Morphological and Transcriptomic Analysis of the Supplemental Boron in the Liver of Ostrich Chicks.

African ostrich chicks (Struthio camelus) were divided into six groups, and each received different levels of boric acid (source of boron) in the drinking water (0, 40, 80, 160, 320, and 640 mg/L respectively) to examine the histological, apoptotic, biochemical, and transcriptomic parameters. Morphological analysis in different groups was assessed by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and terminal deoxynucleotide transferase dUTP Nick-End Labeling (TUNEL) assay. The biochemical profile was evaluated spectrophotometrically. Detailed RNA-Seq of the data was performed using the transcriptomic method. H&E staining showed well-developed liver structure up to the 160 mg/L boric acid (BA) supplement groups, while BA doses (320 mg/L and 640 mg/L) caused changes in hepatocytes and portal triads. PAS staining showed that glycogen levels were optimal in the 80 mg/L BA dose group, but a reduction in glycogen levels was observed after this group, particularly in the 640 mg/L BA supplement group. Cellular apoptosis showed a biphasic pattern, and the BA dose above 160 mg/L enhanced cell death. In addition, serum analysis showed that doses of 80-160 mg BA were beneficial for ostrich liver. Then, the transcriptome analysis of the 80 mg dose also showed mainly positive effects on the liver. These results demonstrated that chronic BA exposure (320-640 mg) can cause significant histological, apoptotic, and biochemical changes in African ostrich liver, while the adequate dose of supplementation (particularly 80 mg BA) promotes liver growth.

Boron Supplementation Promotes Osteogenesis of Tibia by Regulating the Bone Morphogenetic Protein-2 Expression in African Ostrich Chicks.

The present study aimed to explore the effects of supplemental boron on osteogenesis of tibia and to investigate the possible relationship between additional boron and the expression of bone morphogenetic protein-2 (BMP-2) in tibia of ostrich chicks. Therefore, forty-eight African ostrich chicks (15 days old) were supplemented with 0 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, and 640 mg/L of boron in drinking water for 75 days. The paraffin sections of tibia used to measure histomorphometric parameters by hematoxylin and eosin (HE) staining, Masson's staining, and immunohistochemistry (IHC). Enzyme-linked immunosorbent assay was performed to assess the level of BMP-2, osteocalcin (BGP), glucocorticoids (GCs), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) in serum. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique was performed to detect the cell apoptosis. The results indicated that low dose of supplemental boron (40 mg/L-160 mg/L) in drinking water promotes bone development by increasing the mature ossein. The expression of BMP2 on 45 days was higher than 90 days. Serum level of BMP-2, BGP, and GCs changed significantly in groups with low dosage of boron, and OPG/RANKL ratio was upregulated from 0 to 160 mg/L. Cell apoptosis was least in 40 mg/L and 160 mg/L groups. Taken together, low dose of boron supplemented in drinking water could promote osteogenesis and growth and development of tibia by regulating the expression and secretion of BMP-2 and providing a dynamically balanced environment for tibia growth, development, and reconstruction by regulating the concentrations of BGP, GCs, and OPG/RANKL ratio in serum.

Drug Discovery and Development of Novel Therapeutics for Inhibiting TMAO in Models of Atherosclerosis and Diabetes.

