The anti‐diabetic drug and peroxisome proliferator‐activated receptor‐gamma (PPARγ) agonist, rosiglitazone, alters astrocyte activation; however, its mechanism remains less‐known. We hypothesized ...participation of epidermal growth factor receptor (EGFR), known to control astrocyte reactivity. We first detected that rosiglitazone promoted glial fibrillary acidic protein (GFAP) expression in primary astrocytes as well as the mouse cerebral cortex, associated with increased EGFR activation. Screening for EGFR ligands revealed a rosiglitazone‐mediated increase of heparin‐binding epidermal growth factor (HB‐EGF) in astrocytes, resulting in HB‐EGF release into culture medium and mouse cerebrospinal fluid too. Treatment with HB‐EGF‐siRNA and EGFR inhibitors showed that the rosiglitazone‐induced HB‐EGF and p‐EFGR were interdependent, which participated in GFAP increase. Interestingly, we observed that rosiglitazone could induce cellular and secreted‐HB‐EGF in neurons also, contributing toward the activated EGFR‐induced GFAP in astrocytes. Probing whether these effects of rosiglitazone were PPARγ‐linked, revealed potential PPARγ‐responsive elements within HB‐EGF gene. Moreover, gel‐shift, site‐directed mutagenesis, chromatin‐immunoprecipitation and luciferase‐reporter assays demonstrated a PPARγ‐dependent HB‐EGF transactivation. Subsequently, we examined effects of rosiglitazone in a high‐fat diet‐fed diabetes mouse model, and supporting observations in the normal cortical cells, identified a rosiglitazone‐induced GFAP, astrocyte and neuronal HB‐EGF and secreted‐HB‐EGF in the cerebral cortex of diabetic mice. Moreover, assessing relevance of increased HB‐EGF and GFAP revealed an anti‐apoptotic role of rosiglitazone in the cerebral cortex, supported by a GFAP‐siRNA as well as HB‐EGF‐siRNA‐mediated increase in cleaved‐caspase 3 and 9 levels in the rosiglitazone‐treated astrocyte–neuron coculture. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARγ‐dependent HB‐EGF/EGFR signaling and increased GFAP.
The anti‐diabetic drug rosiglitazone, promoted GFAP expression in astrocytes, associated with increased EGFR activation. Rosiglitazone induced a PPARγ‐dependent expression of the EGFR ligand, HB‐EGF, in astrocytes and neurons, contributing towards the activated EGFR‐induced GFAP. Similar findings were observed in the diabetic mice. Additionally, increased HB‐EGF and GFAP resulted in an anti‐apoptotic role of rosiglitazone within the brain. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARγ‐dependent HB‐EGF/EGFR signalling and increased GFAP.
Abstract
Withanolides are the group of active chemical constituents of Withania somnifera (L.) Dunal. Withaferin A, withanolide A and withanone presents three of the biologically most active ...constituents of this herb. These steroidal lactones are isomers of each other and thus, pose significant difficulty in their separation. In present study, a simple, specific and reliable RP-HPLC method has been developed and validated for their separation and simultaneous quantification. Separation was carried out on Lichrocart Purospher STAR RP-18e column (250 × 4.5 mm, 5 µm) using mobile phase, methanol and 0.01 M ammonium acetate buffer (pH 5) in the ratio 60:40, v/v. The calibration curves were linear (r
2 > 0.99) for all the three compounds across concentration range of 1.56–50 µg/mL. The lower limit of quantification for all the analytes was 1.56 µg/mL. The intra-day and inter-day accuracy was between 88.65% and 110.66% and coefficient of variation was between 0.55 and 10.12. The analytes were stable under different storage conditions. The developed method was successfully applied to analyze the samples for simultaneous determination of permeability of the three withanolides in rats using in situ single-pass intestinal perfusion model. Withanolide A and withanone were found to be high permeability compounds while withaferin A could not be detected.
Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate ...therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34
38
progenitors and quiescent CD34
cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC
) of imatinib by >4 logs and remarkably eroding quiescent CD34
cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.
Tea (
Camellia sinensis
) has several reported health benefits, including that on bone health attributed to catechins of which the most abundant is epigallocatechin-3-gallate (EGCG). However, several ...preclinical and clinical studies raise safety concerns about EGCG in tea extract causing acute liver failure. Tea also contains kaempferol, albeit scanty, and it has hepatoprotective and osteogenic effects. Here, we utilized a novel extraction procedure of acid hydrolysis to enhance the osteogenic effect of tea extract while reducing its hepatotoxicity. The resultant extract (USKECSE) has a ~40-fold increase in kaempferol and a 2.5-fold reduction in EGCG content compared with the hydroethanolic extract (USCSE). In a female Sprague Dawley (SD) rat femur osteotomy model, USKECSE (100 mg/kg) but not USCSE promoted bone regeneration. In a rat postmenopausal osteoporosis model induced by bilateral ovariectomy (OVX), USKECSE through an osteogenic mechanism maintained bone mass, strength, and microarchitecture to the levels of ovary-intact rats with no hepatotoxic effect. After a single oral dose (100 mg/kg) of USKECSE to adult rats, kaempferol was detectable for 48 hours, suggesting its significant absorption and distribution in plasma. Peak kaempferol concentration in plasma (C
max
) was 483 ng/ml (2 μM), and at this concentration, kaempferol induces osteoblast differentiation. USKECSE had no genotoxicity, and its safety index assessed by preclinical toxicity studies, including safety pharmacology, was >20-fold. Taken together, we report a novel extraction process that enhanced the osteogenicity and concomitantly reduced hepatotoxicity of tea extract with significant kaempferol bioavailability and a favorable systemic safety profile. Based on these data, we propose assessing the USKECSE effect for postmenopausal osteoporosis treatment.
