Experimental pulmonary fibrosis was suppressed by microRNA-506 through NF-kappa-mediated apoptosis and inflammation Abstract Fibrosis in the lungs usually occurs in the initial phase of acute respiratory distress syndrome (ARDS), which exacerbates poor prognosis among patients. MicroRNAs (miRs) have the ability to modulate the expression profiles of many genes, thus essentially altering cell phenotypes. We hypothesize that miRs may be involved in the development of lung fibrosis in mice. In this study, mice were treated with lipopolysaccharide (LPS) to establish the lung fibrosis animal model. Hematoxylin and eosin (H&E) staining and western blot (WB) were performed to confirm the successful establishment of the model. Quantitative PCR (qPCR) and WB were utilized to monitor the expression of miRs and proteins. A dual-luciferase reporter assay was used to detect the interaction between miR and genes. We observed miR-506 downregulation in lung tissues during lung fibrosis after ARDS rat modeling by LPS exposure. We also observed that its expression level was similar to that observed in TGF-β1-induced human MRC-5 cells. The proportion of apoptotic cells decreased, while levels of inflammatory cytokines were upregulated in lung tissues during lung fibrosis and in fibroblasts after TGF-β1 treatment. In order to elucidate the possible role of miR-506, it was overexpressed in mice with ARDS. It was revealed that miR-506 significantly ameliorated the degree and spread of pulmonary damage stimulated by LPS. miR-506 also induced apoptosis in vivo and in vitro, while also ameliorating the inflammatory response. Notably, p65, a subunit of NF-κB, acts as a target of miR-506. p65 expression was downregulated in TGF-β1-treated MRC-5 cells upon transfection with miR-506 mimic. Indeed, the 3′-UTR of human p65 contained functional human miR-506-responsive sequences. LPS induction and TGF-β1 stimulation in mice led to p65 upregulation. In addition, p65 knockdown in the ARDS mouse model partially ameliorated the severity of lung lesions, induced apoptosis and reduced inflammation in lung tissue. Our findings revealed that miR-506 could be an important modulator of apoptosis and inflammation and a regulator of lung fibrosis.

Expression of prostaglandin (PG) D synthase lipocalin and hematopoietic type and PG D receptor during restart of spermatogenesis following downregulation using a slow release GnRH agonist implant in the dog Abstract Prostaglandin D and the associated prostaglandin D synthases (PGDS) and receptor (DP) are considered to be involved in spermatogenesis. However, the interplay of the PGDS-DP system in male reproduction is far from being understood. The expression of PGDS lipocalin (L) and hematopoietic (H) type and DP was studied in the GnRH agonist–downregulated canine testis (week, w 0) and during recrudescence of spermatogenesis after implant removal (w 3, 6, 9, 12). H-PGDS, L-PGDS and DP were present in the adult (CG), juvenile (JG) and downregulated canine testis at the mRNA level. PGDS immunohistochemistry revealed positive staining in the cytoplasm of Leydig cells (LCs) of all samples i.e., no difference between groups. mRNA expression (ratio) of L-, H-PGDS and DP did not differ between groups w 0–12 and CG. In contrast, significant differences were found for L-PGDS (p = 0.0388), H-PGDS (p < 0.001) and DP (p < 0.001) for the groups at downregulation (w0, suprelorin group, SG, profact group, PRG) compared with the control groups (JG, CG). L-PGDS expression was lowest in JG, whereas H-PGDS was significantly lower in CG compared with JG and at downregulation (p < 0.001 to p < 0.01). The highest ratio for H-PGDS and DP was observed in the dogs treated with buserelin acetate (PRG). Our data show that the PGDS-DP system is expressed in juvenile and adult canine testes and that downregulation of the testicular endocrine and germinative function significantly affects H-PGDS, L-PGDS and DP mRNA expression indicating a role in the regulation of spermatogenesis.