Diabetes mellitus exists as a comorbidity with congestive heart failure (CHF). However, the exact molecular signaling mechanism linking CHF as the major form of mortality from diabetes remains unknown. Type 2 diabetic patients display abnormally high levels of metabolic products associated with gut dysbiosis. One such metabolite, trimethylamine N-oxide (TMAO), has been observed to be directly related with increased incidence of cardiovascular diseases (CVD) in human patients. TMAO a gut-liver metabolite, comes from the metabolic degenerative product trimethylamine (TMA) that is produced from gut microbial metabolism. Elevated levels of TMAO in diabetics and obese patients are observed to have a direct correlation with increased risk for major adverse cardiovascular events. The pro-atherogenic effect of TMAO is attributed to enhancing inflammatory pathways with cholesterol and bile acid dysregulation, promoting foam cell formation. Recent studies have revealed several potential therapeutic strategies for reducing TMAO levels and will be the central focus for the current review. However, few have focused on developing rational drug therapeutics and may be due to the gaps in knowledge for understanding the mechanism by which microbial TMA producing enzymes and hepatic flavin-containing monoxygenase (FMO) can work together in preventing elevation of TMAO levels. Therefore, it is critical to understand the advantages of developing a novel rational drug design strategy that manipulates FMO production of TMAO and TMA production by microbial enzymes. This review will focus on the inspection of FMO manipulation, as well as gut microbiota dysbiosis and its influence on metabolic disorders including cardiovascular disease and describe novel potential pharmacological therapeutic development.

The Cardioprotective Mechanism of Phenylaminoethyl Selenides (PAESe) Against Doxorubicin-Induced Cardiotoxicity Involves Frataxin.

Doxorubicin (DOX) is an anthracycline cancer chemotherapeutic that exhibits cumulative dose-limiting cardiotoxicity and limits its clinical utility. DOX treatment results in the development of morbid cardiac hypertrophy that progresses to congestive heart failure and death. Recent evidence suggests that during the development of DOX mediated cardiac hypertrophy, mitochondrial energetics are severely compromised, thus priming the cardiomyocyte for failure. To mitigate cumulative dose (5 mg/kg, QIW x 4 weeks with 2 weeks recovery) dependent DOX, mediated cardiac hypertrophy, we applied an orally active selenium based compound termed phenylaminoethyl selenides (PAESe) (QIW 10 mg/kg x 5) to our animal model and observed that PAESe attenuates DOX-mediated cardiac hypertrophy in athymic mice, as observed by MRI analysis. Mechanistically, we demonstrated that DOX impedes the stability of the iron-sulfur cluster biogenesis protein Frataxin (FXN) (0.5 fold), resulting in enhanced mitochondrial free iron accumulation (2.5 fold) and reduced aconitase activity (0.4 fold). Our findings further indicate that PAESe prevented the reduction of FXN levels and the ensuing elevation of mitochondrial free iron levels. PAESe has been shown to have anti-oxidative properties in part, by regeneration of glutathione levels. Therefore, we observed that PAESe can mitigate DOX mediated cardiac hypertrophy by enhancing glutathione activity (0.4 fold) and inhibiting ROS formation (1.8 fold). Lastly, we observed that DOX significantly reduced cellular respiration (basal (5%) and uncoupled (10%)) in H9C2 cardiomyoblasts and that PAESe protects against the DOX-mediated attenuation of cellular respiration. In conclusion, the current study determined the protective mechanism of PAESe against DOX mediated myocardial damage and that FXN is implicitly involved in DOX-mediated cardiotoxicity.

RNA-Seq-Based Gene Expression Pattern and Morphological Alterations in Chick Thymus during Postnatal Development.

The thymus is a lobulated unique lymphoid immune organ that plays a critical role in the selection, development, proliferation, and differentiation of T cells. The thymus of developing chickens undergoes continued morphological alterations; however, the biomolecular and transcriptional dynamics of the postnatal thymus in avian species is not clear yet. Therefore, the thymuses from chickens at different stages of development (at weeks 0, 1, 5, 9, 18, and 27) were used in the present study. The RNA-seq method was used to study the gene expression patterns. On average, 24120819 clean reads were mapped, differentially expressed genes (DEGs) were identified on the basis of log values (fold change), including 744 upregulated and 425 downregulated genes. The expression pattern revealed by RNA-seq was validated by quantitative real-time PCR (qPCR) analysis of four important genes, which are PCNA, CCNA2, CCNB2, and CDK1. Thus, the current study revealed that during postnatal development, the thymus undergoes severe atrophy. Thymus structure was damaged and gene expression changed dramatically, especially at the 27th week of age. Moreover, we found significant changes of several signaling pathways such as the cytokine-cytokine receptor interaction and cell cycle signaling pathways. Hence, it may be inferred that those signaling pathways might be closely related to the postnatal chicken thymus development.

