Osteoporosis GWAS-implicated DNM3 locus contextually regulates osteoblastic and chondrogenic fate of mesenchymal stem/progenitor cells through oscillating miR-199a-5p levels.

Genome wide association study (GWAS)-implicated bone mineral density (BMD) signals have been shown to localize in cis-regulatory regions of distal effector genes using 3D genomic methods. Detailed characterization of such genes can reveal novel causal genes for BMD determination. Here, we elected to characterize the "DNM3" locus on chr1q24, where the long non-coding RNA DNM3OS and the embedded microRNA MIR199A2 (miR-199a-5p) are implicated as effector genes contacted by the region harboring variation in linkage disequilibrium with BMD-associated sentinel single nucleotide polymorphism, rs12041600. During osteoblast differentiation of human mesenchymal stem/progenitor cells (hMSC), miR-199a-5p expression was temporally decreased and correlated with the induction of osteoblastic transcription factors RUNX2 and Osterix. Functional relevance of miR-199a-5p downregulation in osteoblastogenesis was investigated by introducing miR-199a-5p mimic into hMSC. Cells overexpressing miR-199a-5p depicted a cobblestone-like morphological change and failed to produce BMP2-dependent extracellular matrix mineralization. Mechanistically, a miR-199a-5p mimic modified hMSC propagated normal SMAD1/5/9 signaling and expressed osteoblastic transcription factors RUNX2 and Osterix but depicted pronounced upregulation of SOX9 and enhanced expression of essential chondrogenic genes ACAN, COMP, and COL10A1. Mineralization defects, morphological changes, and enhanced chondrogenic gene expression associated with miR-199a-5p mimic over-expression were restored with miR-199a-5p inhibitor suggesting specificity of miR-199a-5p in chondrogenic fate specification. The expression of both the DNM3OS and miR-199a-5p temporally increased and correlated with hMSC chondrogenic differentiation. Although miR-199a-5p overexpression failed to further enhance chondrogenesis, blocking miR-199a-5p activity significantly reduced chondrogenic pellet size, extracellular matrix deposition, and chondrogenic gene expression. Taken together, our results indicate that oscillating miR-199a-5p levels dictate hMSC osteoblast or chondrocyte terminal fate. Our study highlights a functional role of miR-199a-5p as a BMD effector gene at the DNM3 BMD GWAS locus, where patients with cis-regulatory genetic variation which increases miR-199a-5p expression could lead to reduced osteoblast activity.

Modular, Vascularized Hypertrophic Cartilage Constructs for Bone Tissue Engineering Applications.

Insufficient vascularization is a main barrier to creating engineered bone grafts for treating large and ischemic defects. Modular tissue engineering approaches have promise in this application because of the ability to combine tissue types and to localize microenvironmental cues to drive desired cell function. In direct bone formation approaches, it is challenging to maintain sustained osteogenic activity, since vasculogenic cues can inhibit tissue mineralization. This study harnessed the physiological process of endochondral ossification to create multiphase tissues that allowed concomitant mineralization and vessel formation. Mesenchymal stromal cells in pellet culture were differentiated toward a cartilage phenotype, followed by induction to chondrocyte hypertrophy. Hypertrophic pellets exhibited increased alkaline phosphatase activity, calcium deposition, and osteogenic gene expression relative to chondrogenic pellets. In addition, hypertrophic pellets secreted and sequestered angiogenic factors, and supported new blood vessel formation by co-cultured endothelial cells and undifferentiated stromal cells. Multiphase constructs created by combining hypertrophic pellets and vascularizing microtissues and maintained in unsupplemented basal culture medium were shown to support robust vascularization and sustained tissue mineralization. These results demonstrate a new in vitro strategy to produce multiphase engineered constructs that concomitantly support the generation of mineralize and vascularized tissue in the absence of exogenous osteogenic or vasculogenic medium supplements.

A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Nutrition, Food and Diet in Health and Longevity: We Eat What We Are.

Nutrition generally refers to the macro- and micro-nutrients essential for survival, but we do not simply eat nutrition. Instead, we eat animal- and plant-based foods without always being conscious of its nutritional value. Furthermore, various cultural factors influence and shape our taste, preferences, taboos and practices towards preparing and consuming food as a meal and diet. Biogerontological understanding of ageing has identified food as one of the three foundational pillars of health and survival. Here we address the issues of nutrition, food and diet by analyzing the biological importance of macro- and micro-nutrients including hormetins, discussing the health claims for various types of food, and by reviewing the general principles of healthy dietary patterns, including meal timing, caloric restriction, and intermittent fasting. We also present our views about the need for refining our approaches and strategies for future research on nutrition, food and diet by incorporating the molecular, physiological, cultural and personal aspects of this crucial pillar of health, healthy ageing and longevity.

