Masson's Trichrome Staining Research Articles

Identification of a Regenerative Protocol for Recellularizing Human Auricular Cartilage Scaffolds.

Utilizing biological scaffolds for cartilage tissue engineering is a promising tool for improving auricular reconstruction. Decellularized auricular scaffolds provide a means of regenerating cartilage for in vivo implantation, but identifying the ideal regenerative mix remains challenging. Human cadaver auricular cartilage was decellularized and recellularized with either auricular chondrocytes alone, auricular chondrocytes with adipose-derived stem cells, or both cells with platelet-rich plasma. Confirmation of decellularization and recellularization was done by hematoxylin and eosin staining. Extracellular matrix preservation and production were determined by Masson's trichrome, Alcian blue, and Verhoeff-van Gieson staining. Collagen II assessments were made using immunohistochemistry. Decellularization of cadaver auricular cartilage was confirmed by the absence of cells, reduction in glycosaminoglycans, and the preservation of collagen and elastin. Recellularization was more efficient when chondrocytes were seeded with adipose-derived stem cells, which was enhanced by adding platelet-rich plasma. Coculture with platelet-rich plasma yielded better total collagen (56% increase) and glycosaminoglycan (47% increase) induction. Moreover, when platelet-rich plasma was added, collagen II induction was significantly increased (42%; P < 0.05). We identified a regenerative protocol that included auricular chondrocytes, adipose-derived stem cells, and platelet-rich plasma, which stimulated chondrogenesis on decellularized auricular cartilage. This finding provides a model to explore cartilage formation and the potential for improving auricular and cartilage-based reconstruction.

CSWT improves ventricular function by promoting angiogenesis through Rho/ROCK/ERK1/2 signal pathway in infarcted myocardium in rats

Abstract Objective This study aimed to investigate the effects of extracorporeal cardiac shock wave therapy (CSWT) on myocardial ischemia and cardiac function in rats with myocardial infarction, and to explore its impact on the expression of myocardial vascular endothelial growth factor and the extent of myocardial fibrosis, thereby elucidating the mechanism of angiogenesis. Methods A rat model of myocardial infarction was induced by ligating the left anterior descending branch of the coronary artery, confirmed by echocardiography. Animals were randomly assigned to five groups: sham operation group, myocardial infarction group, myocardial infarction + CSWT group, myocardial infarction + Y-27632 group, and myocardial infarction + CSWT + Y-27632 group. Cardiac function was evaluated by echocardiography on the 16th and 72nd day post-surgery. Myocardial morphology was assessed by HE staining, and the extent of myocardial fibrosis was determined by Masson's trichrome staining. Immunohistochemical staining for factor VIII was conducted to observe angiogenesis in each infarcted area. Western blotting was employed to detect the protein expression of extracellular regulated kinase (ERK1/2) and pro-angiogenic cytokines in the infarct border zone. Results CSWT treatment significantly reduced myocardial fibrosis and improved cardiac function compared to the myocardial infarction group. Additionally, CSWT upregulated ERK expression and angiogenesis-related factors, promoting angiogenesis. The beneficial effects of CSWT were abolished by inhibiting the Rho/ROCK/ERK1/2 pathway. Furthermore, the area of myocardial fibrosis was significantly reduced in the MI+Y-27632 group compared to the myocardial infarction group (p &amp;lt; 0.01), and left ventricular systolic function index was significantly higher in the myocardial infarction group. Conclusions CSWT may promote angiogenesis post-myocardial infarction by activating the Rho/ROCK/ERK1/2 pathway. Additionally, Y-27632 may improve cardiac function in rats with myocardial infarction, potentially through mitigating myocardial fibrosis.echo images of ratsprotein expression by WB

Complement factor D deficiency ameliorates adverse cardiac remodelling by inhibiting C5a-C5aR mediated ISGs+ neutrophil activation post myocardial infarction

