Elevated glucocorticoid alters the developmental dynamics of hypothalamic neurogenesis in zebrafish.

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analyse the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and survival. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic developmental trajectory, a process crucial for health across the life-course.

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.

Clinical pharmacokinetics and pharmacodynamics of topical non-biological therapies for psoriasis patients.

INTRODUCTION: Psoriasis is a chronic inflammatory cutaneous disease that causes patients psychosocial distress. Topical therapies are utilized for mild-to-moderate disease and for more severe disease in conjunction with systemic therapies. Topical corticosteroids are a cornerstone of treatment for psoriasis, but long-term use can cause stria and cutaneous atrophy and as well as systemic side effects such as topical steroid withdrawal. Non-steroidal topical therapies tend to be safer than topical corticosteroids for long-term use. AREAS COVERED: We conducted a literature review on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of topical therapies for psoriasis. We discuss how the PK and PD characteristics of these therapies inform clinicians on efficacy and toxicity when prescribing for patients. EXPERT OPINION: Topical corticosteroids, used intermittently, are very safe and effective. Long-term, continuous use of topical corticosteroids can cause systemic side effects. Several generic and newly approved non-steroidal options are available, but no head-to-head studies compare the effectiveness of the generics (vitamin D analogs, tacrolimus, pimecrolimus) against the newer therapies (roflumilast, tapinarof). Patients often do not respond to topical therapies due to poor adherence to treatment regimens. For patients resistant to topical treatment, phototherapy or systemic therapy may be an option.

Asiatic acid prevents glucocorticoid-induced femoral head osteonecrosis via PI3K/AKT pathway.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) represents a predominant etiology of non-traumatic osteonecrosis, imposing substantial pain, restricting hip mobility, and diminishing overall quality of life for affected individuals. Centella asiatica (L.) Urb. (CA), an herbal remedy deeply rooted in traditional oriental medicine, has exhibited noteworthy therapeutic efficacy in addressing inflammation and facilitating wound healing. Drawing from CA's historical applications, its anti-inflammatory, anti-apoptotic, and antioxidant attributes may hold promise for managing GIONFH. Asiatic acid (AA), a primary constituent of CA, has been substantiated as a key contributor to its anti-apoptotic, antioxidant, and anti-inflammatory capabilities, showcasing a close association with orthopedic conditions. For the investigation of whether AA could alleviate GIONFH through suppressing oxidative stress, apoptosis, and to delve into its potential cellular and molecular mechanisms, the connection between AA and disease was analyzed through network pharmacology. DEX-induced apoptosis in rat osteoblasts and GIONFH in rat models, got utilized for the verification in vitro/vivo, on underlying mechanism of AA in GIONFH. Network pharmacology analysis reveals a robust correlation between AA and GIONFH in multiple target genes. AA has demonstrated the inhibition of DEX-induced osteoblast apoptosis by modulating apoptotic factors like BAX, BCL-2, Cleaved-caspase3, and cleaved-caspase9. Furthermore, it effectively diminishes the ROS overexpression and regulates oxidative stress through mitochondrial pathway. Mechanistic insights suggest that AA's therapeutic effects involve phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT) pathway activation. Additionally, AA has exhibited its potential to ameliorate GIONFH progression in rat models. Our findings revealed that AA mitigated DEX-induced osteoblast apoptosis and oxidative stress through triggering PI3K/AKT pathway. Also, AA can effectively thwart GIONFH occurrence and development in rats.

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.

Glucocorticoids with or without fludrocortisone in septic shock: a narrative review from a biochemical and molecular perspective.

Two randomised controlled trials have reported a reduction in mortality when adjunctive hydrocortisone is administered in combination with fludrocortisone compared with placebo in septic shock. A third trial did not support this finding when hydrocortisone administered in combination with fludrocortisone was compared with hydrocortisone alone. The underlying mechanisms for this mortality benefit remain poorly understood. We review the clinical implications and potential mechanisms derived from laboratory and clinical data underlying the beneficial role of adjunctive fludrocortisone with hydrocortisone supplementation in septic shock. Factors including distinct biological effects of glucocorticoids and mineralocorticoids, tissue-specific and mineralocorticoid receptor-independent effects of mineralocorticoids, and differences in downstream signalling pathways between mineralocorticoid and glucocorticoid binding at the mineralocorticoid receptor could contribute to this interaction. Furthermore, pharmacokinetic and pharmacodynamic disparities exist between aldosterone and its synthetic counterpart fludrocortisone, potentially influencing their effects. Pending publication of well-designed, randomised controlled trials, a molecular perspective offers valuable insights and guidance to help inform clinical strategies.

