Honokiol Attenuates Choroidal Neovascularization by Inhibiting the Hypoxia-Inducible Factor-α/Vascular Endothelial Growth Factor Axis via Nuclear Transcription Factor-Kappa B Activation.

PURPOSE: Honokiol is a lignan isolated from Magnolia officinalis and exhibits anti-angiogenic properties. This study was conducted to investigate the role of honokiol in choroidal neovascularization. METHODS: C57BL/6 mice were treated with honokiol at 10-20 mg/kg by daily intraperitoneal injection from day 1 to 6 after laser photocoagulation. ARPE-19 cells were cultured under hypoxic conditions with or without the presence of honokiol. After laser photocoagulation and honokiol treatment, hematoxylin and eosin staining, immunofluorescence and fundus fluorescein angiography were used to analyze the effect of honokiol on choroidal neovascularization formation. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay, immunofluorescence, luciferase assay, and chromatin immunoprecipitation were performed to explore the mechanism of honokiol in the pathological process of choroidal neovascularization. Finally, the role of honokiol on the human choroidal vascular endothelial cells was detected by using 5-ethynyl-20-deoxyuridine assay, Transwell and Tube formation assays. RESULTS: The results of hematoxylin and eosin staining and immunofluorescence suggested that honokiol reduced the thickness, length, and area of choroidal neovascularization lesions in laser-induced choroidal neovascularization mouse model. Fundus fluorescein angiography showed that choroidal neovascularization leakage was reduced in honokiol group and the concentration of 20 mg/kg showed better effects. Mechanism studies have shown that honokiol exerted inhibitory effects on choroidal neovascularization by inactivating hypoxia-inducible factor-1α/vascular endothelial growth factor axis through the nuclear transcription factor-kappa B signaling pathway. The same results were obtained in ARPE-19 cells under hypoxic conditions. Furthermore, the conditional medium of retinal pigmented epithelial cells promoted the proliferation, migration, and tube formation of human choroidal vascular endothelial cells, while honokiol reversed these. CONCLUSION: We demonstrated that honokiol attenuated choroidal neovascularization formation by inactivating the hypoxia-inducible factor-1α/vascular endothelial growth factor axis through nuclear transcription factor-kappa B signaling pathway.

Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images.

Immunohistochemistry (IHC) is a well-established and commonly used staining method for clinical diagnosis and biomedical research. In most IHC images, the target protein is conjugated with a specific antibody and stained using diaminobenzidine (DAB), resulting in a brown coloration, whereas hematoxylin serves as a blue counterstain for cell nuclei. The protein expression level is quantified through the H-score, calculated from DAB staining intensity within the target cell region. Traditionally, this process requires evaluation by 2 expert pathologists, which is both time consuming and subjective. To enhance the efficiency and accuracy of this process, we have developed an automatic algorithm for quantifying the H-score of IHC images. To characterize protein expression in specific cell regions, a deep learning model for region recognition was trained based on hematoxylin staining only, achieving pixel accuracy for each class ranging from 0.92 to 0.99. Within the desired area, the algorithm categorizes DAB intensity of each pixel as negative, weak, moderate, or strong staining and calculates the final H-score based on the percentage of each intensity category. Overall, this algorithm takes an IHC image as input and directly outputs the H-score within a few seconds, significantly enhancing the speed of IHC image analysis. This automated tool provides H-score quantification with precision and consistency comparable to experienced pathologists but at a significantly reduced cost during IHC diagnostic workups. It holds significant potential to advance biomedical research reliant on IHC staining for protein expression quantification.

Effects of amyloid ß (Aß)42 and Gasdermin D on the progression of Alzheimer's disease in vitro and in vivo through the regulation of astrocyte pyroptosis.

PURPOSE: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid ß (Aß) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms. METHODS: In vivo, 5 µL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aß42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry. RESULTS: Aß42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1ß, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 ß and IL-18, reduced Aß deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice. CONCLUSION: Aß42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease. PURPOSE: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid ß (Aß) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms. METHODS: In vivo, 5 µL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aß42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry. RESULTS: Aß42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1ß, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 ß and IL-18, reduced Aß deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice. CONCLUSION: Aß42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease.

MiR-590-3p Promotes the Phenotypic Switching of Vascular Smooth Muscle Cells by Targeting Lysyl Oxidase.

