Journal of Molecular Histology最新文献

Laryngocutaneous fistula is one of the most important complications encountered after larynx surgery. Stem cell therapy is a promising treatment approach for the future, both without the need for surgical methods and by assisting surgical methods to close the fistula. 30 female Downey Sprague rats were divided into 5 separate groups and pharyngocutaneous fistula was created. Stem cells were administered subcutaneously to Group 1(SC-SC) after the creation of PCF, and via the tail vein to Group 2 (V-SC). The 3rd group (C) was made as a control group and while fistula formation and subsequent closure characteristics in the neck were monitored without any treatment, the rats in the 4th group (V-M/SH) were given tail vein medium/sham and the 5th group (SC-M/SH) was given as subcutaneous medium/sham group. PCF closure was significantly higher in the group given SC compared to the control group. The differences between the groups were examined by Hematoxilen Eosin staining with light microscopy and evaluated in terms of inflammatory cell infiltration, fibroblastic activity, collagen accumulation and neo-angiogenesis using the Ehrlich Hunt scale. After the experiments, all groups were stained with immunocytochemistry via TGF-β, VEGF and TNF-α antibodies. The statistical analysis was performed using the One-Way ANOVA test. When all these parameters were compared with the control group, a statistically significant difference was found.

This study investigated tempol action on genes and miRNAs related to NFκB pathway in androgen dependent or independent cell lines and in TRAMP model in the early and late-stages of cancer progression. A bioinformatic search was conducted to select the miRNAs to be measured based on the genes of interest from NFκB pathway. The miR-let-7c-5p, miR-26a-5p and miR-155-5p and five target genes (BCL2, BCL2L1, RELA, TNF, PTGS2) were chosen for RT-PCR and gene enrichment analyses. In vitro, PC-3 and LNCaP cells were exposed, respectively, to 1.0 or 2.0 mM of tempol during 48 h. In vivo, five experimental groups were evaluated regarding tempol effects in the early (CT12 and TPL12 groups) and late-stages (CT20, TPL20-I and TLP20-II) of PCa development. TPL groups were treated with 50 mg/kg or 100 mg/kg of tempol. The ventral lobe of the prostate and the plasma was collected. Tempol treatment increased miRs expression in PC-3 and LNCaP. For both cell lines, tempol decreased RELA expression. In TRAMP model, tempol increased miRNA expression in prostate for all treated groups. Tempol upregulated the miRNA expressions related to the NFκB pathway in the prostate tissue and human tumor cell lines. Their increase is mainly linked to increased cell death and delayed CaP aggressivenes. Thus, tempol’s capacity for miRNA-mediated gene silencing to decrease tissue proliferation and cell survival processes is part of its tissue mechanics.

Objective

This study aimed to elucidate the role of pyruvate dehydrogenase kinase-1 (PDK1) in cervical cancer (CC) by investigating its impact on cell proliferation, migration, and epithelial-mesenchymal transition (EMT) under hypoxic conditions.

Methods

PDK1-silenced CC cell lines were established using lentiviral shRNA technology. Cell migration and invasion were assessed through scratch and Transwell assays, respectively. Cellular activity and apoptosis-related protein expression levels were evaluated using MTT assays and western blotting. Transcriptome sequencing elucidates the regulatory pathways impacted by PDK1 silencing, and rescue experiments confirmed the underlying mechanisms. Xenograft models with nude mice were used to validate the effects of PDK1 silencing on CC progression.

Results

PDK1 silencing reduced migration, invasion, and cellular activity under hypoxic conditions while promoting apoptosis. Transcriptomic analysis revealed that PDK1 suppression downregulated the WNT4/β-catenin/FOXO1 pathway, decreasing EMT-related protein expression. Mechanistically, PDK1 enhanced β-catenin stability by inhibiting its phosphorylation through AKT-mediated GSK3β inactivation, promoting EMT and anti-apoptotic gene transcription.

Conclusions

Targeting PDK1 may provide novel therapeutic strategies specifically for CC by modulating the WNT4/β-catenin/FOXO1 pathway and associated EMT and apoptotic processes.

Facial nerve injuries lead to significant functional impairments and psychological distress for affected patients. Effective repair of these injuries remains a challenge. For longer nerve gaps, the regeneration outcomes after nerve grafting remain suboptimal due to limited sources and postoperative immune responses. Tissue engineering techniques are conventional methods for repairing peripheral nerve defects. This study explores the potential of dental pulp cells (DPCs) combined with stem cell factor (SCF) to enhance neurogenic differentiation and improve facial nerve regeneration. DPCs were isolated from rabbit dental pulp, the pluripotency of the cells was identified from three perspectives: osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation. In vivo experiments involved injuring the buccal branch of the facial nerve in New Zealand white rabbits, followed by treatment with PBS, DPCs, SCF, or SCF + DPCs. Functional recovery was assessed over 12 weeks, with SCF + DPCs demonstrating the most significant improvement in whisker movement scores. Histomorphological evaluations revealed enhanced myelinated fiber density and axonal morphology in the SCF + DPCs group. RNA sequencing identified 608 differentially expressed genes, with enrichment in the TGF-β signaling pathway. In in vitro experiments, we demonstrated from multiple angles using Western blot analysis, Real-time quantitative polymerase chain reaction (QPCR) analysis, and immunofluorescence staining that SCF can promote the neurogenic differentiation of DPCs through the TGF-β1 signaling pathway. Our findings indicate that the combination of SCF and DPCs offers a promising strategy for enhancing facial nerve repair.

