Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

Identification of Peucedanum japonicum Thunb. extract components and their protective effects against dexamethasone-induced muscle atrophy.

BACKGROUND: Peucedanum japonicum Thunb. (PJ) is a vegetable widely consumed in East Asia and is known to have anticancer and anti-inflammatory effects. However, the effect of PJ on muscle atrophy remains elusive. PURPOSE: This study aimed to investigate the effect of PJ and its active compound on dexamethasone (DEX)-induced muscle atrophy. METHODS: We performed qualitative and quantitative analysis of PJ using ultra-performance liquid chromatography-mass spectrometry tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively. The efficacy of PJ and its main compound 4-caffeoylquinic acid (CQA) on muscle atrophy was evaluated in DEX-induced myotube atrophy and DEX-induced muscle atrophy in mouse myoblasts (C2C12) and C57BL/6 mice, in vitro and in vivo, respectively. RESULTS: The UPLC-MS/MS and HPLC data showed that the concentration of 4-CQA in PJ was 18.845 mg/g. PJ and 4-CQA treatments significantly inhibited DEX-induced myotube atrophy by decreasing protein synthesis and glucocorticoid translocation to the nucleus in C2C12 myotubes. In addition, PJ enhanced myogenesis by upregulating myogenin and myogenic differentiation 1 in C2C12 cells. PJ supplementation effectively increased muscle function and mass, downregulated atrogenes, and decreased proteasome activity in C57BL/6 mice. Additionally, PJ effectively decreased the nuclear translocation of forkhead transcription factor 3 alpha by inhibiting glucocorticoid receptor. CONCLUSION: Overall, PJ and its active compound 4-CQA alleviated skeletal muscle atrophy by inhibiting protein degradation. Hence, our findings present PJ as a potential novel pharmaceutical candidate for the treatment of muscle atrophy.

Macamide, a component of maca (Lepidium meyenii Walp) lipophilic extract, enhances myogenic differentiation via AKT/p38 signaling and attenuates dexamethasone-induced muscle atrophy.

Maca (Lepidium meyenii) is a plant that grows in the central Andes region of Peru, and it has been reported to have various bioactive functions, such as improving or preventing osteoporosis, sexual dysfunction, and memory impairment. In this study, maca roots of various colors (yellow, red, or black) were extracted using different polar solvents (PE, HEX, or BuOH) to compare their effects on muscle differentiation. Among them, the red maca lipophilic extract, which showed the most effectiveness, was chosen for further investigation. Our results show that RMLE enhances muscle differentiation by inducing MyoD-E2A heterodimerization through the activation of the AKT/p38 pathway. Additionally, RMLE attenuated dexamethasone-induced muscle atrophy by inhibiting nuclear translocation of FoxO3a and expression of E3-ligase (MAFbx and MURF1) in vitro and in vivo. Therefore, based on these results suggest that lipophilic extract of maca, which can abundantly contain nonpolar compounds, macamides, can enhance the functional properties of maca in alleviating muscle homeostasis.

Novel insights into the anti-asthmatic effect of Raphanus sativus L. (Raphani Semen): Targeting immune cells, inflammatory pathways and oxidative stress markers.

