RESUMEN
Osteoporosis is evident in postmenopausal women and is an osteolytic disease characterized by bone loss that further increases the susceptibility to bone fractures and frailty. The use of complementary therapies to alleviate postmenopausal osteoporosis is fairly widespread among women. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. This study aimed to determine whether Cirsium setidens water extracts (CSEs), the component linarin, and its aglycone acacetin blocked ovariectomy (OVX)-induced bone loss. This study employed OVX C57BL/6 female mice as a model for postmenopausal osteoporosis. CSEs, acacetin, or linarin was orally administrated to OVX mice at a dose of 20 mg/kg for 8 weeks. Surgical estrogen loss in mice for 8 weeks reduced bone mineral density (BMD) of mouse femur and serum 17ß-estradiol level and enhanced the serum receptor activator of NF-κB ligand/osteoprotegerin ratio with uterine atrophy. CSEs and linarin reversed such adverse effects and enhanced femoral BMD in OVX mice. Oral administration of CSEs and linarin attenuated tartrate-resistant acid phosphate activity and the induction of αvß3 integrins and proton suppliers in resorption lacunae in femoral bone tissue of OVX mice. In addition, CSEs and linarin curtailed the bone levels of cathepsin K and matrix metalloproteinase-9 responsible for osteoclastic bone resorption. On the other hand, CSEs and linarin enhanced the formation of trabecular bones in estrogen-deficient femur with increased induction of osteocalcin and osteopontin. Further, treatment with CSEs and linarin enhanced the collagen formation-responsive propeptide levels in the circulation along with the increase in the tissue non-specific alkaline phosphatase level in bone exposed to OVX. Supplementing CSEs, acacetin, or linarin to OVX mice elevated the formation of collagen fibers in OVX trabecular bone, evidenced using Picrosirius red staining. Accordingly, CSEs and linarin were effective in retarding osteoclastic bone resorption and promoting osteoblastic bone matrix mineralization under OVX conditions. Therefore, linarin, which is abundant in CSEs, may be a natural compound for targeting postmenopausal osteoporosis and pathological osteoresorptive disorders.
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Resorción Ósea , Cirsium , Osteoporosis Posmenopáusica , Animales , Femenino , Ratones , Densidad Ósea , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/etiología , Colágeno/farmacología , Estrógenos/farmacología , Ratones Endogámicos C57BL , Osteoporosis Posmenopáusica/tratamiento farmacológico , Ovariectomía/efectos adversosRESUMEN
Osteoporosis manifest in postmenopausal women is an osteolytic disease characterized by bone loss, leading to increased susceptibility to bone fractures and frailty. The use of complementary therapies to alleviate postmenopausal osteoporosis is fairly widespread among women. The current study examined that Pangasius hypophthalmus fish skin collagen hydrolysates (fsCH) inhibited ovariectomy (OVX)-induced bone loss by conducting inter-comparative experiments for anti-osteoporotic activity among 206-618 mg/kg fsCH, 2 mg/kg isoflavone, 15 mg/kg glycine-proline-hydroxyproline (GPH) tripeptide, and calcium lactate. Surgical estrogen loss of mice for 8 weeks reduced serum 17ß-estradiol levels with uterus atrophy, which was ameliorated by orally administering fsCH or isoflavone to mice. Similar to isoflavone, fsCH containing GPH-enhanced bone mineral density reduced levels of cathepsin K and proton-handling proteins, and elevated collagen 1 level in OVX bones. The treatment with fsCH and isoflavone enhanced the serum levels of collagen synthesis-related procollagen type 1 carboxy/amino-terminal propeptides reduced by OVX, whereas serum levels of osteocalcin and alkaline phosphatase, as well as collagen breakdown-related carboxy/amino-terminal telopeptides of type 1 collagen were reduced in OVX mice treated with fsCH, isoflavone, and calcium lactate. The trabecular bones were newly formed in OVX bones treated with isoflavone and fsCH, but not with calcium lactate. However, a low-dose combination of fsCH and calcium lactate had a beneficial synergy effect on postmenopausal osteoporosis. Furthermore, similar to isoflavone, 15-70 µg/mL fsCH, with its constituents of GPH and dipeptides of glycine-proline and proline-hydroxyproline, enhanced osteogenesis through stimulating differentiation, matrix mineralization, and calcium deposition of MC3T3-E1 osteoblasts. Accordingly, the presence of fsCH may encumber estrogen deficiency-induced bone loss through enhancing osteoclastogenic differentiation and matrix collagen synthesis. Therefore, fsCH may be a natural compound retarding postmenopausal osteoporosis and pathological osteoresorptive disorders.
