Folia histochemica et cytobiologica最新文献

Introduction: Astrocytes react to microenvironmental changes. Their reactivity is manifested by an increase in glial fibrillary acidic protein (GFAP) and S100b protein levels, hypertrophy and hyperplasia. The aim of the study was to analyse immunoreactive GFAP (GFAP-IR) and S100b (S100b-IR) astrocytes of hippocampal CA1 area in adult rats intragastrically (i.g.) treated with habanero peppers.

Material and methods: Brains from 10 control rats (group C) and 10 rats receiving oil suspension of habanero fruits for 7 days (group I-7) or 28 days (group II-28) were used. Antibodies against GFAP and S100b were used for immunohistochemistry. Morphology and distribution of astrocytes was evaluated under light microscope and their density was quantitatively analysed.

Results: In the CA1 hippocampal area of group II-28 rats, GFAP-IR cells with numerous, branched processes were observed. S100b-IR astrocytes had delicate, single processes in comparison with cells without processes observed in groups I-7 and C. In groups I-7 and II-28, GFAP-IR astrocytes' density significantly increased in SR - stratum radiatum of hippocampal CA1 area. In group I-7, a density of cells with the expression of S100b was significantly increased in SO - stratum oriens layer. In group II-28, the density of S100b-IR astrocytes was decreased.

Conclusions: Habanero peppers administrated to rats, especially for a longer time, caused reactive changes in the astrocytes in hippocampal CA1 area, and thus these glial cells may protect neurons against excitotoxic damage.

Introduction: Naringenin, a dihydro-flavonoid compound that shows chemotactic activity, may have a good application prospect in repairing bone tissue, but its specific mechanism in bone regeneration, especially the osteogenic differentiation of stem cells, needs for a further study. The aim of this study was to investigate the effect of naringenin on the osteogenic differentiation and its roles in the C-X-C chemokine receptor type 4/stromal cell-derived factor 1 (SDF-1/CXCR4) signal pathway of bone marrow-derived mesenchymal stem cells (BMSCs).

Material and methods: BMSCs were harvested from the femurs and tibias of 4-to-6-week-old male Sprague-Dawley rats. Cell Counting kit-8 assay was used to determine cytotoxicity of naringenin. Alkaline phosphatase (ALP) activity was measured in cell's precipitates and alizarin-red staining was performed to determine the osteogenic differentiation capacity of the BMSCs. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting were adopted to determine the expression of genes and proteins.

Results: The cellular morphology was spindle-shaped, and arranged in radial and whorled patterns. The flow cytometric analysis have confirmed the presence of characteristic surface proteins in the harvested BMSCs. Different concentrations (0-200 μg/ml) of naringenin have no influence on the viability and proliferation rate of the BMSCs. The highest ALP activity was found at culture day 7 and 9 when the concentration of naringenin was 75 and 100 μg/ml. Positive red or dark red stained cells with mineralized nodules can be observed on day 14. The expression of ALP, Runt-related transcription factor 2, CXCR4 and SDF-1a at the gene and protein levels in naringenin-treated cells were significantly higher than those in the control cells. Moreover, AMD3100, an inhibitor of CXCR4, suppressed the expression of the studied genes and proteins.

Conclusions: Naringenin does not show toxic effect on BMSCs. Naringenin promotes the expression of the SDF-1a gene and protein via the SDF-1/CXCR4 signaling pathway. A better understanding of the mechanisms of naringenin action would be helpful for developing specific therapeutic strategies to improve bone regeneration after injuries.

Introduction: Osteoarthritis (OA) is the most prevailing musculoskeletal dysfunction triggered by lesions in synovial membranes and articular cartilage. MicroRNAs (miRNAs) have emerged as crucial regulators participated in many biological processes, such as osteoarthritis. This study was undertaken to address the role of miR-25-3p in the apoptosis of rat chondrocytes under an OA-like condition and its underlying mechanism.

Material and methods: OA cellular model was established in rat chondrocytes by TNF-a induction. Then, qRTPCR and Western blotting were utilized for evaluation of the expressions of miR-25-3p and insulin-like growth factor-binding protein 7 (IGFBP7), CCK-8 assay for inspection of chondrocyte viability, flow cytometry for assessment of cell apoptosis rate, Western blotting for the detection of cleaved caspase-3 level and dual-luciferase reporter gene assay for verification of the targeting relationship between miR-25-3p and IGFBP7.

