Muskan Gupta

Aqueous extract from the Withania somnifera leaves as a potential anti- neuroinflammatory agent: a mechanistic study

BackgroundMicroglial-mediated neuroinflammation is a key factor underlying the pathogenesis of various neurodegenerative diseases and also an important target for the development of the neuroinflammation-targeted therapeutics. Conventionally, the nonsteroidal anti-inflammatory drugs (NSAIDs) are prescribed, but they are associated with long-term potential risks. Natural products are the cornerstone of modern therapeutics, and Ashwagandha is one such plant which is well known for its immunomodulatory properties in Ayurveda.MethodsThe current study was aimed to investigate the anti-neuroinflammatory potential of Ashwagandha (Withania somnifera) leaf water extract (ASH-WEX) and one of its active chloroform fraction (fraction IV (FIV)) using β-amyloid and lipopolysaccharide (LPS)-stimulated primary microglial cells and BV-2 microglial cell line. Iba-1 and α-tubulin immunocytochemistry was done to study the LPS- and β-amyloid-induced morphological changes in microglial cells. Inflammatory molecules (NFkB, AP1), oxidative stress proteins (HSP 70, mortalin), apoptotic markers (Bcl-xl, PARP), cell cycle regulatory proteins (PCNA, Cyclin D1), and MHC II expression were analyzed by Western blotting. Mitotracker and CellRox Staining, Sandwich ELISA, and Gelatin Zymography were done to investigate ROS, pro-inflammatory cytokines, and matrix metalloproteinase production, respectively. Ashwagandha effect on microglial proliferation, migration, and its apoptosis-inducing potential was studied by cell cycle analysis, migration assay, and Annexin-V FITC assay, respectively.ResultsASH-WEX and FIV pretreatment was seen to suppress the proliferation of activated microglia by causing cell cycle arrest at Go/G1 and G2/M phase along with decrease in cell cycle regulatory protein expression such as PCNA and Cyclin D1. Inhibition of microglial activation was revealed by their morphology and downregulated expression of microglial activation markers like MHC II and Iba-1. Both the extracts attenuated the TNF-α, IL-1β, IL-6, RNS, and ROS production via downregulating the expression of inflammatory proteins like NFkB and AP1. ASH-WEX and FIV also restricted the migration of activated microglia by downregulating metalloproteinase expression. Controlled proliferation rate was also accompanied by apoptosis of activated microglia. ASH-WEX and FIV were screened and found to possess Withaferin A and Withanone as active phytochemicals.ConclusionsThe current data suggests that ASH-WEX and FIV inhibit microglial activation and migration and may prove to be a potential therapeutic candidate for the suppression of neuroinflammation in the treatment of neurodegenerative diseases.

An Approach for the Selective Detection of Nitric Oxide in Biological Systems: An in vitro and in vivo Perspective.

A naphthalimide-based fluorescent probe, LyNP-NO, was designed and synthesized for the selective detection of exogenously and endogenously generated nitric oxide (NO) in C6 glial cells. In addition, LyNP-NO was also explored for monitoring endogenous NO levels in rat hippocampus at various tissue depths by stimulating the brain with N-methyl-d-aspartate (NMDA).

Tinospora cordifolia ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats.

Sleep deprivation (SD) leads to the spectrum of mood disorders like anxiety, cognitive dysfunctions and motor coordination impairment in many individuals. However, there is no effective pharmacological remedy to negate the effects of SD. The current study examined whether 50% ethanolic extract of Tinospora cordifolia (TCE) can attenuate these negative effects of SD. Three groups of adult Wistar female rats - (1) vehicle treated-sleep undisturbed (VUD), (2) vehicle treated-sleep deprived (VSD) and (3) TCE treated-sleep deprived (TSD) animals were tested behaviorally for cognitive functions, anxiety and motor coordination. TSD animals showed improved behavioral response in EPM and NOR tests for anxiety and cognitive functions, respectively as compared to VSD animals. TCE pretreatment modulated the stress induced-expression of plasticity markers PSA-NCAM, NCAM and GAP-43 along with proteins involved in the maintenance of LTP i.e., CamKII-α and calcineurin (CaN) in hippocampus and PC regions of the brain. Interestingly, contrary to VSD animals, TSD animals showed downregulated expression of inflammatory markers such as CD11b/c, MHC-1 and cytokines along with inhibition of apoptotic markers. This data suggests that TCE alone or in combination with other memory enhancing agents may help in managing sleep deprivation associated stress and improving cognitive functions.