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BACKGROUND: Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD. PURPOSE: The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7ß-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation. METHODS AND RESULTS: Murine macrophages were incubated with 28 µM 7ß-hydroxycholesterol in absence and presence of 1-20 µΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7ß-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7ß-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination. CONCLUSION: These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury.
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Hidroxicolesteroles , Ubiquitina , Animales , Estrés del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico , Macrófagos , RatonesRESUMEN
Diabetes induces bone deterioration, which leads to increased risk of fracture, osteopenia, and osteoporosis. Thus, diabetes-associated bone fragility has been recognized as a diabetic complication. However, the pathophysiological effects of hyperglycemia on bone turnover remain unclear. Literature evidence demonstrates that anti-diabetic medications increase the risk of fractures in individuals with type 2 diabetes. Scopoletin is a naturally occurring hydroxycoumarin potentially exhibiting anti-inflammatory and antioxidant activities and ameliorating insulin resistance as an anti-diabetic agent. However, little is known regarding the effects of scopoletin on the impairment of bone remodeling that is caused by diabetes. The aim of this study was to identify that scopoletin was capable of inhibiting the impairment of bone remodeling and turnover in a mouse model of type 2 diabetes. Submicromolar scopoletin accelerated the formation TRAP-positive multinucleated osteoclasts (40.0 vs. 105.1%) and actin ring structures impaired by 33 mM glucose. Further, 1-20 µM scopoletin enhanced bone resorption and the induction of matrix-degrading enzymes in diabetic osteoclasts. The oral administration of 10 mg/kg scopoletin elevated serum RANKL/OPG ratio and osteocalcin level reduced in db/db mice along with an increase in BMD by ~6-14%; however, it was not effective in lowering blood glucose and hemoglobin glycation. In addition, the supplementation of scopoletin elevated the formation of trabecular bones and collagen fibers in femoral epiphysis and metaphysis with a thicker epiphyseal plate and cortical bones. Furthermore, 1-20 µM scopoletin enhanced ALP activity (4.39 vs. 7.02 nmol p-nitrophenyl phosphate/min/mg protein) and deposits of mineralized bone nodules in cultured osteoblasts reduced by 33 mM glucose. The treatment of diabetic osteoblasts with scopoletin stimulated the cellular induction of BMP-2 and osteopontin and Runx2 transcription. Accordingly, the administration of scopoletin protected mice from type 2 diabetes-associated bone loss through boosting bone remodeling via the robust induction of bone turnover markers of both osteoclasts and osteoblasts. These findings suggest that scopoletin could be a potential osteoprotective agent for the treatment of diabetes-associated bone loss and fractures.
RESUMEN
Collagen hydrolysates have been suggested as a favorable antiaging modality in skin photoaged by persistent exposure to ultraviolet radiation (UV). The current study evaluated the beneficial effect of collagen hydrolysates (fsCH) extracted from Pangasius hypophthalmus fish skin on wrinkle formation and moisture preservation in dorsal skin of hairless mice challenged with UV-B. Inter-comparative experiments were conducted for anti-photoaging among fsCH, retinoic acid (RA), N-acetyl-D-glucosamine (NAG), and glycine-proline-hydroxyproline (GPH). Treating human HaCaT keratinocytes with 100-200 µg/mL fsCH reciprocally ameliorated the expression of aquaporin 3 (AQP3) and CD44 deranged by UV-B. The UV-B-induced deep furrows and skin thickening were improved in parched dorsal skin of mice supplemented with 206-412 mg/kg fsCH as well as RA and GPH. The UV-B irradiation enhanced collagen fiber loss in the dorsal dermis, which was attenuated by fsCH through enhancing procollagen conversion to collagen. The matrix metalloproteinase expression by UV-B in dorsal skin was diminished by fsCH, similar to RA and GPH, via blockade of collagen degradation. Supplementing fsCH to UV-B-irradiated mice decreased transepidermal water loss in dorsal skin with reduced AQP3 level and restored keratinocyte expression of filaggrin. The expression of hyaluronic acid synthase 2 and hyaluronidase 1 by UV-B was remarkably ameliorated with increased production of hyaluronic acid by treating fsCH to photoaged mice. Taken together, fsCH attenuated photoaging typical of deep wrinkles, epidermal thickening, and skin water loss, like NAG, RA, or GPH, through inhibiting collagen destruction and epidermal barrier impairment.
Asunto(s)
Colágeno/farmacología , Proteínas en la Dieta/farmacología , Envejecimiento de la Piel/efectos de los fármacos , Enfermedades de la Piel/tratamiento farmacológico , Piel/efectos de los fármacos , Rayos Ultravioleta/efectos adversos , Animales , Proteínas Filagrina , Masculino , Ratones , Ratones Pelados , Piel/patología , Piel/efectos de la radiación , Envejecimiento de la Piel/patología , Envejecimiento de la Piel/efectos de la radiación , Enfermedades de la Piel/etiología , Enfermedades de la Piel/patologíaRESUMEN
Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10-20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1-20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress-MAPK signaling-inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.
