Alcian Blue Research Articles

Cell Proliferation, Chondrogenic Differentiation, and Cartilaginous Tissue Formation in Recombinant Silk Fibroin with Basic Fibroblast Growth Factor Binding Peptide.

Regeneration of articular cartilage remains a challenge for patients who have undergone cartilage injury, osteochondritis dissecans and osteoarthritis. Here, we describe a new recombinant silk fibroin with basic fibroblast growth factor (bFGF) binding peptide, which has a genetically introduced sequence PLLQATLGGGS, named P7. In this study, we cultured a human mesenchymal cell line derived from bone marrow, UE6E7-16, in wild-type fibroin sponge (FS) and recombinant silk fibroin sponge with P7 peptide (P7 FS). We compared cell proliferation, chondrogenic differentiation and cartilaginous tissue formation between the two types of sponge. After stimulation with bFGF at 3 ng/mL, P7 FS showed significantly higher cell growth (1.2-fold) and higher cellular DNA content (5.6-fold) than did wild-type FS. To promote chondrogenic differentiation, cells were cultured in the presence of TGF-β at 10 ng/mL for 28 days. Immunostaining of P7 FS showed SOX9-positive cells comparable to wild-type FS. Alcian-Blue staining of P7 FS also showed cartilaginous tissue formation equivalent to wild-type FS. A significant increase in cell proliferation in P7 FS implies future clinical application of this transgenic fibroin for regeneration of articular cartilage. To produce cartilaginous tissue efficiently, transgenic fibroin sponges and culture conditions must be improved. Such changes should include the selection of growth factors involved in chondrogenic differentiation and cartilage formation.

Homogeneously assembled covalent organic framework membrane co-stitched by β-cyclodextrin and chitosan for dye separation

Covalent organic frameworks (COFs) based membranes have demonstrated great potential in molecular separation. However, COFs are usually synthesized as powders, and difficult to form large-area and highly crystalline membranes. There normally exists unfavorable crystal flaws and stacking defects in membranes, impairing their rejection performance. The severe reaction conditions of solvothermal synthesis also inhibit their fabrication. Here, a comprehensive strategy was developed to fabricate a homogeneously assembled COF membrane co-stitched by β-cyclodextrin (β-CD) and chitosan (CS). Specifically, the COF crystallites (named TATP) were homogeneously formed and assembled to be films at room temperature under the control of n-propylamine; The aminated CS chemically bonded the TATP crystallites on the substrate and helped assemble them to form dense films. β-CD molecules were subsequently embedded into the TATP layer and crosslinked to further stitch membrane defects. The prepared CD-TATP-NH2CS/nylon membrane demonstrated enhanced excellent rejection performance. It could reject 100 % of R250 (BB), Congo red, Direct blue15, and 99.4 % of Alcian blue. It also could selectively separate Methyl orange (MO) and BB mixed solutions with only MO passing through, owing to the co-stitching of the membrane defects by β-CD and CS. This study provides a significant concept and strategy for fabricating high-performance COF membranes.

Challenges in Biofilm Identification in Diabetic Foot Infections: Review of Literature.

Foot ulcerations are one of the most common complications of diabetes and one of the major initial causes of amputations. The formation of biofilms on wounds significantly contributes to infections and delayed healing. While existing methods for identifying these biofilms have limitations, there is a need for a convenient tool for its clinical application. This literature review aimed to address the problem with current clinical biofilm identification in wound care and a proposal for biofilm-detection-based wound care in diabetic foot ulcer patients. Identifying biofilms is particularly vital due to the absence of typical signs of infection in DFUs. However, current approaches, although effective, often prove invasive and technically intricate. The wound blotting technique, involving attaching a nitrocellulose membrane and subsequent staining, presents an alternative that is swift and non-invasive. Research highlights the applicability of wound blotting with alcian blue staining in clinical scenarios, consistently producing sensitive outcomes. By addressing the critical need for early biofilm detection, wound blotting holds promise for enhancing DFU management and contributing to strategies aimed at preventing amputations.