Results: The miR-25-3p expression was decreased and IGFBP7 was elevated in TNF-a-induced rat chondrocytes. The miR-25-3p inhibited chondrocyte apoptosis and IGFBP7 promoted apoptosis as evidenced by enhanced cell viability and suppressed cell apoptosis in OA chondrocytes after miR-25-3p overexpression or IGFBP7 knockdown. The miR-25-3p facilitated chondrocyte viability and repressed cell apoptosis in OA by negatively regulating IGFBP7.

Conclusions: MiR-25-3p negatively regulates IGFBP7 to promote chondrocyte proliferation and restrain chondrocyte apoptosis. Our findings suggest that the regulation of IGFBP7 by miR-25-3p may emerge as a novel therapeutic regimen for OA.

Introduction: The pathological mechanism of cerebral ischemia/reperfusion (CIR) injury is complicated and unclear. Apart from the involvement of many low-molecular factors it was found that several miRNAs were dysregulated during and after CIR injury in cell models. This study aimed to explore the effects of miR-378a-5p on in vitro model of (CIR) injury-induced neuronal apoptosis and provide a new mechanism of CIR injury.

Material and methods: Primary hippocampal neurons were isolated from newborn Sprague-Dawley rats. Oxygen- glucose deprivation/reoxygenation (OGDR) for 24 h and 48 h was used as an in vitro model of CIR. Cell viability was measured using MTT assay and apoptosis was determined by flow cytometry. Quantitative real time PCR (qRT-PCR) assay and Western blotting were used to examine mRNA and protein expressions, respectively. TargetScan was used to predict the direct target of miR-378a-5p and luciferase assay was used to validate that calmodulin-dependent protein kinase kinase-2 (CAMKK2) was the direct target of miR-378a-5p.

Results: miR-378a-5p expression was significantly increased after OGDR at 24 h and 48 h. After OGDR, cell viability was reduced, which was reversed by miR-378a-5p and enhanced by shCAMKK2 plasmid. Cell apoptosis was increased after OGDR, which was prevented by miR-378a-5p and enhanced by shCAMKK2 plasmid. Results of TargetScan and luciferase assay demonstrated that miR-378a-5p could directly bind to 3'-untranslated region (3'-UTR) of CAMKK2. Both mRNA and protein expression of CAMKK2 were downregulated by miR-378a-5p mimics and upregulated by miR-378a-5p inhibitors. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was positively associated with expression of CAMKK2.

Conclusions: Data of this study indicated that miR-378a-5p was significantly overexpressed after OGDR. miR-378a-5p could bind to 3'-UTR of CAMKK2 to inhibit cell proliferation through regulation of CAMKK2/AMPK pathway providing a new mechanism and biomarker for the diagnosis and potential treatment of CIR injury.

Introduction: Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals.

Material and methods: Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1.

Results: The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals.

Conclusions: The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.

Introduction: In our previous study, it has been confirmed that formaldehyde (FA) not only inhibits the proliferative activity, but also causes DNA-protein crosslinks (DPCs) formation in bone marrow mesenchymal stem cells (BMSCs). The purpose of this study was to detect the protective effect of astragalus polysaccharide (APS) against the cytotoxicity and genotoxicity of BMSCs exposed to FA, and to explore potential molecular mechanisms of APS activity.

Material and methods: Human BMSCs were cultured in vitro and randomly divided into control cells (Ctrl group), FA-treated cells (FA group, 120 μmol/L), and cells incubated with FA and increasing concentrations (40, 100, or 400 μg/mL) of APS (FA + APS groups). Cytotoxicity was measured by MTT assay. DNA strand breakage, DNA-protein crosslinks (DPCs), and micronucleus formation were respectively detected by comet assay, KCl-SDS precipitation assay, and micronucleus assay. The mRNA and protein expression level of xeroderma pigmentosum group A (XPA), xeroderma pigmentosum group C (XPC), excision repair cross-complementation group 1 (ERCC1), replication protein A1 (RPA1), and replication protein A2 (RPA2) were all detected by qRT-PCR and Western Blot.

Results: Compared with the FA group, the cytotoxicity, DNA strand breakage, DPCs, and micronucleus levels were decreased significantly in FA + APS groups (P < 0.01). Meanwhile, the mRNA and protein expression of XPA, XPC, ERCC1, RPA1, and RPA2 were up regulated significantly in the FA + APS groups (P < 0.05) with the most prominent effect of the 100 μg/mL APS.

Conclusions: Our results suggest that APS can protect the cytotoxicity and genotoxicity of human BMSCs induced by FA. The mechanism may be associated with up-regulated expression of XPA, XPC, ERCC1, RPA1, and RPA2 in the nucleotide excision repair (NER) pathway which promotes DNA damage repair.

