos was significantly increased compared to that in G1 embryos. Mitochondrial membrane potential and ATP production were lower in G2 embryos than in G1 embryos. Phosphorylation of Drp1 at Ser 616 increased in G1 embryos during the cleavage stages compared to that in the zygote but was not significantly different in G2 embryos. Then, the effects of Mito-TEMPO were investigated in G2 embryos. Blastocyst formation rate (G2: 19.1 ± 5.1% vs G2 + Mito-TEMPO: 28.8 ± 4.0%; 1.5-fold, p < 0.05) and mitochondrial aggregation were recovered after superoxide reduction by Mito-TEMPO treatment. Thus, we showed that Mito-TEMPO improves blastocyst development by superoxide reduction in porcine embryos in vitro.

78 Mito-TEMPO, a Scavenger for Mitochondria-Derived Reactive Oxygen Species, Enhances Porcine Pre-Implantation Embryo Development

The production of reactive oxygen species (ROS) from mitochondria contributes to redox signalling, mitochondrial functions, and apoptosis. However, the specific effects of mitochondria target superoxide (O2 •–) on porcine embryo development remain unclear. The objective of present study was to examine the differences of mitochondrial functions and dynamics in 2 subpopulations of porcine zygotes (G1 and G2), and to investigate the effects of Mito-TEMPO on porcine embryo development. Porcine embryos were visually classified in 2 groups [Grade (G)1: over 90%, and G2: below 90%] according to the lipid distribution at the zygote stage. The blastocyst development rate was greater in G1 than in G2 embryos (G1: 26.5 ± 5.9% v. G2: 16.2 ± 7.9%; P &lt; 0.05). To evaluate blastocyst quality, we performed a 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL assay. The proportion of TUNEL-positive cells was higher (P &lt; 0.05) in G2 than G1 embryos. We measured superoxide production by MitoSOX staining as mitochondrial superoxide specific fluorescence dye by iRiSTM Digital Cell Image System (Logos Biosystems Inc., Gyeonggi-do, South Korea). Red fluorescence intensity of superoxide in G2 embryos significantly increased (P &lt; 0.05) compared with that in G1. We investigated changes in mitochondrial functions using a Mitotracker JC-1 mitochondrial membrane potential assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and ATP determination kit, respectively. Mitochondria membrane potential and ATP production were lower (P &lt; 0.05) in G2 embryos than in G1 embryos. To confirm the protein levels of mitochondria fission protein DRP1, we performed Western blot analysis (per 40 embryos). Phosphorylation DRP1-Ser616 was increased (P &lt; 0.05) in G1 embryos at cleavage stage compared with that in zygote, but not significantly different in G2 embryos. Thus, G2 embryos showed low development rate until blastocyst via mitochondrial dysfunction, increase in fission protein expression and mitochondrial aggregation according to the elevation of mito-ROS. Subsequently, the effect of the adding superoxide scavenger Mito-TEMPO was investigated in G2 embryos. Blastocyst formation (G2+MitoTempo: 28.8 ± 4.0% v. G2: 19.1 ± 5.1%; P &lt; 0.05) and mitochondrial aggregation were recovered by mito-ROS reduction mediated by Mito-TEMPO. Our observations demonstrated that regulation of superoxide in mitochondria is important in pre-implantation development of porcine embryos. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037-04-2-HD020) through the Rural Development Administration and the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

Investigation of the expression of apoptosis-inducing factor-mediated apoptosis in Hirschsprung's disease.