Dietary Supplementation With Tinospora cordifolia Improves Anxiety-Type Behavior and Cognitive Impairments in Middle-Aged Acyclic Female Rats.

The midlife transition period in women marks the progressive flattening of neurological health along with increased adiposity, dyslipidemia, frailty, and inflammatory responses mainly attributed to the gradual decline in estrogen levels. Conflicting reports of hormone replacement therapy (HRT) necessitate the exploration of novel therapeutic interventions using bioactive natural products having the least toxicity and a holistic mode of action for the preservation of metabolic homeodynamics with aging in women. The present study was planned to investigate the effects of aging and/or a high-fat diet (HFD) on cognitive impairments and anxiety and further their management by dietary supplement with the Tinospora cordifolia stem powder (TCP). Acyclic female rats were included in this study as the model system of the perimenopause phase of women along with young 3-4 months old rats as controls. Rats were fed on with and without TCP supplemented normal chow or HFD for 12 weeks. Animals fed on a TCP supplemented normal chow showed consistent management of body weight over a 12-week regimen although their calorie intake was much higher in comparison to their age-matched controls. Post-regimen, neurobehavioral tests, such as novel object recognition and elevated plus maze, performed on these animals showed improvement in their learning and memory abilities as well as the anxiety-like behavior. Furthermore, due to the presence of multiple components, TCP was observed to modulate the expression of key marker proteins to ameliorate neuroinflammation and apoptosis and promote cell survival and synaptic plasticity in the hippocampus and the prefrontal cortex (PFC) regions of the brain. These findings suggest that TCP supplementation in diet during the midlife transition period in women may be a potential interventional strategy for the management of menopause-associated anxiety and cognitive impairments and healthy aging.

Dietary intervention with Tinospora cordifolia improved aging-related decline in locomotor coordination and cerebellar cell survival and plasticity in female rats.

Reduced bone mineral density, and muscle strength are the hallmark of aging-related motor coordination deficits and related neuropathologies. Since cerebellum regulates motor movements and balance perception of our body, therefore it may be an important target to control the age-related progression of motor dysfunctions. Dry stem powder of Tinospora cordifolia (TCP) was tested as a food supplement to elucidate its activity to attenuate age-associated locomotor dysfunctions. Intact acyclic middle-aged female rats were used in this study as the model system of the transition phase from premenopause to menopause in women along with cycling young adult rats. Normal chow or 30% High Fat Diet (HFD), supplemented with or without TCP was fed to animals for 12 weeks and then tested for locomotor performance on rotarod followed by post-sacrifice protein expression studies. In comparison to young adults, middle-aged animals showed an increase in number of falls and lesser time spent in rotarod performance test, whereas, animals given TCP supplemented feed showed improvement in performance with more pronounced effects observed in normal chow than HFD fed middle-aged rats. Further, due to its multicomponent nature TCP was found to target the expression of various markers of neuroinflammation, apoptosis, cell survival, and synaptic plasticity in the cerebellum region. The current findings suggest that TCP supplementation in the diet may prove to be a potential interventional strategy for the management of frailty and fall-associated morbidities caused by aging-related deterioration of bone mineral density, and muscle strength.

Aging-related changes in metabolic indicators in female rats and their management with Tinospora cordifolia.

Conflicting reports of HRT necessitates exploration of therapeutic interventions with the least side effects to preserve metabolic homeodynamics in women later in life. The current study was designed to elucidate the cumulative effects of aging and/or high fat diet (HFD) on some metabolic indicators and their management by Tinospora cordifolia stem powder (TCP) using middle-aged acyclic and young adult cyclic female rats as the model system. Animals were fed on either normal chow or HFD supplemented with or without TCP. Blood and liver tissue were collected for biochemical, and histological studies as well as for expression of proteins regulating lipid metabolism. Animals fed with TCP supplemented normal chow feed showed bodyweight management over 12-weeks despite their high feed and calories intake compared to young and age-matched controls as well as HFD-fed animals. TCP dose used was not toxic and rather prevented age-associated liver dysfunctions and ameliorated dyslipidemia and oxidative stress, normalized blood glucose, insulin, leptin, and secretary pro-inflammatory cytokines. Further, bodyweight management effect of TCP was observed to target AMPK signalling pathway as the mediator of lipogenesis, sterol biosynthesis, lipolysis, and ß-oxidation of fatty acids. These findings suggest that TCP supplementation in diet may be a potential interventional strategy to ameliorate aging-associated hepatic and metabolic dysfunctions and to promote healthy aging.