Asunto(s)
Quempferoles/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Embolia Pulmonar/prevención & control , Enfisema Pulmonar/prevención & control , Receptores Proteinasa-Activados/antagonistas & inhibidores , Animales , Fumar Cigarrillos/efectos adversos , Evaluación Preclínica de Medicamentos , Quempferoles/farmacología , Masculino , Ratones Endogámicos BALB C , Estrés Oxidativo , Embolia Pulmonar/etiologíaRESUMEN
Particulate matter (PM) is a mixture of solid and liquid air pollutant particles suspended in the air, varying in composition, size, and physical features. PM is the most harmful form of air pollution due to its ability to penetrate deep into the lungs and blood streams, causing diverse respiratory diseases. Aesculetin, a coumarin derivative present in the Sancho tree and chicory, is known to have antioxidant and anti-inflammatory effects in the vascular and immune system. However, its effect on PM-induced airway thickening and mucus hypersecretion is poorly understood. The current study examined whether naturally-occurring aesculetin inhibited airway thickening and mucus hypersecretion caused by urban PM10 (uPM10, particles less than 10 µm). Mice were orally administrated with 10 mg/kg aesculetin and exposed to 6 µg/mL uPM10 for 8 weeks. To further explore the mechanism(s) involved in inhibition of uPM10-induced mucus hypersecretion by aesculetin, bronchial epithelial BEAS-2B cells were treated with 1-20 µM aesculetin in the presence of 2 µg/mL uPM10. Oral administration of aesculetin attenuated collagen accumulation and mucus hypersecretion in the small airways inflamed by uPM10. In addition, aesculetin inhibited uPM10-evoked inflammation and oxidant production in lung tissues. Further, aesculetin accompanied the inhibition of induction of bronchial epithelial toll-like receptor 4 (TLR4) and epidermal growth factor receptor (EFGR) elevated by uPM10. The inhibition of TLR4 and EGFR accompanied bronchial mucus hypersecretion in the presence of uPM10. Oxidative stress was responsible for the epithelial induction of TLR4 and EGFR, which was disrupted by aesculetin. These results demonstrated that aesculetin ameliorated airway thickening and mucus hypersecretion by uPM10 inhalation by inhibiting pulmonary inflammation via oxidative stress-stimulated TLR4 and EGFR. Therefore, aesculetin may be a promising agent for treating airway mucosa-associated disorders elicited by urban coarse particulates.
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Podocyte injury inevitably results in leakage of proteins from the glomerular filter and is vital in the pathogenesis of diabetic nephropathy (DN). The underlying mechanisms of podocyte injury facilitate finding of new therapeutic targets for DN treatment and prevention. Tangeretin is an O-polymethoxylated flavone present in citrus peels with anti-inflammatory and antioxidant properties. This study investigated the renoprotective effects of tangeretin on epithelial-to-mesenchymal transition-mediated podocyte injury and fibrosis through oxidative stress and hypoxia caused by hyperglycemia. Mouse podocytes were incubated in media containing 33 mM glucose in the absence and presence of 1-20 µM tangeretin for up to 6 days. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg tangeretin for 8 weeks. Non-toxic tangeretin inhibited glucose-induced expression of the mesenchymal markers of N-cadherin and α-smooth muscle actin in podocytes. However, the reduced induction of the epithelial markers of E-cadherin and P-cadherin was restored by tangeretin in diabetic podocytes. Further, tangeretin enhanced the expression of the podocyte slit diaphragm proteins of nephrin and podocin down-regulated by glucose stimulation. The transmission electron microscopic images revealed that foot process effacement and loss of podocytes occurred in diabetic mouse glomeruli. However, oral administration of 10 mg/kg tangeretin reduced urine albumin excretion and improved foot process effacement of diabetic podocytes through inhibiting loss of slit junction and adherenes junction proteins. Glucose enhanced ROS production and HIF-1α induction in podocytes, leading to induction of oxidative stress and hypoxia. Similarly, in diabetic glomeruli reactive oxygen species (ROS) production and HIF-1α induction were observed. Furthermore, hypoxia-evoking cobalt chloride induced epithelial-to-mesenchymal transition (EMT) process and loss of slit diaphragm proteins and junction proteins in podocytes, which was inhibited by treating submicromolar tangeretin. Collectively, these results demonstrate that tangeretin inhibited podocyte injury and fibrosis through blocking podocyte EMT caused by glucose-induced oxidative stress and hypoxia.
