RESUMEN
The methionine-choline-deficient (MCD) diet-induced non-alcoholic steatohepatitis (NASH) in mice is a well-established model. Our study aims to elucidate the factors influencing liver pathology in the MCD mouse model by examining physiological, biochemical, and molecular changes using histology, molecular techniques, and OMICS approaches (lipidomics, metabolomics, and metagenomics). Male C57BL/6J mice were fed a standard chow diet, a methionine-choline-sufficient (MCS) diet, or an MCD diet for 10 weeks. The MCD diet resulted in reduced body weight and fat mass, along with decreased plasma triglyceride, cholesterol, glucose, and insulin levels. However, it notably induced steatosis, inflammation, and alterations in gene expression associated with lipogenesis, inflammation, fibrosis, and the synthesis of apolipoproteins, sphingolipids, ceramides, and carboxylesterases. Lipid analysis revealed significant changes in plasma and tissues: most ceramide non-hydroxy-sphingosine lipids significantly decreased in the liver and plasma but increased in the adipose tissue of MCD diet-fed animals. Oxidized glycerophospholipids mostly increased in the liver but decreased in the adipose tissue of the MCD diet-fed group. The gut microbiome of the MCD diet-fed group showed an increase in Firmicutes and a decrease in Bacteroidetes and Actinobacteria. Metabolomic profiling demonstrated that the MCD diet significantly altered amino acid biosynthesis, metabolism, and nucleic acid metabolism pathways in plasma, liver, fecal, and cecal samples. LC-MS data indicated higher total plasma bile acid intensity and reduced fecal glycohyodeoxycholic acid intensity in the MCD diet group. This study demonstrates that although the MCD diet induces hepatic steatosis, the mechanisms underlying NASH in this model differ from those in human NASH pathology.
RESUMEN
Diet-based models are commonly used to investigate obesity and related disorders. We conducted a comparative profiling of three obesogenic diets HFD, high fat diet; HFHF, high fat high fructose diet; and HFCD, high fat choline deficient diet to assess their impact on the gut microbiome and metabolome. After 20 weeks, we analyzed the gut microbiota and metabolomes of liver, plasma, cecal, and fecal samples. Fecal and plasma bile acids (BAs) and fecal short-chain fatty acids (SCFAs) were also examined. Significant changes were observed in fecal and cecal metabolites, with increased Firmicutes and decreased Bacteroidetes in the HFD, HFHF, and HFCD-fed mice compared to chow and LFD (low fat diet)-fed mice. Most BAs were reduced in plasma and fecal samples of obese groups, except taurocholic acid, which increased in HFCD mice's plasma. SCFAs like acetate and butyrate significantly decreased in obesogenic diet groups, while propionic acid specifically decreased in the HFCD group. Pathway analysis revealed significant alterations in amino acid, carbohydrate metabolism, and nucleic acid biosynthesis pathways in obese mice. Surprisingly, even LFD-fed mice showed distinct changes in microbiome and metabolite profiles compared to the chow group. This study provides insights into gut microbiome dysbiosis and metabolite alterations induced by obesogenic and LFD diets in various tissues. These findings aid in selecting suitable diet models to study the role of the gut microbiome and metabolites in obesity and associated disorders, with potential implications for understanding similar pathologies in humans.
Asunto(s)
Microbioma Gastrointestinal , Humanos , Animales , Ratones , Insulina/metabolismo , Ratones Endogámicos C57BL , Obesidad/metabolismo , Dieta Alta en Grasa/efectos adversos , MetabolomaRESUMEN
We evaluated the effects of select herbal extracts (Tinospora cordifolia [TC], Tinospora cordifolia with Piper longum [TC + PL], Withania somnifera [WS], Glycyrrhiza glabra [GG], AYUSH-64 [AY-64], and Saroglitazar [S]) on various parameters in a diet-induced obesity mouse model. After 12 weeks of oral administration of the herbal extracts in high-fat diet (HFD)-fed C57BL/6J mice, we analyzed plasma biochemical parameters, insulin resistance (IR), liver histology, and the expression of inflammatory and fibrosis markers, along with hepatic lipidome. We also used a 3D hepatic spheroid model to assess their impact on profibrotic gene expression. Among the extracts, TC + PL showed a significant reduction in IR, liver weight, TNF-α, IL4, IL10 expression, and hepatic lipid levels (saturated triglycerides, ceramides, lysophosphocholines, acylcarnitines, diglycerides, and phosphatidylinositol levels). Saroglitazar reversed changes in body weight, IR, plasma triglycerides, glucose, insulin, and various hepatic lipid species (fatty acids, phospholipids, glycerophospholipids, sphingolipids, and triglycerides). With the exception of GG, Saroglitazar, and other extracts protected against palmitic acid-induced fibrosis marker gene expression in the 3D spheroids. TC + PL and Saroglitazar also effectively prevented HFD-induced insulin resistance, inflammation, and specific harmful lipid species in the liver.