Updated measurement method for transparent exopolymer particles (TEPs) and their precursors with insights into efficient monitoring

Transparent exopolymer particles (TEPs) and their precursors play a critical role in various environmental processes, including contaminant transport and membrane fouling. This study presents an improved Alcian blue (AB) staining-based TEP measurement method and investigates spectroscopic proxies to enhance analysis convenience and reproducibility. The proposed method employs deionized (DI) water and sonication to extract particulate TEPs (TEP0.4μm) and particulate TEPs and their precursors (TEP10kDa), thus eliminating the need for concentrated sulfuric acid and simplifying the procedure with an integrated standard curve. This AB-DI method had a low detection limit of 5 μg Xeq/L for a 1 L sample volume, allowing precise measurements to be made across a broad concentration range. When used in wastewater treatment and microfiltration membrane filtrates, the AB-DI method provided insights into the removal mechanisms of TEPs and their precursors. This study also identified effective spectroscopic proxies by correlating TEP concentrations with optical properties, achieving remarkable estimation accuracy using the UV254 absorbance of TEP-extracted solutions. The UV254 and fluorescence intensities of the protein-like peaks in raw water samples demonstrated significant potential as proxies for TEPs and their precursors. The proposed AB-DI method represents a viable approach for the real-time TEP monitoring for desalination and water reuse processes.

Correlation between the Long Head of the Biceps Microscopic Degeneration and Extent of Apoptotic Process.

Objectives: The purpose of this study was to determine the correlation between microscopic degeneration in the long head of the biceps tendon (LHBT) and the apoptotic process. Methods: This study included 26 consecutive patients who had undergone arthroscopic biceps tenodesis or tenotomy for symptomatic LHBT with or without concomitant rotator cuff tears (RCTs). Histological examination of the specimens under a light microscope was conducted after staining with hematoxylin, eosin, and the Alcian blue. Histopathological changes were assessed using the original Bonar score and the modified Bonar score and then correlated with the expression of the subsequent apoptosis markers: activated caspase-3 (casp3), tumor protein p53 (p53), and B-cell lymphoma 2 (BCL-2). Results: The mean original Bonar score was 8.65 (range 5-11), while the modified Bonar score was 7.61. There was no correlation between the original Bonar score and the age of the patients, but a positive correlation was found between the modified Bonar score and the age of the patients (p = 0.0022). There was no correlation between the age of patients and the expression indexes of BCL-2 and casp3. However, the expression of the p53 index showed a positive correlation with patient aging (p = 0.0441). Furthermore, there was no correlation observed between the expression of apoptotic indexes and both the original and modified Bonar scale. Conclusions: In LHB tendinopathy, the expression of apoptosis does not seem to directly correlate with the extent of degeneration, particularly in the late stages of tendinopathy. However, the transformations observed in collagen and ground substance were significantly associated with age, as well as tendinous tissue degeneration quantified according to modified Bonar score. The age of patients was also linked with the expression of the p53 index, as an increased apoptosis in the studied population.

Insulin promotes the bone formation capability of human dental pulp stem cells through attenuating the IIS/PI3K/AKT/mTOR pathway axis

BackgroundInsulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms.MethodsCell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining.ResultsInsulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10− 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10− 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10− 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation.ConclusionsInsulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

House dust mite-induced asthma exacerbates Alzheimer’s disease changes in the brain of the AppNL-G-F mouse model of disease

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques, neuroinflammation, and neuronal death. Besides aging, various comorbidities increase the risk of AD, including obesity, diabetes, and allergic asthma. Epidemiological studies have reported a 2.17-fold higher risk of dementia in asthmatic patients. However, the molecular mechanism(s) underlying this asthma-associated AD exacerbation is unknown. This study was designed to explore house dust mite (HDM)-induced asthma effects on AD-related brain changes using the AppNL-G-F transgenic mouse model of disease. Male and female 8–9 months old C57BL/6J wild type and AppNL-G-F mice were exposed to no treatment, saline sham, or HDM extract every alternate day for 16 weeks for comparison across genotypes and treatment. Mice were euthanized at the end of the experiment, and broncho-alveolar lavage fluid (BALF), blood, lungs, and brains were collected. BALF was used to quantify immune cell phenotype, cytokine levels, total protein content, lactate dehydrogenase (LDH) activity, and total IgE. Lungs were sectioned and stained with hematoxylin and eosin, Alcian blue, and Masson’s trichrome. Serum levels of cytokines and soluble Aβ1-40/42 were quantified. Brains were sectioned and immunostained for Aβ, GFAP, CD68, and collagen IV. Finally, frozen hippocampi and temporal cortices were used to perform Aβ ELISAs and cytokine arrays, respectively. HDM exposure led to increased levels of inflammatory cells, cytokines, total protein content, LDH activity, and total IgE in the BALF, as well as increased pulmonary mucus and collagen staining in both sexes and genotypes. Levels of serum cytokines increased in all HDM-exposed groups. Serum from the AppNL-G-F HDM-induced asthma group also had significantly increased soluble Aβ1-42 levels in both sexes. In agreement with this peripheral change, hippocampi from asthma-induced male and female AppNL-G-F mice demonstrated elevated Aβ plaque load and increased soluble Aβ 1–40/42 and insoluble Aβ 1–40 levels. HDM exposure also increased astrogliosis and microgliosis in both sexes of AppNL-G-F mice, as indicated by GFAP and CD68 immunoreactivity, respectively. Additionally, HDM exposure elevated cortical levels of several cytokines in both sexes and genotypes. Finally, HDM-exposed groups also showed a disturbed blood–brain-barrier (BBB) integrity in the hippocampus of AppNL-G-F mice, as indicated by decreased collagen IV immunoreactivity. HDM exposure was responsible for an asthma-like condition in the lungs that exacerbated Aβ pathology, astrogliosis, microgliosis, and cytokine changes in the brains of male and female AppNL-G-F mice that correlated with reduced BBB integrity. Defining mechanisms of asthma effects on the brain may identify novel therapeutic targets for asthma and AD.