Transcriptional Study Revealed That Boron Supplementation May Alter the Immune-Related Genes Through MAPK Signaling in Ostrich Chick Thymus.

The objective of this study is to construct a digital gene expression tag profile to identify genes potentially related to immune response in the ostrich. Exposure to boron leads to an immune response in the ostrich, although the underlying mechanism remains obscure. Thus, a dire need of biological resource in the form of transcriptomic data for ostriches arises to key out genes and to gain insights into the function of boron on the immune response of thymus. For this purpose, RNA-Seq analysis was performed using the Illumina technique to investigate differentially expressed genes in ostrich thymuses treated with different boric acid concentrations (0, 80, and 640 mg/L). Compared with the control group, we identified 309 upregulated and 593 downregulated genes in the 80 mg/L treated sample and 228 upregulated and 1816 downregulated genes in 640 mg/L treated sample, respectively. Trend analysis of these differentially expressed genes uncovers three statistically significant trends. Functional annotation analysis of the differentially expressed genes verifies multiple functions associated with immune response. When ostrich thymuses were treated with boron, expression changes were observed in genes predominantly associated with MAPK and calcium signaling pathways. The results of this study provide all-inclusive information on gene expression at the transcriptional level that further enhances our apprehension for the molecular mechanisms of boron on the ostrich immune system. The calcium and MAPK signaling pathways might play a pivotal role in regulating the immune response of boron-treated ostriches.

Effects of Boron Supplementation on Expression of Hsp70 in the Spleen of African Ostrich.

Increased synthesis of heat shock protein 70 (Hsp70) occurs in prokaryotes and eukaryotes in response to physiological, environmental, and chemical exposures, thus allowing the cell survival from fatal conditions. Hsp70 cytoprotective properties may be clarified by its anti-apoptotic function. Boron has been reported to play an essential role in various organ developments and metabolisms. However, it is not known if boron is also able to modulate the Hsp70. In the present study, the actions of boron on ostrich spleen and expression level of Hsp70 were investigated. Thirty healthy ostrich chicks were randomly assigned to six groups: groups I, II, III, IV, V, and VI and fed the basal diet spiked with 0-, 40-, 80-, 160-, 320-, and 640-mg boric acid (BA)/L, respectively, in drinking water. The histomorphological examination in the spleen was done by hematoxylin and eosin (HE) staining. The expression level of Hsp70 was analyzed by immunohistochemistry (IHC) and western blotting, and mRNA expression of Hsp70 was investigated by quantitative real-time PCR (qPCR). In order to investigate apoptosis, TUNEL assay reaction in all treatment groups was analyzed. Our results showed that the histological structure of spleen up to 160 mg/L BA supplementation groups well developed. The Hsp70 expression level first induced at low-dose groups (up to group IV) and then inhibited dramatically in high-dose groups (V and VI) while comparing with the group I (0 mg BA). The TUNEL assay reaction revealed that the cell apoptosis amount was decreased in group IV, but in group V and especially in group VI, it was significantly increased (P < 0.01). Taken altogether, proper dietary boron treatment might stimulate ostrich chick spleen development by promoting the Hsp70 expression level and inhibiting apoptosis, while a high amount of boron supplementation would impair the ostrich spleen structure by inhibiting Hsp70 expression level and promoting cell apoptosis.

Electroacupuncture Attenuates Visceral Hypersensitivity by Inhibiting JAK2/STAT3 Signaling Pathway in the Descending Pain Modulation System.