RESUMEN
Diet-induced obesity mouse models are widely utilized to investigate the underlying mechanisms of dyslipidemia, glucose intolerance, insulin resistance, hepatic steatosis, and type 2 diabetes mellitus (T2DM), as well as for screening potential drug compounds. However, there is limited knowledge regarding specific signature lipids that accurately reflect dietary disorders. In this study, we aimed to identify key lipid signatures using LC/MS-based untargeted lipidomics in the plasma, liver, adipose tissue (AT), and skeletal muscle tissues (SKM) of male C57BL/6J mice that were fed chow, LFD, or obesogenic diets (HFD, HFHF, and HFCD) for a duration of 20 weeks. Furthermore, we conducted a comprehensive lipid analysis to assess similarities and differences with human lipid profiles. The mice fed obesogenic diets exhibited weight gain, glucose intolerance, elevated BMI, glucose and insulin levels, and a fatty liver, resembling characteristics of T2DM and obesity in humans. In total, we identified approximately 368 lipids in plasma, 433 in the liver, 493 in AT, and 624 in SKM. Glycerolipids displayed distinct patterns across the tissues, differing from human findings. However, changes in sphingolipids, phospholipids, and the expression of inflammatory and fibrotic genes showed similarities to reported human findings. Significantly modulated pathways in the obesogenic diet-fed groups included ceramide de novo synthesis, sphingolipid remodeling, and the carboxylesterase pathway, while lipoprotein-mediated pathways were minimally affected. This study provides a tissue-specific comparison of lipid composition, highlighting the usefulness of DIO models in preclinical research. However, caution is warranted when extrapolating findings from these models to dyslipidemia-associated pathologies and their complications in humans.
Asunto(s)
Diabetes Mellitus Tipo 2 , Dislipidemias , Hígado Graso , Intolerancia a la Glucosa , Humanos , Masculino , Ratones , Animales , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/prevención & control , Insulina , Diabetes Mellitus Tipo 2/complicaciones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Dieta , Hígado Graso/metabolismo , Fosfolípidos/metabolismo , Esfingolípidos , Dislipidemias/complicacionesRESUMEN
Objective: The largest pandemic in history, the COVID-19 pandemic, has been declared a doomsday globally. The second wave spreading worldwide has devastating consequences in every sector of life. Several measures to contain and curb its infection have forged significant challenges for the education community. With an estimated 1.6 billion learners, the closure of schools and other educational institutions has impacted more than 90% of students worldwide from the elementary to tertiary level. Methods: In a view to studying impacts on student's fraternity, this article aims at addressing alternative ways of educating-more specifically, online education-through the analysis of Google trends for the past year. The study analyzed the platforms of online teaching and learning systems that have been enabling remote learning, thereby limiting the impact on the education system. Thorough text analysis is performed on an existing dataset from Kaggle to retrieve insight on the clustering of words that are more often looked at during this pandemic to find the general patterns of their occurrence. Findings: The results show that the coronavirus patients are the most trending patterns in word search clustering, with the education system being at the control and preventive measures to bring equilibrium in the system of education. There has been significant growth in online platforms in the last year. Existing assets of educational establishments have effectively converted conventional education into new-age online education with the help of virtual classes and other key online tools in this continually fluctuating scholastic setting. The effective usage of teaching tools such as Microsoft Teams, Zoom, Google Meet, and WebEx are the most used online platforms for the conduction of classes, and whiteboard software tools and learning apps such as Vedantu, Udemy, Byju's, and Whitehat Junior have been big market players in the education system over the pandemic year, especially in India. Conclusions: The article helps to draw a holistic approach of ongoing online teaching-learning methods during the lockdown and also highlights changes that took place in the conventional education system amid the COVID pandemic to overcome the persisting disruption in academic activities and to ensure correct perception towards the online procedure as a normal course of action in the new educational system. To fill in the void of classroom learning and to minimize the virus spread over the last year, digital learning in various schools and colleges has been emphasized, leading to a significant increase in the usage of whiteboard software platforms.