ETHNOPHARMACOLOGICAL RELEVANCE: Raphanus sativus L. is a well-known medicinal plant with traditional therapeutic applications in various common ailments including inflammation and asthma. AIMS OF THE STUDY: This study aimed to evaluate the chemical composition and anti-asthmatic potential of the hydro-methanolic extract of the leaves of R. sativus L. (Rs.Cr) using various in vitro and in vivo investigations. MATERIALS AND METHODS: The Rs.Cr was subjected to preliminary phytochemical analysis and HPLC profiling. The safety was assessed through oral acute toxicity tests in mice. The antiasthmatic effect of the extract was studied using milk-induced leukocytosis and ovalbumin (OVA)-induced allergic asthma models established in mice. While mast cell degranulation and passive paw anaphylaxis models were established in rats. Moreover, effect of the extract was studied on various oxidative and inflammatory makers. The antioxidant effect of the extract was also studied by in vitro DPPH method. RESULTS: The HPLC profiling of Rs.Cr showed the presence of important polyphenols in a considerable quantity. In toxicity evaluation, Rs.Cr showed no sign of morbidity or mortality with LD50 < 2000 mg/kg. The extract revealed significant mast cell disruption in a dose-dependent manner compared to the intoxicated group. Similarly, treatment with Rs.Cr and dexamethasone significantly (p < 0.001) reduced paw edema volume. Subcutaneous injection of milk at a dose of 4 mL/kg, after 24 h of its administration, showed an increase in the leukocyte count in the intoxicated group. Similarly, mice treated with dexamethasone and Rs.Cr respectively showed a significant decrease in leukocytes and eosinophils count in the ovalbumin-induced allergic asthma model. The extract presented a significant (p˂0.001) alleviative effect on the levels of SOD and GSH, MDA, IL-4, IL-5, and IL-13 in a dose-dependent manner as compared to the intoxicated group. Furthermore, the histological evaluation also revealed a notable decrease in inflammatory and goblet cell count with reduced mucus production. CONCLUSION: The current study highlights mechanism-based novel insights into the anti-asthmatic potential of R. sativus that also strongly supports its traditional use in asthma.

Dexamethasone upregulates macrophage PIEZO1 via SGK1, suppressing inflammation and increasing ROS and apoptosis.

The side effects of high-dose dexamethasone in anti-infection include increased ROS production and immune cell apoptosis. Dexamethasone effectively activates serum/glucocorticoid-regulated kinase 1 (SGK1), which upregulates various ion channels by activating store-operated calcium entry (SOCE), leading to Ca2+ oscillations. PIEZO1 plays a crucial role in macrophages' immune activity and function, but whether dexamethasone can regulate PIEZO1 by enhancing SOCE via SGK1 activation remains unclear. The effects of dexamethasone were assessed in a mouse model of sepsis, and primary BMDMs and the RAW264.7 were treated with overexpression plasmids, siRNAs, or specific activators or inhibitors to examine the relationships between SGK1, SOCE, and PIEZO1. The functional and phenotypic changes of mouse and macrophage models were detected. The results indicate that high-dose dexamethasone upregulated SGK1 by activating the macrophage glucocorticoid receptor, which enhanced SOCE and subsequently activated PIEZO1. Activation of PIEZO1 resulted in Ca2+ influx and cytoskeletal remodelling. The increase in intracellular Ca2+ mediated by PIEZO1 further increased the activation of SGK1 and ORAI1/STIM1, leading to intracellular Ca2+ peaks. In the context of inflammation, activation of PIEZO1 suppressed the activation of TLR4/NFκB p65 in macrophages. In RAW264.7 cells, PIEZO1 continuous activation inhibited the change in mitochondrial membrane potential, accelerated ROS accumulation, and induced autophagic damage and cell apoptosis in the late stage. CaMK2α was identified as a downstream mediator of TLR4 and PIEZO1, facilitating high-dose dexamethasone-induced macrophage immunosuppression and apoptosis. PIEZO1 is a new glucocorticoid target to regulate macrophage function and activity. This study provides a theoretical basis for the rational use of dexamethasone.

Physiological roles of propolis and red ginseng nanoplatforms in alleviating dexamethasone-induced male reproductive challenges in a rat model.

BACKGROUND: Red ginseng and propolis are well-known antioxidants that have been related to a reduction in oxidative stress. OBJECTIVE: This study evaluated the efficiency of red ginseng and propolis, either in powder or as nano-forms against dexamethasone-induced testicular oxidative challenges in adult male albino rats. METHODS: Forty rats were divided into 8 equal groups including control negative group that was given vehicle (DMSO), control positive group that was administered dexamethasone in addition to the nano-propolis, nano-ginseng, nano-propolis + dexamethasone, nano ginseng+dexamethasone, propolis+dexamethasone and ginseng + dexamethasone groups. Serum, semen and tissue samples were obtained. RESULTS: Lower testosterone levels, higher levels of MDA, and lower levels of total antioxidant capacity in serum, as well as impaired semen quality and a disturbed histopathological picture of both the testis and seminal glands, were all observed as significant negative effects of dexamethasone. These findings were confirmed by lower gene expression profiles of CYP11A1, StAR, HSD-3b, Nrf-2 and ACTB-3b in testicular and seminal gland tissues. The most powerful anti-dexamethasone effects were obtained with either propolis in nanoform or conventional ginseng. CONCLUSION: Propolis nano-formulation and ginseng in conventional form could be considered excellent candidates to ameliorate the oxidative stress provoked by dexamethasone, however, neither nano-ginseng nor conventional propolis showed such effects.