One of the widely accepted hypotheses of Hirschsprung's disease (HD) is that the absence of ganglion cells in the distal part of the intestine is caused by the death of enteric neural crest-derived cells following migration. Although a caspase-dependent pathway has not yet been detected in the HD bowel, it is unclear whether a caspase-independent pathway contributes toward aganglionosis. In the current study, we observed highly condensed marginal heterochromatin in nuclei only in the transitional segment using electron microscopy and a high proportion of TUNEL-positive cells were observed in the transitional segment. Activation of caspase was not observed in any segments of the HD bowel upon characterization of the apoptotic pathway. Rather, real-time PCR results showed that apoptosis-inducing factor (AIF) and calpain-1 mRNAs were highly expressed in the transitional segment, whereas autophagy protein 5 (Atg5) was highly expressed in the narrow segment. Western blot results were consistent with mRNA levels, with increased AIF, calpain-1, and Atg5 expressions in the transitional segment compared with the dilated segment. Furthermore, correlation analysis indicated an inverse correlation between calpain-1 and Atg5 mRNA levels in both the narrow segment and the transitional segment. These results indicated that apoptosis occurs in the HD bowel. The detection of related genes indicates that the AIF-mediated apoptotic pathway may be responsible for the absence of ganglion cells in HD and calpain-1 may act as the regulatory switch between autophagy and apoptosis.

Isoquercetin ameliorates tunicamycin-induced apoptosis in rat dorsal root ganglion neurons via suppressing ROS-dependent endoplasmic reticulum stress.

Recent studies showed that Isoquercetin (Iso), a novel extracts of plants and fruits could protect neuronal cells from neurotoxicity and neuro-inflammation. However, its role in acute spinal cord injury (ASCI) have not been elucidated. In the present study, we investigated whether Iso could prevent Tunicamycin (TUN)-induced rat dorsal root ganglion (DRG) neurons from apoptosis and endoplasmic reticulum (ER) stress. DRG neurons were pre-treated with different doses of Iso for 24h (h) and then were stimulated with TUN (0.75μg/ml) for 24h. The cytotoxic effects and apoptosis were detected by MTT assay and TUNEL staining. The protein and mRNA expression levels were detected by Real Time PCR and Western blot, respectively. The localization of cleaved caspase-12 was evaluated by immunofluorescent staining and Western blot. The activation of caspase were measured by colorimetric assays and Western blot. Lactate Dehydrogenase (LDH) and Malondialdehyde (MDA) leakage were detected by the LDH or MDA Detection Kit PLUS. Iso protected TUN-associated DRG neurons from being damaged by cytotoxicity and apoptosis in a dose-dependent manner. Increased LDH and MDA leakage and proportion of TUNEL-positive cells, activation of caspase-3 and -9, increased Bcl-2 Assaciated X protein (Bax)/B cell lymphoma/lewkmia-2 (Bcl-2) ratio and mRNA levels of p53 Upregulated Modulator Of Apoptosis (PUMA) and DP5, and mitochondrial Cytochrome c release. Additionally, Iso down-regulated mRNA levels of ER stress genes, such as glucose-regulated protein 78 (GRP78), GRP94, C/EBP homologous protein (CHOP), and cleaved caspase-12 in TUN-induced DRG neurons. Moreover, Iso blocked the activation of three key branches of unfolded protein response in DRG neurons, including phosphorylation of pancreatic ER stress kinase (PERK), eukaryotic initiation factor 2 alpha (eIF2α), inositol-requiring enzyme 1α (IRE1α), and transcription factor 6 (ATF6). Collectively, Iso prevented TUN-induced DRG neurons apoptosis by inhibiting mitochondrial and ER stress-associated apoptotic signaling cascades, suggesting that Iso possessed neuro-protective ability which could be beneficial in the prevention of ASCI.

Bacillary replication and macrophage necrosis are determinants of neutrophil recruitment in tuberculosis