RESUMEN
The SET1 family of enzymes are well known for their involvement in the histone 3 lysine 4 (H3K4) methylation, a conserved trait of euchromatin associated with transcriptional activation. These methyltransferases are distinct, and involved in various biological functions in the cell. Impairment in the function of SET1 family members leads to a number of abnormalities such as skeletal and neurological defects, leukaemogenesis and even lethality. Tremendous progress has been made in understanding the unique biological roles and the mechanism of SET1 enzymes in context with H3K4 methylation/canonical functions. However, in recent years, several studies have indicated the novel role of SET1 family proteins, other than H3K4 methylation, which are equally important for cellular functions. In this review, we focus on these non-canonical function of SET1 family members.
Asunto(s)
Histonas , Proteínas de Saccharomyces cerevisiae , Metilación de ADN , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Mural cells synthesize and deposit laminin to the basement membrane. To investigate the function of mural cell-derived laminin, we generated a mutant mouse line lacking mural cell-derived laminin (termed PKO). In a previous study, we showed that the PKO mice were grossly normal under homeostatic condition, but developed blood-brain barrier (BBB) breakdown with advanced age (> 8 months), suggesting that these mutants are intrinsically weak. Based on these findings, we hypothesized that PKO mice have exacerbated injuries in pathological conditions. METHODS: Using collagenase-induced intracerebral hemorrhage (ICH) as an injury model, we examined various stroke outcomes, including hematoma volume, neurological function, neuronal death, BBB integrity, paracellular/transcellular transport, inflammatory cell infiltration, and brain water content, in PKO mice and their wildtype littermates at young age (6-8 weeks). In addition, transmission electron microscopy (TEM) analysis and an in vitro ICH model were used to investigate the underlying molecular mechanisms. RESULTS: Compared to age-matched wildtype littermates, PKO mice display aggravated stroke outcomes, including larger hematoma size, worse neurological function, increased neuronal cell death, enhanced BBB permeability, increased transcytosis, and elevated inflammatory cell infiltration. These mutants also exhibit high baseline brain water content independent of aquaporin-4 (AQP4). In addition, mural cell-derived laminin significantly reduced caveolin-1 without affecting tight junction proteins in the in vitro ICH model. CONCLUSIONS: These results suggest that mural cell-derived laminin attenuates BBB damage in ICH via decreasing caveolin-1 and thus transcytosis, regulates brain water homeostasis, and plays a beneficial role in ICH.
Asunto(s)
Membrana Basal/metabolismo , Barrera Hematoencefálica/metabolismo , Hemorragia Cerebral/patología , Laminina/metabolismo , Animales , Barrera Hematoencefálica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcitosis/fisiologíaRESUMEN
Brain pericytes synthesize and deposit laminin at the blood-brain barrier (BBB). The function of pericyte-derived laminin in BBB maintenance remains largely unknown. In a previous study, we generated pericytic laminin conditional knockout (PKO) mice, which developed BBB breakdown and hydrocephalus in a mixed genetic background. However, since hydrocephalus itself can compromise BBB integrity, it remains unclear whether BBB disruption in these mutants is due to loss of pericytic laminin or secondary to hydrocephalus. Here, we report that, in C57Bl6 dominant background, the PKO mice fail to show hydrocephalus, have a normal lifespan, and develop BBB breakdown in an age-dependent manner. Further mechanistic studies demonstrate that abnormal paracellular transport, enhanced transcytosis, decreased pericyte coverage, and diminished AQP4 level are responsible for BBB disruption in PKO mice. These results suggest that pericyte-derived laminin plays an indispensable and age-dependent role in the maintenance of BBB integrity under homeostatic conditions.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Laminina/metabolismo , Pericitos/metabolismo , Factores de Edad , Animales , Acuaporina 4/metabolismo , Células Endoteliales/metabolismo , Femenino , Laminina/genética , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Skeletal muscle regeneration is a highly orchestrated process and involves the activation of many cellular and molecular pathways. Although satellite cells (SCs) are the major cell type responsible for muscle regeneration, pericytes show remarkable myogenic potential and various advantages as cell therapy in muscular disorders. This chapter first introduces the structure, marker expression, origin, and category of pericytes. Next, we discuss their functions in muscular dystrophy and/or muscle injuries, focusing on their myogenic, adipogenic, fibrogenic, chondrogenic, and osteogenic activities. Understanding this knowledge will promote the development of innovative cell therapies for muscle disorders, including muscular dystrophy.