Abstract Background Exaggerated inflammatory response after myocardial infarction (MI) exacerbates myocardial injury and adverse cardiac remodeling, leading to heart failure. Current management of MI involves myocardial protection and antifibrotic therapy but do not target the inflammatory response per se. Complement factor D (CFD) is an upstream rate-limiting enzyme of complement system activation, which is the early event of inflammatory response post MI. We hypothesize that CFD can be an important modulator of the inflammation-remodeling cascade post MI, but the specific mechanism remains elusive. Purpose To investigate the role and mechanism of CFD in regulating inflammatory expansion and adverse remodeling post MI. Methods We generated Cfd-AKO (Cfdfl/fl Adipoq-cre) mice using the Cre-LoxP recombination system, as CFD is mainly secreted by adipocytes. Immune status and cardiac remodeling progress were assessed by cardiovascular magnetic resonance (CMR), histology, flow cytometry, immunofluorescence, and ELISA. Single cell RNA sequencing (scRNA-seq) analyzed non­cardiomyocytes isolated from Cfd-AKO and WT mice post MI or sham operation. Genetic ablation and co-culture experiments dissected the functional molecular properties and intercellular communication. An CFD inhibitor ACH-4471 was used to explore the role of CFD in ischemia-reperfusion (IR) injury. Results The survival of Cfd-AKO mice was strongly improved, while ventricular arrhythmia induction rates and number of episodes were significantly reduced compared with those of WT mice. CMR imaging showed that Cfd-AKO mice had higher ejection fraction and reduced left ventricular dilation than WTs. Masson trichrome and picrosirius red staining revealed that besides smaller infarct size, the extent of fibrosis in the border and distal remote area decreased in Cfd-AKO mice. Cfd loss reduced myocardial injury, inhibited the release of inflammatory factors, and limits the amplification of complement components C3a and C5a post MI. ScRNA-seq revealed that CFD modulated neutrophil chemotactic ability and pro-inflammatory function by inducing interferon-stimulated genes expressing (ISG+) neutrophil CXCL10 expression. Mechanistically, CFD activates the ISG transcriptional profile via the C5a/C5aR dependent STAT1/2-IRF9 pathway. Activated neutrophils promote myofibroblast transformation through IL1β/IL1R dependent intercellular communication, thus facilitating pathological fibrosis. Early pharmacological blocking of CFD in mice IR injury models inhibited inflammation, reduced infarct size and ameliorated cardiac dysfunction. Conclusions We demonstrate the crucial role of CFD in cardiac remodeling post MI. This process is critically regulated by C5a-C5aR, an important component of complement-inflammation cascade. This CFD-C5a mediated neutrophil pro-inflammatory and pro-fibrotic dual activity represents a novel pathway and an attractive therapeutic window for heart failure post MI.CFD in cardiac remodelling post MI

Heritable heart failure traits in mice undergoing early life stress

Abstract Introduction Adverse childhood experiences, known as early life stress (ELS), are associated with increased cardiovascular disease risk in later life, yet the underlying mechanisms remain elusive. Recent evidence indicates that parental life experiences can be transmitted to the offspring. Whether ELS exerts transgenerational effects on the heart is unknown. Purpose To investigate the effects of ELS on cardiac structure and function in exposed parents and in their offspring, across 3 generations. Methods We used a mouse model of ELS (MSUS) that mimics the exposure to traumatic experiences during childhood in humans. Mouse pups (F1) were separated from their mother (F0) unpredictably each day for 3 hours from postnatal day 1 (PND1) to PND14. During separation, dams were exposed to an additional unpredictable stressor by being subjected to either, a forced swim (18°C water for 5 minutes) or a 20-minute physical tube restraint, anytime (unpredictably) during the 3 hours. From PND15, mice are left undisturbed with their mother until PND21, are then raised normally until adulthood. Control litters were raised normally. A high-resolution Micro-Ultrasound System (Vevo 3100, Visualsonics) was used for the echocardiographic assessment at 6, 12 and 18 months both in the exposed animals (F0) as well as in their offspring (F1) and grandoffspring (F2). Both male and female mice were studied. Heart weight/tibia length and left ventricle weight/tibia length were used to assess cardiac mass while myocardial fibrosis was assessed by Masson's Trichrome and Picriosirius Red stainings. Lung congestion was assessed as lung wet/dry weight ratio. Single cell RNA sequencing (scRNAseq) was used in LV specimens from MSUS mice to unveil cell-specific transcriptional changes. Finally, MSUS mice underwent environmental enrichment (cages containing running wheels, maze) to investigate any rescuing effect on cardiac function. Results F1 mice displayed increased LV mass, impaired diastolic function, myocardial fibrosis and lung congestion. A time-dependent worsening of cardiac performance was observed from 6 to 18 months, both in males and females. ScRNAseq unveiled dysregulation of transcriptional programs underlying inflammation and lipotoxicity in the cardiomyocyte and endothelial cell clusters. The MSUS offspring did not show changes of cardiac function at 6 months, however diastolic dysfunction and lung congestion were observed at 12 and 18 months. A similar impairment of cardiac function was observed in the MSUS grandoffspring (F3). Of interest, 6-week exposure to an environmental enrichment protocol was able to improve LV mass, diastolic function and lung congestion in 12 months-old MSUS mice. Conclusions ELS induces a transgenerational transmission of cardiac phenotypic alterations which can be rescued by EE. Our results shed light on the potential role of ELS on heart failure development and potential mitigation strategies.

Allicin Ameliorated High-glucose Peritoneal Dialysis Solution-induced Peritoneal Fibrosis in Rats via the JAK2/STAT3 Signaling Pathway.