We previously determined that burst size necrosis is the chief mode of mononuclear cell death in the lungs of mice with tuberculosis. The present study explored the link between infection-induced necrosis of mononuclear phagocytes and neutrophil accumulation in the lungs of mice challenged with one of four Mycobacterium tuberculosis strains of increasing virulence (RvΔphoPR mutant, H37Ra, H37Rv and Erdman). At all time points studied, Erdman produced the highest bacterial load and the highest proportion and number of M. tuberculosis-infected neutrophils. These parameters, and the proportion of TUNEL-positive cells, tracked with virulence across all strains tested. Differences in neutrophil infection were not reflected by levels of chemoattractant cytokines in bronchoalveolar lavage fluid, while interferon-γ (reported to suppress neutrophil trafficking to the lung in tuberculosis) was highest in Erdman-infected mice. Treating Erdman-infected mice with ethambutol decreased the proportion of mononuclear phagocytes with high bacterial burden and the ratio of infected neutrophils to infected mononuclear cells in a dose-dependent manner. We propose that faster replicating M. tuberculosis strains cause more necrosis which in turn promotes neutrophil recruitment. Neutrophils infected with M. tuberculosis constitute a biomarker for poorly controlled bacterial replication, infection-induced mononuclear cell death, and increased severity of immune pathology in tuberculosis.

HIF‐1α‐induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression.

STAT1-caspase 3 pathway in the apoptotic process associated with steroid-induced necrosis of the femoral head

Osteocyte apoptosis is the main manifestation of steroid-induced avascular necrosis of the femoral head (SANFH). STAT1 and caspase 3 participate in the process of apoptosis and STAT1 upregulates the expression of caspase 3. We examined the relationship between the STAT1-caspase 3 pathway and apoptosis in SANFH. All specimens were divided into four groups: the negative control group, Ficat I-II group, Ficat III group, and Ficat IV-V group, and examined histologically, with a TUNEL assay, immunohistochemically, with a caspase 3 activity assay, with ELISAs of STAT1 and phospo-STAT1 (p-STAT1), with a western blotting analysis of p-STAT1 and with real-time RT-PCR. The proportion of empty lacunae increased significantly with the development of SANFH. The proportion of TUNEL-positive cells and immunohistochemical analysis of caspase 3 also increased significantly, although the Ficat I-II group did not differ significantly from the negative control group. Immunohistochemical analysis of STAT1 and p-STAT1, caspase 3 activity all showed significant differences among the groups. An ELISA and a western blotting analysis of p-STAT1 showed significant differences among the groups. An ELISA of STAT1, real-time RT-PCR analysis of caspase 3 and STAT1 all showed significant differences among the groups except between the Ficat I-II and negative control groups. The correlation analysis showed strong positive relationships between the proportion of empty lacunae and the proportion of TUNEL-positive cells between caspase 3 activity and the proportion of TUNEL-positive cells and between the levels of p-STAT1 protein and caspase 3 mRNA. The apoptotic process in SANFH develops with the upregulated expression of caspase 3 via the expression and activation of STAT1. The STATI-caspase 3 pathway plays a critical role in the development of SANFH.

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types-notochordal cells (NCs) and non-notochordal chondrocyte-like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI-positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, identified as NCs. The proportion of TUNEL-positive cells, which predominantly comprised non-NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up-regulation of ADAMTS-5 in the 1-day loaded group and MMP-3 in the 7-day loaded group. Aggrecan-1 and collagen type 2α-1 mRNA levels were down-regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

Abstract 106: A Novel Small Molecule Protects Against Myocardial Ischemia and Doxorubicin-Induced Cardiomyopathy Through Heat Shock Response and Proteostasis Regulation In Vivo