A phosphate glass reinforced composite acrylamide gradient scaffold for osteochondral interface regeneration

The bone-cartilage interface is defined by a unique arrangement of cells and tissue matrix. Injury to the interface can contribute to the development of arthritic joint disease. Attempts to repair osteochondral damage through clinical trials have generated mixed outcomes. Tissue engineering offers the potential of integrated scaffold design with multiregional architecture to assist in tissue regeneration, such as the bone-cartilage interface. Challenges remain in joining distinct materials in a single scaffold mass while maintaining integrity and avoiding delamination. The aim of the current work is to examine the possibility of joining two closely related acrylamide derivatives such as, poly n-isopropyl acrylamide (pNIPAM) and poly n‑tert‑butyl acrylamide (pNTBAM). The target is to produce a single scaffold unit with distinct architectural regions in the favour of regenerating the osteochondral interface. Longitudinal phosphate glass fibres (PGFs) with the formula 50P2O5.30CaO.20Na2O were incorporated to provide additional bioactivity by degradation to release ions such as calcium and phosphate which are considered valuable to assist the mineralization process. Polymers were prepared via atom transfer radical polymerization (ATRP) and solutions cast to ensure the integration of polymers chains. Scaffold was characterized using scanning electron microscope (SEM) and Fourier transform infra-red (FTIR) techniques. The PGF mass degradation pattern was inspected using micro computed tomography (µCT). Biological assessment of primary human osteoblasts (hOBs) and primary human chondrocytes (hCHs) upon scaffolds was performed using alizarin red and colorimetric calcium assay for mineralization assessment; alcian blue staining and dimethyl-methylene blue (DMMB) assay for glycosaminoglycans (GAGs); immunostaining and enzyme-linked immunosorbent assay (ELISA) to detect functional proteins expression by cells such as collagen I, II, and annexin A2. FTIR analysis revealed an intact unit with gradual transformation from pNIPAM to pNTBAM. SEM images showed three distinct architectural regions with mean pore diameter of 54.5 µm (pNIPAM), 16.5 µm (pNTBAM) and 118 µm at the mixed interface. Osteogenic and mineralization potential by cells was observed upon the entire scaffold's regions. Chondrogenic activity was relevant on the pNTBAM side of the scaffold only with minimal evidence in the pNIPAM region. PGFs increased mineralization potential of both hOBs and hCHs, evidenced by elevated collagens I, X, and annexin A2 with reduction of collagen II in PGFs scaffolds. In conclusion, pNIPAM and pNTBAM integration created a multiregional scaffold with distinct architectural regions. Differential chondrogenic, osteogenic, and mineralized cell performance, in addition to the impact of PGF, suggests a potential role for phosphate glass-incorporated, acrylamide-derivative scaffolds in osteochondral interface regeneration.