Asunto(s)
Transición Epitelial-Mesenquimal , Flavonas/farmacología , Glucosa/metabolismo , Hipoxia/metabolismo , Estrés Oxidativo , Podocitos/metabolismo , Animales , Línea Celular Transformada , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Podocitos/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
For the optimal resorption of mineralized bone matrix, osteoclasts require the generation of the ruffled border and acidic resorption lacuna through lysosomal trafficking and exocytosis. Coumarin-type aesculetin is a naturally occurring compound with anti-inflammatory and antibacterial effects. However, the direct effects of aesculetin on osteoclastogenesis remain to be elucidated. This study found that aesculetin inhibited osteoclast activation and bone resorption through blocking formation and exocytosis of lysosomes. Raw 264.7 cells were differentiated in the presence of 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and treated with 1-10 µM aesculetin. Differentiation, bone resorption, and lysosome biogenesis of osteoclasts were determined by tartrate-resistance acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, immunocytochemical analysis, and LysoTracker staining. Aesculetin inhibited RANKL-induced formation of multinucleated osteoclasts with a reduction of TRAP activity. Micromolar aesculetin deterred the actin ring formation through inhibition of induction of αvß3 integrin and Cdc42 but not cluster of differentiation 44 (CD44) in RANKL-exposed osteoclasts. Administering aesculetin to RANKL-exposed osteoclasts attenuated the induction of autophagy-related proteins, microtubule-associated protein light chain 3, and small GTPase Rab7, hampering the lysosomal trafficking onto ruffled border crucial for bone resorption. In addition, aesculetin curtailed cellular induction of Pleckstrin homology domain-containing protein family member 1 and lissencephaly-1 involved in lysosome positioning to microtubules involved in the lysosomal transport within mature osteoclasts. These results demonstrate that aesculetin retarded osteoclast differentiation and impaired lysosomal trafficking and exocytosis for the formation of the putative ruffled border. Therefore, aesculetin may be a potential osteoprotective agent targeting RANKL-induced osteoclastic born resorption for medicinal use.
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Resorción Ósea/metabolismo , Lisosomas/metabolismo , Osteoclastos/metabolismo , Umbeliferonas/farmacología , Animales , Antígenos de Diferenciación/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/patología , Lisosomas/patología , Ratones , Osteoclastos/patología , Células RAW 264.7RESUMEN
Hyperglycemia elicits tight junction disruption and blood-retinal barrier breakdown, resulting in diabetes-associated vison loss. Eucalyptol is a natural compound found in eucalyptus oil with diverse bioactivities. This study evaluated that eucalyptol ameliorated tight junctions and retinal barrier function in glucose/amyloid-ß (Aß)-exposed human retinal pigment epithelial (RPE) cells and in db/db mouse eyes. RPE cells were cultured in media containing 33 mM glucose or 5 µM Aß for 4 days in the presence of 1-20 µM eucalyptol. The in vivo animal study employed db/db mice orally administrated with 10 mg/kg eucalyptol. Nontoxic eucalyptol inhibited the Aß induction in glucose-loaded RPE cells and diabetic mouse eyes. Eucalyptol reversed the induction of tight junction-associated proteins of ZO-1, occludin-1 and matrix metalloproteinases in glucose- or Aß-exposed RPE cells and in diabetic eyes, accompanying inhibition of RPE detachment from Bruch's membrane. Adding eucalyptol to glucose- or Aß-loaded RPE cells, and diabetic mouse eyes reciprocally reversed induction/activation of apoptosis-related bcl-2, bax, cytochrome C/Apaf-1 and caspases. Eucalyptol attenuated the generation of reactive oxygen species and the induction of receptor for advanced glycation end products in Aß-exposed RPE cells and diabetic eyes. Eucalyptol may ameliorate RPE barrier dysfunction in diabetic eyes through counteracting Aß-mediated oxidative stress-induced RPE cell apoptosis.
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BACKGROUND: Since enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PURPOSE: This study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. METHODS: This study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. RESULTS: Linarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. CONCLUSION: These observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvß3 integrin and core-linked CD44.