This study tested whether combined ceftriaxone and adipose-derived mesenchymal stem cells (ADMSCs) would defend the spinal cord against acute spinal infection (ASI) in rodent. Adult-Male-SD rats were grouped into groups 1 (SC)/2 (ASI)/3 (ASI + ceftriaxone from days 2 to 28 after ASI induction)/4 (ASI + allogenic ADMSCs from day 2 for a total of 3 doses/3 consecutive intervals by intravenous injection)/5 (ASI + combined ceftriaxone and ADMSC) and spinal cord tissues were harvested by day 28. Circulatory levels of TNF-α/IL-6 at days 7 and 28, and these two parameters in spinal fluid at day 28 were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3/4, and significantly lower in group 3 than in group 4 (all p < 0.0001). The day-28 bacterial colony formation unit (CFU) in vertebral bone and circulatory WBC counts at the time points of days 7/14/28, and the protein expressions of upstream (TRL-2/TLR-4/MYD88/TRAF6/IKKα/IKKβ /IKBβ/p-NF-κB) and downstream (IL-1β/IL-6/TNF-α/IFN-γ/iNOS) inflammatory signalings displayed a similar pattern of inflammatory biomarkers in spinal fluid among the groups (all p < 0.0001). By day 28, the bone injury score/bone marrow density/ratio of bone volume (BV) to the bone tissue volume (TV)/ratio of bone surface (BS) to BV/ratio of BS to bone TV/trabecular number exhibited an opposite, whereas the trabecular space exhibited an alike pattern of inflammatory biomarkers among the groups (all p < 0.0001). Combined ceftriaxone and ADMSCs therapy offered an additional benefit on protecting the vertebral bone/spinal cord against ASI damage.

Autism spectrum disorder (ASD) is a group of severe neurodevelopmental disorders. This study aimed to elucidate the potential ameliorating effect of postnatal administration of MSCs-derived Exo in a rat model of ASD. Male pups were divided into control (Cont), (VPA); pups of pregnant rats injected with VPA subcutaneously (S.C.) at embryonic day (ED) 13, and (VPA + Exo); pups were intravenously (I.V.) injected with MSCs-derived Exo either at postnatal day (P) 21 (adolescent VPA + Exo) or P70 (adult VPA + Exo). They were evaluated for physiological, histopathological and immunohistochemical changes of cerebellar structure, and genetic expression of PI3k and mTOR. The VPA adult group showed increased locomotor activity and impaired social activity, and anxiety. The cerebellar histological structure was disrupted in VPA groups. VPA + Exo groups showed preservation of the normal histological structure of the cerebellum. Immunohistochemical studies revealed enhanced expression of caspase-3, GFAP, Nestin, and VEGF in VPA groups beside modifying PI3K and mTOR genetic expression. MSCs-derived Exo ameliorated most of the rat cerebellar histopathological alterations and behavioral changes. Their mitigating effect could be established through their antiapoptotic, anti-inflammatory and anti-neurogenesis effect besides modifying PI3k-mTOR signaling.

Celiac disease (CD) is a chronic autoimmune disease of the small bowel mucosa that develops because of the altered immune response to gluten, which leads to intestinal epithelium damage and villous atrophy. However, studies on regeneration of the damaged small bowel mucosa and density of intestinal stem cells (ISC) in CD persons are still scarce. We aimed to evaluate the number of small bowel mucosa cells positive for LGR5, CD138/Syndecan-1, CD71 and CXCR3 in CD and in controls with normal bowel mucosa; to find relationship between these markers and degree of small intestinal atrophy and to compare these results with our previous data about the number of CD103 + , IDO + DCs, FOXP3 + Tregs, enterovirus (EV) density and serum zonulin level. The paraffin sections of the small bowel biopsies were obtained from 26 children with CD (median age 6.5 years), and from 20 controls with normal intestinal mucosa (median age 14.2 years) and from the tissue bank of the Department of Pathology of Tartu University Hospital (from 18 participants with CD including 14 children (median age 13.2 years) and from 11subjects with normal small bowel mucosa, including one child aged 4.8 years. The number of LGR5 + , CD71 + , CD138 + , and CXCR3 + cells was evaluated using immunohistochemistry. The median number of CD138 + and CXCR3 + cells was significantly higher in the small bowel mucosa in CD compared with normal mucosa (p = 0.0002 for CD138 and p = 0.006 for CXCR3). The median number of CD71 + cells was significantly higher in normal small bowel mucosa (p = 0.005). The number of LGR5 + cells did not differ between persons with CD and those with normal small bowel mucosa (p = 0.7). A markedly increased number of CD138 + and CXCR3 + cells in the small bowel mucosa of participants with CD confirms their role in the pathogenesis of this disease. There was no expected marked difference in the density of any of the studied markers between lower or higher grade of small bowel atrophy and level of tTG-IgA in CD.