Asunto(s)
Desarrollo de Músculos , Músculo Esquelético/citología , Pericitos/citología , Diferenciación Celular , Humanos , Distrofias Musculares , Mioblastos/citología , RegeneraciónRESUMEN
Endothelial cells make laminin-411 and laminin-511. Although laminin-411 is well studied, the role of laminin-511 remains largely unknown due to the embryonic lethality of lama5-/- mutants. In this study, we generated endothelium-specific lama5 conditional knockout (α5-TKO) mice and investigated the biological functions of endothelial lama5 in blood-brain barrier (BBB) maintenance under homeostatic conditions and the pathogenesis of intracerebral hemorrhage (ICH). First, the BBB integrity of α5-TKO mice was measured under homeostatic conditions. Next, ICH was induced in α5-TKO mice and their littermate controls using the collagenase model. Various parameters, including injury volume, neuronal death, neurological score, brain edema, BBB integrity, inflammatory cell infiltration, and gliosis, were examined at various time points after injury. Under homeostatic conditions, comparable levels of IgG or exogenous tracers were detected in α5-TKO and control mice. Additionally, no differences in tight junction expression, pericyte coverage, and astrocyte polarity were found in these mice. After ICH, α5-TKO mice displayed enlarged injury volume, increased neuronal death, elevated BBB permeability, exacerbated infiltration of inflammatory cells (leukocytes, neutrophils, and mononuclear cells), aggravated gliosis, unchanged brain edema, and worse neurological function, compared to the controls. These findings suggest that endothelial lama5 is dispensable for BBB maintenance under homeostatic conditions but plays a beneficial role in ICH.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Lesiones Encefálicas/metabolismo , Hemorragia Cerebral/metabolismo , Células Endoteliales/metabolismo , Laminina/metabolismo , Animales , Astrocitos/metabolismo , Edema Encefálico/metabolismo , Lesiones Encefálicas/etiología , Hemorragia Cerebral/etiología , Colagenasas/efectos adversos , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Noqueados , Pericitos/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Stroke is a cerebrovascular disorder that affects many people worldwide. In addition to the well-established functions of astrocytes and microglia in stroke pathogenesis, pericytes also play an important role in stroke progression and recovery. As perivascular multi-potent cells and an important component of the blood-brain barrier (BBB), pericytes have been shown to exert a large variety of functions, including serving as stem/progenitor cells and maintaining BBB integrity. Here in this review, we summarize the roles of pericytes in stroke pathogenesis, with a focus on their effects in cerebral blood flow, BBB integrity, angiogenesis, immune responses, scar formation and fibrosis.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Pericitos/metabolismo , Accidente Cerebrovascular/patología , Cicatriz/patología , Humanos , Neovascularización Patológica , Pericitos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFRß, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.
Asunto(s)
Citometría de Flujo/métodos , Músculo Esquelético/citología , Pericitos/citología , Animales , Diferenciación Celular/fisiología , Ratones , Músculo Esquelético/metabolismo , Pericitos/metabolismoRESUMEN
OBJECTIVE: Atherogenic diet (AD) and high fat diet (HFD) cause deleterious effect on bone micro-architecture and this phenomenon prompts aortic calcification. This study aims to show the effects of Caviunin ß-d-glucopyranoside (CAFG), against bone loss and its associated aortic calcification in presence of AD and HFD challenged diets. METHODS: Five groups of C57BL/6 male mice with 8 animals in each group, comprising of chow, AD, HFD, AD+CAFG and HFD+CAFG were fed with respective diets for 16 weeks. At the end of the treatment period, preventive effects of CAFG on bone tissue were analyzed by assessing the osteogenic potential of bone marrow cells, bone micro-architecture, ability of new bone formation and histomorphometry studies. Aortic calcification was assessed by transcription and translation analysis of osteogenic key markers in aortic tissue and assessment of aortic endothelial function. Plasma lipid profiling was done to assess the effects of diets as its role in both bone loss and aortic calcification. RESULTS: Bone marrow stromal cell (BMSC's) dynamics showed that AD and HFD decreased osteoblast number that led to bone loss, deterioration in bone micro-architecture with up-regulated bone resorptive genes that lead to increase in aortic calcification. CAFG treatment rescued the bone health by modulating BMSC's towards osteogenic lineage. It increased the osteogenic gene expression with simultaneous decrease in osteoclastic genes thus stabilized the receptor activator of nuclear factor-kappa-B ligand/osteoprotegerin ratio that eventually reduced the amount of calcification in aorta. Biochemical studies showed that CAFG reduced the TC, TG and LDL-C content with no marked changes in HDL-C. Moreover, CAFG decreased the osteogenic key markers in the aortic tissue and enhanced endothelial function. CONCLUSION: Overall, this study indicates that CAFG protected against physiologically challenged diet induced bone loss with associated vascular calcification in mice. Moreover, data revealed that atherogenic diet is more detrimental as compared to the excess fatty acid diet to the bone and aorta.