ABSTRACT: We investigated the clinical characteristics of patients with acute aortic dissection (AAD) and miR-590-3p levels in serum, tissue, and vascular smooth muscle cells. The effect of miR-590-3p on the vascular smooth muscle cell phenotype was assessed, and the regulation of lysyl oxidase by miR-5903p was determined. C57BL/6 mice were used to investigate the incidence of AAD and effects of miR-5903p on AAD. The miR-590-3p levels were measured in the aortae of mice, and hematoxylin and eosin staining and Masson staining were performed to identify the morphological features of the aorta. Comparative analysis revealed significant differences in clinical characteristics between patients with AAD and healthy control subjects, with most patients with AAD exhibiting concomitant hypertension and nearly 50% having atherosclerosis. Lysyl oxidase was a direct target of miR-590-3p. Lysyl oxidase overexpression inhibited switching of the vascular smooth muscle cell phenotype from contractile to synthetic, but miR-590-3p overexpression significantly reversed this change. In the mouse model, miR-590-3p upregulation increased the incidence of AAD to 93.3%, and its incidence decreased to 13.3% after miR-590-3p inhibition. Hematoxylin and eosin and Masson staining revealed that the miR-590-3p agomiR group had a greater loss of the contractile phenotype in the dissected aortic wall and an increased number of muscle fibers in the aortic wall, which contributed to thickening of the aortic wall and the formation of a false lumen in aortic dissection. miR-590-3p might be pivotal in the pathogenesis of AAD. Thus, targeting miR-590-3p or its downstream pathways could represent a therapeutic approach for AAD.

Association of Nonpuerperal Mastitis with Cytokines Related to Helper T Cells TH1/TH2 and TH17/Treg.

Objective: To investigate the association of nonpuerperal mastitis with cytokines related to the helper T cells TH1/TH2 and TH17/Treg and associated immune balance. Methods: From 2016 to 2021, we included 40 patients with non-puerperal mastitis who underwent surgery at China-Japan Friendship Hospital and compared them with 40 control patients with benign non-infectious breast disease. Hematoxylin-eosin staining detects inflammatory infiltrates of breast tissue. The expression of interferon γ and interleukin 4 in breast tissue was detected by immunofluorescence imaging, and the relative protein expression of TH1/TH2 and TH17/Treg cell-associated cytokines in CD4+ T cells was detected by western blotting. CD4+ T cells were isolated by fluorescence-activated cell sorting for detection of the relative protein expression of interferon γ and interleukin 4 in CD4+ T cells. Results: Hematoxylin-eosin staining showed that the nonpuerperal mastitis group had significantly greater inflammatory infiltration than the control group. Immunofluorescence images showed the relative fluorescence intensity of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative fluorescence intensity of interleukin 4 did not significantly differ between the 2 groups (P = .0686). Western blotting revealed that the relative protein expression of interferon γ, interleukin 2, and interleukin 17 was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 (P = .0512), interleukin 10 (P = .3088), and transforming growth factor ß (P = .0653) did not significantly differ between the 2 groups. Flow cytometry of isolated CD4+ T cells showed the relative protein expression of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 did not significantly differ between the 2 groups (P = .0680). Conclusion: The expression of the TH1 cytokines interferon γ and interleukin 2 and the TH17 cytokine interleukin 17 was significantly higher in patients with nonpuerperal mastitis, while the TH2 cytokine interleukin 4 and the Treg cytokines interleukin 10 and transforming growth factor ß were expressed at lower levels. This study provides new research ideas for the treatment of mastitis.

Lupenone improves type 2 diabetic nephropathy by regulating NF-κB pathway-mediated inflammation and TGF-ß1/Smad/CTGF-associated fibrosis.