Synergistic effects of stromal cell-derived factor-1α and bone morphogenetic protein-2 treatment on odontogenic differentiation of human stem cells from apical papilla cultured in the VitroGel 3D system Abstract Pulp-dentin regeneration in the apical region of immature permanent teeth represents a significant clinical challenge. Tissue engineering approaches using bioactive molecules and scaffolds may have the potential to regenerate the natural apical structure of these teeth, representing a superior alternative to existing treatment regimens. The aims of this study are (i) to evaluate the VitroGel 3D system, an animal origin-free polysaccharide hydrogel, as a possible injectable scaffold for pulp-dentin regeneration and (ii) to investigate the effects of stromal cell-derived factor-1α (SDF-1α) and bone morphogenetic protein-2(BMP-2) cotreatment on odontogenic differentiation of human stem cells from apical papilla (SCAP) cultured in the VitroGel 3D system. The morphology, viability and proliferation of SCAP cultured in the VitroGel 3D system were measured via scanning electron microscopy (SEM), live and dead cell staining and CCK-8 assays. Alkaline phosphatase (ALP) activity, real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and Western blot analysis were further used to evaluate the odontogenic differentiation of SCAP cultured in the VitroGel 3D system in vitro. Finally, the odontogenic differentiation was assessed in vivo through ectopic subcutaneous injection. The results showed that SCAP cultured in 3D hydrogel demonstrated favorable viability and proliferation. SDF-1α and BMP-2 cotreatment enhanced odontogenic differentiation-related gene and protein expression in vitro and promoted odontogenic differentiation of SCAP in vivo. In conclusion, the present study demonstrated that the VitroGel 3D system promoted SCAP proliferation and differentiation. Moreover, SDF-1α cotreatment had synergistic effects on BMP-2-induced odontogenic differentiation of human SCAP cultured in the VitroGel 3D system both in vitro and in vivo.

Protective role of exogenous recombinant peroxiredoxin 6 under ischemia-reperfusion injury of kidney Abstract Peroxiredoxin 6 (Prx6) is an important antioxidant enzyme with various functions in the cell. Prx6 reduces a wide range of peroxide substrates, playing a leading role in maintaining the redox homeostasis of mammalian cells. In addition to the peroxidase activity, a phospholipase A2-like activity was demonstrated for Prx6, which plays an important role in the metabolism of membrane phospholipids. Besides that, due to its peroxidase and phospholipase activities, Prx6 participates in intracellular and intercellular signal transduction, thus triggering regenerative processes in the cell, suppressing apoptosis caused by various factors, including ischemia-reperfusion injuries. A nephroprotective effect of exogenous recombinant Prx6 administered before ischemia-reperfusion injury was demonstrated on an animal model. Exogenous Prx6 effectively alleviates the severeness of renal ischemia-reperfusion injuries and facilitates normalization of their structural and functional conditions. Infusion of exogenous Prx6 increases the survival rate of experimental animals by almost 3 times. Application of exogenous Prx6 can be an effective approach in the prevention and treatment of renal ischemia-reperfusion kidney lesions and in preserving isolated kidneys during transplantation.

Immunofluorescent characterization of innervation and nerve-immune cell neighborhood in mouse thymus Abstract The central nervous system impacts the immune system mainly by regulating the systemic concentration of humoral substances, whereas the peripheral nervous system (PNS) communicates with the immune system specifically according to local “hardwiring” of sympathetic/parasympathetic (efferent) and sensory (afferent) nerves to the primary and secondary lymphoid tissue/organs (e.g., thymus spleen and lymph nodes). In the present study, we use immunofluorescent staining of neurofilament-heavy to reveal the distribution of nerve fibers and the nerve-immune cell neighborhood inside the mouse thymus. Our results demonstrate (a) the presence of an extensive meshwork of nerve fibers in all thymic compartments, including the capsule, subcapsular region, cortex, cortico-medullary junction and medulla; (b) close associations of nerve fibers with blood vessels (including the postcapillary venules), indicating the neural control of blood circulation and immune cell dynamics inside the thymus; (c) the close proximity of nerve fibers to various subsets of thymocytes (e.g., CD4+, CD8+ and CD4+CD8+), dendritic cells (e.g., B220+, CD4+, CD8+ and F4/80+), macrophages (Mac1+ and F4/80+) and B cells. Our novel findings concerning thymic innervation and the nerve-immune cell neighborhood in situ should facilitate the understanding of bi-directional communications between the PNS and primary lymphoid organs. Since the innervation of lymphoid organs, including the thymus, may play essential roles in the pathogenesis and progression of some neuroimmune, infectious and autoimmune diseases, better knowledge of PNS-immune system crosstalk should benefit the development of potential therapies for these diseases.