Asunto(s)
Aorta/patología , Aterosclerosis/tratamiento farmacológico , Huesos/patología , Calcinosis/tratamiento farmacológico , Dieta Alta en Grasa , Glicósidos/uso terapéutico , Isoflavonas/uso terapéutico , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Fenómenos Biomecánicos/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Calcinosis/patología , Hueso Esponjoso/patología , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glicósidos/química , Isoflavonas/química , Lípidos/sangre , Hígado/patología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones Endogámicos C57BL , Obesidad/patología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fosfatasa Ácida Tartratorresistente/metabolismo , Microtomografía por Rayos XRESUMEN
Balance between adipocyte and osteoblast differentiation is the key link of disease progression in obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and adipocytes prompted us to analyse the effect of FNT on adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced obesity. The anti-obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT suppresses the adipogenic differentiation of 3T3-L1 fibroblasts, through down-regulation of key adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of ß-catenin were attributed to the anti-adipogenic action of FNT. In vivo, 12 weeks of FNT treatment inhibited the development of obesity in mice by attenuating HFD-induced body weight gain and visceral fat accumulation. The anti-obesity effect of FNT results from increased energy expenditure. FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT's rescuing action against obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased adipogenic potentials. Our findings suggest that FNT inhibits adipogenesis through AMPK/ß-catenin signal transduction pathways and protects against HFD-induced obesity and bone loss.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adipogénesis/efectos de los fármacos , Isoflavonas/farmacología , Obesidad/prevención & control , Osteoporosis/prevención & control , beta Catenina/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Resorción Ósea/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Osteoporosis/etiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Desacopladora 1/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Laminin, a major basement membrane component that has direct contact with pericytes under physiological conditions, actively regulates the proliferation and differentiation/fate determination of pericytes. Recently, two types of pericytes (type I and type II) with different molecular markers and functions have been identified in skeletal muscles. Whether laminin differentially regulates the proliferation and differentiation of these two subpopulations remains unclear. METHODS: Wild-type and pericytic laminin-deficient mice under Nestin-GFP background were used to determine if laminin differentially regulates the proliferation and differentiation of type I and type II pericytes. Specifically, type I and type II pericytes were isolated from these mice, and their proliferation and differentiation were examined in vitro. Moreover, in vivo studies were also performed. RESULTS: We demonstrate that, although laminin inhibits the proliferation of both type I and type II pericytes in vitro, loss of laminin predominantly induces proliferation of type II pericytes in vivo. In addition, laminin negatively regulates the adipogenic differentiation of type I pericytes and positively regulates the myogenic differentiation of type II pericytes in vitro. CONCLUSIONS: Laminin differentially regulates the proliferation and differentiation of type I and type II pericytes.