Peritoneal fibrosis (PF) is one of the most serious complications of peritoneal dialysis (PD) and is the greatest obstacle to the clinical application of PD. Chinese herbal monomers have been effective in the prevention and treatment of PF. The aim of this study was to observe the effect of allicin on PF in rats induced by high glucose and to investigate its molecular mechanism of action. A rat model of PF was established by using a 4.25% glucose-based standard peritoneal dialysis solution. The degree of peritoneal pathological damage was assessed by Hematoxylin and eosin (H&E) staining. Peritoneal collagen deposition was detected by Masson's trichrome staining. The levels of Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in the serum were measured by Enzyme Linked Immunosorbent Assay (ELISA). The expression levels of TGF-β, α-smooth muscle actin (α-SMA) and collagen I were examined by western blotting and immunohistochemistry. The protein expression levels and mRNA levels of E-cadherin, N-cadherin, vimentin, janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in peritoneal tissue were determined by western blotting and qRT-PCR. TGF-β1 stimulated human peritoneal mesothelial cells (HPMCs), and the cells were treated with allicin and the JAK2/STAT3 pathway activator colivelin alone or in combination. A cell counting kit-8 (CCK-8) assay was used to measure cell viability. The role of JAK2/STAT3 in the effects of allicin was confirmed via in vitro mechanistic research by western blotting, wound healing assays and Transwell assays. Allicin relieves the inflammatory response by downregulating the levels of IL-1β, IL-6, MCP-1 and TNF-α. Furthermore, allicin decreased the expression of TGF-β, α-SMA and collagen I. Allicin also alleviated epithelial-to-mesenchymal transition (EMT), as specifically manifested by increased E-cadherin and reduced N-cadherin and vimentin. Further studies revealed that allicin reduced the protein levels of JAK2, STAT3, p-JAK2, and p-STAT3. The results of the cellular experiments verified the above results. The ability of allicin to inhibit fibrosis and the EMT process was significantly attenuated after HPMCs were treated with colivelin. Taken together, these findings suggest that allicin inhibits inflammation and EMT, thereby improving PF, and this protective effect may be achieved by inhibiting the JAK2/STAT3 signaling pathway.

Effect of Local Zoledronic Acid Application in Alveolar Bone Healing: An Experimental Study.

This experimental study aimed to assess the three-dimensional and histological changes of the alveolar socket with local application of ZA. Nine male New Zealand white rabbits were randomly allocated into 4-, 8-, and 12-week groups after the extraction of 4 incisor teeth. The upper and lower right sockets were filled with ZA + collagen sponge (ZA-CS group), while the left sockets were filled with collagen sponge alone (CS group) postoperatively. At 4, 8, and 12 weeks (T1, T2, and T3) after the extraction, radiographic and histomorphometric evaluations were conducted for both groups. The increase in alveolar bone density of the ZA-CS group at T2 and T3 was significantly higher than the CS group (P＜0.01). Three-dimensional evaluations demonstrated no statistically significant differences in ridge height and width between the 2 groups at T2 and T3. On histological evaluation, the ZA-CS group included significantly fewer TRAP-positive cells than the CS group at T1 (P＜0.05). In ALP staining, all cases in both groups were classified as positive at each time point. Masson trichrome staining showed significantly higher mean red collagen volume fraction in the ZA-CS group (76.09%, 79.64%) than in the CS group (66.17%, 69.22%) at T2 and T3, respectively (P＜0.05 and ＜0.01). Although local ZA application with collagen sponge did not reduce alveolar ridge contraction, it improved the bone density and maturity of newly formed bone after tooth extraction.

Metformin Attenuates Vocal Fold Fibrosis via AMPK Signaling.

Vocal fold fibrosis is a challenging condition with no clear consensus on effective treatment methods. Given the demonstrated efficacy of metformin in treating various fibrotic diseases, we hypothesized that metformin could reduce vocal fold fibrosis via the AMPK signaling pathway. In our study, we induced vocal fold injury in rabbits and administered metformin intraperitoneally at a dose of 250mg/kg two weeks post-injury. Four weeks after the injury, vocal folds were excised and analyzed for fibrosis using Masson's trichrome staining, immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), and Western blotting. In vitro, vocal fold fibroblasts treated with metformin (10μM) ± TGF-β1 (10ng/mL) were utilized to assess metformin's antifibrotic effects, with Compound C (10μM) employed to inhibit AMPK signaling. Our results demonstrate that metformin significantly improved the structural integrity of the vocal fold lamina, reduced collagen deposition, and decreased the expression levels of COL1A1 and α-SMA. Furthermore, metformin activated the AMPK signaling pathway in vocal fold fibroblasts, resulting in decreased expression of COL1A1, α-SMA, TGF-β, Smad2, and Smad3. These findings suggest that metformin attenuates vocal fold fibrosis by modulating the AMPK signaling pathway, providing a foundation for developing new therapeutic options for vocal fold fibrosis.

Hydrogel encapsulating gold nanoparticles for targeted delivery of nitroglycerin to reduce post-cardiac dysfunction inflammation by inhibiting the Wnt/β-catenin signaling pathway.