BACKGROUND: Type 2 diabetic nephropathy is a common diabetic complication and the main cause of death in patients with diabetes. Research has aimed to find an ideal drug with minimal side effects for treating this disease. Banana peel has been shown to be anti-diabetic, with lupenone isolated from banana peel exhibiting antidiabetic and anti-inflammatory activities; However, the effects of lupenone on type 2 diabetic nephropathy are largely unknown. PURPOSE: This study aimed to investigate the ameliorative effect of lupenone on type 2 diabetic nephropathy, and its mechanism from both anti-inflammatory and anti-fibrotic perspectives. METHODS: Spontaneous type 2 diabetic nephropathy db/db mouse models were given three levels of lupenone (24 or 12 or 6 mg/kg/d) via intragastric administration for six weeks, and irbesartan treatment was used for the positive control group. We explored the effects and mechanism of lupenone action using enzyme-linked immunosorbent assay, automatic biochemical analyzer, hematoxylin-eosin and Masson staining, real time-PCR, and western blotting. Concurrently, a high-sugar and high-fat diet combined with a low-dose streptozotocin-induced type 2 diabetic nephropathy rat model was used for confirmatory research. RESULTS: Lupenone administration maintained the fasting blood glucose; reduced glycosylated hemoglobin, insulin, and 24 h proteinuria levels; and markedly regulated changes in biochemical indicators associated with kidney injury in serum and urine (including 24 h proteinuria, micro-albumin, N-acetyl-ß-d-glucosaminidase, α1-micro-globulin, creatinine, urea nitrogen, uric acid, total protein, and albumin) of type 2 diabetic nephropathy mice and rats. Hematoxylin-eosin and Masson staining as well as molecular biology tests revealed that inflammation and fibrosis are the two key processes affected by lupenone treatment. Lupenone protected type 2 diabetic nephropathy kidneys by regulating the NF-κB-mediated inflammatory response and TGF-ß1/Smad/CTGF pathway-associated fibrosis. CONCLUSION: Lupenone has potential as an innovative drug for preventing and treating diabetic nephropathy. Additionally, it has great value for the utilization of banana peel resources.

Salidroside Inhibits Ganglion Cell Apoptosis by Suppressing the Müller Cell Inflammatory Response in Diabetic Retinopathy.

PURPOSE: This study aimed to investigate the role of salidroside (SAL) in the cellular communication between Müller cells and retinal ganglion cells in diabetic mice. METHODS: The diabetes mellitus (DM) animal models were established by the intraperitoneal injection of streptozotocin and treatment with SAL via gavage or by the injection of IL-22BP into the vitreous cavity. Immunohistochemistry was used to measure the expression of the glial fibrillary acidic protein in Müller cells. The expression of IL-22 and IL-22Rα1 in retinal tissues was assessed by immunofluorescence. Western blotting was used to measure the expression of inflammatory and apoptosis-related proteins. Hematoxylin-eosin staining, TUNEL staining, and flow cytometry were used to analyze the apoptosis of retinal ganglion cells. The effect of cellular interactions was explored by Transwell assays. RESULTS: Western blotting showed that glial fibrillary acidic protein, IL-22 protein expression was significantly upregulated in the DM animal models compared with the control mice. Immunofluorescence showed that IL-22 was highly expressed in Müller cells and IL-22Rα1 was expressed in ganglion cells in the retina of DM mice. Hematoxylin-eosin and TUNEL staining results showed an increase in the number of ganglion cells apoptotic in DM. However, SAL reversed these phenomena. Meanwhile, after coculture with Müller cells, Western blotting suggested that ganglion cells secreted p-STAT3, and c-caspase3 protein expression was increased. More interestingly, the treatment of IL-22BP and SAL inhibited the expression of the p-STAT3 and c-caspase3 proteins. Flow cytometry indicates that compared with the control group, the apoptosis rate of ganglion cells was increased in the high glucose group, while the apoptosis rate of cells in the recombinant IL-22 protein group was significantly increased, while the SAL inhibited ganglion cells apoptosis. CONCLUSION: SAL inhibits the apoptosis of retinal ganglion cells via the IL-22/STAT3 pathway in Müller cells.

Immune activity of Ki-67 during nasal polyp development.

OBJECTIVE: Nasal polyps are non-cancerous, soft painless growth of nasal mucosa. In this study, our aim was to investigate the Ki-67 expression level in nasal polyps by immunohistochemical method. PATIENTS AND METHODS: 30 patients with nasal polyps were included in this study. Nasal polyps were processed for paraffin wax embedding protocol. Samples were fixed and embedded in paraffin blocks. 5 µm sections were stained with Hematoxylin-Eosin dye and immune stained with Ki-67 antibody. Sections were analyzed under light microscope. RESULTS: Blood parameters showed that white blood cells, hematocrit and platelet were higher than normal range. In sections of hematoxylin-eosin staining, elevated basal cells, thin basement membrane, leukocyte infiltration, collagen fibers degeneration were observed. Masson trichrome staining revealed that degenerative epithelial cells, detached basement membrane and edema were observed. Ki-67 expression was observed in mucosal epithelial cells, vascular endothelial cells and plasma cells in immune staining. CONCLUSIONS: Epithelial degeneration in nasal polyps and leukocyte infiltration induce nasal adenoma. Ki-67 expression may be a diagnostic tool for epithelial leukocyte formation.