S009-0629 methyl-8-(methylthio)-2-phenyl-6-p-tolyl-4,5-dihydro-2H-benzoeindazole-9-carboxylate is a novel antidiabetic agent with PTP1B inhibitory activity. In this study, we have investigated the in ...vitro metabolic stability, plasma protein binding, blood partitioning, and oral pharmacokinetic study of S009-0629 in rats. The plasma protein binding, blood partitioning, and metabolic stability were determined by HPLC method. The oral pharmacokinetic study was analyzed by liquid chromatography coupled mass spectrometry (LC-MS/MS) method. The plasma protein binding of S009-0629 using modified charcoal adsorption method at 5 and 10 µg/mL was 80.58 ± 1.04% and 81.95 ± 1.15%, respectively. The K
of S009-0629 was independent of concentration and time. The in-vitro half-life of S009-0629 at 5 and 10 µM using rat liver microsomes was determined as 273 ± 24.46 and 281.67 ± 26.53 min, respectively. After oral administration, S009-0629 exhibited C
55.51 ± 1.18 ng/mL was observed at 18 hr (t
). S009-0629 was found to have the large apparent volume of distribution (1,894.93 ± 363.67 L/kg). Oral in-vivo t
of S009-0629 was found to be 41.23 ± 5.96 hr. A rapid and highly sensitive LC-MS/MS method was validated for S009-0629 in rat plasma. S009-0629 has high plasma protein binding and low hepatic extraction. S009-0629 has no affinity with human P-gp and BCRP in ATPase assay. After oral dosing, S009-0629 has slow absorption and elimination in rats.
Type 2 diabetes is associated with increased fracture risk and delayed facture healing; the underlying mechanism, however, remains poorly understood. We systematically investigated skeletal pathology ...in leptin receptor-deficient diabetic mice on a C57BLKS background (db). Compared with wild type (wt), db mice displayed reduced peak bone mass and age-related trabecular and cortical bone loss. Poor skeletal outcome in db mice contributed high-glucose- and nonesterified fatty acid-induced osteoblast apoptosis that was associated with peroxisome proliferator-activated receptor ... coactivator 1-α (PGC-1a) downregulation and upregulation of skeletal muscle atrogenes in osteoblasts. Osteoblast depletion of the atrogene muscle ring finger protein-1 (MuRF1) protected against gluco- and lipotoxicity-induced apoptosis. Osteoblast-specific PGC-1α upregulation by 6-C-β-d-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF), an adiponectin receptor 1 (AdipoR1) agonist, as well as metformin in db mice that lacked AdipoR1 expression in muscle but not bone restored osteopenia to wt levels without improving diabetes. Both GTDF and metformin protected against gluco- and lipotoxicity-induced osteoblast apoptosis, and depletion of PGC-1α abolished this protection. Although AdipoR1 but not AdipoR2 depletion abolished protection by GTDF, metformin action was not blocked by AdipoR depletion. We conclude that PGC-1α upregulation in osteoblasts could reverse type 2 diabetes-associated deterioration in skeletal health. (ProQuest: ... denotes formulae/symbols omitted.)
Adiponectin is an adipocytokine that signals through plasma membrane-bound adiponectin receptors 1 and 2 (AdipoR1 and -2). Plasma adiponectin depletion is associated with type 2 diabetes, obesity, ...and cardiovascular diseases. Adiponectin therapy, however, is yet unavailable owing to its large size, complex multimerization, and functional differences of the multimers. We report discovery and characterization of 6-C-β-D-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF) as an orally active adiponectin mimetic. GTDF interacted with both AdipoRs, with a preference for AdipoR1. It induced adiponectin-associated signaling and enhanced glucose uptake and fatty acid oxidation in vitro, which were augmented or abolished by AdipoR1 overexpression or silencing, respectively. GTDF improved metabolic health, characterized by elevated glucose clearance, β-cell survival, reduced steatohepatitis, browning of white adipose tissue, and improved lipid profile in an AdipoR1-expressing but not an AdipoR1-depleted strain of diabetic mice. The discovery of GTDF as an adiponectin mimetic provides a promising therapeutic tool for the treatment of metabolic diseases.
The methanol extract of the rhizome of Bergenia ciliata Sternb. (Saxifragaceae) has been evaluated for anti‐inflammatory potential using two acute rat models (carrageenan‐ and serotonin ...(5‐HT)‐induced rat paw oedema) and a chronic rat model (cotton pouch‐induced granuloma). Phenylbutazone (100 mg kg−1), a non‐steroidal anti‐inflammatory agent, was used as a standard. The methanol extract (100, 200 or 300 mg kg−1) exhibited significant (P < 0.05) anti‐inflammatory activity in all the animal models. At 300 mg kg−1 the methanol extract exhibited maximum inhibition of 32.4 ± 2.89% in carrageenan‐induced rat paw oedema while the standard showed an inhibition of 44.1 ± 2.7% after 3 h of drug treatment. In the serotonin‐induced rat paw oedema model, 300 mg kg−1 methanol extract suppressed oedema by 45.33 ± 2.09%, whereas the standard produced an inhibition of 53.5 ± 4.3%. In the cotton pouch granuloma model the methanol extract inhibited significantly (P < 0.001) the granuloma weight in a dose‐dependent manner. In this model, 300 mg kg−1 extract produced a maximum inhibition of 31.4 ± 1.09% in granuloma weight compared with 41.1 ± 1.32% reduction in granuloma weight for the standard. The methanol extract of B. ciliata exhibited significant anti‐inflammatory potential at the dose levels examined.
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