The discovery of nitric oxide's role in biological processes like platelet function, vasodilation, cell permeability, and inflammation has advanced our understanding of organic nitrate therapy's hemodynamic and nonhemodynamic effects. Short-term use of organic nitrates prevents left ventricular enlargement and infarct expansion. However, information on their long-term impact on LV remodeling in post-acute cardiac dysfunction patients is limited. In this study, we utilized an innovative active hydrogel with gelatin (Gel)/polyethylene glycol (PEG)/polylactic acid (PLA) encapsulating gold nanoparticles (AuNPs)-based drug delivery system for the sustained release of nitroglycerin (NTG). Gel/PEG/PLA/NTG/AuNPs hydrogel-based system is a non-transplant surgical method that can adhere to the surface of the heart and deliver the drug directly to the epicardium. Cardiac dysfunction was induced by ligating the left anterior descending coronary artery. Echocardiograms were used to study the pre- and post-operative hemodynamics. Hematoxylin and eosin (H&E) and Masson's trichrome stain (MTS) stainingrevealed that the acute myocardial infarction (AMI) rats' group had irregularly shaped fibers and a lack of transverse striations, whereas Gel/PEG/PLA/NTG/AuNPs hydrogel group showed significant improvement. Rats in the Gel/PEG/PLA hydrogel group demonstrated marked vasodilation, compared to the AMI group. Mechanistically, we determined that hydrogel disrupts the initiation of post-cardiac dysfunction via inhibiting Wnt/β-catenin transcriptional activation. Hence, the Gel/PEG/PLA/NTG/AuNPs hydrogel group effectively protected against ischemic injury and inflammation in AMI, demonstrating a novel method for treating acute cardiac dysfunction.

Comparison of Different Types of Poly-L-lactic Acid Microspheres In Vitro and In Vivo Studies

Abstract Background Biodegradable polymers are commonly used as dermal fillers in plastic surgery. Among these, poly-L-lactic acid (PLLA) distinguishes itself owing to its good biocompatibility, degradability, and ability to act as a collagen stimulator. Objectives In this study, the differential behavior of PLLA microspheres with varying microscopic morphology and surface hydrophilicity was investigated both in vitro and in vivo. Methods The introduction of short hydrophilic polyethylene glycol (PEG) chains into the PLLA molecule was employed to modify the morphology and enhance the surface hydrophilicity of the microspheres. The morphology and physicochemical properties of the PLLA and PLLA-b-PEG microspheres were characterized. Irregular PLLA particles, PLLA and PLLA-b-PEG microspheres were implanted into the subcutaneous tissue of rabbit models, and at 4, 26 and 52 weeks after implantation, biopsy samples were collected for hematoxylin and eosin (H&amp;E) and Masson's trichrome staining to evaluate differences in the tissue response between different implants. Results The results of in vitro research demonstrated that while the addition of short-chain hydrophilic PEG afforded a smoother surface for the microspheres, it had no significant effect on the molecular weight and degradation rate of PLLA. The histological examination revealed that the PLLA-b-PEG microspheres exhibited enhanced biocompatibility compared to the pure PLLA microspheres, while the irregular PLLA particles showed the highest inflammatory response among the three materials. Conclusions In this study, we found that the properties of PLLA were improved upon modification by short-chain PEG without reducing the collagen regeneration ability, thereby affording a better histocompatibility.

Intervention effects of Naoxintong capsules on psychological and cardiac status in depressed rats after heart failure

Abstract Background Depression is a common clinical phenomenon in the patients with heart failure (HF). In traditional Chinese medicine (TCM), diseases in the brain and heart are thought to be correlated and interact. Naoxintong capsules (NXT) has been used for treating cardio-cerebrovascular diseases, while its therapeutic effect on depression after HF remains unclear. Objective The aim of the study is to evaluate the intervention effect of NXT on depression after HF. Methods Sprague-Dawley rats were assigned into the following 5 groups: sham, model, NXT (250, 1000 mg/kg), and valsartan (8 mg/kg). Coronary artery occlusion was performed to induce HF and subsequent depression in rats. The cardiac function was evaluated by echocardiography, hematoxylin-eosin staining, and Masson trichrome staining. The sucrose preference test and Morris water maze test were carried out to assess the depressive behaviors in rats. The ultrastructure of hippocampal CA1 neurons was observed and the levels of corticotropin-releasing hormone in the hypothalamus, brain-derived neurotrophic factor in the cortex, and adrenocorticotropic hormone (ACTH) in the plasma were determined by enzyme-linked immunosorbent assay. The levels of dopamine, 5-hydroxytryptamine, norepinephrine, and γ-aminobutyric acid in the hippocampus were measured by UPLC-QQQ-MS. Results NXT reduced myocardial injury and pathological changes in the cardiac tissue and increased the left ventricular ejection fraction, left ventricular fractional shortening, and cardiac output. NXT increased the sugar preference rate and number of crossings and shortened the escape latency. Furthermore, the NXT treatment restored the levels of corticotropin-releasing hormone, adrenocorticotropic hormone, brain-derived neurotrophic factor, dopamine, and γ-aminobutyric acid to the baseline values. Conclusions NXT not only demonstrates cardioprotective effect but also attenuates depression in the rats after HF. It may exert the antidepressant effect by inhibiting the hyperactivity of the hypothalamic-pituitary-adrenal axis and recovering the levels of neurotrophic factors and neurotransmitters.