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.

1,25(OH)2D3 and its analogue calcipotriol inhibit the migration of human synovial and mesenchymal stromal cells in a wound healing model - A comparison with glucocorticoids.

Vitamin D analogue calcipotriol is currently used in the local treatment of psoriasis. However, it also has antiproliferative and anti-inflammatory effects in the cells of the joint - suggesting a possible benefit in local treatment of arthritis. In this study, calcipotriol was studied in different in vitro methods to find out its effect on synovial and mesenchymal stromal cells. Primary human cell lines of osteoarthritis or rheumatoid arthritis patients (five mesenchymal stromal cells, MSC, and four synovial stromal cells, SSC) were cultured to study migration and proliferation of the cells in a wound healing model. The media was supplemented with calcipotriol, 1,25(OH)2D3, dexamethasone, betamethasone, methylprednisolone or control solution in 1-100 nM concentrations. To see possible toxic effects of calcipotriol, concentrations up to 10 µM in SSCs and MSCs were studied in apoptosis and necrosis assays in four cell lines. Calcipotriol and 1,25(OH)2D3, as well as the three glucocorticoids, reduced the migration of both SSCs and MSCs. In SSCs, the effect of calcipotriol and 1,25(OH)2D3 was at least as effective as with glucocorticoids, while with MSCs, the glucocorticoids were stronger inhibitors of migration. The antimigratory of calcipotriol and 1,25(OH)2D3 was consistently maintained in 10 µM and 1 µM. Calcipotriol was not toxic to MSCs and SSCs up to concentrations of 10 µM. Calcipotriol, as well as 1,25(OH)2D3, exerts antimigratory and antiproliferative effects on human SSCs and MSCs of the joint. These effects are not caused by apoptosis or necrosis. Both calcipotriol and 1,25(OH)2D3 have similar effects as glucocorticoids without apparent toxicity, suggesting that calcipotriol might be an eligible candidate to the local treatment of arthritis with a broad therapeutic window.

Effects of OmniGen AF feed supplementation on glucocorticoids, blood leukocyte messenger RNA abundance, and energy metabolism in overstocked Holstein dairy cows.

The physiological consequences of overstocking require more investigation, and no research has explored whether dietary supplements could mitigate the anticipated negative physiological effects. OmniGen AF (OG, Phibro Animal Health Corporation, Teaneck, NJ, USA) is a nutritional supplement that has been shown to support the immune system of cattle following internal and environmental stressors. This study aimed to determine if a 45-day period of OG feed supplementation would influence whole blood leukocyte messenger RNA abundance, energy metabolism and glucocorticoid concentration, during a two-week period of overstocking. Two stocking density treatments (control: one headlock and lying stall per cow; overstocked: 0.5 headlocks and 0.5 lying stalls per cow) and two diet treatments (control: no added supplement; and OG: 56 g/cow per day) were investigated. Four pens of 15 cows were fed their assigned diet (two pens per diet; control stocking density) for 45 days after which each stocking density treatment was applied for a 14-day period using a cross-over design; this study design was replicated twice. During each 14-day period, blood was collected on day four to measure whole blood leukocyte messenger RNA abundance (cluster of differentiation 80, interleukin 8 receptor-beta, interleukin 10 receptor-beta and L-selectin) and fecal samples were collected every two days to measure fecal cortisol metabolite concentration (11,17-dioxoandrostanes). At the end of each 14-day period, eight cows from each pen were selected for an intravenous glucose tolerance test; glucose, insulin and non-esterified fatty acids were measured. There were no effects of diet or stocking density on leukocyte messenger RNA abundance. Fecal cortisol metabolite concentrations were highest for overstocked cows on the control diet on day four of the stocking density treatment; however, by day 10, overstocked cows fed OG had the highest fecal cortisol metabolite concentrations. Overstocked cows, regardless of diet, had an attenuated insulin response during the glucose tolerance test, represented by a lower area under the curve estimate. Cows fed OG but not overstocked, had a lower non-esterified fatty acid nadir during the glucose challenge, compared to all the other treatments. In conclusion, overstocking prompts a physiological stress response and alters energy metabolism by decreasing the insulin response to an intravenous glucose challenge. Feeding OG during overstocking delayed the increase in fecal cortisol metabolites by several days; however, it is unclear if this altered glucocorticoid response benefited the cow, as OG had no effect on insulin responses or immune parameters.