Study of the nerve endings and mechanoreceptors of the medial meniscotibial ligament of the knee: A structural and distribution analysis.

BACKGROUND: The aim of the present study is to describe the morphology and distribution of the nerve endings of the meniscotibial ligament (MTL) of the knee, in order to understand the interaction between the proprioceptive system and knee mechanics. METHODS: Twenty medial MTLs were obtained from deceased organ donors. The ligaments were measured, weighed and cut. Sections (10 mm) were prepared on hematoxylin and eosin-stained slides for analysis of tissue integrity, and 50 mm sections were submitted to immunofluorescence with the protein gene product (PGP) 9.5 as primary antibody and Alexa Fluor 488 as secondary antibody, followed by microscopic analysis. RESULTS: The medial MTL was identified in 100% of the dissections, with average length, width, thickness and weight of 7.07 ± 1.34 mm, 32.25 ± 3.09 mm, 3.53 ± 0.27 mm and 0.67 ± 0.13 g, respectively. The hematoxylin and eosin-stained histological sections exhibited typical ligament structure, with dense well-organized collagen fibers and vascular tissue. All the specimens analyzed contained type I (Ruffini) mechanoreceptors and free (type IV) nerve endings, varying from parallel to intertwined fibers. Nerve endings not classified with different irregular shapes were also found. Most type I mechanoreceptors were found close to the MTL insertions on the tibial plateau, while the free nerve endings were found adjacent to the capsule. CONCLUSION: The medial MTL showed a peripheral nerve structure, primarily type I and IV mechanoreceptors. These findings suggest that the medial MTL is important for proprioception and medial knee stabilization.

Involvement of a novel cAMP signaling mediator for beige adipogenesis.

BACKGROUND: Exposure to cold temperature stimulates the sympathetic nervous system that activates ß-adrenergic receptor signals in brown and beige adipocytes, leading to the induction of adaptive thermogenesis in mammals. Prominin-1 (PROM1) is a pentaspan transmembrane protein that is widely identified as a marker for stem cells, although the role of this protein as a regulator of many intracellular signaling cascades has been recently delineated. The main focus of the current study is to identify the previously unknown role of PROM1 in beige adipogenesis and adaptive thermogenesis. METHODS: Prom1 whole body knockout (Prom1 KO) mice, Prom1 adipogenic progenitor (AP) cell-specific knockout (Prom1 APKO) mice and Prom1 adipocyte-specific knockout (Prom1 AKO) mice were constructed and were subject for the induction of adaptive thermogenesis. The effect of systemic Prom1 depletion was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. Flow cytometric analysis was performed to determine the identity of PROM1-expressing cell types, and the resultant cells were subject to beige adipogenesis in vitro. The potential role of PROM1 and ERM in cAMP signaling was also assessed in undifferentiated AP cells in vitro. Finally, the specific effect of Prom1 depletion on AP cell or mature adipocytes on adaptive thermogenesis was evaluated by hematoxylin and eosin staining, immunostaining, and biochemical analysis in vivo. RESULTS: Prom1 KO mice displayed an impairment in cold- or ß3-adrenergic agonist-induced adaptive thermogenesis in subcutaneous adipose tissues (SAT) but not in brown adipose tissues (BAT). By fluorescence-activated cell sorting (FACS) analysis, we identified that PROM1 positive cells are enriched in PDGFRα+Sca1+ AP cells from SAT. Interestingly, Prom1 knockout stromal vascular fractions showed reduced PDGFRα expression, suggesting a role of PROM1 in beige adipogenic potential. Indeed, we found that Prom1-deficient AP cells from SAT showed reduced potential for beige adipogenesis. Furthermore, AP cell-specific depletion of Prom1, but not adipocyte-specific depletion of Prom1, displayed defects in adaptive thermogenesis as evidenced by resistance to cold-induced browning of SAT and dampened energy expenditure in mice. CONCLUSION: We found that PROM1 positive AP cells are essential for the adaptive thermogenesis by ensuing stress-induced beige adipogenesis. Identification of PROM1 ligand might be useful in the activation of thermogenesis that could be potentially beneficial in combating obesity.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.

Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xianglian Pill (XLP) is a classical Chinese medicine prescription applied for controlling ulcerative colitis (UC). Whereas, the underlying mechanism remains unclear. AIM OF THE STUDY: The present work was aimed to investigate the mechanism of XLP in dextran sulfate sodium (DSS)-induced UC via the Toll Like Receptor 4 (TLR4)/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-B (NF-κB) signaling in mice. MATERIALS AND METHODS: The major components of XLP were detected by high-performance liquid chromatography-diode array detection (HPLC-DAD). The ulcerative colitis model was induced by DSS in mice. 5-Amino Salicylic Acid (5-ASA) group and XLP group were intragastrically treated. Disease activity index (DAI) and colon length were monitored and hematoxylin-eosin (HE) staining was conducted. Gasdermin D (GSDMD)-N and TLR4 expressions in colon tissues were visualized by immunofluorescence. TLR4 mRNA was measured by Real Time Quantitative PCR (RT-qPCR). The expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), active-caspase-1, GSDMD-N, TLR4, MYD88, NF-κB, p-NF-κB, and the ubiquitination of TLR4 in colon tissues were detected by Western blot. Myeloperoxidase (MPO) enzyme activity was examined and serum inflammatory factors Interleukin (IL)-1ß, IL-6, Tumor Necrosis Factor-α (TNF-α), and IL-18 were determined by Enzyme-linked Immunosorbent Assay (ELISA). TLR4-/- mice were applied for verifying the mechanism of XLP attenuated DSS symptoms. RESULTS: The XLP treatment extended colon length, reduced DAI, and attenuated histopathological alteration in DSS-induced mice. XLP administration suppressed MPO activity and reduced the content of IL-1ß, IL-6, TNF-α and IL-18 in serum. XLP also inhibited the expression levels of GSDMD-N, TLR4, NLRP3, active-caspase-1, MyD88, p-NF-κB/NF-κB in colon tissues of DSS-induced mice. TLR4-/- mice proved that TLR4 was involved in XLP-mediated beneficial effect on DSS-induced ulcerative colitis. CONCLUSIONS: XLP might treat ulcerative colitis by regulating the TLR4/MyD88/NF-κB signaling pathway.

Swimming exercise and nano-l-arginine supplementation improve oxidative capacity and some autophagy-related genes in the soleus muscle of aging rats.

The present research aims to evaluate the effect of swimming exercise and chitosan-coated l-arginine on mitochondrial oxidation, BCL2 Interacting Protein 3 (Bnip3), NIP-like protein × (Nix), B-cell lymphoma-extra-large (Bcl-xL) and autophagy-related protein light chain 3(LC3) expression in soleus muscle of aging rats. In this experimental research, 25 male Wistar rats were assigned into five groups randomly: young, old, old + Nano l-arginine (Nano L-a), old + exercise (Ex), and old + Nano l-arginine (Nano L-a) + exercise (Ex) (n = 5 in each). They performed a swimming exercise program five days a week for six weeks. To determine the relative strength for rats before and after performing these interventions, the 1repetition maximum (1RM) test was done as a pre and post-test. The exercise program started with 20 min and after four sessions, gradually increased to 60 min and this time was maintained until the completion of the training period. l-arginine coated with chitosan nanoparticles was given to the rats in the l-arginine-supplemented group via gavage at a dosage of 500 mg/kg/day, five days a week, for six weeks. Additionally, the rats in all groups were fed a normal diet (2.87 kcal/g and 15 % energy from fat). Upon the completion of the protocol implementation, the rats were sacrificed and the soleus muscle was fixed and frozen to determine hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), gene expression analysis, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC). The results from the present research indicated that swimming exercise and Nano l-arginine improve the strength and histology of muscle tissue in old rats (p < 0.05). Aging significantly increased the expression of Nix and Bnip3 (p < 0.05) and reduced the Bcl-xL gene expression (p < 0.05). The expression of LC3 protein also increased with aging (p < 0.05). Therapeutic interventions, such as combined treatment (old + Nano L-a + Ex) for old animals, reduced the amount of this protein in soleus muscle (p < 0.05). The ROS values also showed a significant reduction only in the old + Nano L-a + Ex group compared to the old group. Moreover, TAC values show a significant decrease in the old and old + Ex groups in comparison to the young group. The use of arginine supplement, especially in nano form, along with swimming exercise seems to reduce the oxidative damage to the elderly muscle tissue, which has a positive effect on the structure and function of the soleus muscle. Since these interventions only had a significant effect on LC3 protein, further studies with more diverse measurement methods for autophagy are suggested.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect. AIM OF THE STUDY: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). RESULTS: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. CONCLUSION: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Analysis of the relationship between placental histopathological aspects of preterm and term birth.