Electroacupuncture (EA) has been used for treating visceral hypersensitivity (VH). However, the underlying molecular mechanism remains unclear. This study was aim to testify the effect of EA on ileitis-provoked VH, and to confirm whether EA attenuates VH through Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathway in the periaqueductal gray (PAG)-the rostral ventromedial medulla (RVM)-the spinal cord dorsal horn (SCDH) axis. Methods: Goats were anesthetized and laparotomized for injecting 2,4,6-trinitro-benzene-sulfonic acid (TNBS)-ethanol solution (30mg TNBS dissolved in 40% ethanol) into the ileal wall to induce VH. EA was treated for 30min from day 7, then every 3 days for six times. VH was assessed by visceromotor response (VMR) and pain behavior response to 20, 40, 60, 80, and 100 mmHg colorectal distension pressures at day 7, 10, 13, 16, 19, and 22. The spinal cord in the eleventh thoracic vertebra and the brain were collected at day 22. The protein and mRNA levels of IL-6, JAK2, and STAT3 in the SCDH were detected with western blot and qPCR, respectively. The distribution of these substances was observed with immunohistochemistry in the ventrolateral PAG (vlPAG), RVM (mainly the nucleus raphe magnus, NRM), SCDH, the nucleus tractus solitaries (NTS) and the dorsal motor nucleus of vagi (DMV). Results: Goats administered with TNBS-ethanol solution showed diarrhea, enhanced VMR and pain behavior response, and increased IL-6, phosphorylated JAK2 and STAT3 (pJAK2 and pSTAT3) in the vlPAG, NRM, NTS and DMV, and their protein and mRNA levels in the SCDH. EA relieved diarrhea, VMR and pain behavior response, decreased IL-6, pJAK2 and pSTAT3 levels in the vlPAG, NRM, SCDH, NTS, and DMV except for pSTAT3 in the DMV, but did not affect mRNA level of these three substances in the SCDH. Conclusion: EA attenuates VH probably through inhibiting JAK2/STAT3 signaling pathway in the PAG-RVM-SCDH axis.

Toll like receptor 4 signaling pathway participated in Salmonella lipopolysaccharide-induced spleen injury in young chicks.

Spleen is one of the crucial sites for cellular and humoral immunity but it easily damaged during pathogenic infections resulting in immunosuppression. The current study was therefore performed to explore the mechanism of acute spleen injury induced by salmonella lipopolysaccharide (LPS) in young chicks. Healthy one-day-old Cobb strain broiler chicks were intra-peritoneally injected with saline or LPS. LPS treatment caused significant decreases in body and spleen weights at 36 and 72 h. Histological analysis showed the changes of ellipsoid structures with beginning of nuclear pyknosis and karyolysis similar to steatosis at 12 h, maximum histopathological lesions were seen at 36 h, however these were disappeared at 72 h post LPS stimulation. Cell proliferation was decreased (low PCNA positivity) and apoptosis increased (high ssDNA positivity) in the spleen at 12 and 36 h after LPS treatment. The expression levels of mRNA for caspase-3, caspase-8, B-cell lymphoma 2 (BCL-2), tumor protein p53 or p53 and Bcl-2 homologous antagonist killer (BAK) showed slight increase at some time points following LPS stimulation. LPS treatment also induced significant up-regulation in toll like receptor 4 (TLR4) at 36 h post LPS stimulation and slight increase in expressions of its downstream molecules (MyD88 and NF-κB) at 12 h post LPS treatment. The keystone cytokines (TNF-α and IL-6) exhibited significant up-regulation at 12 h following LPS stimulation. Our findings provided novel information about the histopathological as well as apoptotic and proliferative alterations in spleen mediated by TLR4 signaling induced by Salmonella LPS in avian species.

Lipopolysaccharide mediates immuno-pathological alterations in young chicken liver through TLR4 signaling.