Yeast mixture supplementation modulates faecal microbiota and ileum morphology of weaning pigs

Different yeast strains benefit postweaning piglets by promoting intestinal health. The objective of this study was to investigate the effect of a yeast mixture containing Kluyveromyces marxianus fragilis, Pichia guilliermondii, and Saccharomyces cerevisiae (Vetoquinol italia s.r.l., Italy) on gut health parameters and growth performance traits of weaned piglets. Forty-eight postweaning castrated male piglets (27 ± 1.7 days, 7.19 ± 0.54 kg) were randomly allocated to two homogeneous experimental groups and involved in a 28-day trial. Both the groups received a basal diet with (yeast mixture, YM) or without (control, CTR) the inclusion of 0.8% yeast mixture during weeks 1 and 2, and 0.6% during weeks 3 and 4. Individual BW and box feed intake were determined on days 0, 14, and 28, and average daily gain and Gain:Feed ratio were subsequently calculated for each administration period (0–14, 14–28). Individual faecal samples were collected for microbiota analysis on days 4, 14, 21, and 28, and faecal score was evaluated on the same days. At the end of the trial, 12 piglets for each group were sacrificed, and ileal tissue was sampled for morphological analysis and the evaluation of mucins profile, using Alcian-Blue/Periodic Acid-Shiff (PAS) staining. On ileum samples, dividing and differentiated epithelial cells were also identified using proliferating cell nuclear antigen and alkaline phosphatase expression, respectively. Differences in the means between the experimental groups were determined by ANOVA, while the metataxonomics analyses were performed by sequencing for V3 and V4 hypervariable regions of the 16S rRNA gene. Growth performance traits were not different among the two experimental groups when considering the whole trial period, while treated animals showed increased faecal consistency on weeks 1 and 4 (P = 0.036 and 0.021, respectively). Yeast mixture administration increased the abundance of Bifidobacterium (P = 0.006) and Coprococcus 2 (P = 0.015), and decreased Clostridium Sensu Stricto 1 (P = 0.019) at all the considered timepoints. Ileum villous height, villous width, and crypt depth were significantly increased by yeast mixture supplementation (P = 0.019; P = 0.013; P = 0.036, respectively), while no differences were observed for the villous:crypt ratio among the groups. The mucin profile showed no differences among experimental groups for acid and neutral glycoconjugates. However, a higher presence of PAS-positive mucins was highlighted in the villi of YM piglets (P < 0.001) compared to CTR. Overall, the administration of a yeast mixture to postweaning piglets showed positive effects on gut health when compared to piglets not receiving the tested product, improving beneficial genera and intestinal morphology.

Polystyrene nanoplastics-induced intestinal barrier disruption via inflammation and apoptosis in zebrafish larvae (Danio Rerio)

Plastics are one of the most pervasive materials on Earth, to which humans are exposed daily. Polystyrene (PS) is a common plastic packaging material. However, the impact of PS on human health remains poorly understood. Therefore, this study aimed to identify intestinal damage induced by PS nanoplastics (PS-NPs) in zebrafish larvae which have a high homology with humans. Four days post fertilization (dpf), zebrafish larvae were exposed to 0-, 10-, and 50-ppm PS-NPs for 48 h Initially, to ascertain if 100 nm PS-NPs could accumulate in the gastrointestinal (GI) tract of zebrafish larvae, the larvae were exposed to red fluorescence-labeled PS-NPs, and at 6 dpf, the larvae were examined using a fluorescence microscope. Analysis of the fluorescence intensity revealed that the GI tract of larvae exposed to 50-ppm exhibited a significantly stronger fluorescence intensity than the other groups. Nonfluorescent PS-NPs were then used in further studies. Scanning electron microscopy (SEM) confirmed the spherical shape of the PS-NPs. Fourier-transform infrared spectroscopy (FT-IR) analysis revealed chemical alterations in the PS-NPs before and after exposure to larvae. The polydispersity index (PDI) value derived using a Zetasizer indicated a stable dispersion of PS-NPs in egg water. Whole-mount apoptotic signal analysis via TUNEL assay showed increased apoptosis in zebrafish larval intestines exposed to 50-ppm PS-NPs. Damage to the intestinal tissue was assessed by Alcian blue (AB) and hematoxylin and eosin (H&E) staining. AB staining revealed increased mucin levels in the zebrafish larval intestines. Thin larval intestinal walls with a decrease in the density of intestinal epithelial cells were revealed by H&E staining. The differentially expressed genes (DEGs) induced by PS-NPs were identified and analyzed. In conclusion, exposure to PS-NPs may damage the intestinal barrier of zebrafish larvae due to increased intestinal permeability, and the in vivo gene network may change in larvae exposed to PS-NPs.

Multiple Administration of Dexamethasone Possesses a Deferred Long-Term Effect to Glycosylated Components of Mouse Brain.

Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.

Yiqi Jiedu Huayu decoction inhibits precancerous lesions of chronic atrophic gastritis by inhibiting NLRP3 inflammasome-mediated pyroptosis.

Chronic atrophic gastritis (CAG) is a prevalent chronic gastritis usually accompanied by precancerous lesions such as intestinal metaplasia and dysplasia. The increasing application of traditional Chinese medicine in CAG treatment has shown promising results with low side effects and significant efficacy. To investigate the pharmacological effects of Yiqi Jiedu Huayu decoction (YJHD) on precancerous lesions of CAG. A CAG rat model was established by Helicobacter pylori bacteria solution combined with N-methyl-N'-nitro-N-nitrosoguanidine. Histopathological measurements were conducted by hematoxylin-eosin and alcian blue and periodic acid-Schiff staining. Serum levels of inflammatory factors and gastric mucosal-related factors were examined using enzyme-linked immunosorbent assay. Protein and mRNA levels were quantified via western blot and quantitative real-time polymerase chain reaction analysis, respectively. Molecular interaction was verified by chromatin immunoprecipitation (ChIP) assay. YJHD greatly attenuated pathological changes in the gastric mucosa and precancerous lesions in CAG rats. Meanwhile, YJHD treatment reduced serum levels of inflammatory factors [interleukin (IL)-6, tumor necrosis factor-α and C-reactive protein] and increased serum levels of gastric mucosal-related factors (gastrin, pepsin, somatostatin and prostaglandin E2) in CAG rats. In addition, YJHD administration suppressed NLRP3 inflammasome-mediated cell pyroptosis, as well as the activation of TLR4/NF-κB and IL-6/STAT3 signaling pathways. Mechanically, ChIP experiments confirmed that NLRP3 transcription was regulated by TLR4/NF-κB and IL-6/STAT3 signaling. Taken together, YJHD alleviated NLRP3 inflammasome formation and pyroptosis of epithelial cells in CAG, potentially through the inactivation of TLR4/NF-κB and IL-6/STAT3 pathways.

A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Underlying Mechanism of Fluoride Inhibits Colonic Gland Cells Proliferation by Inducing an Inflammation Response.

The integrity of colonic gland cells is a prerequisite for normal colonic function and maintenance. To evaluate the underlying injury mechanisms in colonic gland cells induced by excessive fluoride (F), forty-eight female Kunming mice were randomly allocated into four groups and treated with different concentrations of NaF (0, 25, 50, and 100 mg F-/L) for 70 days. As a result, the integrity of the colonic mucosa and the cell layer was seriously damaged after F treatment, as manifested by atrophy of the colonic glands, colonic cell surface collapse, breakage of microvilli, and mitochondrial vacuolization. Alcian blue and periodic acid Schiff staining revealed that F decreased the number of goblet cells and glycoprotein secretion. Furthermore, F increased the protein expression of TLR4, NF-κB, and ERK1/2 and decreased IL-6, interfered with NF-κB signaling, following induced colonic gland cells inflammation. The accumulation of F inhibited proliferation via the JAK/STAT signaling pathway, as characterized by decreased mRNA and protein expression of JAK, STAT3, STAT5, PCNA, and Ki67 in colon tissue. Additionally, the expression of CDK4 was up-regulated by increased F concentration. In conclusion, excessive F triggered colonic inflammation and inhibited colonic gland cell proliferation via regulation of the NF-κB and JAK/STAT signaling pathways, leading to histopathology and barrier damage in the colon. The results explain the damaging effect of the F-induced inflammatory response on the colon from the perspective of cell proliferation and provide a new idea for explaining the potential mechanism of F-induced intestinal damage.

Examining the Synergic Effect of Exosomes Derived from Mouse Mesenchymal Stem Cells and Low-frequency Electromagnetic Field on Chondrogenic Differentiation