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Resorción Ósea/tratamiento farmacológico , Flavonas/farmacología , Adhesiones Focales/efectos de los fármacos , Glicósidos/farmacología , Osteoclastos/efectos de los fármacos , Actinas/metabolismo , Animales , Matriz Ósea/efectos de los fármacos , Matriz Ósea/metabolismo , Resorción Ósea/metabolismo , Cirsium/química , Adhesiones Focales/metabolismo , Receptores de Hialuranos/metabolismo , Integrina alfaVbeta3/metabolismo , Ratones , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Células RAW 264.7RESUMEN
Pulmonary fibrosis is a disease in which lung tissues become fibrous and thereby causes severe respiratory disturbances. Various stimuli induce infiltration of macrophages to the respiratory tract, secreting inflammatory cytokines, which subsequently leads to the development of pulmonary fibrosis. Aesculetin, a major component of the sancho tree and chicory, is known to biologically have antioxidant and anti-inflammatory effects. Human alveolar epithelial A549 cells were cultured for 24 h in conditioned media of THP-1 monocyte-derived macrophages (mCM) with 1-20 µM aesculetin. Micromolar aesculetin attenuated the cytotoxicity of mCM containing inflammatory tumor necrosis factor-α (TNF)-α and interleukin (IL)-8 as major cytokines. Aesculetin inhibited alveolar epithelial induction of the mesenchymal markers in mCM-exposed/IL-8-loaded A549 cells (≈47-51% inhibition), while epithelial markers were induced in aesculetin-treated cells subject to mCM/IL-8 (≈1.5-2.3-fold induction). Aesculetin added to mCM-stimulated A549 cells abrogated the collagen production and alveolar epithelial CXC-chemokine receptor 2 (CXCR2) induction. The production of matrix metalloproteinase (MMP) proteins in mCM-loaded A549 cells was reduced by aesculetin (≈52% reduction), in parallel with its increase in tissue inhibitor of metalloproteinases (TIMP) proteins (≈1.8-fold increase). In addition, aesculetin enhanced epithelial induction of tight junction proteins in mCM-/IL-8-exposed cells (≈2.3-2.5-fold induction). The inhalation of polyhexamethylene guanidine (PHMG) in mice accompanied neutrophil predominance in bronchoalveolar lavage fluid (BALF) and macrophage infiltration in alveoli, which was inhibited by orally administrating aesculetin to mice. Treating aesculetin to mice alleviated PHMG-induced IL-8-mediated subepithelial fibrosis and airway barrier disruption. Taken together, aesculetin may antagonize pulmonary fibrosis and alveolar epithelial barrier disruption stimulated by the infiltration of monocyte-derived macrophages, which is typical of PHMG toxicity, involving interaction of IL-8 and CXCR2. Aesculetin maybe a promising agent counteracting macrophage-mediated inflammation-associated pulmonary disorders.
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Células Epiteliales Alveolares/efectos de los fármacos , Interleucina-8/metabolismo , Macrófagos/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Umbeliferonas/farmacología , Células A549 , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibrosis , Humanos , Masculino , Ratones Endogámicos BALB C , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/prevención & control , Células THP-1RESUMEN
Accumulating evidence demonstrates that the risk of osteoporotic fractures increases in patients with diabetes mellitus. Thus, diabetes-induced bone fragility has recently been recognized as a diabetic complication. As the fracture risk is independent of the reduction in bone mineral density, deterioration in bone quality may be the main cause of bone fragility. Coumarin exists naturally in many plants as phenylpropanoids and is present in tonka beans in significantly high concentrations. This study investigated whether coumarin ameliorated the impaired bone turnover and remodeling under diabetic condition. The in vitro study employed murine macrophage Raw 264.7 cells differentiated to multinucleated osteoclasts with receptor activator of nuclear factor-κΒ ligand (RANKL) in the presence of 33 mM glucose and 1-20 µM coumarin for five days. In addition, osteoblastic MC3T3-E1 cells were exposed to 33 mM glucose for up to 21 days in the presence of 1-20 µM coumarin. High glucose diminished tartrate-resistant acid phosphatase activity and bone resorption in RANKL-differentiated osteoclasts, accompanying a reduction of cathepsin K induction and actin ring formation. In contrast, coumarin reversed the defective osteoclastogenesis in diabetic osteoclasts. Furthermore, high glucose diminished alkaline phosphatase activity and collagen type 1 induction of osteoblasts, which was strongly enhanced by submicromolar levels of coumarin to diabetic cells. Furthermore, coumarin restored the induction of RANK and osteoprotegerin in osteoclasts and osteoblasts under glucotoxic condition, indicating a tight coupling of osteoclastogenesis and osteoblastogenesis. Coumarin ameliorated the impaired bone turnover and remodeling in diabetic osteoblasts and osteoclasts by suppressing the interaction between advanced glycation end product (AGE) and its receptor (RAGE). Therefore, coumarin may restore optimal bone turnover of osteoclasts and osteoblasts by disrupting the hyperglycemia-mediated AGE-RAGE interaction.