Effects of genistein aglycone in glucocorticoid induced osteoporosis: A randomized clinical trial in comparison with alendronate.

Glucocorticoid-induced osteoporosis (GIO) complicates the clinical management of patients subjected to long-term glucocorticoid use. This study explored the effects of genistein on bone loss in a randomized double-blind alendronate-controlled trial in postmenopausal women with GIO. 200 postmenopausal women (taking at least 5 mg of prednisone equivalents) since 3 months, or more, and expected to continue for at least other 12 months, were randomized to receive genistein (54 mg/day daily) or alendronate (70 mg once a week) for 24 months. Both groups received also Calcium and Vitamin D3 supplementation. Median bone mineral density (BMD) at the antero-posterior lumbar spine significantly increased from 0.75 g/cm2 at baseline to 0.77 g/cm2 at 1 year and 0.79 g/cm2 at 2 years in alendronate-treated patients and from 0.77 g/cm2 at baseline to 0.79 g/cm2 at 12 months and to 0.80 g/cm2 at 24 months in genistein recipients. No difference was observed between the two treatments. Median BMD at the femoral neck increased from 0.67 g/cm2 at baseline to 0.68 g/cm2 at 1 year and 0.69 g/cm2 at 2 years in alendronate-treated patients and from 0.68 g/cm2 at baseline to 0.70 g/cm2 at 12 months and to 0.71 g/cm2 at 24 months in genistein recipients. No difference was observed between alendronate and genistein groups in BMD. Regarding bone markers genistein and alendronate statistically decreased c-terminal telopeptide, while osteocalcin, bone-ALP, and sclerostin showed greater changes in genistein treated patients. This randomized clinical trial suggests that genistein aglycone represents an additional therapeutic option for patients with GIO.

An Artemisia scoparia extract attenuates glucocorticoid-induced lipolysis in adipocytes.

OBJECTIVE: Prescription glucocorticoid (GC) use is widespread across developed countries for the treatment of several inflammatory conditions. Elevated GCs are known to promote lipolysis and metabolic disorders. An extract of Artemisia scoparia (SCO) has been shown to reduce lipolysis and promote metabolic health but has not been investigated in the context of excess GCs. Our aim was to examine the effects of SCO on GC-induced lipolysis. METHODS: Mature adipocytes were pretreated with vehicle or SCO, then exposed to either the synthetic GC dexamethasone (DEX) or tumor necrosis factor alpha (TNFα). Medium was collected and assayed for glycerol and fatty acids as measures of lipolysis. The expression of several lipolytic genes and proteins was assessed, and the involvement of glucocorticoid receptor (GR) in SCO's effects was also interrogated. RESULTS: SCO significantly attenuated DEX-induced lipolysis but did not interfere with DEX-mediated changes in inflammatory gene profiles in adipocytes. SCO treatment resulted in significant reductions in monomeric phosphodiesterase (PDE) protein levels while elevating PDE multimeric complex formation, but other canonical lipolytic mediators were unaltered. SCO attenuated lipolysis even when GR expression was significantly knocked down. Finally, it was demonstrated that SCO was distinct from rosiglitazone in its antilipolytic effects. CONCLUSIONS: SCO attenuates GC-induced lipolysis independently of GR activity. Future studies are needed to elucidate underlying mechanisms.

Acute Ozone-Induced Transcriptional Changes in Markers of Oxidative Stress and Glucocorticoid Signaling in the Rat Hippocampus and Hypothalamus Are Sex-Specific.