This study was to investigate the role of microRNA (miR)-330-5p derived from mesenchymal stem cells-secreted exosomes (MSCs-Exo) in cerebral ischemia-reperfusion injury (CI/RI) through targeting lysine N-methyltransferase SET domain containing 7 (SETD7). MSCs-Exo were separated and identified. MSCs-Exo were used to treat the middle cerebral artery occlusion (MCAO) rat model. By using the nerve injury score, Nissl, hematoxylin and eosin, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, the neural function, pathological alterations, and neuronal death in MCAO rats were examined. Using an enzyme-linked immunosorbent test, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in brain homogenate were tested. Rat brain expression levels of SETD7 and miR-330-5p were examined. Subsequently, the effects of MSCs-Exo, miR-330-5p, and SETD7 on neurological function and pathological alterations were assessed using gain and loss function tests. miR-330-5p expression was decreased and SETD7 expression was increased in the brain tissue of MCAO rats. Both MSCs-Exo and MSCs-Exo-derived miR-330-5p reduced inflammation in MCAO rats. miR-330-5p targeted SETD7, and SETD7 upregulation blocked the therapeutic effect of MSCs-Exo-derived miR-330-5p on MCAO rats. MSCs-Exo-derived miR-330-5p targets SETD7 to reduce inflammation in MCAO rats, providing a new therapeutic target for CI/RI therapy.

Traumatic brain injury (TBI) is a common neurosurgical emergency. As a macrophage in brain, microglia involves in secondary TBI injury. UCF-101, an Omi/HtrA2 inhibitor, protects against neurological disorders. This study aims to investigate the effects of UCF-101 in TBI and its mechanism. Mouse microglia cell BV2 cells were exposed to 1 µg/mL LPS to construct TBI in vitro models. Following CCK8 assay, cells were treated with LPS + UCF-101 (2, 5, 10 µM), LPS + Compound C (AMPK inhibitor, 20 µM), and LPS + UCF-101 + Compound C groups. With lactate dehydrogenase (LDH) content detection, ELISA and qRT-PCR assays were used to measure proinflammatory factors. Biomarkers of M1 (CD16/32 and iNOS) and M2 phenotypes (CD206), as well as AMPK/NF-κB pathway-related protein expression were assessed by flow cytometry, immunofluorescence, and Western blot methods. There was a decrease in M1 phenotype biomarkers and an increase in M2 phenotype biomarkers after UCF-101 treatment. UCF-101 exposure reduced TNF-α, LDH, IL-1β, IL-6, IL-8, p-NF-κB p65/NF-κB p65, and activated p-AMPK α (T172)/AMPK α (T172) expression. Importantly, further Compound C treatment counteracted these effects of UCF-101. In conclusion, UCF-101 ameliorates TBI by promoting microglia M2 polarization via AMPK/NF-κB pathways in LPS-induced BV2 cells, providing solid scientific foundation for clinical application of UCF-101 in TBI treatment.

The effect of rabies virus infection on dendritic morphology and on the expression of the MAP2 protein in Purkinje cells in the cerebellum of mice was studied. ICR mice were inoculated with rabies virus, and six days later, the mice were sacrificed, the cerebellum was removed and processed for Golgi-Cox staining or MAP2 immunohistochemistry. Infection with rabies virus altered the dendritic pattern of Purkinje cells ranged from moderate changes to accentuated retraction in the dendritic tree of some Purkinje cells. The loss of dendritic branches in the samples of mice infected with RABV was also reflected in a decrease in intersections quantified using the Sholl technique, thus suggesting dendritic pathology. Immunoreactivity to MAP2 protein in the molecular layer of the cerebellum of control mice was mainly distributed in dendrites of Purkinje cells. Some somas were faintly stained. In infected mice immunoreactivity to MAP2 was intense in somas and dendrites of Purkinje cells and in some interneurons. These results are consistent with similar findings we previously reported for the cerebral cortex and spinal cord of rabies-infected mice. But they differ from studies in other pathologies where an association between dendritic pathology and loss of MAP2 immunoreactivity has been found. Our studies in rabies contribute to suggestion that MAP2 overexpression may also be associated with alterations in dendritic morphology. MAP2 protein contributes to maintaining cytoskeleton stability. However, in rabies, increased MAP2 expression here only determined by immunohistochemistry could destabilize the cytoskeleton of dendrites. Golgi staining is considered the gold standard for the study of dendritic morphology. Its association with changes in MAP2 expression appears to provide molecular support for the concept of dendritic pathology. These results contribute to the understanding of the effect of rabies virus infection on dendritic morphology. They therefore reinforce the idea that rabies not only has a dysfunctional effect on neurons, as some authors claim, but also affects their structure.