A bodipy based fluorescent probe for evaluating and identifying cancer, normal and apoptotic C6 cells on the basis of changes in intracellular viscosity

The applications of a bodipy based probe 1 for the identification of diseased cell population out of normal cells on the basis of changes in intracellular viscosity have been explored. Probe 1 works on the principle of restriction of rotation in viscous medium and the molecular rotor nature of probe 1 is supported by low temperature 1H NMR and variable dihedral angle DFT and TD-DFT studies. More importantly, probe 1 is the first probe which shows its practical application in monitoring micro-viscosity changes in a cell based model system of undifferentiated, differentiated and apoptotic C6 glial cells. Further, probe 1 can effectively monitor the apoptosis pathway by showing an increase in fluorescence intensity from cancerous cells to apoptotic cells via real time live-cell video imaging. Moreover, the viscosity changes in living cells were proved by fluorescence lifetime imaging (FLIM) studies, flow cytometry using Annexin-V and Bcl-xl expression by immunocytofluorescence (ICC) and western blot analysis.

A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: in vitro and in vivo Applications

A fluorescein-based fluorescent probe has been designed and synthesised that selectively detects H2 S in aqueous medium, among various analytes tested. This fluorescein-based fluorescent probe has also been successfully utilised for real-time imaging of exo- and endogenously produced H2 S in cancer cells and normal cells. Moreover, the probe can also detect H2 S in the rat brain hippocampus at variable depths and in living nematodes.

Obesity and Neuroinflammation

Obesity is the core and baseline component of metabolic syndrome and is a major risk factor for many diseases like Type II diabetes, cardiovascular diseases, hypertension, stroke and neurodegenerative diseases. Sedentary lifestyle, unhealthy eating habits and genetic predisposition are responsible for the increasing prevalence of obesity worldwide. Chronic overnutrition causes low-grade inflammation in several peripheral tissues as well as central nervous system, particularly hypothalamus. Activation of various proinflammatory pathways such as IKKβ/NF-κB, JNK and PKR are thought to be the major players in the induction of systemic and central inflammation. Further, neuroinflammation causes intracellular disturbances and exacerbates various stresses such as oxidative stress, ER stress and autophagic defects leading to impaired neurohormonal signalling as well as autonomic regulation of nutrient metabolism and energy balance. As obesity poses major health threat, effective therapies to minimize obesity-related comorbidities are surely needed. By targeting the inflammatory component, the progression of obesity can be slowed down. In vivo studies from our lab suggest that Withania somnifera helps to reduce hypothalamic inflammation triggered by high-fat-diet-induced obesity. Various lifestyle interventions along with herbal supplementation may effectively help to prevent obesity and its associated pathologies.

Stitching of tyrosine and 10H-acridin-9-one: turn-ON fluorescence in the narrow pH range 7.4–8.5 and intracellular labelling of cancer cells

We tailored 10H-acridin-9-one and (S)-tyrosine into 3-(4-hydroxyphenyl)-2-[(9-oxo-9,10-dihydroacridine-4-carbonyl) amino]propionic acid (2). 2 underwent pH dependent protonation/deprotonation and the effect was harnessed in terms of change in the fluorescence. The characteristic fluorescence change in the molecule in the pH 7.5 ± 1 range and its cell permeability allowed us to label cancer cells.

Neuromodulatory Role of Withania somnifera

Withania somnifera, commonly known as Ashwagandha, is one of the important herbs being used in Ayurveda since times immemorial. It has been classified as a ‘rasayana’ herb owing to its adaptogenic and rejuvenating properties. Both root and leaf extracts of the plant have been used for the treatment of various disorders such as cancer, anxiety, inflammation and various neurological disorders due to its wide array of properties. This chapter focuses on the effects of W. somnifera in various CNS disorders. Various pre-clinical studies investigating the use of W. somnifera in modulation of neuroplasticity, anxiety, neuroinflammation and neuroprotection have been discussed in detail. A plethora of studies confirm the use of W. somnifera and its active phytochemicals (Withaferin-A, Withanone, Withanoside IV, Withanolide A, sitoindosides VII-X) alone or in combination as potential therapeutic agents. W. somnifera can be incorporated as an important dietary supplement for management of anxiety and associated cognitive and functional impairments.