Exposure to a prototypic air pollutant ozone (O3) has been associated with the activation of neuroendocrine stress response along with neural changes in oxidative stress (OS), inflammation, and Alzheimer's disease-like pathologies in susceptible animal models. We hypothesized that neural oxidative and transcriptional changes induced by O3 in stress responsive regions are sex-dependent. Male and female adult Long-Evans rats were exposed to filtered air or O3 for two consecutive days (0.8 ppm, 4 h/day) and brain regions were flash-frozen. Activities of cerebellar OS parameters and mitochondrial complex I, II, and IV enzymes were assessed to confirm prior findings. We assessed transcriptional changes in hypothalamus (HYP) and hippocampus (HIP) for markers of OS, microglial activity and glucocorticoid signaling using qPCR. Although there were no O3 or sex-related differences in the cerebellar activities of OS and mitochondrial enzymes, the levels of protein carbonyls and complex II activities were higher in females regardless of O3. There were no statistical differences in baseline expression of genes related to OS (Cat, Dhcr24, Foxm1, Gpx1, Gss, Nfe2l2, Sod1) except for lower HYP Sod1 expression in air-exposed females than males, and higher HIP Gss expression in O3-exposed females relative to matched males. Microglial marker Aif1 expression was higher in O3-exposed females relative to males; O3 inhibited Itgam only in males. The expression of Bdnf in HIP and HYP was inhibited by O3 in both sexes. Genes related to glucocorticoid signaling (Fkbp4, Fkbp5, Hsp90aa1, Hspa4, nr3c1, nr3c2) showed sex-specific effects due to O3 exposure. Baseline expression of HIP Fkbp4 was higher in females relative to males. O3 inhibited Nr3c1 in female HIP and male HYP, but Nr3c2 was inhibited in male HYP. Fkbp4 expression was higher in O3-exposed females when compared to matched males, whereas Fkbp5 was expressed at higher levels in both brain regions of males and females. These results indicate that sex-specific brain region responses to O3 might, in part, be caused by OS and regulation of glucocorticoid signaling.

Novel Peptides from Sea Cucumber Intestinal Enzyme Hydrolysates Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells via Phosphorylation of PPARγ at Serine 112.

SCOPE: Osteo-adipogenic differentiation imbalance of bone mesenchymal stem cells (BMSCs) has been linked to a variety of pathophysiological processes such as obesity and osteoporosis. Recent studies report that the phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ) Ser112 affects the fate decision of BMSCs. Novel peptides from the sea cucumber intestinal peptide (SCIP) have been proved to promote the growth of longitudinal bone. However, it is unclear the effect of SCIP on BMSCs differentiation fate. METHODS AND RESULTS: BMSCs in vitro and glucocorticoid induced mice are employed to investigate the effects of SCIP on osteo-adipogenic differentiation of BMSCs. In vitro results show that SCIP supplement significantly promotes the proliferation and osteogenic differentiation of BMSCs, upregulates the expression of osteogenic marker. In vivo results show that SCIP supplement ameliorates the osteo-adipogenic differentiation imbalance in glucocorticoid-treated mice, decreases bone marrow fat, and elevates bone mineral density. Mechanistically, SCIP supplement promotes and maintains the phosphorylation of PPARγ Ser112 through AMPK/ERK and TAZ signals, thereby inducing the osteogenic differentiation of BMSCs. CONCLUSION: Supplement with SCIP promotes BMSCs to differentiate into osteoblasts. These results suggest that SCIP has potential as a functional food to improve obesity and osteoporosis.

Allolobophora caliginosa coelomic fluid and extract alleviate glucocorticoid-induced osteoporosis in mice by suppressing oxidative stress and regulating osteoblastic/osteoclastic-related markers.

Allolobophora calignosa (Ac) is a folk medicine for millennia, as it possesses many biological activities. This study aimed to investigate the chemo-preventive activity of A.calignosa coelomic fluid (AcCF) and A.calignosa extract (AcE) on glucocorticoid-induced osteoporosis (GIOP) in mice. Characterization and in vitro biological activity of AcE and AcCF has been assessed. Male CD-1 mice were subcutaneously received dexamethasone (DEX) (1 mg/kg, 5 times/week) and concurrently intraperitoneally treated with either AcCF (20 mg/kg) or AcE (45 mg/kg) every other day for 28 days. Serum and bone homogenates were subjected for qPCR and biochemical analysis. AcE and AcCF treatment significantly increased bone mineral density (BMD), bone mineral content (BMC), calcium (Ca), phosphorus (P), and calcitonin levels, whereas activity of serum alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), serum acidic phosphatase (ACP), bone acidic phosphatase (BACP) and parathyroid hormone (PTH) levels were significantly reduced compare with untreated GIOP mice. Treatment with AcE and AcCF modulates oxidative stress and downregulated Rank and Mmp9 expression, as well as increased glycosaminoglycan content in the organic bone matrix, resulting in osteoclastogenesis inhibition. Overall, AcCF and AcE show a chemo-preventive activity against GIOP by inhibiting oxidative stress and regulating expression and/or activity of osteoblast/osteoclast-related markers.