Nerve/glial antigen 2 is crucially involved in the revascularization of freely transplanted pancreatic islets Abstract Pancreatic islets are highly vascularized endocrine units. Accordingly, their adequate revascularization is of major importance for successful islet transplantation. The proteoglycan, nerve/glial antigen 2 (NG2) expressed in pericytes is a crucial regulator of angiogenesis. Therefore, we herein analyze whether this surface protein contributes to the revascularization of grafted islets. Islets were isolated from NG2+/+ (wild-type) and NG2−/− mice and their cellular composition was analyzed by immunohistochemical detection of insulin, glucagon, somatostatin and CD31. Moreover, insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). In addition, isolated islets were transplanted into dorsal skinfold chambers of wild-type mice and their revascularization was determined by intravital fluorescence microscopy and immunohistochemistry. NG2+/+ and NG2−/− islets did not differ in their cellular composition and insulin secretion. However, transplanted NG2−/− islets exhibited a significantly lower functional capillary density and a reduced number of CD31-positive microvessels. These findings demonstrate that the loss of NG2 impairs the revascularization of transplanted islets, underlining the importance of this pericytic proteoglycan for islet engraftment.

Placental cell death patterns exhibit differences throughout gestation in two strains of laboratory mice Abstract Cell death is an essential physiological process required for the proper development and function of the human placenta. Although the mouse is a commonly used animal model for development studies, little is known about the extent and distribution of cell death in the mouse placenta throughout development and its physiological relevance. In the present study, we report the results of a systematic and quantitative assessment of cell death patterns in the placentae of two strains of laboratory mice commonly used for developmental studies—ICR and C57Bl/6. TUNEL staining revealed that ICR and C57Bl/6 placentae exhibited similar cell death patterns to those reported in human placentae during pregnancy, with comparatively infrequent death observed during early gestation, which increased and became more organized towards term. Interestingly, when comparing strain differences, increased cell death was observed in almost all regions of the inbred C57Bl/6 placentae compared to the outbred ICR strain. Finally, since Bcl-2 ovarian killer (Bok) has been reported to be a key player in human placental cell death, we examined its expression in murine placentae throughout gestation. Bok protein expression was observed in all placental regions and increased towards term in both strains. The results of this study indicate that although strain-specific differences in placental cell death exist, the overall rates and patterns of cell death during murine placentation parallel those previously described in humans. Thus, the murine placenta is a useful model to investigate molecular pathways involved in cell death signaling during human placentation.

Immunolocalization of calcium sensing and transport proteins in the murine endolymphatic sac indicates calciostatic functions within the inner ear Abstract An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere’s disease.

The role of oxidative stress in ovarian toxicity induced by haloperidol and clozapine—a histological and biochemical study in albino rats Abstract Oxidative stress has been implicated in reproductive toxicity induced by antipsychotics (APs). This study aims to further investigate the role of AP-induced oxidative stress in reproductive dysfunction. Thirty adult female albino rats were divided into three groups including a control group (n = 10) receiving distilled water, HAL group (n = 10) receiving haloperidol (HAL) (2 mg/kg/day), and CLZ group (n = 10) receiving clozapine (CLZ) (20 mg/kg/day). After 28 days, the rats were anesthetized, blood was withdrawn from their hearts, and ovaries were removed before they were sacrificed. Serum prolactin concentrations were measured. For each rat, one ovary was used for biochemical studies including mitochondrial complexes I and III activities and oxidative stress markers (lipid peroxidation, super oxide dismutase [SOD], catalase [CAT], and reduced glutathione [GSH]). The other ovary was used for histopathological examination and immunohistochemistry staining for p53 and Ki-67. HAL-treated rats showed significantly (p < 0.001) higher serum prolactin concentrations compared with other groups. HAL significantly inhibited complexes I (p < 0.001) and III activities (p < 0.05), while CLZ inhibited only complex I (p < 0.001). Lipid peroxidation was increased by HAL (p < 0.001) and CLZ (p < 0.01). HAL caused significant (p < 0.001) reductions in SOD, CAT, and GSH. CLZ caused a significant decrease in SOD (p < 0.001) and GSH (p < 0.01) with no effect on CAT. Histopathological studies of CLZ- and HAL-treated ovaries showed features suggestive of hyperprolactinemia and oxidative stress. Ki-67- and P53-immunostained sections were suggestive of disruption of cellular proliferation. These findings support the hypothesis that HAL and CLZ induce reproductive dysfunction through mechanisms involving ovarian mitochondrial dysfunction and oxidative stress.

Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes Abstract In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish β-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.