Baicalin ameliorates insulin resistance and regulates hepatic glucose metabolism via activating insulin signaling pathway in obese pre-diabetic mice.

BACKGROUND: Diabetes belongs to the most prevalent metabolic diseases worldwide, which is featured with insulin resistance, closely associated with obesity and urgently needs to be treated. Baicalin, belonging to natural flavonoids, has been reported to inhibit oxidative stress or inflammatoin. PURPOSE: This study investigated the properties of baicalin on modulating abnormal glucolipid metabolism, as well as the underlying in-vitro and in-vivo mechanisms. METHODS: Insulin-resistant (IR)-HepG2 cells were stimulated by dexamethasone (20 µM) and high glucose (50 mM) for 48 h and incubated with or without baicalin or metformin for another 16 h. Male C57BL/6 J mice were fed with a high-fat diet (HFD, 60 % kcal% fat) during the total 14 weeks. Obese mice were then administered with baicalin (50 and 100 mg/kg) or vehicle solution everyday through oral gavage during the last 4-week period. Moreover, baicalin metabolisms in vitro and in vivo were determined using UPLC/MS/MS to study its metabolism situation. RESULTS: Exposure to dexamethasone and high glucose damaged the abilities of glycogen synthesis and glucose uptake with elevated oxidative stress and increased generation levels of advanced glycation end-products (AGEs) in HepG2 cells. These impairments were basically reversed by baicalin treatment. Four-week oral administration with baicalin ameliorated hyperglycemia and dyslipidemia in HFD-induced obese and pre-diabetic mice. Downregulation of IRS/PI3K/Akt signaling pathway accomplished with reduced GLUT4 expression and enhanced GSK-3ß activity was observed in insulin resistant HepG2 cells as well as liver tissues from pre-diabetic mice; and such effect was prevented by baicalin. Moreover, baicalin and its matabolites were detected in IR-HepG2 cells and mouse plasma. CONCLUSION: The study illustrated that baicalin alleviated insulin resistance by activating insulin signaling pathways and inhibiting oxidative stress and AGEs production, revealing the potential of baicalin to be a therapeutic natural flavonoid against hepatic insulin and glucose-lipid metabolic disturbance in pre-diabetes accompanied with obesity.

Chitosan derived chito-oligosaccharides promote osteoblast differentiation and offer anti-osteoporotic potential: Molecular and morphological evidence from a zebrafish model.

This study delves into the potential of chito-oligosaccharides (COS) to promote osteoblast differentiation and prevent osteoporosis, utilizing experiments with mouse MSCs and the zebrafish model. The preliminary biocompatibility study affirms the non-toxic nature of COS across various concentrations. In the osteoblast differentiation study, COS enhances ALP activity and calcium deposition at the cellular level. Moreover, COS induces the upregulation of molecular markers, including Runx2, Type I collagen, ALP, osteocalcin, and osteonectin in mouse MSCs. Zebrafish studies further demonstrate COS's anti-osteoporotic effects, showcasing its ability to expedite fin fracture repair, vertebral mineralization, and bone mineralization in dexamethasone-induced osteoporosis models. The scale regenerative study reveals that COS mitigates the detrimental effects of dexamethasone induced osteoclastic activity, reducing TRAP and hydroxyproline levels while elevating the expression of Runx2a MASNA isoform, collagen2α, OC, and ON mRNAs. Additionally, COS enhances calcium and phosphorus levels in regenerated scales, impacting the bone-healthy calcium-to­phosphorus ratio. The study also suggests that COS modulates the MMP3-Osteopontin-MAPK signaling pathway. Overall, this comprehensive investigation underscores the potential of COS to prevent and treat osteoporosis. Its multifaceted cellular and molecular effects, combined with in vivo bone regeneration and repair, propose that COS may be effective in addressing osteoporosis and related bone disorders. Nonetheless, further research is imperative to unravel underlying mechanisms and optimize clinical applications.