Background: Cartilage has intrinsically limited healing power, and regeneration of cartilage damages has remained a challenge. Secreted products of mesenchymal stem cells have shown a new therapeutic strategies for cartilage injuries. Also it has been observed that low frequency electromagnetic field plays a key role in biological processes. Objective: This research was performed to investigate the synergic effect of mesenchymal stem cell-derived exosomes and low frequency electromagnetic field on chondrogenic differentiation. Methods: In this in vitro study, mesenchymal stem cell-derived exosomes were identified using AFM, SEM, TEM microscopy, and DLS method. Cells were treated in chondrogenic medium by exosomes, low frequency electromagnetic field, and the synergy of both. The cell survival was examined using MTT and Annexin methods, and cartilage differentiation was confirmed by Alcian blue staining. The expression of Sox-9, Acan, Col 2a1 and Col 10a1 genes was examined via Real- Time PCR technique on day 14 post-treatment. Results: The results confirmed the presence of exosomes with an approximate size of less than 100 nm. The results of Alcian blue revealed greater expression of glycosaminoglycans in the synergic treatment group compared to the other groups. Real-time PCR showed a significant increase in the expression of Sox-9, Acan, and Col 2a1 genes, as well as a significant reduction of Col 10a1 gene expression in the synergic treatment group compared to other groups. Conclusion: This study indicated that the synergic effect of exosome and low-frequency electromagnetic fields would lead to enhanced chondrogenic differentiation, which can be further explored in future clinical studies

Changes in human skin composition due to intrinsic aging: a histologic and morphometric study

Skin represents the main barrier against the external environment, but also plays a role in human relations, as one of the prime determinants of beauty, resulting in a high consumer demand for skincare-related pharmaceutical products. Given the importance of skin aging in both medical and social spheres, the present research aims to characterize microscopic changes in human skin composition due to intrinsic aging (as opposed to aging influenced by external factors) via histological analysis of a photoprotected body region. Samples from 25 autopsies were taken from the periumbilical area and classified into four age groups: group 1 (0–12 years), group 2 (13–25 years), group 3 (26–54 years), and group 4 (≥ 55 years). Different traditional histological (hematoxylin–eosin, Masson’s trichrome, orcein, toluidine, Alcian blue, and Feulgen reaction) and immunohistochemical (CK20, CD1a, Ki67, and CD31) stains were performed. A total of 1879 images photographed with a Leica DM3000 optical microscope were morphometrically analyzed using Image ProPlus 7.0 for further statistical analysis with GraphPad 9.0. Our results showed a reduction in epidermis thickness, interdigitation and mitotic indexes, while melanocyte count was raised. Papillary but not reticular dermis showed increased thickness with aging. Specifically, in the papillary layer mast cells and glycosaminoglycans were expanded, whereas the reticular dermis displayed a diminution in glycosaminoglycans and elastic fibers. Moreover, total cellularity and vascularization of both dermises were diminished with aging. This morphometric analysis of photoprotected areas reveals that intrinsic aging significantly influences human skin composition. This study paves the way for further research into the molecular basis underpinning these alterations, and into potential antiaging strategies.

A Periodic Comparison of Harderian Gland in Henna Partridge (Alectoris chukar) According to Different Developmental Stages

Background: The present study aimed at determining histological structure and histochemical properties of the henna partridge (Alectoris chukar) Harderian gland periodically. Objectives: For this purpose, the Harderian glands of 12 (6 females + 6 males) healthy henna partridges aged 3 and 6 months were used. Methods: Totally removed Harderian gland tissue samples were kept in 10% neutral formolin for 36 hours and fixed. After washing, dehydrating, and polishing with known histological techniques, tissue pieces were blocked in paraffin. Results: We found that a thin connective tissue capsule surrounds the Harderian gland of the henna partridge and that the capsule sends septums into the gland and divides the gland into lobes and lobules. It was observed that the corpus glandulae consist of low or high prismatic epithelial cells. Within each lobe, there is a single main draining channel which has a very large lumen along with primary draining channels. Primary and main draining channel epithelial cells have a single-layered cuboidal structure. By Gordon-Sweet staining, it was found that reticular fiber bundles started from the connective tissue capsule that surrounded the organ and spread by branching. The reticular fiber bundles extended in thin strands to the periphery of the corpus glandulae. These reticular fiber bundles in the regions where the connective tissue septums widened are thicker and form a network. The reticular fiber bundles are seen in the basal part of the epithelial cells forming the main draining channel and especially in the crypt areas. Reticular fibers are also found around blood vessels. Strong Alcian blue (AB)-positive epithelial cells are observed in the corpus glandulae in AB pH=2.5 staining, but no periodic acid-Schiff (PAS)-positive epithelial cells are observed in PAS staining. While the majority of primary draining channel cells show a strong AB-positive reaction, a few cells showed a weak PAS-positive reaction. Regarding the main draining channel epithelium, goblet cells show a weak and strong AB-positive reaction in AB pH=2.5 staining method, respectively and a weak PAS-positive reaction in PAS staining method. Conclusion: In line with these findings, no significant difference was found in terms of general histological structure and histochemical properties of the 3- and 6-month-old henna partridge Harderian gland.