Heat shock protein 70 promotes the progression of type 2 diabetic nephropathy by inhibiting T-cell immunoglobulin and mucin domain-3 and thereby promoting Th17/Treg imbalance.

Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. We aimed to investigate the role of regulatory T cells (Tregs) and helper T cells 17 (Th17) in the development and progression of DN. A mouse type 2 diabetic nephropathy (T2DN) model was established. Immunohistochemistry was used to detect the expression of HSP70 and Tim-3 in mouse kidney tissues, and western blotting was used to detect the expression levels of HSP70 and Tim-3. PAS staining and Masson's trichrome staining were used to detect the degree of kidney injury. Flow cytometry was used to detect the number of Th17 and Treg cells in blood and kidney tissues. The expression levels of interleukin 17 (IL-17) and interleukin 10 (IL-10) in the serum were measured via ELISA. The expression of HSP70 was significantly increased while the expression of Tim-3 was significantly decreased in the kidneys of mice in the T2DN group compared with those in the control (NC) group. Additionally, the inhibition of HSP70 upregulated the expression of Tim-3 in T2DN mice. The Th17/Treg ratio was significantly greater in the blood and kidneys of the mice in the T2DN group than in those of the NC group, the expression of serum IL-17 was increased, and the expression of IL-10 was decreased. Increased HSP70 inhibits Tim-3 expression in T2DN mouse kidney tissues, and subsequently causes a Th17/Treg imbalance and an inflammatory response, ultimately leading to kidney injury. The inhibition of HSP70 may alleviate the progression of T2DN.

Diacerein and myo-inositol alleviate letrozole-induced PCOS via modulation of HMGB1, SIRT1, and NF-kB: A comparative study.

Polycystic ovary syndrome (PCOS) is the most prevalent cause of anovulatory infertility in women. Myo-inositol supplementation has displayed effectiveness in curing PCOS patients. Diacerein, an anti-inflammatory medication, has not been extensively studied in the context of reproductive disorders. This study aimed to compare the role of myo-inositol and diacerein in PCOS and the probable mechanisms mediating their actions. Forty adult female rats were divided equally into the following: control, PCOS, PCOS+Myo-inositol, and PCOS+Diacerein groups. Rats were subjected to arterial blood pressure (ABP), electromyography (EMG), and uterine reactivity measurements. Blood samples were collected for measuring hormonal assays, glycemic state, lipid profile, oxidative stress, and inflammatory markers. Ovaries and uteri were extracted for histological examination, including hematoxylin and eosin staining, Masson's trichrome staining, immunohistochemistry, and rt-PCR analysis of ovarian tissues. PCOS was associated with significant increases in ABP, uterine frequency and amplitude of contraction, luteinizing hormone, testosterone, lipid, glycemic and inflammatory markers, malondialdehyde, high-mobility group box 1 (HMGB1), nuclear factor kappa (NF-kB), ovarian fibrosis, and endometrial thickening. In contrast, there was a significant reduction in follicular stimulating hormone, reduced glutathione, and Sirtuin 1 (SIRT1) when compared with control group. Both myo-inositol and diacerein counteract PCOS changes; but diacerein's effectswere superiorto myo-inositol's for all parameters, except for lipid and glycemic markers. Diacerein possessed anti-inflammatory properties and showed significant efficacy in mitigating the endocrinal, metabolic, and ovarian structural alterations linked to PCOS. Its beneficial actions likely stem from reducing oxidative stress, dyslipidemia, and hyperglycemia, potentially through the modulation of HMGB1, SIRT1, and NF-kB pathways.

Bone-targeted ultrasound-responsive nanobubbles for siRNA delivery to treat osteoporosis in mice