OBJECTIVES: This study aims to establish a correlation between placental histopathological and immunohistochemical (IHC) changes and preterm birth with fetal growth restriction (FGR, formerly called intrauterine growth restriction - IUGR). PATIENTS, MATERIALS, AND METHODS: This prospective study was performed on a group of 30 parturients, with singleton gestation, of which 15 patients gave birth at term, and the other 15 patients gave birth prematurely. After the statistical correlation of the clinical and demographic data with premature birth (PB) and term birth (TB), we performed histological and IHC research on the respective placentae. To observe normal and pathological microscopic placental structures, we used the Hematoxylin-Eosin (HE) and Periodic Acid Schiff-Hematoxylin (PAS-H) classical stainings, but also special immunostaining with anti-cluster of differentiation 34 (CD34) and anti-vascular endothelial growth factor (VEGF) antibodies. RESULTS: We found a statistically significant difference between the TB∕PB categories and the age of the patients, their antepartum weight, the weight of the newborns, and the placenta according to the sex of the newborn. Histological analysis revealed in the case of TB, small areas of perivillous amyloid deposition, with the significant extension of these areas both intravillous and perivillous in the case of PB. Massive intravillous calcifications, syncytial knots, and intravillous vascular thrombosis were also frequently present in PB. With PAS-H staining were highlighted the intra∕extravillous vascular basement membranes, but especially the massive fibrin deposits rich in glycosaminoglycans. By the IHC technique with the anti-CD34 antibody, we noticed the numerical vascular density, higher in the case of TB, but in the case of PB, there were large areas of placental infarction, with a lack of immunostaining in these areas. Through the anti-VEGF antibody, we observed the presence of signal proteins that determined and stimulated the formation of neoformation vessels in the areas affected by the lack of post-infarction placental vascularization. We observed a highly significant difference between placental vascular density between TB∕PB and newborn weight, sex, or placental weight. CONCLUSIONS: Any direct proportional link between the clinical maternal-fetal and histological elements yet studied must be considered. Thus, establishing an antepartum risk group can prevent a poor pregnancy outcome.

Enhanced Apoptosis in Choroidal Tissues in Lens-Induced Myopia Guinea Pigs by Activating the RASA1 Signaling Pathway.

Purpose: This study aimed to explore the role of the RAS p21 protein activator 1 (RASA1) signaling pathway in apoptosis in choroid tissues from guinea pigs with negative lens-induced myopia (LIM). Methods: Biometric measurements were performed to examine refractive status, ocular parameters, and choroidal thickness (ChT) after myopia induction. The choroidal morphology was observed by hematoxylin and eosin (H&E) staining and TUNEL assay. The expression of the RASA1 signaling pathway at the mRNA and protein levels in choroidal tissues was measured by real-time quantitative PCR (qPCR) and western blot assays. Results: Compared with the normal control (NC) group, the ocular length of the guinea pigs in LIM increased remarkably, as did the myopic refraction. ChT decreased after myopia induction. H&E staining showed that the thickness and laxity of the choroidal tissues in LIM were strikingly reduced. The number of apoptotic cells in the LIM eyes was increased. Moreover, qPCR and western blot assays showed that the expression levels of both RASA1 and BCL-2-associated agonist of cell death (BAD) were higher in the LIM group than in the NC group, whereas the expression level of B-cell lymphoma 2 (BCL-2) was decreased after 2 weeks of experimental myopia. However, the trend of RASA1, BAD, and BCL-2 expression was reversed after 4 weeks of experimental myopia compared with levels after 2 weeks of experimental myopia. Conclusions: Results showed that the RASA1 signaling pathway is activated in choroid tissues in myopic guinea pigs. Activated RASA1 signaling induces high BAD expression and low BCL-2 expression, which in turn promotes apoptosis and ultimately causes ChT thinning in myopic guinea pigs.

[Kindlin-2 regulates endometrium development via mTOR and Hippo signaling pathways in mice].