Inhaled paraquat-induced lung injury in rat, improved by the extract of Zataria multiflora boiss and PPARγ agonist, pioglitazone.

The effects of a PPAR-γ agonist, pioglitazone and Zataria multiflora (Z. multiflora) on inhaled paraquat (PQ)-induced lung oxidative stress, inflammation, pathological changes and tracheal responsiveness were examined. The study was carried out in control rats exposed to normal aerosol of saline, PQl and PQh groups exposed to aerosols of 27 and 54 mg/m3 PQ, groups exposed to high PQ concentration (PQh) and treated with 200 and 800 mg/kg/day Z. multiflora, 5 and 10 mg/kg/day pioglitazone, low doses of Z. multiflora + pioglitazone, and 0.03 mg/kg/day dexamethasone. Increased tracheal responsiveness, transforming growth factor beta (TGF-ß) and lung pathological changes due to PQh were significantly improved by high doses of Z. multiflora and pioglitazone, dexamethasone and extract + pioglitazone, (p < 0.05 to p < 0.001). In group treated with low doses of the extract + pioglitazone, the improvements of most measured variables were significantly higher than the low dose of two agents alone (p < 0.05 to p < 0.001). Z. multiflora improved lung injury induced by inhaled PQ similar to dexamethasone and pioglitazone which could be mediated by PPAR-γ receptor.

Jakyak-gamcho-tang, a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, ameliorates dexamethasone-induced muscle atrophy and muscle dysfunction.

BACKGROUND: Although chronic treatment with glucocorticoids, such as dexamethasone, is frequently associated with muscle atrophy, effective and safe therapeutics for treating muscle atrophy remain elusive. Jakyak-gamcho-tang (JGT), a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, has long been used to relieve muscle tension and control muscle cramp-related pain. However, the effects of JGT on glucocorticoid-induced muscle atrophy are yet to be comprehensively clarified. PURPOSE: The objective of the current study was to validate the protective effect of JGT in dexamethasone-induced muscle atrophy models and elucidate its underlying mechanism through integrated in silico - in vitro - in vivo studies. STUDY DESIGN AND METHODS: Differential gene expression was preliminarily analyzed using the RNA-seq data to determine the effects of JGT on C2C12 myotubes. The protective effects of JGT were further validated in dexamethasone-treated C2C12 myotubes by assessing cell viability, myotube integrity, and mitochondrial function or in C57BL/6 N male mice with dexamethasone-induced muscle atrophy by evaluating muscle mass and physical performance. Transcriptomic pathway analysis was also performed to elucidate the underlying mechanism. RESULTS: Based on preliminary gene set enrichment analysis using the RNA-seq data, JGT regulated various pathways related to muscle differentiation and regeneration. Dexamethasone-treated C2C12 myotubes and muscle tissues of atrophic mice displayed substantial muscle protein degradation and muscle loss, respectively, which was efficiently alleviated by JGT treatment. Importantly, JGT-mediated protective effects were associated with observations such as preservation of mitochondrial function, upregulation of myogenic signaling pathways, including protein kinase B/mammalian target of rapamycin/forkhead box O3, inhibition of ubiquitin-mediated muscle protein breakdown, and downregulation of inflammatory and apoptotic pathways induced by dexamethasone. CONCLUSION: To the best of our knowledge, this is the first report to demonstrate that JGT could be a potential pharmaceutical candidate to prevent muscle atrophy induced by chronic glucocorticoid treatment, highlighting its known effects for relieving muscle spasms and pain. Moreover, transcriptomic pathway analysis can be employed as an efficient in silico tool to predict novel pharmacological candidates and elucidate molecular mechanisms underlying the effects of herbal medications comprising diverse biologically active ingredients.