Acute Sleep Deprivation-Induced Anxiety and Disruption of Hypothalamic Cell Survival and Plasticity: A Mechanistic Study of Protection by Butanol Extract of Tinospora cordifolia.

Since sleep is a key homeostatic phenomenon of the body, therefore understanding the complex etiology of the neurological outcome of sleep deprivation (SD) such as anxiety, depression, cognitive dysfunctions, and their management is of utmost importance. The findings of the current study encompass the neurobehavioral as well as hormonal, and neuroinflammatory changes in serum and hypothalamus region of the brain as an outcome of acute SD and their amelioration by pre-treatment with butanol extract of Tinospora cordifolia. SD group animals showed anxiety-like behavior as evident from Elevated Plus Maze data and higher serum cortisol levels, whereas, pre-treatment with B-TCE showed anxiolytic activity and also reduced cortisol levels which was corroborated by an increase in leptin and insulin levels. Further, SD induced elevation of serum pro-inflammatory cytokines IL-6, TNF-α, IL-1ß, and MCP-1 and subsequent activation of astroglial cells in the hypothalamus was suppressed in B-TCE pre-treated animals. The current findings suggest that besides the cortical structures, hypothalamus region's synaptic plasticity and cell survival are adversely impacted by acute SD. Further active ingredients present in B-TCE may be useful for the management of SD-induced anxiety, systemic inflammation, and neuroinflammation by targeting hypothalamic BDNF-TrkB/PI3K-Akt pathways.

Butanol Extract of Tinospora cordifolia Alleviates Acute Sleep Deprivation-Induced Impairments in Cognitive Functions and Neuromuscular Coordination in Middle-Aged Female Rats.

Sleep deprivation due to present-day lifestyle and late-hours work commitments are associated with a broad spectrum of neurobehavioral complications. Moreover, women, as they age, become prone to the cumulative effects of menopause such as sleep disturbances, adiposity, and inflammation which are attributed to a compromised immuno-neuro-endocrine axis. So far, no effective therapeutic remedy is available to mitigate the adverse effects of SD. The current study was aimed to elucidate the neuroprotective potential of n-Butanol fraction obtained from hydroalcoholic extract of Tinospora cordifolia stem (B-TCE). Four groups of female rats are (1) Vehicle-undisturbed sleep, (2) Vehicle-sleep deprived (between 6 a.m. and 6 p.m.), (3) B-TCE oral feeding for 2 weeks and sleep deprivation, and (4) B-TCE alone undisturbed sleep group. Novel Object Recognition test was used to study cognitive impairments and Rotarod for motor coordination. Rats were then sacrificed to study the expression of various marker proteins in the hippocampus and piriform cortex regions of the brain by western blotting. SD was observed to impair the exploratory behavior and neuromuscular coordination, whereas, B-TCE pre-treatment was observed to ameliorate these behavioral functions'- impairments and further suppressed the changes in the expression of markers for synaptic plasticity, inflammation, cell survival, and apoptosis pathways. The current data suggest that B-TCE may be effective in the management of acute SD-associated impairments in learning and memory functions and neuromuscular coordination.

In-vitro antitumor activity of compounds from Glycyrrhiza glabra against C6 glioma cancer cells: identification of natural lead for further evaluation.

A HP20 resin-based unique method was adopted to get an active fraction of the hydroalcoholic extract of G. glabra roots. The fraction showed potent cytotoxicity against cancer cell line and was further subjected to detailed phytochemical investigation to obtain ten biomarkers. The isolated compounds were also tested for the cytotoxicity against the C6 glioma cell line in vitro using MTT assay. Among the isolated compounds, glycyrrhetic acid (1), glabrol (6), and glabridin (9) exhibited significant cytotoxicity. The compounds showed a dose-dependent decrease in cell viability. The active compounds were subjected to molecular docking study against topoisomerase I and topoisomerase II to support the mechanism of antitumor activity.