Background: A potentially new non-cell-based therapy is the systemic delivery of small molecules to revive or protect injured myocardium. By screening a drug library, we identified a novel small molecule, sk421, and tested its effectiveness in two myocardial injury models in vivo: a myocardial infarction model and doxorubicin (DOX)-induced cardiomyopathy model. Results: Sk421 significantly increased cellular viability of DOX-injured cardiomyocytes by preventing apoptosis in vitro, demonstrated by assays for MTT (p=0.042), annexin-V, and active caspase-3 (p=0.004). Sk421 increased the concentrations of the GATA4 and the heat shock proteins (HSPs) in cardiomyocytes (by 1.5-fold), and fibroblasts (by 1.2-fold), but was unable to rescue DOX-injured fibroblast cells depleted of HSPs. Sk421 maintained the total proteosome by a 3.5-fold inhibition of ubiquitinized GATA4 degradation, as shown by immunoprecipitation assays. Oral administration of sk421(4mg/kg) rescued DOX-injured mice by significantly preserving cardiac function, weight gain, cardiomyocyte lesion size and abundance, and by reducing overall mortality (20% in treated groups) compared to control DOX-injured mice (80% in untreated groups, p&lt;0.03). The levels of GATA4 and HSPs were increased in the sk421-rescued mice. In the MI model, (n=5/group), sk421 reduced myocardial infarct size, and protected heart function evaluated by ejection fraction 45.8 ± 2.4 vs. 34.8 ± 3.3, p=0.005 (day7), and 44.3 ± 2.8 vs. 34.4 ± 4.1, p=0.011 (day28). Sk421 treatment of MI rats resulted in a higher fraction of viable tissue (within the LV (70.20 ± 0.5024, n=3) as compared to only MI group (56.38 ± 3.879%, n=3) and the infiltration of CD68+ cells was found to be more in the infarct region of LV of MI group rats as compared to MI+CEL. Quantitative analysis showed a significantly higher proportion of TUNEL positive cells and mRNA levels of IL-1β, IL-6, TNF-α and MMP2 in the myocardium of MI rats compared to MI+SK421 rats. Conclusion: This study demonstrates the broad therapeutic benefit of sk421 in animal model of myocardial injury by mechanism of inducing the heat shock response and preserving GATA levels. The ease of oral delivery makes sk421 a potentially clinically useful adjunct for myocardial preservation.

Effects of spironolactone on atrial structural remodelling in a canine model of atrial fibrillation produced by prolonged atrial pacing

Suppression of the renin-angiotensin-aldosterone system can prevent atrial fibrillation (AF) by attenuating atrial structural remodelling but the role of aldosterone in AF prevention has not been investigated thoroughly. We explored whether the aldosterone antagonist, spironolactone, could improve atrial structural remodelling in long-term rapid pacing-induced AF. Three groups of dogs were used, sham-operated, control and spironolactone-treated groups. Dogs in the control and spironolactone groups had right atrial pacing for 6 weeks. The spironolactone group was given spironolactone 1 week before and during the atrial pacing. After 6 weeks of pacing, atrial structural and functional changes were assessed by echocardiography, haemodynamic parameters by cardiac catheterization, histopathological changes by light and electron microscopy and cardiomyocyte apoptosis by TUNEL. Caspase-3, Bcl-2, bax, calpain I, calpastatin, matrix metalloproteinase (MMP)-9 and tissue inhibitors of metalloproteinase (TIMP)-1 were analysed by immunohistochemistry and Western blotting. The inducibility and duration of AF were measured by atrial burst pacing. After atrial pacing, the proportion of TUNEL positive cells, myolysis, atrial fibrosis and dilatation were all significantly increased and these changes were inhibited by spironolactone. Spironolactone treatment reversed the increased expression of caspase-3, bax, calpain I and MMP-9 and the decreased level of Bcl-2, calpastatin and TIMP-1, induced by chronic atrial pacing. Also spironolactone prevented the increased inducibility and duration of AF, induced by tachypacing. Treatment with spironolactone prevented myocardial apoptosis, myolysis, atrial fibrosis and dilatation, suggesting a possible beneficial effect of aldosterone antagonism on atrial structural remodelling in AF.

Caffeine enhances radiosensitization to orthotopic transplant LM3 hepatocellular carcinoma in vivo