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Remodelación Ósea/efectos de los fármacos , Cumarinas/farmacología , Glucosa/efectos adversos , Productos Finales de Glicación Avanzada/metabolismo , Osteoblastos/citología , Osteoclastos/citología , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Ratones , Modelos Biológicos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ligando RANK/farmacología , Células RAW 264.7RESUMEN
Macrophage polarization has been implicated in the pathogenesis of metabolic diseases such as obesity, diabetes, and atherosclerosis. Macrophages responsiveness to polarizing signals can result in their functional phenotype shifts. This study examined whether high glucose induced the functional transition of M2 macrophages, which was inhibited by asaronic acid, one of purple perilla constituents. J774A.1 murine macrophages were incubated with 40 ng/mL interleukin (IL)-4 or exposed to 33 mM glucose in the presence of 1-20 µΜ asaronic acid. In macrophages treated with IL-4 for 48 h, asaronic acid further accelerated cellular induction of the M2 markers of IL-10, arginase-1, CD163, and PPARγ via increased IL-4-IL-4Rα interaction and activated Tyk2-STAT6 pathway. Asaronic acid promoted angiogenic and proliferative capacity of M2-polarized macrophages, through increasing expression of VEGF, PDGF, and TGF-ß. In glucose-loaded macrophages, there was cellular induction of IL-4, IL-4 Rα, arginase-1, and CD163, indicating that high glucose skewed naïve macrophages toward M2 phenotypes via an IL-4-IL-4Rα interaction. However, asaronic acid inhibited M2 polarization in diabetic macrophages in parallel with inactivation of Tyk2-STAT6 pathway and blockade of GLUT1-mediated metabolic pathway of Akt-mTOR-AMPKα. Consequently, asaronic acid deterred functional induction of COX-2, CTGF, α-SMA, SR-A, SR-B1, and ABCG1 in diabetic macrophages with M2 phenotype polarity. These results demonstrated that asaronic acid allayed glucose-activated M2-phenotype shift through disrupting coordinated signaling of IL-4Rα-Tyk2-STAT6 in parallel with GLUT1-Akt-mTOR-AMPK pathway. Thus, asaronic acid has therapeutic potential in combating diabetes-associated inflammation, fibrosis, and atherogenesis through inhibiting glucose-evoked M2 polarization.
Asunto(s)
Benzoatos/farmacología , Glucosa/metabolismo , Activación de Macrófagos/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Polaridad Celular/efectos de los fármacos , Diabetes Mellitus/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/efectos de los fármacos , Ratones , FenotipoRESUMEN
Pulmonary emphysema is characterized by a loss of alveolar integrity due to prolonged cigarette smoking and inhaled irritants. Dried yeast extracts (YE) are employed as food additives, savory flavorings, or creation of umami taste sensations. Despite being rich in nutrition, their application as nutraceuticals and functional foods is not investigated much and little is known about the inhibition of pulmonary emphysema. This study examined whether YE ameliorated pulmonary emphysema in mice is evoked by cigarette smoke (CS) and ovalbumin (OVA). Mice were orally administrated with 25-100 mg/kg YE for 8 weeks. Alveolar epithelial A549 cells exposed to lipopolysaccharide or CS extracts (CSE) were supplemented with 10-100 µg/mL YE. Oral YE administration reduced bronchoalveolar lavage fluid leukocytosis in CS-/OVA-exposed mice. YE reduced induction of inflammatory mediators and MMP-12, and diminished reactive oxygen species production and emphysematous alterations in CS-challenged airways. The YE treatment blunted bax/bcl-2 ratio and activation of p53 and caspases in CS-exposed lungs. Apoptotic death was dampened in CSE-loaded YE-supplemented A549 cells. YE curtailed tissue levels of MMP-12 in inflammatory OVA-exposed lungs. YE abrogated the secretion of TNF-α and MCP-1 through blocking NF-κB signaling in endotoxin-loaded A549 cells. Thus, the antioxidant YE may therapeutically ameliorate oxidative stress and inflammatory tissue destruction in emphysematous diseases.