Asunto(s)
Membrana Basal/metabolismo , Laminina/genética , Nestina/genética , Pericitos/metabolismo , Animales , Membrana Basal/citología , Diferenciación Celular , Proliferación Celular , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Laminina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Nestina/metabolismo , Pericitos/clasificación , Pericitos/citologíaRESUMEN
Leaves of Dalbergia sissoo is known to have protective actions against postmenopausal bone loss in rat. In this study, we have evaluated the fracture healing properties of ethanolic extract (EE) of Dalbergia sissoo leaves. To observe the fracture healing property in the drill-hole injury model, we randomly divided total 32 adult female Sprague Dawley rats (180±200g) into 4 groups: (i) Control operated group; (ii) EE (250mg/kg/day); (iii) EE (500mg/kg/day) and (iv) EE (1000mg/kg/day). The right femora were fractured at the mid-diaphysis region and each group of rats received their respective treatment for 15days. Ethanol extract dose dependently induced bone regeneration at the fracture site assessed by fluorochrome labeling. All of three doses, 250mg/kg/day dose significantly increased bone volume fraction, trabecular thickness, trabecular number, and connectivity density and decreased trabecular separation in bone. Furthermore, the extract induced the expression of osteogenic genes including BMP-2, BMP-4, RunX-2 and COL-1 compared to the control group. The EE improved fracture healing much earlier (day 15) than the normal healing process, as assessed by the increased callus volumes and mineralized nodule formation. This extract is found beneficial in fracture healing of rat.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Hueso Cortical/efectos de los fármacos , Dalbergia , Etanol/farmacología , Curación de Fractura/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Regeneración Ósea/fisiología , Células Cultivadas , Hueso Cortical/lesiones , Hueso Cortical/fisiología , Modelos Animales de Enfermedad , Femenino , Fémur/efectos de los fármacos , Fémur/lesiones , Fémur/fisiología , Curación de Fractura/fisiología , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Extractos Vegetales/aislamiento & purificación , Ratas , Ratas Sprague-DawleyRESUMEN
Laminin, a major component of the basement membrane, plays an important role in blood brain barrier regulation. At the neurovascular unit, brain endothelial cells, astrocytes, and pericytes synthesize and deposit different laminin isoforms into the basement membrane. It has been shown that laminin α4 (endothelial laminin) regulates vascular integrity at embryonic/neonatal stage, while astrocytic laminin maintains vascular integrity in adulthood. Here, we investigate the function of pericyte-derived laminin in vascular integrity. Using a conditional knockout mouse line, we report that loss of pericytic laminin leads to hydrocephalus and BBB breakdown in a small percentage (10.7%) of the mutants. Interestingly, BBB disruption always goes hand-in-hand with hydrocephalus in these mutants, and neither symptom is observed in the rest 89.3% of the mutants. Further mechanistic studies show that reduced tight junction proteins, diminished AQP4 expression, and decreased pericyte coverage are responsible for the BBB disruption. Together, these data suggest that pericyte-derived laminin is involved in the maintenance of BBB integrity and regulation of ventricular size/development.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Laminina/metabolismo , Animales , Acuaporina 4/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Desmina/metabolismo , Hidrocefalia/metabolismo , Hidrocefalia/patología , Laminina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Pericitos/citología , Pericitos/metabolismo , Fenotipo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
OBJECTIVE: This study aims to evaluate the skeletal effects of dalbergin (DBN), isolated from Dalbergia sissoo heartwood, in ovariectomized (OVx) BALB/c mice, a postmenopausal osteoporosis model of bone loss. METHODS: Adult BALB/c mice were used and randomly assigned in to six groups with 6 animals (n=6) in each group: sham (surgery operated without ovariectomy) with vehicle, ovariectomy with vehicle, ovariectomy (OVx) with estradiol (E2 5.0µgkg-1day-1), or ovariectomy with dalbergin at three different doses of DBN (1.0, 5.0 and10mgkg-1day-1). Daily oral administration of the vehicle, estradiol, or DBN was started 8 weeks post-surgery and continued for 8 weeks. At the end of experiment, mice were sacrificed and assessed for trabecular bone structure of tibia, lumbar vertebra (L5) and alterations in biochemical and uterine parameters, pharmacokinetic profile and gene expression were monitored for each group. RESULTS: Treatment with DBN prevented trabecular bone loss in cancellous bone in the tibial metaphysis and lumbar vertebra region of the ovariectomized mice. Micro-CT data showed that mice treated with DBN at 1.0mgkg-1day-1 exhibited improved bone micro-architecture that was sustained with decreased expression of bone resorption markers like TRAP and RANK and caused an increase in osteogenic markers like RUNX2, BMP2 and OPG/RANKL ratio compared with OVx+vehicle treated mice. Moreover, DBN treatment induced no uterine estrogenicity and significantly lowered the osteocalcin amount in serum when compared with OVx+V group. DBN reached its maximum concentration (Cmax) 238.49±21.37ngml-1 in serum as early as 1h of administration. Overall, DBN (1.0mgkg-1day-1) treatment exhibited similar bone conserving effect against bone-loss as estradiol treatment. CONCLUSION: Daily oral administration of DBN for 8 weeks showed significant anabolic effects on bone micro-architectural parameters along with down regulation of bone resorptive markers without compromising safety at uterine level. Therefore, our study provides basis for DBN as a therapeutic candidate against postmenopausal osteoporosis.