This project aimed to study the efficacy of a bone-targeted ultrasound-responsive nanobubble (NB) platform to deliver gene-silencing cathepsin K (CTSK) siRNA into the bone for osteoporosis treatment using in vitro and in vivo studies. To this end, characterization of CTSK siRNA loaded NB functionalized with alendronate (NB-CTSK siRNA-AL) was performed using transmission electron microscopy (TEM) imaging, and a release profile was obtained through fluorescent spectroscopy. In vitro studies were conducted by culturing NB-CTSK siRNA-AL with osteoclasts to evaluate siRNA uptake, CTSK expression, and the expression of tartrate-resistant acid phosphatase (TRAP). A control group and an NB-CTSK siRNA-AL treated group of ovariectomized (OVX) mice (n = 4) were tested. The OVX group that received treatment underwent weekly sessions for 4 weeks, during which they were exposed to low-intensity pulsed ultrasound (LIPUS) stimulation following administration of NB-CTSK siRNA-AL, prior to being sacrificed. Both groups underwent a series of tests to evaluate the bone targeting, safety, and efficacy of the nanoplatform. These tests included biodistribution studies conducted at 4 h and 24 h post-injection, a 3-point bending test of the femurs, nano-computed tomography analysis, as well as Hematoxylin & Eosin histological staining, Masson's Trichrome staining, and CTSK staining. The biodistribution showed the accumulation of NB-CTSK siRNA-AL in the bone and liver. Results showed that the OVX mice treated with NB-CTSK siRNA-AL had increased distal cortical bone thickness (174.4 ± 5.28 μm vs. 144.3 ± 10.66 μm, p > 0.05)) and bone volume fraction (16.5 ± 3.96 % vs. 6.55 ± 0.13 % (p > 0.05)). A reduced collagen degradation and downregulated CTSK expression were evident in the staining procedures. No adverse effects were recorded within histological assessments on the liver, kidney, and heart post-treatment. Morphology was shown to be normal and healthy within muscle cells post-LIPUS stimulation of NB-CTSK siRNA-AL. From these results, it can be concluded that an ultrasound-mediated NB-CTSK siRNA-AL can serve as a reliable, safe CTSK siRNA carrier to bone-specific targets for in vivo osteoporosis treatment.

Silybin Meglumine Mitigates CCl4-Induced Liver Fibrosis and Bile Acid Metabolism Alterations.

Altered patterns of bile acids (BAs) are frequently present in liver fibrosis, and BAs function as signaling molecules to initiate inflammatory responses. Silybin meglumine (SLB-M) is widely used in treating various liver diseases including liver fibrosis. However, research on its effects on bile acid (BA) metabolism is limited. This study investigated the therapeutic effects of SLB-M on liver fibrosis and BA metabolism in a CCl4-induced murine model. A murine liver fibrosis model was induced by CCl4. Fibrosis was evaluated using HE, picrosirius red, and Masson's trichrome staining. Liver function was assessed by serum and hepatic biochemical markers. Bile acid (BA) metabolism was analyzed using LC-MS/MS. Bioinformatics analyses, including PPI network, GO, and KEGG pathway analyses, were employed to explore molecular mechanisms. Gene expression alterations in liver tissue were examined via qRT-PCR. SLB-M treatment resulted in significant histological improvements in liver tissue, reducing collagen deposition and restoring liver architecture. Biochemically, SLB-M not only normalized serum liver enzyme levels (ALT, AST, TBA, and GGT) but also mitigated disruptions in both systemic and hepatic BA metabolism by increased unconjugated BAs like cholic acid and chenodeoxycholic acid but decreased conjugated BAs including taurocholic acid and taurodeoxycholic acid, compared to that in CCl4-induced murine model. Notably, SLB-M efficiently improved the imbalance of BA homeostasis in liver caused by CCl4 via activating Farnesoid X receptor. These findings underscore SLB-M decreased inflammatory response, reconstructed BA homeostasis possibly by regulating key pathways, and gene expressions in BA metabolism.

Facilitation of diabetic wound healing by far upstream element binding protein 1 through augmentation of dermal fibroblast activity.

Diabetes mellitus (DM) often leads to wound healing complications, partly attributed to the accumulation of advanced glycosylation end products (AGEs) that impair fibroblast function. Far Upstream Element Binding Protein 1 (FUBP1) regulates cell proliferation, migration, and collagen synthesis. However, the impact of FUBP1 on diabetic wound healing remains unknown. This study is designed to explore the function and mechanisms of FUBP1 in diabetic wound healing. Eighteen Sprague-Dawley rats (weighing 220-240g) were randomly assigned to three groups (n = 6): a control group (NC) of healthy rats, a model group (DM) of untreated diabetic rats, and a treatment group (DM + FUBP1) of diabetic rats accepting FUBP1 treatment. A 10mm diameter circular full-thickness skin defect was created on the back of each rat. On days 1 and 7, rats in the treatment group received local injections of 5µg FUBP1 protein at the wound site, whereas the control group and model group were administered saline. Wound healing was documented on days 0, 3, 7, 10, and 14, with tissue samples from the wound areas collected on day 14 for histological analysis, including H&E staining, Masson's trichrome staining, and immunohistochemistry. Western blot analysis was utilized to assess the expression of GSK-3β, Wnt3a, and β-catenin. In vitro, the effects of various concentrations of AGEs on cell viability and FUBP1 expression were examined in human dermal fibroblasts (HDF). Cells were genetically modified to overexpress FUBP1 using lentiviral vectors and were cultured for 48h in media with or without AGEs. The impacts on fibroblast proliferation, migration, and Wnt/β-catenin signaling were evaluated using CCK-8, scratch assays, and Western blot analysis. Animal investigation revealed that from day 7 onwards, the wound healing rate of the treatment group was higher than that of the model group but lower than the control group. On day 14, the wound healing rates were as follows: control group (0.97 ± 0.01), model group (0.84 ± 0.03), and treatment group (0.93 ± 0.01). These differences were statistically significant. Histological analysis indicates that FUBP1 promotes granulation tissue formation, re-epithelialization, and collagen deposition in treatment group. Additionally, FUBP1 protein expression decreased in dermal fibroblasts when exposed to AGEs. Overexpression of FUBP1 significantly enhanced fibroblast proliferation and migration, activating the Wnt/β-catenin pathway and mitigating the inhibitory effects of AGEs. Our results suggest that FUBP1 can be a promising therapeutic target for diabetic wound healing, potentially counteracting the detrimental effects of AGEs on dermal fibroblasts through the Wnt/β-catenin pathway.