Tinospora cordifolia ameliorates brain functions impairments associated with high fat diet induced obesity.

Obesity is a rapidly growing health problem worldwide and its prevalence has increased markedly in both the developing and developed nations. It is associated with a range of co-morbidities such as cardiovascular disease, type 2 diabetes mellitus, and cognitive dysfunctions. Therefore, the need for a safe and effective treatment has led to the exploration of natural products for the management of obesity. In the present study, we tested the anxiolytic, anti-apoptotic, and anti-neuroinflammatory potential of Tinospora cordifolia in a high fat diet-induced obesity rat model system. Young female Wistar albino rats were divided into three groups: (1) Low fat diet (LFD), fed on normal chow feed; (2) High fat diet (HFD), fed on diet containing 30% fat by weight; and (3) High fat diet containing extract (HFDE), fed on high fat diet supplemented with the stem powder of T. cordifolia (TCP). The rats from each group were kept on their respective feeding regimen for 12 weeks. The body weight and calorie intake were recorded weekly. The elevated plus maze test and rotarod performance test were performed to evaluate the anxiety-like behavior and locomotor coordination, respectively. The levels of serum cytokines (IL-6 and TNF-α) were estimated and various markers for inflammation, synaptic plasticity, apoptosis, and energy homeostasis were studied by western blotting. The HFDE rats showed reduced anxiety-like behavior and improved locomotor behavior as compared to HFD-induced obese rats. The TCP supplementation in high fat diet suppressed the expression of inflammatory molecules, including serum cytokines (IL-6 and TNF-α), and modulated apoptosis and synaptic plasticity. TCP was found to be effective in managing body weight in HFD-fed rats by maintaining energy metabolism and cellular homeostasis. T. cordifolia may be recommended as a potential therapeutic agent to prevent the adverse effects of obesity and obesity-associated brain dysfunctions.

PSA mimetic 5-nonyloxytryptamine protects cerebellar neurons against glutamate induced excitotoxicity: An in vitro perspective.

PSA-NCAM is a molecule of therapeutic interest for its key role in promoting neuritogenesis and synaptic plasticity. The current study was aimed to investigate the neuroregenerative potential of 5-nonyloxytryptamine (5-NOT) as PSA mimetic compound against glutamate induced excitotoxicity. 2D and 3D cultures of cerebellar neurons challenged with glutamate were used to ascertain the effect of 5-NOT on neurite outgrowth, migration and expression of neuronal plasticity markers. Glutamate excitotoxicity is one of the major underlying pathological factor responsible for neurodegeneration in various neurological disorders such as trauma, stroke, ischemia, epilepsy and neurodegenerative diseases.5-NOT treatment was observed to promote axonal growth and defasiculation in glutamate challenged neurons as well as promoted the migration of cerebellar neurons in both wound scratched area and cerebellar explant cultures. Further, 5-NOT treatment upregulated the expression of synaptic plasticity and cell survival pathway proteins which showed reduced expression after glutamate induced excitotoxicity. Thus, this preliminary data reveals thatPSA-mimetic,5-NOT may prove to be a potential neuroprotective candidate for neurodegenerative diseases.

From ayurvedic folk medicine to preclinical neurotherapeutic role of a miraculous herb, Tinospora cordifolia.

In Ayurveda, the age-old Indian traditional system of medicine, health is considered to be achieved as equilibrium of physical and mental wellbeing and brain related ailments are recognized as 'Vatavyadi'. Rasayana herbs were mainly used for pharmacological treatment of neurological diseases and Tinospora cordifolia is one of the popular Rasayana herbs of Ayurveda. The traditional claims of therapeutic activity of this herb for treatment of fever, diabetes, anxiety, immunodeficiency, memory deficit and psychological problems have been explored by different research groups using reverse pharmacology and advance technological approaches. The aim of current review is to compile and discuss the neurotherapeutic potential of T. cordifolia in the light of various preclinical and clinical studies from literature. This review summarizes the information about different extracts of this herb and decoctions used for various neuro-related problems such as neurodegenerative diseases, neuroinflammation, sleep disorders, neural cancers, memory and cognition deficits and psychological problems besides other potential activities. The review also provides the knowledge of underlying therapeutic mechanism of T. cordifolia and its active phytoconstituents.

The neuroplasticity marker PSA-NCAM: Insights into new therapeutic avenues for promoting neuroregeneration.