BACKGROUND: Lipopolysaccharide (LPS) induces acute liver injury and the complex mechanisms include the activation of toll like receptor 4 (TLR4) signaling pathway in many species. However, immuno-pathological changes during TLR4 signaling under LPS stress in acute liver injury is poorly understood in avian species. The present investigation was therefore carried out to evaluate these alterations in TLR4 signaling pathway during acute liver injury in young chickens. RESULTS: After intraperitoneal injection of LPS or saline, liver samples were harvested at 0, 2, 6, 12, 24, 36, 72 and 120 h (n = 6 at each time point) and the microstructures were analyzed by hematoxylin and eosin (H&E) staining. Alanine aminotransferase (ALT) and caspase-3 enzyme activity was assessed by enzyme-linked immunosorbent assay (ELISA). Proliferative cell nuclear antigen (PCNA), single stranded DNA (ssDNA) and TLR4 protein expressions were determined by immunohistochemistry. Gene expressions of PCNA, caspase-3, caspase-8, TLR4 and its downstream molecules were analyzed by quantitative polymerase chain reaction (qPCR). LPS injection induced significantly higher ALT activity, severe fatty degeneration, necrotic symptoms, ballooning degeneration, congestion, enhanced inflammatory cell infiltration in liver sinusoids, decreased proliferation, increased apoptosis and significant up-regulation in TLR4 and its downstream molecules (MyD88, NF-κB, TNF-α, IL-1ß and TGF-ß) expression at different time points. CONCLUSIONS: This study indicated that TLR4 signaling and its downstream molecules along with certain cytokines play a key role in acute liver injury in young chickens. Hence, our findings provided novel information about the histopathological, proliferative and apoptotic alterations along with changes in ALT and caspase-3 activities associated with acute liver injury induced by Salmonella LPS in avian species.

Lipopolysaccharide induces acute bursal atrophy in broiler chicks by activating TLR4-MAPK-NF-κB/AP-1 signaling.

We investigated the mechanisms that induce atrophy of the chicken bursa of Fabricius (BF) upon lipopolysaccharide (LPS) treatment in young chicks. LPS treatment resulted in ∼36% decrease in bursal weight within 36 h (P < 0.01). Histological analysis showed infiltration of eosinophilic heterophils and nucleated oval shaped RBCs in or near blood vessels of the BF from LPS-treated chicks. Scanning electron micrographs showed severe erosion and breaks in the mucosal membrane at 12 h and complete exuviation of bursal mucosal epithelial cells at 36 h. We observed decreased cell proliferation (low PCNA positivity) and increased apoptosis (high TUNEL and ssDNA positivity) in the BF 12-72 h after LPS treatment. RNA-seq analysis of the BF transcriptome showed 736 differentially expressed genes with most expression changes (637/736) 12 h after LPS treatment. KEGG pathway analysis identified TLR4-MAPK-NF-κB/AP-1 as the key signaling pathway affected in response to LPS stimulation. These findings indicate LPS activates the TLR4-MAPK-NF-κB/AP-1 signaling pathway that mediates acute atrophy of the chicken bursa of Fabricius by inducing inflammation and apoptosis.

Electroacupuncture Inhibits the Activation of p38MAPK in the Central Descending Facilitatory Pathway in Rats with Inflammatory Pain.

The mitogen-activated protein kinases (MAPKs), especially p38MAPK, play a pivotal role in chronic pain. Electroacupuncture (EA) relieves inflammatory pain underlying the descending pathway, that is, the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal cord dorsal horn (SCDH). However, whether EA antagonizes inflammatory pain through regulation of p38MAPK in this descending facilitatory pathway is unclear. Complete Freund's adjuvant (CFA) was injected into the hind paw of rats to establish inflammatory pain model. EA was administrated for 30 min at Zusanli and Kunlun acupoints at 0.5, 24.5, 48.5, and 72.5 h, respectively. The paw withdrawal threshold (PWT), paw edema, and Phosphor-p38MAPK-Immunoreactivity (p-p38MAPK-IR) cells were measured before (0 h) and at 1, 3, 5, 7, 25, and 73 h after CFA or saline injection. EA increased PWT at 1, 3, 25, and 73 h and inhibited paw edema at 25 and 73 h after CFA injection. Moreover, the increasing number of p-p38MAPK-IR cells which was induced by CFA was suppressed by EA stimulation in PAG and RVM at 3 and 5 h and in SCDH at 5, 7, 25, and 73 h. These results suggest that EA suppresses inflammation-induced hyperalgesia probably through inhibiting p38MAPK activation in the descending facilitatory pathway.