Niclosamide attenuates erectile dysfunction and corporal fibrosis via reversal of Smad signaling in diabetic rat model.

Diabetes mellitus-induced erectile dysfunction (DMED) is a common urological complication of diabetes, and current drugs often fail to provide an effective treatment. Smad2/3 signaling-mediated corporal fibrosis has a critical role in the molecular basis of DMED. We investigated the effect of Niclosamide (Nic), an antihelmintic drug with antifibrotic effects, on erectile function in a rat DMED model. Male Sprague Dawley rats were injected intraperitoneally (i.p) with streptozotocin (75mg/kg) to induce diabetes. At week 8, both diabetic and nondiabetic rats were treated with Nic (10mg·kg-1/day; i.p) or vehicle for 4weeks. At week 12, erectile function was evaluated as intracavernous pressure (ICP) response to the electrical stimulation of the cavernous nerve (CN). Penile tissues were harvested for Masson's trichrome staining or western blotting to determine corporal fibrosis and Smad2/3 pathway-related protein expression, respectively. At the end of the experimental protocol, in vivo erectile function was assessed by measuring the ratio of ICP/ mean arterial pressure (MAP) and total ICP following CN stimulation. Smooth muscle content and collagen fibers were evaluated by Masson's trichrome staining of the penile tissues. The expressions of fibrosis-related proteins (Smad2, Smad3, fibronectin) were determined using western blotting in the penile tissues. Erectile function, as determined by the maximum ICP/MAP and total ICP/MAP ratios, was drastically decreased in diabetic rats. Corporal tissues of diabetic rats were severely fibrotic with a significant increase in collagen fibers and a marked reduction in smooth muscle content. Also, the protein expressions of phosphorylated (p-)Smad2, p-Smad3 and fibronectin were significantly increased in the penis of diabetic rats. Both functional and molecular alterations in DMED were effectively reversed by Nic-treated diabetic rats without a glycemic alteration. Nic could be a promising candidate for the treatment of DMED due to its antifibrotic effects. The present study provides the first evidence that Nic has beneficial effect on erectile dysfunction by attenuating corporal fibrosis in a rat model of DMED. The effect of Nic on penile endothelial function and the other potential underlying mechanisms needs to be further elucidated. Nic improved erectile function in DMED rats possibly suppressing penile fibrosis by inhibiting Smad2/3 signaling. These results suggest a potential therapeutic repurposing of Nic as an adjuvant treatment in DMED.

Phosphatidylserine Topically Attenuates Imiquimod-induced Psoriasis Through Inflammation Inhibition in Mice.

Psoriasis is a chronic skin condition that is associated with persistent inflammation and skin lesions. Topical therapy has been a promising approach to the alleviation of psoriasis through the application of anti-inflammatory agents. Phosphatidylserine (PS) administration has shown anti-inflammatory effects in the trials. Consequently, the objective of this study was to evaluate the effects of topical PS on the potential improvement of an imiquimod (IMQ)-induced psoriasis model. Additionally, cyclosporine A was utilized as a comparative anti-psoriatic agent in our study. The psoriasis model was established by topically applying IMQ to the dorsal skin of mice once daily for five consecutive days. The efficacy of topical PS was assessed using the Psoriasis Area and Severity Index (PASI) score to evaluate skin lesions. Subsequently, the skin samples were analyzed using Baker's scoring system, Masson's trichrome staining, immunohistochemistry, and real-time PCR analysis. IMQ-induced plaque-type psoriasis resulted in a significant increase (P<0.05) in dermal thickness, hyperkeratosis, PASI score, and inflammatory cytokines at the lesion site. The topical PS and cyclosporine A significantly (P<0.05) reduced PASI score and dermal thickness, while also alleviating erythema and scaling when compared to untreated mice. Furthermore, biomolecular assessments revealed that PS significantly (P<0.05) inhibited the gene expression of IL-17, IL-23, and TNF-α cytokines in the IMQ-induced lesions. Topical PS may pointedly alleviate psoriasis through the inhibition of inflammation. The beneficial effects of the PS recommend further investigation in both experimental and clinical studies in the control of skin psoriasis.

AGK2, a SIRT2 inhibitor, ameliorates D-galactose-induced liver fibrosis by inhibiting fibrogenic factors.