Tongluo Shenggu capsule promotes angiogenesis to ameliorate glucocorticoid-induced femoral head necrosis via upregulating VEGF signaling pathway.

BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined. PURPOSE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH. METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a "drug targets-disease genes" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs). RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro. CONCLUSION: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.

Salidroside alleviates dexamethasone-induced inhibition of bone formation via transforming growth factor-beta/Smad2/3 signaling pathway.

Glucocorticoid-induced osteoporosis is the third epidemic osteoporosis following postmenopausal and senileosteoporosis. According to one study, salidroside made ovariectomized rats' bones strong. Salidroside's potential for treating glucocorticoid-induced osteoporosis remains unproven. This study aimed to investigate the protective effect and mechanism of salidroside on dexamethasone-induced osteogenic differentiation and bone formation in MC3T3-E1 cells and zebrafish. The study proved that salindroside had no harmful impact on MC3T3E1 cells. Salidroside significantly relieved dexamethasone-induced inhibition of ALP (alkaline phosphatase) activity and mineralization in MC3T3-E1 cells, and promoted osteogenic differentiation of cells. Salidroside increased the expression of osteopontin (OPN), runt-related transcription factor 2 (Runx2), osterix (Osx), transforming growth factor-beta (TGF-ß) proteins and promoted the phosphorylation of Smad2/3 in MC3T3-E1 cells treated with dexamethasone. In addition, the effect of salidroside in relieving dexamethasone-induced inhibition of osteogenic differentiation in MC3T3-E1 cells can be blocked by TGF-ß receptor type I/II inhibitor (LY2109761). At the same time, we found that salidroside significantly alleviated the inhibition of dexamethasone-induced bone formation in zebrafish and promoted the mineralization of zebrafish skulls. LY2109761 reversed the protective impact of salidroside on dexamethasone-mediated bone impairment in zebrafish. These findings suggested that salidroside alleviated dexamethasone-induced inhibition of osteogenic differentiation and bone formation via TGF-ß/Smad2/3 signaling pathway.

Icariin Improves Glucocorticoid Resistance in a Murine Model of Asthma with Depression Associated with Enhancement of GR Expression and Function.

Icariin, a flavonoid glycoside isolated from Epimedium brevicornum, exerts a variety of biological activities. However, its effects on depression-induced glucocorticoid resistance in asthma and the underlying mechanisms have not been elucidated. In this study, a murine model of asthma with depression was established by exposure to ovalbumin combined with chronic unpredictable mild stress, and icariin was given orally during ovalbumin challenge and chronic unpredictable mild stress exposure. Depression-like behaviors were assessed by the open field test, forced swim test, and tail suspension test. The characteristic features of allergic asthma, including airway hyperreactivity, histopathology, inflammatory cytokine levels in bronchoalveolar lavage fluid, and immunoglobulin E and corticosterone levels in serum, were examined. Following splenocyte isolation in vitro, the inhibitory effects of corticosterone on the proliferation and cytokine secretion of splenocytes, glucocorticoid receptor DNA-binding activity, and expression of p-glucocorticoid receptor s226, glucocorticoid receptor α, and p-p38 mitogen-activated protein kinase in splenocytes were determined. We found that icariin had limited effects on depression-like behaviors, however, it markedly suppressed airway hyperresponsiveness, inflammatory infiltration in lung tissues, levels of interleukin-4, interleukin-5, and interleukin-6 in bronchoalveolar lavage fluid, and immunoglobulin E in serum. Furthermore, icariin improved the inhibitory effects of corticosterone on lipopolysaccharide-stimulated splenocytes, increased the glucocorticoid receptor expression and glucocorticoid receptor DNA-binding activity, and inhibited the phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase. Taken together, icariin improved glucocorticoid resistance in a murine model of asthma with depression associated with enhancement of glucocorticoid receptor function and glucocorticoid receptor expression, and its effects on the glucocorticoid receptor function were related to decreased phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase.