Exploring the Mechanisms of Electroacupuncture-Induced Analgesia through RNA Sequencing of the Periaqueductal Gray.

Electroacupuncture (EA) can relieve various pains. However, its mechanism in terms of the transcriptome is still not well-known. To explore the full profile of EA-induced molecular modification in the central nerve system, three twins of goats were selected for a match-paired experiment: EA stimulation (60 Hz, 30 min) and none-EA (control). Goats in the EA group showed an increased (p < 0.05) nociceptive threshold compared with the control goats. Experimental goats were sacrificed at 4 h of the experiment, and the periaqueductal grays were harvested for RNA sequencing. As a result, 2651 differentially expressed genes (1803 up-regulated and 848 down-regulated genes) were found and enriched in 30 Kyoto Encyclopedia of Genes and Genomes pathways and 149 gene ontology terms. EA-regulated five neuropeptide genes (proenkephalin, proopiomelanocortin, preprodynorphin, diazepam-binding inhibitor and proprotein convertase 1 inhibitor) were validated with quantitative PCR. Furthermore, up-regulated glutamate receptors, glutamate transporters, γ-aminobutyric acid (GABA) receptors, GABA transporters, synaptotagmins or mitogen-activated protein kinase (MAPK) genes might contribute to EA-induced analgesia through regulating the glutamatergic synapse, GABAergic synapse, MAPKs, ribosome or ubiquitin-proteasome pathways. Our findings reveal a full profile of molecular modification in response to EA and provide a solid experimental framework for exploring the mechanisms underlying EA-induced analgesia.

Transcriptome analysis indicated that Salmonella lipopolysaccharide-induced thymocyte death and thymic atrophy were related to TLR4-FOS/JUN pathway in chicks.

BACKGROUND: Thymus is the crucial site for T cell development and once believed to be immune privileged. Recently, thymus has gained special attention as it is commonly targeted by infectious agents which may cause pathogenic tolerance and subsequent immunosuppression. RESULTS: We analyzed thymic responses to the challenge with Salmonella typhimurium (STm) or lipopolysaccharide (LPS) derived from STm in chicks. Newly hatched chicks were injected intraperitoneally with 5 × 10(4) CFU/mL STm or 50 mg/kg LPS. After LPS treatment, maximum thymocyte death (3 ~ 5-fold change) compared to controls was found at 12 h, and maximum loss of thymic weight (35 %) and reduced thymic index (20 %) were found at 36 h. After STm infection, maximum thymocyte death and thymic atrophy occurred at 36 and 72 h, respectively. No significant changes of thymic structure, chT1+ and CD4+/CD8+ T cell ratio were observed in thymus or spleen tissues after LPS treatment. Furthermore, transcriptome analysis revealed important roles for the TLR4-FOS/JUN signaling pathway in thymic injury. Thus, the major process of thymic atrophy in this study first involved activation of transcriptional factors FOS/JUN upon LPS binding to TLR4 that caused release of inflammatory factors, thereby inducing inflammatory responses and DNA damage and ultimately cell cycle arrest and thymic injury. CONCLUSIONS: STm and Salmonella LPS could induce acute chick thymic injury. LPS treatment acted faster than STm. TLR4-FOS/JUN pathway may play an important role in LPS induced chick thymic injury.

Effects of lipopolysaccharide on the histomorphology and expression of toll-like receptor 4 in the chicken trachea and lung.