Neuroplastic alterations are the key processes involved in adaptation and rehabilitation after all neurological injuries and pathologies. Being the central contributor to the developmental and adult neuroplasticity, the polysialylated form of Neural Cell Adhesion Molecule (PSA-NCAM) may prove to be a potential target to facilitate repair/regeneration after CNS injury and disease. Over the years, several experimental approaches have been developed to exploit the therapeutic potential of PSA-NCAM. Broadly, the studies focused on cell-transplantation strategies to alter PSA-NCAM properties at the injury site, injection of peptide based as well as synthetic PSA mimetics directly into the injury site or the application of PSA containing hydrogels and scaffolds as biomaterials. A comprehensive understanding of the PSA-based experimental approaches, as well as their pros and cons, is urgently required for successful implementation of this molecule in therapeutics. The current review, therefore, has been designed to give the readers a thorough account of all the diverse roles of PSA in the adult nervous system and the recent progress that has been made in developing PSA-based therapeutic approaches for neuroregeneration.

Anti-neuroinflammatory potential of Tylophora indica (Burm. f) Merrill and development of an efficient in vitro propagation system for its clinical use.

Neuroinflammation is a major risk factor associated with the pathogenesis of neurodegenerative diseases. Conventional non-steroidal anti-inflammatory drugs are prescribed but their long term use is associated with adverse effects. Thus, herbal based medicines are attracting major attraction worldwide as potential therapeutic candidates. Tylophora indica (Burm. f) Merrill is a valuable medicinal plant well known in Ayurvedic practices for its immunomodulatory, anti-oxidant, anti-asthmatic and antirheumatic activities. The present study aimed to elucidate the anti-neuroinflammatory potential of water and hydroalcoholic leaf extracts of micropropagated plants of T. indica using BV-2 microglia activated with lipopolysaccharide as an in vitro model system and development of an efficient reproducible protocol for its in vitro cloning. Non cytotoxic doses of the water and hydroalcoholic extracts (0.2µg/ml and 20µg/ml, respectively) were selected using MTT assay. α-Tubulin, Iba-1 and inflammatory cascade proteins like NFκB, AP1 expression was studied using immunostaining to ascertain the anti-neuroinflammatory potential of these extracts. Further, anti-migratory activity was also analyzed by Wound Scratch Assay. Both extracts effectively attenuated lipopolysaccharide induced microglial activation, migration and the production of nitrite via regulation of the expression of NFκB and AP1 as the possible underlying target molecules. An efficient and reproducible protocol for in vitro cloning of T. indica through multiple shoot proliferation from nodal segments was established on both solid and liquid Murashige and Skoog's (MS) media supplemented with 15µM and 10µM of Benzyl Amino Purine respectively. Regenerated shoots were rooted on both solid and liquid MS media supplemented with Indole-3-butyric acid (5-15µM) and the rooted plantlets were successfully acclimatized and transferred to open field conditions showing 90% survivability. The present study suggests that T. indica may prove to be a potential anti-neuroinflammatory agent and may be further explored as a potential therapeutic candidate for the management of neurodegenerative diseases. Further, the current study will expedite the conservation of T. indica ensuring ample supply of this threatened medicinal plant to fulfill its increasing demand in herbal industry.

Butanol Extract of Tinospora cordifolia Ameliorates Cognitive Deficits Associated with Glutamate-Induced Excitotoxicity: A Mechanistic Study Using Hippocampal Neurons.

Overstimulation of glutamate receptors leads to development of excitotoxicity, which is implicated as final destructive pathway in neurodegenerative diseases. Development of alternative therapeutic strategies effective against glutamate-induced excitotoxicity is much in demand. Herbal drug development is emerging as a major research area for the treatment of various debilitating diseases due to multimodal action and least side effects of herbal products. The current study was aimed to investigate neuroprotective potential of butanol extract of Tinospora cordifolia (B-TCE) against glutamate-induced excitotoxicity using primary hippocampal neurons as in vitro and Wistar strain albino rats as in vivo model systems. Molecular and behavioral parameters were studied to elucidate the underlying mechanism of beneficial effects of B-TCE. B-TCE treatment was also effective in prevention of anxiety, cognition, and motor-coordination deficits induced by glutamate. B-TCE pre-treatment protected the hippocampal neurons from glutamate-induced neurodegeneration and impaired plasticity. At molecular level, B-TCE was observed to attenuate overactivation of glutamate receptors. B-TCE promoted upregulation of ERK and AKT pathways of synaptic plasticity and cell survival in the hippocampus region of brain. This study provides first evidence of neuroprotective potential of B-TCE against glutamate-induced excitotoxicity in hippocampus region and suggests that B-TCE may act as a potential candidate for neuroprotective therapeutic approaches. A single compound 'tinosporicide' was further isolated from B-TCE, which was found to be effective at 800× lower concentration against glutamate-induced neurodegeneration under in vitro conditions.