OBJECTIVE: To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice. METHODS: Cdh16-Cre tool mice and Kindlin-2flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway. RESULTS: The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated. CONCLUSION: Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.

miR-124-3p sabotages lncRNA MALAT1 stability to repress chondrocyte pyroptosis and relieve cartilage injury in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a prevalent inflammatory joint disorder. microRNAs (miRNAs) are increasingly involved in OA. AIM: Our study is proposed to clarify the role of miR-124-3p in chondrocyte pyroptosis and cartilage injury in OA. METHODS: OA mouse model was established via the treatment of destabilization of the medial meniscus (DMM), and the in vitro cell model was also established as mouse chondrocytes were induced by lipopolysaccharide (LPS). Mouse cartilage injury was assessed using safranin-O-fast green staining, hematoxylin-eosin staining, and OARSI grading method. Expressions of miR-124-3p, MALAT1, KLF5, and CXCL11 were determined. Cartilage injury (MMP-13, osteocalcin), inflammation (IL-6, IL-2, TNF-, IL-1ß, and IL-18)- and pyroptosis-related factors (Cleaved Caspase-1 and GSDMD-N) levels were detected. Mechanically, MALAT1 subcellular localization was confirmed. The binding relationships of miR-124-3p and MALAT1 and MALAT1 and KLF5 were verified. MALAT1 half-life period was detected. Then, miR-124-3p was overexpressed using agomiR-124-3p to perform the rescue experiments with oe-MALAT1 or oe-CXCL11. RESULTS: miR-124-3p was downregulated in DMM mice and LPS-induced chondrocytes where cartilage injury, and increased levels of inflammation- and pyroptosis-related factors were found. miR-124-3p overexpression relieved cartilage injury and repressed chondrocyte pyroptosis. miR-124-3p bounds to MALAT1 to downregulate its stability and expression, and MALAT1 bounds to KLF5 to enhance CXCL11 transcription. Overexpression of MALAT1 or CXCL11 annulled the repressive function of miR-124-3p in chondrocyte pyroptosis. CONCLUSION: miR-124-3p reduced MALAT1 stability and inhibited the binding of MALAT1 and KLF5 to downregulate CXCL11, thereby suppressing chondrocyte pyroptosis and cartilage injury in OA.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes.

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.

Spatial transcriptomics tools allow for regional exploration of heterogeneous muscle pathology in the pre-clinical rabbit model of rotator cuff tear.

BACKGROUND: Conditions affecting skeletal muscle, such as chronic rotator cuff tears, low back pain, dystrophies, and many others, often share changes in muscle phenotype: intramuscular adipose and fibrotic tissue increase while contractile tissue is lost. The underlying changes in cell populations and cell ratios observed with these phenotypic changes complicate the interpretation of tissue-level transcriptional data. Novel single-cell transcriptomics has limited capacity to address this problem because muscle fibers are too long to be engulfed in single-cell droplets and single nuclei transcriptomics are complicated by muscle fibers' multinucleation. Therefore, the goal of this project was to evaluate the potential and challenges of a spatial transcriptomics technology to add dimensionality to transcriptional data in an attempt to better understand regional cellular activity in heterogeneous skeletal muscle tissue. METHODS: The 3' Visium spatial transcriptomics technology was applied to muscle tissue of a rabbit model of rotator cuff tear. Healthy control and tissue collected at 2 and 16 weeks after tenotomy was utilized and freshly snap frozen tissue was compared with tissue stored for over 6 years to evaluate whether this technology is retrospectively useful in previously acquired tissues. Transcriptional information was overlayed with standard hematoxylin and eosin (H&E) stains of the exact same histological sections. RESULTS: Sequencing saturation and number of genes detected was not affected by sample storage duration. Unbiased clustering matched the underlying tissue type-based on H&E assessment. Connective-tissue-rich areas presented with lower unique molecular identifier counts are compared with muscle fibers even though tissue permeabilization was standardized across the section. A qualitative analysis of resulting datasets revealed heterogeneous fiber degeneration-regeneration after tenotomy based on (neonatal) myosin heavy chain 8 detection and associated differentially expressed gene analysis. CONCLUSIONS: This protocol can be used in skeletal muscle to explore spatial transcriptional patterns and confidently relate them to the underlying histology, even for tissues that have been stored for up to 6 years. Using this protocol, there is potential for novel transcriptional pathway discovery in longitudinal studies since the transcriptional information is unbiased by muscle composition and cell type changes.