Abstract
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.
Highlights
Microglial-mediated neuroinflammation is a key factor underlying the pathogenesis of various neurodegenerative diseases and an important target for the development of the neuroinflammation-targeted therapeutics
We further studied the effect of ASH-WEX and fraction IV (FIV) on various inflammatory pathways like nuclear factor-kB (NFkB), activator protein 1 (AP1), and pAktSER-473 which are known to mediate the production of various proinflammatory mediators like reactive nitrogen species (RNS), reactive oxygen species (ROS), inflammatory cytokines, and matrix metalloproteinases (MMPs) as a result of microglial cell activation
Standardization of ASH-WEX and FIV effective concentration To determine the effective dose of ASH-WEX and FIV, BV-2 microglial cells were treated with different concentrations of ASH-WEX (0.1–2 %) and FIV (5–15 μg/ml) for 48 h and the cell viability was quantified by the conversion of yellow MTT into formazan crystals
Summary
Microglial-mediated neuroinflammation is a key factor underlying the pathogenesis of various neurodegenerative diseases and an important target for the development of the neuroinflammation-targeted therapeutics. In response to infection or injury, they readily become activated and converted in the form of M1 phenotype displaying a variety of surface receptors, including the MHCs and complement receptors [2] They undergo dramatic morphological changes from resting ramified cells to activated amoeboid microglia [3]. Chronic activation of microglia results in neuroinflammation by secreting various neurotoxic and pro-inflammatory mediators causing demyelination and neuronal death [4]. These mediators include PGE2, monocyte chemoattractant protein-1 (MCP-1), inflammatory cytokines such as interleukin-1 β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) and free radicals such as nitric oxide (NO) and superoxide, fatty acid metabolites such as eicosanoids, and quinolinic acid [5]
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