5-Nonyloxytryptamine oxalate-embedded collagen-laminin scaffolds augment functional recovery after spinal cord injury in mice.

Polysialic acid (PSA) is crucial for the induction and maintenance of nervous system plasticity and repair after injury. In order to exploit the immense therapeutic potential of PSA, previous studies have focused on the identification and development of peptide-based or synthetic PSA mimetics. 5-Nonyloxytryptamine (5-NOT) has been previously reported as a PSA-mimicking compound for promoting functional recovery after spinal cord injury in mice. In order to explore the neuroregeneration potential of 5-NOT, the current study was based on a biomaterial approach using collagen-laminin (C/L) scaffolds. In in vitro studies, 5-NOT was observed to promote neurite outgrowth, migration, and fasciculation in cerebellar neuronal cells, whereas in 3D cell cultures it showed more ramification and complex Sholl profiles. 5-NOT promoted the survival and neurite length of cortical neurons when cocultured with glutamate-challenged astrocytes. In in vivo studies, spinal cord compression injury mice were used with immediate application of C/L hydrogels impregnated with 5-NOT. C/L + 5-NOT-treated mice demonstrated ∼75% of motor recovery 14 days after injury. Furthermore, this effect was shown to be dependent on the ERK-MAPK pathway and augmentation of cell survival. Thus, based on a biomaterial approach, our current study provides new insight for 5-NOT-containing hydrogels as a promising candidate to speed up recovery after central nervous system injuries.

Withania somnifera (L.) Dunal ameliorates neurodegeneration and cognitive impairments associated with systemic inflammation.

BACKGROUND: Systemic inflammation driven neuroinflammation is an event which correlates with pathogenesis of several neurodegenerative diseases. Therefore, targeting peripheral and central inflammation simultaneously could be a promising approach for the management of these diseases. Nowadays, herbal medicines are emerging as potent therapeutics against various brain pathologies. Therefore, in this contemporary study, the neuroprotective activity of Ashwagandha (Withania somnifera) was elucidated against the inflammation associated neurodegeneration and cognitive impairments induced by systemic LPS administration using in vivo rat model system. METHODS: To achieve this aim, young adult wistar strain male albino rats were randomized into four groups: (i) Control, (ii) LPS alone, (iii) LPS + ASH-WEX, (iv) ASH-WEX alone. Post regimen, the animals were subjected to Rotarod, Narrow Beam Walking and Novel Object Recognition test to analyze their neuromuscular coordination, working memory and learning functions. The rats were then sacrificed to isolate the brain regions and expression of proteins associated with synaptic plasticity and cell survival was studied using Western blotting and Quantitative real time PCR. Further, neuroprotective potential of ASH-WEX and its active fraction (FIV) against inflammatory neurodegeneration was studied and validated using in vitro model system of microglial conditioned medium-treated neuronal cultures and microglial-neuronal co-cultures. RESULTS: Orally administered ASH-WEX significantly suppressed the cognitive and motor-coordination impairments in rats. On the molecular basis, ASH-WEX supplementation also regulated the expression of various proteins involved in synaptic plasticity and neuronal cell survival. Since microglial-neuronal crosstalk is crucial for maintaining CNS homeostasis, the current study was further extended to ascertain whether LPS-mediated microglial activation caused damage to neurons via direct cell to cell contact or through secretion of inflammatory mediators. ASH-WEX and FIV pretreatment was found to restore neurite outgrowth and protect neurons from apoptotic cell death caused by LPS-induced neuroinflammation in both activated microglial conditioned medium-treated neuronal cultures as well as microglial-neuronal co-cultures. CONCLUSION: This extensive study using in vivo and in vitro model systems provides first ever pre-clinical evidence that ASH-WEX can be used as a promising natural therapeutic remedial for the prevention of neurodegeneration and cognitive impairments associated with peripheral inflammation and neuroinflammation.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 µM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.