Introduction: Endothelial dysfunction is a critical part of heart failure (HF) pathophysiology. It is not clear, however, whether it is present at the similar level in the early and late HF stages.

Material and methods: von Willebrand factor (vWF) and its mRNA levels in biopsies of non-ischemic patients with HF secondary to dilated cardiomyopathy were studied. Consecutive patients with HF were divided into two groups: group A with disease duration ≤ 12 months (n = 59) and group B with disease duration > 12 months (n = 68). The immunoreactivity of the vWF was compared with autopsy sections of 19 control cases. Tissue vWF gene expression was analyzed at the mRNA level by RT-PCR.

Results: In the group A, there was lower vWF immunoreactivity in the coronary microvessels compared to the group B [1.5 (1.0-2.0) vs. 2.0 (1.5-2.4), P = 0.001]. In the control group, only weak vWF expression was observed. Protein expression was not accompanied by vWF mRNA whose levels were significantly higher in the Group A as compared to the Group B [14671 (4932-51561) vs. 3643 (185.3-9030.8), P = 0.005]. Protein vWF expression was inversely associated with its mRNA levels (r = -0.34, P = 0.04).

Conclusions: High myocardial protein expression of vWF in patients with long-lasting HF symptoms may highlight the persistent nature of endothelial dysfunction in such a cohort of patients.

Introduction: The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism.

Material and methods: The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis.

Results: The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed.

Conclusions: TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

Introduction: Soyasaponins are triterpenoid glycosides discovered in soybean and have anti-cancer properties. Soyasaponin A was reported to repress estrogen-insensitive breast cancer cell proliferation. This study intends to explore the role of one isomer of soyasaponin A, i.e. soyasaponin Ag (Ssa Ag), in triple-negative breast cancer (TNBC) development.

Material and methods: Bioinformatic databases were used to predict DUSP6 expression in breast cancer (BC) as well as the correlation between the expression of DUSP6 (or MAPK1, MAPK14) with the prognosis of patients with BC. The expression of DUSP6/MAPK signaling-related genes (DUSP6, MAPK1, and MAPK14) in TNBC cell lines was assessed via Western blot analysis and RT-qPCR. Levels of cell apoptosis proteins (Bax and Bcl-2) in TNBC cells were assessed via Western blot analysis. CCK-8 assay, colony formation assay, and flow cytometry analysis were conducted for the measurement of TNBC cell growth and apoptosis. In vivo xenograft assay was employed for investigating the biological influence of Ssa Ag on tumor growth.

Results: The poor prognosis of BC patients was linked to the aberrant expression of DUSP6/MAPK pathway genes. Low expression of DUSP6 or high expression of MAPK1 (or MAPK14) was correlated to poor prognosis. DUSP6 was downregulated while MAPK1 and MAPK14 were upregulated in TNBC cells versus normal cells. Ssa Ag upregulated DUSP6 expression while downregulated MAPK1 and MAPK14 expression, inhibiting the MAPK signaling pathway. Additionally, Ssa Ag promoted in vitro TNBC cell apoptosis and restrained cell growth, and repressed in vivo tumor growth.

Conclusions: Ssa Ag inhibited TNBC progression via upregulating DUSP6 and inactivating the MAPK signaling pathway.

Introduction: The aim of the study was to investigate the clinical significance of Ly-1 antibody reactive clone (LYAR) in non-small-cell lung cancer (NSCLC).

Material and methods: The expressions of LYAR at the protein level in representative paired NSCLC tumor tissues and adjacent non-tumor tissues were measured by Western blot and immunohistochemistry. Kaplan-Meier method was used to calculate the survival curve of patients with NSCLC. Cell Counting Kit-8 assay and flow cytometry were used to estimate the cell proliferation and cell cycle, respectively. Terminal-deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed to detect cell apoptosis.

Results: LYAR was dramatically overexpressed in NSCLC tissues which were closely related to the survival of patients with NSCLC. In clinical studies, the expression of LYAR was related to the clinical stage, histological differentiation, and Ki-67 expression. A positive correlation was found between LYAR and Ki-67 expression by Spearman's correlation test. After serum starvation for 72 h, serum re-addition significantly increased the expression of LYAR, PCNA, and Cyclin A and promoted the cell cycle progression. LYAR knockdown inhibited the proliferation and induced the G0/G1 cell cycle arrest and apoptosis of A549 cells.

Conclusions: The present study revealed the clinical significance of LYAR in NSCLC. LYAR might serve as a tumor promoter in NSCLC progression by promoting the proliferation and inhibiting the apoptosis of NSCLC cells. Inhibiting the expression of LYAR was considered as a potential novel therapeutic strategy for NSCLC.