The aim of this study was to determine whether caffeine enhanced radiosensitization in an orthotopic transplant of LM3 human hepatocellular cancer in nude mice. LM3 hepatocellular carcinoma cells were infected with red fluorescent protein and irradiated, and cell cycle distribution and survival fraction were detected. A nude mouse model of orthotopic transplant of red fluorescent protein-expressing LM3 hepatocellular cancer was established. Nude mice were divided into four groups: control (NS); caffeine (Caff) alone; irradiation (IR) alone; and caffeine + IR (Caff + IR). Tumor growth curves were described. Expression of cyclin and apoptosis were evaluated by analysis of phosphorylated cyclin dependent kinase 1 (CDC2) Tyr15 (CDC2-Tyr15-P), cyclinB1, TUNEL staining, and caspase-3. Caffeine abrogated IR-induced G(2) phase arrest and decreased survival of irradiated LM3 cells. Caffeine enhanced radiosensitivity of LM3 hepatocellular cancer in vivo. Tumor growth delay time in the Caff + IR group was 14.3 days compared with the NS group, 14.1 days compared with the Caff alone group, and 7.2 days compared with the IR alone group. At 15 Gy, expression of CDC2-Tyr15-P in the Caff + IR group (26.0 +/- 8.9%) was significantly lower than in the IR alone group (68.4 +/- 10.6%), expression of cyclinB1 and proportion of TUNEL-positive cells in the Caff + IR group (30.4 +/- 8.7% and 59.2 +/- 9.5%, respectively) was significantly higher than in the IR alone group (7.0 +/- 3.7% and 24.2 +/- 7.2%, respectively), expression of caspase-3 was consistent with the TUNEL staining results. This study suggested that caffeine might enhance the radiosensitivity of LM3 hepatocellular cancer in vivo, and may be feasible for further clinical applications.

339 DIFFERENTIAL MECHANISM OF LEPTIN ACTION IMPROVING NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

Leptin is a pleiotrophic peptide that has been implicated in the events associated with oocyte maturation and acquisition of developmental competence. Previous studies indicated that leptin supplementation during oocyte maturation has long-term effects, increasing blastocyst development and reducing the proportion of TUNEL-positive cells per blastocyst. Moreover, blastocysts derived from leptin-treated oocytes showed increased BIRC4 and reduced BAX expression (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). The objective of the current study was to determine the mechanism of leptin action during bovine oocyte maturation. In the first series of experiments cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0, 1, or 10 ng/mL leptin or 10% estrous cow serum (ECS) as positive control. Leptin reduced the proportion of cumulus cells undergoing cell death through apoptosis as determined by TUNEL staining (6.8 � 0.4, 1.8 � 0.4, 1.5 � 0.4, and 6.3 � 0.5% for 0, 1, or 10 ng/mL leptin or 10% ECS, respectively; P &lt; 0.0001), but had no effect on the frequency of apoptotic oocytes. Nuclear maturation was also enhanced by leptin. The proportion of oocytes with extruded polar bodies (64.0 � 2.9, 75.7 � 2.9, 73.8 � 2.9, and 63.3 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P &lt; 0.05) and the proportion of DAPI-stained metaphase II oocytes (72.9 � 2.9, 90.5 � 2.9, 85.8 � 2.9, and 82.0 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P &lt; 0.05) were increased by 1 and 10 ng/mL leptin. There was no effect of ECS in any of the parameters examined. A second series of experiments tested whether the maturation-promoting activity of leptin was mediated by cumulus cells. Denuded oocytes (DO) and COCs were matured in serum-free medium containing 0 or 10 ng/mL leptin. The percentage of oocytes with extruded polar bodies (COCs: 59.1 � 3.7% vs. 75.7 � 3.7%, and DO: 45.5 � 3.7% vs. 54.8 � 3.7% for 0 or 10 ng/mL leptin; P &lt; 0.01) was increased by leptin regardless of cumulus cells. Even though leptin did not affect cleavage rate, it increased blastocyst development. The proportion of COCs that developed to the blastocyst stage increased from 22.3 � 4.6% in the control group to 35.2 � 4.1% in the leptin-treated group. On the other hand, DO matured without cumulus cells did not acquire developmental competency, and this was not reversed by leptin supplementation (1.0 � 5.0% vs. 1.0 � 4.7% for 0 or 10 ng/mL leptin; P &lt; 0.05; leptin � oocyte interaction P &lt; 0.05). In conclusion, leptin enhanced oocyte maturation by acting directly in the oocyte and indirectly through cumulus cells. This suggests that leptin modulates the release of cumulus-derived factors secreted in the oocyte through gap junction coupling and in the extracellular environment.