ЭКСПЕРИМЕНТАЛЬНОЕ ИССЛЕДОВАНИЕ СОСТОЯНИЯ КОЖИ ОБЛАСТИ РТА ЖИВОТНЫХ ПОСЛЕ ВОЗДЕЙСТВИЯ СТОМАТОЛОГИЧЕСКИХ ДИОДНЫХ ФОТОПОЛИМЕРИЗАТОРОВ

Aim of the study: studying the effect of light radiation from dental photopolymerizers on the soft tissues of the mouth area (regio oralis A01.2.01.018). Materials and methods. Experimental studies were carried out on 50 eight months male rats, randomly divided into 4 groups: 3 main (15 animals each), the fourth - control (5 animals). Under Zoletil anesthesia, the incisors of the lower jaw of experimental animals were exposed twice to light (wavelength 395 - 480 nm) from a dental photopolymerization device. Three photopolymerization modes were used: main group No. 1 – diode photopolymerizer with a luminous flux power of 1000 mW/cm2, main group No. 2 – diode photopolymerizer with a luminous flux power of 1400 mW/cm2; main group No. 3 – diode photopolymerizer with a luminous flux power of 3200 mW/cm2. Animals in the control group were not exposed to light radiation from dental photopolymerizers. After one, three and seven days, animals from groups No. 1 – 3, seven days – animals from the control group were subjected to ether anesthesia and removed from the experiment by decapitation. For pathomorphological studies, a fragment of skin from the mouth area was isolated. The material was fixed with a 15% aqueous neutral solution of formalin and embedded in paraffin. Histological sections were stained with hematoxylin and eosin, according to the van Gieson method, and Alcian blue; performed the CHIC reaction. The vascular bed and its elements were studied using the Gabu-Dybanu method. A standardized comparative microscopic study of skin tissue adjacent to the irradiation zone was carried out using the ZEISS Axio Imager A2 hardware and software complex at magnifications of 1:100 and 1:200. A descriptive method was used to evaluate the results obtained. Results. Microscopy of preparations of the skin of the lips of experimental animals allowed us to come to the conclusion that the primary reaction of tissues to the effects of light radiation from photopolymerizers is of the same type and does not depend on their technical characteristics. Hyperkeratosis and acanthosis of the epithelium, congestion of the microvasculature, the presence of edema and diffuse infiltration of the dermis were detected. Quantitative and qualitative changes in mast cells were observed - an increase in the degree of their degranulation and the number of process forms, which is a marker of tissue damage in response to irradiation. The described changes, which in general are pathomorphological signs of acute inflammation, were determined on the first and third days after irradiation. Further dynamics indicated a decrease in the described reactive changes by the third day when using the photopolymerization mode: wavelength 395 – 480 nm, power 1000 mW/cm2, exposure 10 sec. When using the photopolymerization mode: wavelength 395 - 480 nm, power 1400/3200 mW/cm2, exposure 3 seconds - by the seventh day of observation there was a positive dynamics in the relief of pathological processes, but signs of inflammation remained. Conclusion. Experimental data indicate the possible development of negative photochemical reactions in the skin of the lips after exposure to light from dental photopolymerizers during the treatment of patients with pathology of hard dental tissues. The results obtained can be used to substantiate the prevention of pathological processes in the tissues of the perioral area. Further in vitro, in vivo, and clinical studies are needed to determine the optimal irradiation intensity, irradiation time, and other aspects to ensure the safety and effectiveness of light curing agents.

Sargassum polysaccharide attenuates osteoarthritis in rats and is associated with the up-regulation of the ITGβ1-PI3K-AKT signaling pathway