Endotoxin or lipopolysaccharide (LPS) exposure can cause injury to the respiratory airways and in response, the respiratory epithelia express toll-like receptors (TLRs) in many species. However, its role in the innate immunity in the avian respiratory system is poorly understood. The aim of the present study was to evaluate the effects of LPS on the chicken trachea and lung. After intraperitoneal LPS or saline injection, the trachea and lungs were harvested at 0, 12, 36 and 72 h (n = 6 at each time point) and histopathologically analysed using haematoxylin and eosin and periodic acid-Schiff staining, while TLR4 expression was determined by immunohistochemistry and secretory Immunoglobulin A (SIgA) levels by enzyme-linked immunosorbent assay. After LPS stimulation, we observed a remarkable decrease in the number of goblet cells along with obvious disruption and desquamation of the ciliated epithelium in the trachea, blurring of the boundary between pulmonary lobules, narrowed or indistinguishable lumen of the pulmonary atria and leukostasis in the lungs. Following LPS stimulation, TLR4 protein expression was up-regulated in both the trachea and the lungs and was found on the ciliated columnar cells as well as in the submucosa of the trachea, and in the lungs on parenchymal and immune cells. However, SIgA levels were only up-regulated in the trachea at 12 h following LPS stimulation. Hence, this report provides novel information about the effects of LPS on the microstructure of the lower respiratory tract and it is concluded that its intra-peritoneal administration leads to TLR4-mediated destruction of the tracheal epithelium and pulmonary inflammation along with increased SIgA expression in the tracheal mucosa.

Adiponectin downregulation is associated with volume overload-induced myocyte dysfunction in rats.

AIM: Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats. METHODS: Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca(2+) transient. RESULTS: A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca(2+) transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 µg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats. CONCLUSION: These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure.

Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased ßMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors, including S-nitrosocysteine, have advantages over other bioactive molecules for use in scalable culture systems in driving cardiac differentiation, since they are inexpensive and the diffusivity of nitric oxide is relatively high. By enabling maintenance of spontaneous contraction in suspension culture and progressing electrophysiological function of resulting SC-CMs toward a more mature phenotype, long-term application of S-nitrosocysteine was shown to be beneficial during cardiac differentiation. Taken together, these results demonstrate the efficiency of nitric oxide as a signaling compound, with implications in the improvement of pluripotent stem cell-derived cardiomyocyte maturation in large-scale culture systems.

Effect of Boric Acid Supplementation on the Expression of BDNF in African Ostrich Chick Brain.

The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks' brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of ostrich chick brain by inhibiting the BDNF expression and increasing cell apoptosis. Taken together, the 160 mg/L boric acid supplementation may be the optimal dose for the brain development of ostrich chicks.

Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich.

B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds.

The role of frataxin in doxorubicin-mediated cardiac hypertrophy.

Doxorubicin (DOX) is a highly effective anti-neoplastic agent; however, its cumulative dosing schedules are clinically limited by the development of cardiotoxicity. Previous studies have attributed the cause of DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the ensuing reactive oxygen species (ROS) formation. The present study investigates the role of frataxin (FXN), a mitochondrial iron-sulfur biogenesis protein, and its role in development of DOX-mediated mitochondrial dysfunction. Athymic mice treated with DOX (5 mg/kg, 1 dose/wk with treatments, followed by 2-wk recovery) displayed left ventricular hypertrophy, as observed by impaired cardiac hemodynamic performance parameters. Furthermore, we also observed significant reduction in FXN expression in DOX-treated animals and H9C2 cardiomyoblast cell lines, resulting in increased mitochondrial iron accumulation and the ensuing ROS formation. This observation was paralleled in DOX-treated H9C2 cells by a significant reduction in the mitochondrial bioenergetics, as observed by the reduction of myocardial energy regulation. Surprisingly, similar results were observed in our FXN knockdown stable cell lines constructed by lentiviral technology using short hairpin RNA. To better understand the cardioprotective role of FXN against DOX, we constructed FXN overexpressing cardiomyoblasts, which displayed cardioprotection against mitochondrial iron accumulation, ROS formation, and reduction of mitochondrial bioenergetics. Lastly, our FXN overexpressing cardiomyoblasts were protected from DOX-mediated cardiac hypertrophy. Together, our findings reveal novel insights into the development of DOX-mediated cardiomyopathy.