Asunto(s)
Cumarinas/uso terapéutico , Dalbergia/química , Fémur/patología , Flavonoides/uso terapéutico , Osteoporosis/tratamiento farmacológico , Ovariectomía , Sustancias Protectoras/uso terapéutico , Administración Oral , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Fenómenos Biomecánicos/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Remodelación Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Hueso Esponjoso/efectos de los fármacos , Hueso Esponjoso/patología , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/farmacocinética , Modelos Animales de Enfermedad , Femenino , Fémur/efectos de los fármacos , Fémur/fisiopatología , Flavonoides/administración & dosificación , Flavonoides/química , Flavonoides/farmacocinética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Ratones Endogámicos BALB C , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocalcina/sangre , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoporosis/diagnóstico por imagen , Osteoporosis/genética , Osteoporosis/patología , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/química , Sustancias Protectoras/farmacocinética , Útero/efectos de los fármacos , Útero/patología , Microtomografía por Rayos XRESUMEN
Dalbergia sissoo Roxb. is a well known medicinal plant of India, enriched with various flavonoids used for treating multiple diseases. Earlier, we have shown that extract of Dalbergia sissoo Roxb. leaves mitigate ovariectomy induced bone loss and pure compounds (neoflavonoids) isolated from it, promote osteoblastogenesis in primary calvarial osteoblasts cells in vitro. Here, we hypothesize that dalsissooal (DSL), a novel neoflavonoid isolated from the heartwood of Dalbergia sissoo Roxb. is an important constituent of the extract that imparts bone forming effects. Treatment with DSL enhanced trabecular bone micro-architecture parameters, biomechanical strength, increased bone formation rate and mineral apposition rate in OVx mice comparable to 17ß-estradiol. It increased bone formation by enhancing osteoblast gene expression and reduced bone turnover by decreasing osteoclastic gene expressions. Interestingly, we observed that DSL has no uterine estrogenic effects. At cellular levels, DSL promoted differentiation of bone marrow cells as well as calvaria osteoblast cells towards osteoblast lineage by enhancing differentiation and mineralizing ability to form mineralizing nodules via stimulating BMP-2 and RunX-2 expressions. Overall, our data suggest that oral supplementation of a novel neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. exhibited bone anabolic action by improving structural property of bone, promoting new bone formation and reducing bone turnover rate in post-menopausal model for osteoporosis with no uterine hyperplasia.
Asunto(s)
Acroleína/análogos & derivados , Dalbergia/química , Flavonoides/farmacología , Osteogénesis/efectos de los fármacos , Osteoporosis/fisiopatología , Fenoles/farmacología , Acroleína/aislamiento & purificación , Acroleína/farmacología , Animales , Calcificación Fisiológica/efectos de los fármacos , Hueso Esponjoso/efectos de los fármacos , Hueso Esponjoso/patología , Hueso Esponjoso/fisiopatología , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estrógenos/deficiencia , Femenino , Flavonoides/aislamiento & purificación , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteocalcina/sangre , Osteoporosis/metabolismo , Osteoporosis/patología , Ovariectomía , Fenoles/aislamiento & purificación , Hojas de la Planta/química , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
Terpenoids were isolated using chromatographic purification through solvent purification technique and identified as Azadirone (1), Epoxyazadiradione (2) Azadiradione (3) Gedunin (4) Nimbin (5) Salannin (6) Azadirachtin A (7) and Azadirachtin B (8) from Azadirachta indica. Out of eight compounds, only three compounds had osteogenic activity and enhanced osteoblast proliferation, differentiation and mineralization in osteoblast cells. Active compounds stimulated osteogenic genes ALP, RunX-2 and OCN expressions in vitro, but Azadirachtin A had a maximum ability to stimulate osteoblast differentiation and mineralization compared to other two active compounds. For in vivo study, Azadirachtin A injected subcutaneously in pups, which enhanced osteogenic gene expressions and promoted bone formation rate significantly. Here, we conclude that active compounds of Azadirachta indica have osteogenic activity and Azadirachtin A has a beneficial effects on bone.