BackgroundOsteoarthritis (OA) presents a formidable challenge, characterized by as-yet-unclear mechanical intricacies within cartilage and the dysregulation of bone homeostasis. Our preliminary data revealed the encouraging potential of a Sargassum polysaccharide (SP), in promoting chondrogenesis. The aim of our study is to comprehensively assess the therapeutic effects of SP on OA models and further elucidate its potential mechanism. MethodsThe protective effects of SP were initially evaluated in an inflammation-induced human chondrocyte (C28) cell model. CCK-8 assays, Alcian blue staining, RT-qPCR and Western blotting were used to verify the chondrogenesis of SP in vitro. To assess the efficacy of SP in vivo, surgically induced medial meniscus destabilization (DMM) OA rats underwent an 8-week SP treatment. The therapeutic effects of SP in OA rats were comprehensively evaluated using X-ray imaging, micro-computed tomography (μ-CT), histopathological analysis, as well as immunohistochemical and immunofluorescent staining. Following these assessments, we delved into the potential signaling pathways of SP in inflammatory chondrocytes utilizing RNA-seq analysis. Validation of these findings was conducted through RT-qPCR and western blotting techniques. ResultsSP significantly enhance the viability of C28 chondrocytes, and increased the secretion of acidic glycoproteins. Moreover, SP stimulated the expression of chondrogenic genes (Aggrecan, Sox9, Col2a1) and facilitated the synthesis of Collagen II protein in C28 inflammatory chondrocytes. In vivo experiments revealed that SP markedly ameliorated knee joint stenosis, alleviated bone and cartilage injuries, and reduced the histopathological scores in the OA rats. μ-CT analysis confirmed that SP lessened bone impairments in the medial femoral condyle and the subchondral bone of the tibial plateau, significantly improving the microarchitectural parameters of the subchondral bone. Histopathological analyses indicated that SP notably enhanced cartilage quality on the surface of the tibial plateau, leading to increased cartilage thickness and area. Immunohistochemistry staining and immunofluorescence staining corroborated these findings by showing a significant promotion of Collagen II expression in OA joints treated with SP. RNA-seq analysis suggest that SP's effects were mediated through the regulation of the ITGβ1-PI3K-AKT signaling axis, thereby stimulating chondrogenesis. Verification through RT-qPCR and Western blot analyses confirmed that SP significantly upregulated the expression of ITGβ1, p110δ, AKT1, ACAN, and Col2a1. Notably, knock-down of ITGβ1 using siRNA in C28 chondrocytes inhibited the expression of ITGβ1, p110δ, AKT1, and ACAN. However, these inhibitory effects were not completely reversed by supplemental SP intervention. ConclusionsIn summary, our findings reveal that SP significantly enhances chondrogenesis both in vitro and in vivo, alleviating OA progression both in bone and cartilage. The observed beneficial effects are intricately linked to the activation of the ITGβ1-PI3K-AKT signaling axis. The translational potential of this articleOur research marks the first instance unveiling the advantageous effects and underlying mechanisms of SP in OA treatment. With its clinical prospects, SP presents compelling new evidence for the advancement of a next-generation polysaccharide drug for OA therapy.

Highly permeable carbon nanotube membranes for efficient surface water treatment through separation and adsorption–electrochemical regeneration

Membrane-based processes feature many advantages for surface water treatment over other technologies; however, the trade-off between membrane selectivity and permeability usually results in the failure to obtain high-quality filtrate at a high permeance. In this study, we report a highly permeable sodium alginate/glutaraldehyde cross-linked carbon nanotube (CNT/SA-GA) membrane with high adsorption ability for surface water treatment. The CNT/SA-GA membranes have a well-formed hollow fiber morphology and good conductivity of 166–261 S/m. They could reject >98 % of large-sized molecules (e.g., Alcian Blue 8GX and Congo Red) at high permeances of 137–227 L/(m2·h·bar). Moreover, they could remove ~100 % of smaller molecules (e.g., Rhodamine B (RhB)) through dynamic adsorption with a capacity of ~57.3 mg/g. Benefiting from their good conductivity, the CNT/SA-GA membranes could easily decompose the pollutants after adsorption saturation through electrochemical oxidation and then regenerated themselves. Though the switch between adsorption and electrochemical oxidation (2.0 V), the CNT/SA-GA membranes could remove ~100 % RhB at a high permeance of 342 L/(m2·h·bar), which is one order of magnitude higher than those of nanofiltration membranes. When used for surface water treatment, the CNT/SA-GA membranes exhibited a much higher TOC removal rate than commercial poly(acrylonitrile) (PAN) membranes (56.2 % vs. 7.2 %). LC-MS/MS analysis showed that many small molecules such as caffeine could be effectively removed by the CNT/SA-GA membranes. In addition, they exhibited a higher fouling resistance than PAN membranes, evidenced by their lower permeance decline ratio (10.1 % vs. 43.7 %). This study will promote the development and applications of the CNT-based membranes for water purification.