Examining the Potential of Vitamin C Supplementation in Tissue-Engineered Equine Superficial Digital Flexor Tendon Constructs.

Because equine tendinopathies are slow to heal and often recur, therapeutic strategies are being considered that aid tendon repair. Given the success of utilizing vitamin C to promote tenogenesis in other species, we hypothesized that vitamin C supplementation would produce dose-dependent improvements in the tenogenic properties of tendon proper (TP) and peritenon (PERI) cells of the equine superficial digital flexor tendon (SDFT). Equine TP- and PERI-progenitor-cell-seeded fibrin three-dimensional constructs were supplemented with four concentrations of vitamin C. The gene expression profiles of the constructs were assessed with 3'-Tag-Seq and real-time quantitative polymerase chain reaction (RT-qPCR); collagen content and fibril ultrastructure were also analyzed. Moreover, cells were challenged with dexamethasone to determine the levels of cytoprotection afforded by vitamin C. Expression profiling demonstrated that vitamin C had an anti-inflammatory effect on TP and PERI cell constructs. Moreover, vitamin C supplementation mitigated the degenerative pathways seen in tendinopathy and increased collagen content in tendon constructs. When challenged with dexamethasone in two-dimensional culture, vitamin C had a cytoprotective effect for TP cells but not necessarily for PERI cells. Future studies will explore the effects of vitamin C on these cells during inflammation and within the tendon niche in vivo.

Tea polyphenol carrier-enhanced dexamethasone nanomedicines for inflammation-targeted treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial inflammation, cartilage damage and bone erosion. In the progression of RA, the inflammatory mediators including ROS, NO, TNF-α, and IL-6 play important roles in the aggravation of inflammation. Hence, reducing the generation and release of inflammatory mediators is of great importance. However, the high dose and frequent administration of clinical anti-inflammatory drugs such as glucocorticoids (GCs) usually lead to severe side effects. The development of nanotechnology provides a promising strategy to overcome these issues. Here, polyphenol-based nanoparticles with inherent anti-oxidative and anti-inflammatory activities were developed and used as a kind of nanocarrier to deliver dexamethasone (Dex). The in vitro experiments confirmed that the nanoparticles and drugs could act synergistically for suppressing inflammatory mediators in the LPS/INF-γ-induced inflammatory cell model. After intravenous administration, the Dex-loaded nanoparticles with good biosafety showed effective accumulation in inflamed joints and improved therapeutic efficacy by inducing anesis of synovial inflammation and cartilage destruction over free Dex in a collagen-induced arthritis (CIA) mouse model. The results demonstrated that polyphenol-based nanoparticles with therapeutic functions may serve as an innovative platform to synergize with chemotherapeutic agents for enhanced treatment of inflammatory diseases.

Inonotus obliquus upregulates muscle regeneration and augments function through muscle oxidative metabolism.

Skeletal muscle wasting related to aging or pathological conditions is critically associated with the increased incidence and prevalence of secondary diseases including cardiovascular diseases, metabolic syndromes, and chronic inflammations. Much effort is made to develop agents to enhance muscle metabolism and function. Inonotus obliquus (I. obliquus; IO) is a mushroom popularly called chaga and has been widely employed as a folk medicine for inflammation, cardiovascular diseases, diabetes, and cancer in Eastern Europe and Asia. However, its effect on muscle health has not been explored. Here, we aimed to investigate the beneficial effect of IO extract in muscle regeneration and metabolism. The treatment of IO in C2C12 myoblasts led to increased myogenic differentiation and alleviation of dexamethasone-induced myotube atrophy. Network pharmacological analysis using the identified specific chemical constituents of IO extracts predicted protein kinase B (AKT)-dependent mechanisms to promote myogenesis and muscle regeneration. Consistently, IO treatment resulted in the activation of AKT, which suppressed muscle-specific ubiquitin E3 ligases induced by dexamethasone. IO treatment in mice improved the regeneration of cardiotoxin-injured muscles accompanied by elevated proliferation and differentiation of muscle stem cells. Furthermore, it elevated the mitochondrial content and muscle oxidative metabolism accompanied by the induction of peroxisome proliferator-activated receptor γ coactivator α (PGC-1α). Our current data suggest that IO is a promising natural agent in enhancing muscle regenerative capacity and oxidative metabolism thereby preventing muscle wasting.

Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts.

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, ß-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, ß-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, ß-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L-1 of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L-1 ß-glycerolphosphate, 75 µmol L-1 ascorbic acid and 10 nmol L-1 dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and regeneration.

Combined Therapy of Experimental Autoimmune Uveitis by a Dual-Drug Nanocomposite Formulation with Berberine and Dexamethasone.

Purpose: Autoimmune uveitis is a kind of sight-threatening ocular and systemic disorders. Recent treatments on autoimmune uveitis still remain many limitations due to extreme complexity and undetermined pathogenesis. In this study, a novel dual-drug nanocomposite formulation is developed to treat experimental autoimmune uveitis by a combined and sustained therapy method. Methods: The dual-drug nanocomposite formulation is constructed by integrating berberine (BBR)-loaded mesoporous silica nanoparticles (MSNs) into dexamethasone (DEX)-loaded thermogel (BBR@MSN-DEX@Gel). The BBR@MSN-DEX@Gel is characterized by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectrometer and rheometer. The in vitro drug release profile, cytotoxicity and anti-inflammation effectiveness of BBR@MSN-DEX@Gel on lipopolysaccharide-stimulated human conjunctival epithelial cells are investigated. After the in vivo drug release profile and biosafety of the dual-drug nanocomposite formulation are confirmed, its treatment effectiveness is fully assessed based on the induced experimental autoimmune uveitis (EAU) Lewis rat's model. Results: The dual-drug nanocomposite formulation has good injectability and thermosensitivity, suitable for administration by an intravitreal injection. The BBR@MSN-DEX@Gel has been found to sustainably release both drugs for up to 4 weeks. The carrier materials have minimal in vitro cytotoxicity and high in vivo biosafety. BBR@MSN-DEX@Gel presents obviously anti-inflammatory effectiveness in vitro. After administration of BBR@MSN-DEX@Gel into Lewis rat's eye with EAU by an intravitreal injection, the nanocomposite formulation significantly suppresses inflammatory reaction of autoimmune uveitis via a dual-drug combined and sustained therapy method, compared with the equivalent dose of single-component formulations. Conclusion: BBR@MSN-DEX@Gel serves as a promising dual-drug nanocomposite formulation for future treatment of autoimmune uveitis.

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells.

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

Oral cortisol and dexamethasone intake: Differential physiology and transcriptional responses in the marine juvenile Sparus aurata.

This study approached the long-term oral administration of cortisol (F) and dexamethasone (DEX), two synthetic glucocorticoids, compared to a control group (CT) in the juveniles of a marine teleost, the gilthead seabream (Sparus aurata). Physiologically, DEX treatment impaired growth, which appears to be linked to carbohydrate allocation in muscle and liver, hepatic triglycerides depletion, and reduced hematocrit. Hypophyseal gh mRNA expression was 2-fold higher in DEX than in CT or F groups. Similarly, hypothalamic trh and hypophyseal pomcb followed this pattern. Plasma cortisol levels were significantly lower in DEX than in CT, while F presented intermediate levels. In the posterior intestine, measured short circuit-current (Isc) was more anion absorptive in CT and F compared to the DEX group, whereas Isc remained unaffected in the anterior intestine. The derived transepithelial electric resistance (TEER) significantly differed between intestinal regions in the DEX group. These results provide new insights to understand better potential targeted biomarkers indicative of the differential glucocorticoid or mineralocorticoid-receptors activation in fish.

Yinlai Decoction Protects Microstructure of Colon and Regulates Serum Level of D-Lactic Acid in Pneumonia Mice Fed with High-Calorie and High-Protein Diet.