In our study, we aimed to investigate the effect of SIRT2 inhibition on function, fibrosis and inflammation in liver fibrosis induced by D-Galactose (D-Gal) administration. A total of 32 3-month-old Sprague Dawley rats were used in the study. Rats were divided into 4 groups as Control, d-Gal, Solvent+d-Gal, d-Gal+AGK2+Solvent. d-Gal (150 mg/kg/day), AGK-2 (10 µM/bw) as a specific SIRT2 inhibitor, 4%DMSO + PBS as a solvent was applied to the experimental groups and physiological saline was applied to the control group for 10 weeks. All applications were performed subcutaneously. Histological fibrotic changes were studied in the liver tissues by Masson's trichrome staining, hematoxylin and eosin staining and immunohistochemistry and the levels of selected factors were determined by quantitative reverse transcription-polymerase chain reaction, western blot analysis, and immunohistochemical analysis. Biochemical parameters and Paraoxonase levels were determined in the plasma. d-Galactose administration increased AST, AST-ALT Ratio, APRI, SIRT2 protein expression, IL1β, TGF β, β-catenin, Type I collagen, Type III collagen and α-SMA, collagen fiber density and histopathological score. ALT and lipid panels were not changed and paraxonase plasma level was shown to decrease. These effects were largely blocked by the SIRT2 inhibitor AGK2. These findings suggest that SIRT2 inhibition attenuates d-Gal-induced liver injury and that this protection may be due to its antifibrotic and anti-inflammatory activities.

Comparison of the Results of Modeling Pulmonary Fibrosis in Sprague Dawley Rats by Intratracheal Administration of Bleomycin in the Form of Sulfate and Chloride at a Dose of 3 mg/kg.

Background/Objectives: Intratracheal administration of bleomycin (BLM) to laboratory rodents is a standard, widely used technique used to model pulmonary fibrosis (PF). BLM, as a modeling agent, is produced mainly in the form of two salts-sulfate and chloride. We compared the results of modeling PF in SD rats by intratracheal administration of BLM sulfate and BLM chloride. Methods: Healthy mature male SD rats were used. PF was modeled by intratracheal administration of BLM sulfate and BLM chloride at a dose of 3 mg/kg. The criteria for the development of PF included body weight gain, changes in respiratory parameters, relative lung weight, cellular composition of broncho-alveolar fluid (BALF), histological assessment of the severity of PF with trichrome Masson staining. Results: Intratracheal administration of both BLM salts led to the development of pronounced PF, which was determined by changes in all of the measured parameters relative to control animals. There were no significant differences between the BLM sulfate and BLM chloride groups in body weight gain, hydroxyproline content, and histological evaluation. However, significant differences were identified in the cellular composition of BALF-a significant increase in alveolar macrophages and neutrophils levels in animals treated with BLM sulfate. Conclusions: Intratracheal administration of both BLM salts led to the development of severe PF; however, the inflammatory process in animals receiving BLM sulfate was more pronounced and prolonged than in animals receiving BLM chloride, which in the former, when observed more than 21 days after modeling, can lead to more severe PF.

Nootkatone mitigates periodontal inflammation and reduces alveolar bone loss via Nrf2/HO-1 and NF-κB pathways in rat model of periodontitis.

Periodontitis (PD) is a chronic inflammatory disease leading to alveolar bone loss. This study investigated the effect of nootkatone and regulatory mechanism in reducing periodontal inflammation and alveolar bone loss in a rat model. Twenty male Sprague-Dawley rats were divided into control, periodontitis, and nootkatone-treated groups (45 or 90 mg/kg). Ligature induction method was adopted to establish the PD model. After 21 days, rats received daily gavage of either saline or nootkatone for 10 days. Alveolar bone loss was assessed using micro-CT. Histological analyses included hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Masson's trichrome stainings. Immunohistochemistry for heme oxygenase 1 (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2) were performed in periodontal tissues. Content of inflammatory cytokines IL-1β, IL-6, and TNF-α in gingival tissues around ligature were assessed using ELISA kits. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were analyzed and Western blot for NF-κB expression in gingival tissues were performed. Nootkatone significantly reduced the distance from cementoenamel junction to alveolar bone crest (CEJ-ABC), enhanced bone mineral density (BMD), bone volume (BV), and BV/total volume (TV) ratio in ligature-induced rats. Higher dose of nootkatone (90 mg/kg) did not show more significant therapeutic effect than lower dose (45 mg/kg). Histological staining showed decreased osteoclasts' number and improved bone architecture in the nootkatone group. Content of IL-1β, IL-6, and TNF-α and inflammatory cell infiltration level in gingival tissues around the ligature were decreased in the nootkatone-treatment rats. Nootkatone increased Nrf2 and HO-1 protein expression and decreased NF-κB protein level, suppressing MDA levels and enhancing SOD activity. In a rat model, nootkatone effectively mitigates periodontal inflammation and alveolar bone loss through the Nrf2/HO-1 and NF-κB pathways. These findings suggest nootkatone as a promising therapeutic agent for the treatment of periodontitis.