RESUMEN
SCOPE: Podocytes are a component of glomerular filtration barrier with interdigitating foot processes. The podocyte function depends on the dynamics of actin cytoskeletal and focal adhesion crucial for foot process structure. This study investigates the renoprotective effects of eucalyptol on the F-actin cytoskeleton formation and focal adhesion assembly in glucose-loaded podocytes and diabetic kidneys. METHODS AND RESULTS: Eucalyptol at 1-20 µm reverses the reduction of cellular level of F-actin, ezrin, cortactin, and Arp2/3 in 33 mm glucose-loaded mouse podocytes, and oral administration of 10 mg kg-1 eucalyptol elevates tissue levels of actin cytoskeletal proteins reduced in db/db mouse kidneys. Eucalyptol inhibits podocyte morphological changes, showing F-actin cytoskeleton formation in cortical regions and agminated F-actin along the cell periphery. Eucalyptol induces focal adhesion proteins of paxillin, vinculin, talin1, FAK, and Src in glucose-exposed podocytes and diabetic kidneys. Additionally, GTP-binding Rac1, Cdc42, Rho A, and ROCK are upregulated in glucose-stimulated podocytes and diabetic kidneys, which is attenuated by supplying eucalyptol. Rho A gene depletion partially diminishes GSK3ß induction of podocytes by glucose. CONCLUSION: Eucalyptol ameliorates F-actin cytoskeleton formation and focal adhesion assembly through blockade of the Rho signaling pathway, entailing partial involvement of GSK3ß, which may inhibit barrier dysfunction of podocytes and resultant proteinuria.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Nefropatías Diabéticas/tratamiento farmacológico , Eucaliptol/farmacología , Adhesiones Focales/efectos de los fármacos , Glucosa/toxicidad , Podocitos/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Animales , Células Cultivadas , Membrana Basal Glomerular/fisiología , Glucógeno Sintasa Quinasa 3 beta/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Macrophage polarization has been implicated in the pathogenesis of obesity and type 2 diabetes, which are recognized as chronic proinflammatory diseases. This study investigated that high level of glucose, similar to lipopolysaccharide (LPS), activated macrophages toward M1 phenotypes and 1-20 µM asaronic acid (AA) counteracted diabetic macrophage activation. AA reduced the LPS-promoted secretion of proinflammatory interleukin (IL)-6 and monocyte chemoattractant protein-1. The LPS markedly elevated the macrophage induction of the M1 markers of Toll-like receptor 4 (TLR4), CD36, and CD68, which was attenuated by AA. Also, the LPS significantly enhanced the nuclear factor (NF)-κB transactivation, signal transducers, and activators of transcription 1 (STAT1)/STAT3 activation and suppressor of cytokine signaling 3 (SOCS3) induction in macrophages. However, AA highly suppressed the aforementioned effects of LPS. Glucose-stimulated macrophages expressed advanced glycation end products (AGEs) and receptor for AGE (RAGE). Administration of 20 µM AA to macrophages partly but significantly attenuated such effects (1.65 ± 0.12 vs 0.95 ± 0.25 times glucose control for AGE; 2.33 ± 0.31 vs 1.40 ± 0.22 times glucose control for RAGE). Furthermore, glucose enhanced the macrophage induction of TLR4 and inducible nitric oxide synthase and IL-6 production, while it demoted the production of anti-inflammatory arginase-1 and IL-10. In contrast, AA reversed the induction of these markers in glucose-loaded macrophages. AA dose-dependently and significantly encumbered NF-κB transactivation, Janus kinase 2 (JAK2) and STAT1/STAT3 activation, and SOCS3 induction upregulated in glucose-supplemented macrophages. These results demonstrated for the first time that AA may limit diabetic macrophage activation toward the M1 phenotype through the inhibition of TLR4-/IL-6-mediated NF-κB/JAK2-STAT signaling entailing AGE-RAGE interaction.
Asunto(s)
Benzoatos/farmacología , Glucosa/inmunología , Janus Quinasa 2/inmunología , Macrófagos/efectos de los fármacos , FN-kappa B/inmunología , Extractos Vegetales/farmacología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT3/inmunología , Animales , Línea Celular , Interleucina-6/genética , Interleucina-6/inmunología , Janus Quinasa 2/genética , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , FN-kappa B/genética , Perilla/química , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Advanced glycation end products (AGE) play a causative role in the development of aberrant phenotypes of intraglomerular mesangial cells, contributing to acute/chronic glomerulonephritis. The aim of this study was to explore mechanistic effects of the flavonoid chrysin present in bee propolis and herbs on actin dynamics, focal adhesion, and the migration of AGE-exposed mesangial cells. The in vitro study cultured human mesangial cells exposed to 33 mM glucose and 100 µg/mL AGE-bovine serum albumin (AGE-BSA) for up to 5 days in the absence and presence of 1â»20 µM chrysin. The in vivo study employed db/db mice orally administrated for 10 weeks with 10 mg/kg chrysin. The presence of ≥10 µM chrysin attenuated mesangial F-actin induction and bundle formation enhanced by AGE. Chrysin reduced the mesangial induction of α-smooth muscle actin (α-SMA) by glucose, and diminished the tissue α-SMA level in diabetic kidneys, indicating its blockade of mesangial proliferation. The treatment of chrysin inhibited the activation of vinculin and paxillin and the induction of cortactin, ARP2/3, fascin-1, and Ena/VASP-like protein in AGE-exposed mesangial cells. Oral administration of chrysin diminished tissue levels of cortactin and fascin-1 elevated in diabetic mouse kidneys. Mesangial cell motility was enhanced by AGE, which was markedly attenuated by adding chrysin to cells. On the other hand, chrysin dampened the induction of autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to AGE and in diabetic kidneys. Furthermore, chrysin reduced the mTOR activation in AGE-exposed mesangial cells and diabetic kidneys. The induction of mesangial F-actin, cortactin, and fascin-1 by AGE was deterred by the inhibition of autophagy and mTOR. Thus, chrysin may encumber diabetes-associated formation of actin bundling and focal adhesion and mesangial cell motility through disturbing autophagy and mTOR pathway.