OBJECTIVE: To investigate the effect of Yinlai Decoction (YD) on the microstructure of colon, and activity of D-lactic acid (DLA) and diamine oxidase (DAO) in serum of pneumonia mice model fed with high-calorie and high-protein diet (HCD). METHODS: Sixty male Kunming mice were randomly divided into 6 groups by the random number table method: normal control, pneumonia, HCD, HCD with pneumonia (HCD-P), YD (229.2 mg/mL), and dexamethasone (15.63 mg/mL) groups, with 10 in each group. HCD mice were fed with 52% milk solution by gavage. Pneumonia mice was modeled with lipopolysaccharide inhalation and was fed by gavage with either the corresponding therapeutic drugs or saline water, twice daily, for 3 days. After hematoxylin-eosin staining, the changes in the colon structure were observed under light microscopy and transmission electron microscope, respectively. Enzyme-linked immunosorbent assay was used to detect the protein levels of DLA and DAO in the serum of mice. RESULTS: The colonic mucosal structure and ultrastructure of mice in the normal control group were clear and intact. The colonic mucosal goblet cells in the pneumonia group tended to increase, and the size of the microvilli varied. In the HCD-P group, the mucosal goblet cells showed a marked increase in size with increased secretory activity. Loose mucosal epithelial connections were also observed, as shown by widened intercellular gaps with short sparse microvilli. These pathological changes of intestinal mucosa were significantly reduced in mouse models with YD treatment, while there was no significant improvement after dexamethasone treatment. The serum DLA level was significantly higher in the pneumonia, HCD, and HCD-P groups as compared with the normal control group (P<0.05). Serum DLA was significantly lower in the YD group than HCD-P group (P<0.05). Moreover, serum DLA level significantly increased in the dexamethasone group as compared with the YD group (P<0.01). There was no statistical significance in the serum level of DAO among groups (P>0.05). CONCLUSIONS: YD can protect function of intestinal mucosa by improving the tissue morphology of intestinal mucosa and maintaining integrity of cell connections and microvilli structure, thereby reducing permeability of intestinal mucosa to regulate the serum levels of DLA in mice.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Salix babylonica L. mitigates pancreatic damage by regulating the Beclin-P62/SQSTM1 autophagy pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Salix babylonica L. belongs to the genus Salix, family Salicaceae. It is traditionally used as an antipyretic, antirheumatic, antidiabetic and for the treatment of ulcers and parasite skin diseases. It also has a range of pharmacological effects, such as anti-inflammatory, anti-tumor, antioxidant, and antibacterial effects. However, there are no reports on the phytochemical profile and efficacy of its leaves extract to modulate dexamethasone induced pancreatic damage. AIM OF THE STUDY: The present study was performed to annotate the phytoconstituents of Salix babylonica leaf extract and explore whether and how it could modulate dexamethasone-induced pancreatic damage and the role of oxidative stress and autophagy in mediating its protective effects. MATERIALS AND METHODS: Wistar rats were used for this study. Salix babylonica in two dose levels (100 and 200 mg/kg) or metformin (50 mg/kg) was given by oral gavage concurrently with dexamethasone which was injected SC in a dose of 10 mg/kg for 4 consecutive days. RESULTS: LC-MS analysis furnished 84 secondary metabolites belonging to phenolic acids, salicinoids, proanthocyanidins, flavonoids, cyclohexanediol glycosides, and hydroxy fatty acids. S. babylonica at both dose levels and metformin decreased the elevated pancreatic beclin while elevated the decreased pancreatic P62/SQSTM1 content compared to dexamethasone. These effects were associated with improved histopathological changes, glycemic and lipid parameters indicating that there might be a connection between autophagy and dexamethasone-induced pancreatic damage. Given that the level of GSH was negatively correlated with the levels of beclin and positively correlated with P62/SQSTM1, while both MDA and NO levels were positively correlated with beclin and negatively correlated with P62/SQSTM1, it seems that dexamethasone induced autophagy may be attributed to dexamethasone induced pancreatic oxidative stress. CONCLUSION: Our results indicate that S. babylonica protects pancreatic tissues against dexamethasone-induced damage by decreasing oxidative stress and its associated autophagy. Our study reveals a new mechanism for dexamethasone effects on pancreas and shows the potential therapeutic role of S. babylonica in mitigating dexamethasone adverse effects on pancreas and establishes the groundwork for future clinical applications.