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

The Nutraceutical Genistein-Lycopene Combination Improves Bone Damage Induced by Glucocorticoids by Stimulating the Osteoblast Formation Process.

Chronic glucocorticoid (GC) therapy is the most common cause of iatrogenic osteoporosis and represents an important risk factor for osteoporosis and bone fractures. New therapeutic approaches are required in order to treat osteoporosis and reduce the side effects related to the use of anti-osteoporotic drugs. In this context, previous studies reported the efficacy of some isoflavones and carotenoids, such as lycopene and genistein, on the reduction of the risk of fracture related to osteoporosis. The aim of this study was to investigate the effects of a combined oral treatment, consisting of genistein and lycopene, in an experimental model of glucocorticoid-induced osteoporosis (GIO). GIO was induced by subcutaneous injection of methylprednisolone (MP, 30 mg/kg) for 60 days, whereas the control group (Sham) received saline solution only. Following induction, MP animals randomly were assigned to receive alendronate, genistein, lycopene, or the association of genistein and lycopene or saline solution for additional 60 days together with MP. Femurs obtained from the Sham group were used for osteoblasts extraction; they were then incubated with dexamethasone (DEX) for 24 h to be then treated with lycopene or genistein or the association of lycopene and genistein for an additional 24 h. Treatments with lycopene and genistein restored the impaired mineralization of cells observed following DEX treatment and stimulated osteoblast differentiation by increasing the depressed expression of bALP and RUNX2 (p < 0.0001). Wnt5a, ß-catenin, and Nrf-2 expression were significantly increased following genistein and lycopene treatment (p < 0.0001), thus confirming their antioxidant activity as well as their ability in stimulating osteoblast function, mostly when genistein and lycopene were used in association. The combined treatment of genistein and lycopene improved the bone damage induced by glucocorticoids and significantly restored the normal architecture of bones as well as adequate interconnectivity of bone trabeculae, thus increasing bone mineral density parameters. The obtained data demonstrated that genistein and lycopene but in particular their association might prevent GC's adverse effects, thus stimulating bone formation and reducing bone resorption, improving bone structure and microarchitecture, through different molecular pathways, such as the Wnt/ß-catenin and the Nrf-2 signaling.

Cutaneous Permeation of a Percutaneously Applied Glucocorticoid Using Plant-Based Anionic Phospholipids in Hydrogenated Vegetable Oil: A Preliminary Study.

Purpose: It is important that, when corticosteroids are used therapeutically, concentrations be reduced as much as possible to mitigate potential adverse events and side effects. This preliminary study compares the permeation for the delivery of a corticosteroid in a 1% hydrocortisone-supplemented topical cream containing anionic polar phospholipids (APP) in hydrogenated vegetable oil (triglyceride) versus a market-leading 1% hydrocortisone in a mineral hydrocarbon-based skin cream. Methods: Using the Franz diffusion cell method with cadaveric skin, the permeation of a 1% hydrocortisone-supplemented cream containing APP (test preparation) was compared with a commercially available 1% hydrocortisone cream (control preparation). The principal APP in the test preparation were phosphatidylinositol, phosphatidylserine and phosphatidylglycerol. Permeation was determined at 4 and 8 h time intervals. Results: The permeation values for the 1% hydrocortisone supplemental APP cream (test preparation) were comparatively very high 1180 ng/cm2 at 4 h and 2173 ng/cm2 at 8 h, in contrast to the 1% hydrocortisone cream (control preparation) values of 13 ng/cm2 at 4 h and 98 ng/cm2 at 8 h. Permeation of skin cream increased significantly from 0 to 4 and 8 h, when comparing the APP test preparation with the control preparation (p < 0.001). This translates, respectively, into the 90-fold greater and a 20-fold greater penetration of the test preparation APP cream over the 1% hydrocortisone cream at 4 h and 8 h time points. Conclusions: This preliminary study demonstrates the enhanced permeation of 1% hydrocortisone when applied topically to the skin in an APP skin cream vehicle. This enhanced permeation suggests the potential use of APP technology to deliver therapeutically effective hydrocortisone treatment to the skin at markedly reduced concentrations of steroid. As such, APP technology may offer an improved approach to the treatment of dermatoses associated with inflammatory diseases and conditions requiring prolonged topical corticosteroid therapy.