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Autofagia , Diabetes Mellitus Experimental , Flavonoides/administración & dosificación , Adhesiones Focales/efectos de los fármacos , Productos Finales de Glicación Avanzada/farmacología , Riñón/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Cortactina/genética , Cortactina/metabolismo , Adhesiones Focales/metabolismo , Humanos , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMEN
Diabetes-associated visual cycle impairment has been implicated in diabetic retinopathy, and chronic hyperglycemia causes detrimental effects on visual function. Chrysin, a naturally occurring flavonoid found in various herbs, has anti-inflammatory, antioxidant, and neuroprotective properties. The goal of the current study was to identify the retinoprotective role of chrysin in maintaining robust retinoid visual cycle-related components. The in vitro study employed human retinal pigment epithelial (RPE) cells exposed to 33 mM of glucose or advanced glycation end products (AGEs) in the presence of 1â»20 µM chrysin for three days. In the in vivo study, 10 mg/kg of chrysin was orally administrated to db/db mice. Treating chrysin reversed the glucose-induced production of vascular endothelial growth factor, insulin-like growth factor-1, and pigment epithelium-derived factor (PEDF) in RPE cells. The outer nuclear layer thickness of chrysin-exposed retina was enhanced. The oral gavage of chrysin augmented the levels of the visual cycle enzymes of RPE65, lecithin retinol acyltransferase (LRAT), retinol dehydrogenase 5 (RDH5), and rhodopsin diminished in db/db mouse retina. The diabetic tissue levels of the retinoid binding proteins and the receptor of the cellular retinol-binding protein, cellular retinaldehyde-binding protein-1, interphotoreceptor retinoid-binding protein and stimulated by retinoic acid 6 were restored to those of normal mouse retina. The presence of chrysin demoted AGE secretion and AGE receptor (RAGE) induction in glucose-exposed RPE cells and diabetic eyes. Chrysin inhibited the reduction of PEDF, RPE 65, LRAT, and RDH5 in 100 µg/mL of AGE-bovine serum albumin-exposed RPE cells. The treatment of RPE cells with chrysin reduced the activation of endoplasmic reticulum (ER) stress. Chrysin inhibited the impairment of the retinoid visual cycle through blocking ER stress via the AGE-RAGE activation in glucose-stimulated RPE cells and diabetic eyes. This is the first study demonstrating the protective effects of chrysin on the diabetes-associated malfunctioned visual cycle.
Asunto(s)
Retinopatía Diabética/prevención & control , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Flavonoides/farmacología , Glucosa/toxicidad , Productos Finales de Glicación Avanzada/toxicidad , Sustancias Protectoras/farmacología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Albúmina Sérica Bovina/toxicidad , Visión Ocular/efectos de los fármacos , Animales , Línea Celular , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas del Ojo/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/fisiopatología , Transducción de Señal/efectos de los fármacosRESUMEN
The purpose of this study was to determine the factors that influence fill weight and weight variability of capsules produced on the In-Cap and to assess any differences in terms of capsule defects between gelatin and HPMC (Quali-V) shells. The In-Cap is an automatic tamping type capsule-filling machine and the low output of approximately 3000 capsules/hour makes it ideal for early formulation development and phase I/IIa clinical supplies manufacture. Four commonly used excipients (Avicel PH101, Avicel PH302, A-Tab, and Prosolv HD90) and a poorly flowing drug blend were encapsulated at various pin settings and powder bed heights. The average fill weight and coefficient of weight variation were determined. The percentage of defective capsules formed during encapsulation was calculated. Results of the study showed that pin setting was critical for controlling the fill weight and the weight variation. The order of pin setting with pin 1 (closer to the powder chute) set to a relatively higher position and pin 4 (before ejection) set to a lower position was found to give higher fill weights with relatively lower weight variability. The powder bed height influenced the fill weight for poorly flowing powders. The capsule machine speed did not appear to significantly influence the fill weight. The fill weight and weight variation were found to depend on the flow property of the material. A large percentage of defective capsules was obtained using HPMC shell size #00. Some of the commonly observed defects included split caps and improperly closed filled capsules. In general, appropriate selection of pin settings and bed height can reduce the weight variability seen, especially with poorly flowing high-dose formulations.